PROGRAMMED FUNCTION OF THE TAMMAR MAMMARY GLAND; The role of milk proteins in gut development
by
Sanjana Kuruppath Master of Science (MSc)
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Deakin University
February, 2014
Acknowledgements
Thank You Almighty!
I thank my family, friends and colleagues for their support without which
completion of this thesis would not have been possible.
First and most of all, I would like to express my sincere and deepest gratitude to my PhD supervisor Professor Kevin R Nicholas, for his constant guidance and support. I would not have been in a position to write this if he had not selected me as a student, given an opportunity to come to Australia and pursue my career in the field of scientific research. I assure, I had the best supervisor for my PhD. Under him, I had the freedom to manage and execute my project with his continuous support and timely guidance because of which I gradually grew as a confident researcher. I was always surprised to see him welcoming his students with a smiling and encouraging face, anytime they knock at his door! I greatly appreciate his commitment and passion towards research and the magical ability to continuously and convincingly promote his students to grow independently. I also thank him, from the bottom of my heart, for all the support and courage he gave me in my personal life too.
I thank my co-supervisors, Dr Christophe Lefevre and Dr Julie A Sharp. I thank Christophe for his advice and assistance, mainly in the field of bioinformatics.
Without him this would have been totally impossible. And above all, for always reminding to hold on to the smile, whatever happens in life! I also thank Julie for all
4 the technical advice she gave during my PhD. I thank both of them for proof-reading my research work, and for all the useful comments and remarks which helped to improve my thesis.
I thank Dr Helen E Abud for welcoming me to her lab in Monash University.
The collaboration with her group gave guidance and assistance for a major part of my research. I also extend my deepest gratitude to Dr Kenneth McColl for the much needed advice and assistance in histology which was a new area for me. For introducing him, I thank Tamsyn Crowley. I would like to thank Professor Norman
Saunders lab of Melbourne University for assisting in getting samples for my project.
I thank all my lab mates (Stephen, Laurine, Alicia, Sugeetha, Ashalyn,
Ashwanth, Swathi, Amit, Akihiko, Gunveen, Venky, Meagan) for all the help and support. Their presence always motivated me to come back to the lab and work. I have gathered a lot of nice memories, especially during our lab lunches and during the wallaby “hunts”! I thank Liz for all the support. Her energy and smile has always been a motivation for me. I would also like to thank my lab mates in Monash
University (Thierry, Helen, Lisa, Katja and Sivan) who guided me in different aspects.
My friends…Without whom, this whole PhD and thesis would have been impossible. I thank all of them, for their presence, tolerance, patience and support. I thank Suneel and Nagu, for their constant support, even when they were far away from me. I personally thank Laurine, Adarsh, Swathi, Rajesh, Raju, Swapna, Mohan,
Madhu and Ananthi. I thank “Almond family”, “Terracall family’ and “DeWaard
5 family”. Their presence never made me feel that I was away from my near and dear ones.
I thank GATES foundation for the funding provided for a significant part of my PhD project. I thank Deakin University for providing scholarship during my
PhD.
And last but not the least, my family - Thanks for everything!
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Communications
Publications under submission
Kuruppath, S., Sharp, J., Lefevre, C., Murphy, R. M. and Nicholas, K. R. (2014).Comparative analysis of Caveolins of mouse and tammar wallaby: Role in regulating mammary gland function.
List of publications Kuruppath, S., Bisana, S., Sharp, J. A., Lefevre, C., Kumar, S. and Nicholas, K. R. (2012). Monotremes and marsupials: comparative models to better understand the function of milk. Journal of Biosciences 37(4): 581-588.
Kuruppath, S., Kumar, A., Modepalli, V. N.,Ngo, K. P., Gras, S. L. and Lefevre, C. Buffalo Milk Transcriptomics. Submitted to the 10th WORLD Buffalo Congress 2013, Phuket Thailand
Book Chapters Published Kumar, A., Buscara, L., Kuruppath, S., Ngo, K. P., Nicholas, K. R. and Lefèvre, C. The emerging role of miRNAs in the lactation process. Lactation: Natural Processes, Physiological Responses and Role in Maternity, Eds: Lisa M. Reyes Cruz and Douglas C. Ortiz Gutierrez, Nova Science Publishers.
Poster presentation at conferences
Sanjana Kuruppath, Julie A Sharp, Christophe M Lefevre and Kevin R Nicholas, A comparative study of the potential role of Caveolins in mouse and tammar wallaby lactation. Poster presented in “Gordon Research Conference on Mammary Gland Biology”, 12th-17th June 2011, Salve Regina University, Newport, RI.
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Sanjana Kuruppath, Julie A Sharp, Christophe M Lefevre and Kevin R Nicholas, A study of the potential role of Caveolins in mouse and tammar wallaby lactation. Poster presented in “8th Annual Symposium on Milk Genomics and Human Health”, 15th-17th November 2011, Melbourne, Australia.
Sanjana Kuruppath, Julie A Sharp, Christophe M Lefevre and Kevin R Nicholas, A study of the potential role of Caveolins in mouse and tammar wallaby lactation. Poster presented in “ASMR Victorian Student Research Symposium”, 4th June 2012, Bio21 Institute, Parkville, VIC, Australia.
Sanjana Kuruppath, Helen E Abud, Julie A Sharp, Christophe Lefevre and Kevin R Nicholas, Tammar Wallaby: a marsupial model to understand the role of milk in regulating stomach development. Poster presented at the 10th “International Milk Genomics and Human Health Annual Symposium”, October 1-3, 2013 in Davis, California.
Oral presentation at conferences Sanjana Kuruppath, Helen E Abud, Julie A Sharp, Christophe Lefevre and Kevin R Nicholas, Tammar Wallaby: a marsupial model to understand the role of milk in regulating stomach development. Presented at the 10th “International Milk Genomics and Human Health Annual Symposium”, October 1-3, 2013 in Davis, California. Student travel award.
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Table of Contents
CHAPTER 1: General Introduction and Literature Review...... 29 1.1 Introduction...... 30 1.2 Evolution of Mammals...... 31 1.3 Mammary gland structure and lactation...... 35 1.4 Lactation: comparison between eutherians and marsupials...... 35 1.4.1 Eutherians...... 35 1.4.2 Marsupials...... 36 1.5 Milk and its components...... 40 1.5.1 Carbohydrate...... 40 1.5.2 Protein...... 40 1.5.3 Fat...... 42 1.5.4 Growth factors...... 43 1.5.5 MicroRNA...... 43 1.6 Regulation of lactation...... 44 1.6.1 Hormones...... 46 1.6.1.1 Insulin...... 46 1.6.1.2 Glucocorticoid...... 47 1.6.1.3 Prolactin...... 47 1.6.1.4 Hormonal regulation of milk protein gene expression: comparison between mouse and tammar wallaby...... 48 1.6.2 Extracellular Matrix...... 49 1.6.2.1 Eutherians...... 50 1.6.2.2 Marsupials...... 50 1.6.3 Different cell types...... 51 1.6.3.1 Myoepithelial cells...... 51 1.6.3.2 Adipocytes and stromal fibroblasts...... 52 1.6.3.3 Caveolins; potential influence on mammary function by a mechanism of cell-cell interaction...... 52
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1.6.4 Milk factors...... 53 1.7 GIT development and maturation in mammals...... 54 1.7.1 Stomach development in mammals...... 55 1.7.1.1 Stomach development in mouse...... 58 1.7.1.1.1 Gastric mucosa development...... 58 1.7.1.1.2 Muscle layer development...... 59 1.7.1.2 Stomach development in Opossum and Tammar Wallaby.....59 1.8 Role of milk in GIT development...... 61 1.9 Aim and scope of the study...... 65
CHAPTER 2: Comparative analysis of caveolins in mouse and tammar Wallaby: Role in regulating mammary gland function...... 67 2.1 Introduction...... 68 2.2 Material and Methods...... 70 2.2.1 Animals...... 70 2.2.2 Tissue culture...... 70 2.2.3 RNA extraction and reverse transcription PCR...... 71 2.2.4 Quantitative polymerase chain reaction...... 71 2.2.5 Sequence Analysis...... 72 2.2.6 Preparation of mouse mammary gland sections and immunohistochemistry...... 72 2.2.7 Statistical analysis...... 72 2.3 Results...... 74 2.3.1 A time course for hormonal regulation of Caveolin 1 and 2 genes in mouse mammary gland explants...... 74 2.3.2 Localisation of Caveolin 1 in mouse mammary gland...... 76 2.3.3 Comparative genome organisation and protein alignment of Caveolin 1 and 2 of tammar wallaby and mouse...... 79 2.3.4 Expression profile of Caveolin 1 and 2 across tammar wallaby lactation cycle...... 82
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2.3.5 Hormonal regulation of Caveolin 1 and 2 genes in tammar wallaby mammary gland explants...... 82 2.4 Discussion...... 86
CHAPTER 3: Effect of tammar wallaby milk on in vitro stomach models...... 92 3.1 Introduction...... 93 3.2 Material and methods...... 97 3.2.1. Animal ethics and sample collection...... 97 3.2.2 Milk collection and processing...... 97 3.2.3 Analysis of gene expression by RT-PCR...... 98 3.2.4 Imaging and microscopy...... 98 3.2.5 Effect of phase 2B tammar wallaby milk on embryonic stomach explant culture...... 99 3.2.5.1 Embryonic stomach explant culture...... 99 3.2.5.2 Histology...... 102 3.2.5.3 Analysis of phase 2B tammar wallaby milk on gastric gene marker expression and stomach epithelial cell proliferation...... 102 3.2.5.3.1 BrdU incorporation assay...... 102 3.2.5.3.2 Immunohistochemistry...... 103 3.2.5.4 Analysis of tammar wallaby 2A milk on gastric gene marker...... 104 3.2.6 Analysis of phase 2A tammar wallaby milk on gastric organoid model...... 104 3.2.6.1 Gastric gland unit isolation and culture conditions...... 104 3.2.7 Statistical analysis...... 105 3.3 Results...... 107 3.3.1 Effect of tammar wallaby milk on embryonic stomach explants...... 107 3.3.1.1 Establishment of embryonic stomach explant culture...... 107
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3.3.1.2 Effect of phase 2B tammar wallaby milk on stomach explant culture...... 109 3.3.1.2.1 Effect of phase 2B tammar wallaby milk on gastric gene markers...... 109 3.3.1.2.2 Effect of phase 2B tammar wallaby milk on pattern of proliferation and apoptosis in embryonic stomach explants...... 110 3.3.1.3 Effect of phase 2A tammar wallaby milk on stomach explant culture...... 112 3.3.2 Effect of phase 2A tammar wallaby milk on gastric organoid culture system...... 115 3.3.2.1 Development of gastric organoid culture system...... 115 3.3.2.2 Gene expression analysis in gastric organoids...... 115 3.3.2.3 Effect of phase 2A tammar wallaby milk on gene expression in gastric organoids...... 118 3.4 Discussion...... 119
CHAPTER 4: Putative role of milk in stomach development of gray short-tailed opossum (Monodelphis domestica)...... 128 4.1 Introduction...... 129 4.1.1 Stomach development in mammals; Monodelphis domestica as a new model...... 132 4.2 Material and methods...... 134 4.2.1 Animal Ethics...... 134 4.2.2 Stomach tissue collection...... 134 4.2.3 Histochemistry...... 135 4.3 Results...... 135 4.4 Discussion...... 149 4.4.1 Stomach development in gray short-tailed opossum...... 149 4.4.2 Possible role of milk in stomach development...... 151
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CHAPTER 5: Regulation of gut development; a bioinformatic approach to identify putative bioactives of early phase tammar wallaby milk also present in human amniotic fluid and placenta...... 156
5.1 Introduction...... 157 5.2 Material and Methods...... 160 5.2.1 Microarray based transcriptome profiling of tammar wallaby mammary gland across lactation...... 160 5.2.1.1 Identification of secreted proteins of phase 2A tammar wallaby lactation...... 160 5.2.2 RNA-seq based transcriptome profiling of tammar wallaby mammary gland across lactation...... 161 5.2.2.1 Mammary gland collection and RNA extraction...... 161 5.2.2.2 RNA-seq...... 162 5.2.2.3 RNA-seq data analysis...... 162 5.2.2.4 Identification of genes encoding secreted proteins expressed during phase 2A tammar wallaby lactation...... 163 5.2.3 Proteome and transcriptome datasets of human amniotic fluid and placenta...... 164 5.2.4 Venn diagram – Comparison of tammar wallaby and human bioactives...... 165 5.3 Results...... 165 5.3.1 Microarray based transcriptome profiling of tammar wallaby mammary gland across lactation...... 165 5.3.1.1 Genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland identified by microarray transcriptome profiling...... 165 5.3.1.2 Venn diagram depicting the number of bioactives of tammar wallaby phase 2A lactation and/or human amniotic fluid/ placenta...... 167
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5.3.1.3 Comparison of genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland with genes/proteins of human amniotic fluid/placenta...... 167 5.3.2 RNA-seq based transcriptome profiling of tammar wallaby mammary gland across lactation...... 171 5.3.2.1 Milk protein gene expression...... 171 5.3.2.2 Genes expressed in tammar wallaby mammary gland during the lactation cycle...... 171 5.3.2.3 Genes encoding secreted proteins expressed during tammar wallaby phase 2A lactation...... 183 5.3.2.4 Venn diagram depicting the number of bioactives of tammar wallaby phase 2A lactation and/or human amniotic fluid/ placenta...... 185 5.3.2.5 Comparison of genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland with genes/proteins of human amniotic fluid/placenta...... 185 5.4 Discussion...... 189 5.4.1 Gene transcriptome profiling to identify putative bioactives in tammar wallaby phase 2A milk...... 190 5.4.2 Putative bioactive proteins with plausible role in regulating gut development...... 194 5.4.3 Putative bioactive proteins with no known role in regulating gut development...... 201 5.4.4 Conclusion...... 205
CHAPTER 6: General Discussion...... 207 6.1 Regulation of mammary gland function in tammar wallaby...... 209 6.2 Role of tammar wallaby milk in stomach development...... 213 6.3 Gray short-tailed opossum: An alternative marsupial model to understand the putative role of milk in stomach development...... 217
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6.4 A bioinformatic approach to identify milk bioactives of tammar wallaby...... 222 6.5 Tammar wallaby as a legitimate biomedical research model...... 223
BIBLIOGRAPHY...... 225
APPENDICES...... 274
Appendix 1: Expression profiles of secreted proteins of phase 2A...... 275
Appendix 2: Expression profiles of phase 2B specific genes...... 289
Appendix 3: Expression profiles of phase 3 specific genes...... 293
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List of Figures and Tables
CHAPTER 1
Figure 1.1: Evolution of mammals and lactation...... 34
Figure 1.2: Tammar Wallaby (Macropus eugenii)...... 38
Figure 1.3: The lactation cycle of tammar wallaby...... 39
Figure 1.4: Components and regulators of mammary gland function...... 45
Figure 1.5: Diagrammatic representation of the gastric gland...... 57
CHAPTER 2
Table 2.1 Primer used in qPCR...... 73
Figure 2.1: The levels of ȕ-Casein, Caveolin 1 and Caveolin 2 expression in mouse mammary explants treated with lactogenic hormones...... 75
Figure 2.2: The level of expression of genes in mouse mammary gland treated with lactogenic hormones...... 77
Figure 2.3: Localisation of Caveolin 1 by immunohistochemistry in mouse mammary gland during pregnancy...... 78
Figure 2.4: Comparative genome organisation of Caveolin 1 and 2 of tammar wallaby and mouse...... 80
Figure 2.5: Protein sequence alignment of tammar wallaby and mouse Caveolin 1 and 2...... 81
Figure 2.6: qPCR analysis of gene expression levels in tammar wallaby mammary gland tissue during the lactation cycle...... 83
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Figure 2.7: qPCR analysis of gene expression levels on day 2 and day 4 in tammar wallaby mammary explants treated with lactogenic hormones...... 85
CHAPTER 3
Table 3.1: Primer sequences used for qPCR...... 99
Figure 3.1: Protocol for culturing of mouse embryonic stomach...... 101
Figure 3.2: Experimental design for stomach explant culture model...... 101
Figure 3.3: Protocol for isolation of gastric glands for stomach organoid culture...106
Figure 3.4: Experimental design for gastric organoid model...... 106
Figure 3.5: E14 embryonic stomach explants cultured for three days in media containing 0.5% to 10% FCS...... 108
Figure 3.6: Gene expression analysis by qPCR in day 0 and day 3 embryonic stomach explants...... 109
Figure 3.7: Gene expression in E14 embryonic stomach explants at day 3 following treatment with phase 2B milk...... 110
Figure 3.8: Effect of phase 2B tammar wallaby milk on pattern of proliferation in
E15 embryonic stomach explants at 24 h...... 111
Figure 3.9: Effect of phase 2B tammar wallaby milk on epithelial cell death in E15 embryonic stomach explants at 24 h...... 112
Figure 3.10: Gene expression analysis by qPCR in embryonic stomach explants at day 0 and day 2 of treatment with tammar wallaby milk...... 114
Figure 3.11: Growth of gastric organoids...... 116
Figure 3.12: Gene expression analysis in gastric organoids by qPCR...... 117
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Figure 3.13: Effect of phase 2A tammar wallaby milk on gene expression levels in gastric organoids...... 119
CHAPTER 4
Figure 4.1: Reproductive cycle and developmental stages of gray short-tailed opossum...... 131
Figure 4.2: Milk composition of gray short-tailed opossum...... 131
Figure 4.3: Gastrointestinal tract of gray short-tailed opossum...... 134
Figure 4.4: Transverse section of an eight day old opossum stomach...... 137
Figure 4.5: Transverse section of a 14 day old opossum stomach (PAS stain)...... 138
Figure 4.6: Transverse section of a 35 day old opossum stomach (lower magnification)...... 138
Figure 4.7: Transverse section of a 35 day old opossum stomach (higher magnification)...... 139
Figure 4.8: Transverse section of a 35 day old opossum stomach (H and E stain)...... 140
Figure 4.9: Transverse section of a 45 day old opossum stomach...... 141
Figure 4.10: Transverse section of a 45 day old opossum stomach (PAS stain).....142
Figure 4.11: Transverse section of a 45 day old opossum stomach (H and E stain)...... 143
Figure 4.12: Transverse section of a 61 day old opossum stomach...... 144
Figure 4.13: Transverse section of a 75 day old opossum stomach...... 145
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Figure 4.14: Transverse section of a 75 day old opossum stomach (PAS stain)...... 146
Figure 4.15: Transverse section of stomach of an adult opossum...... 147
Figure 4.16: Transverse section of stomach of an adult opossum (PAS stain)...... 148
Figure 4.17: Stomach development in tammar wallaby...... 155
CHAPTER 5
Table 5.1: Genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland identified by microarray transcriptome profiling...... 166
Table 5.2: Comparison of genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland with genes/proteins of human amniotic fluid/placenta...... 170
Table 5.3: Data store report of data aligned to Monodelphis domestica genome...... 174
Table 5.4: Genes expressed in the tammar wallaby mammary gland during phase 2A lactation identified by RNA-seq data analysis...... 175
Table 5.5: Genes expressed in the tammar wallaby mammary gland during phase 2B lactation identified by RNA-seq data analysis...... 181
Table 5.6: Genes expressed in the tammar wallaby mammary gland during phase 3 lactation identified by RNA-seq data analysis...... 182
Table 5.7: Genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland identified by RNA-seq transcriptome profiling...... 184
Table 5.8: Comparison of genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland with genes/proteins of human amniotic fluid/placenta...... 188
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Figure 5.1: Venn diagram depicting the common proteins in human amniotic fluid
(proteome and transcriptome list) and tammar wallaby phase 2A milk (microarray transcriptome list)...... 168
Figure 5.2: Venn diagram depicting the proteins common to the human placenta
(proteome and transcriptome list) and tammar wallaby phase 2A milk (microarray transcriptome list)...... 169
Figure 5.3: Milk protein gene expression level...... 173
Figure 5.4: A 2D-area graph representing the expression profile of genes expressed in the tammar wallaby mammary gland during phase 2A lactation...... 180
Figure 5.5: A 2D-area graph representing the expression profile of genes expressed in the tammar wallaby mammary gland during phase 2B lactation...... 181
Figure 5.6: A 2D-area graph representing the expression profile of genes expressed in the tammar wallaby mammary gland during phase 3 lactation...... 182
Figure 5.7: Venn diagram depicting the number of common proteins between human amniotic fluid (proteome and transcriptome list) and tammar wallaby phase 2A milk
(RNA-seq based transcriptome list)...... 186
Figure 5.8: Venn diagram depicting the number of common proteins between human placenta (proteome and transcriptome list) and tammar wallaby phase 2A milk
(RNA-seq based transcriptome list)...... 187
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Abbreviations
%Percentage °C Degree Celsius ACL Asynchronous Concurrent Lactation Į-lac Alpha-lactalbumin BLG Beta lactoglobulin BSA Bovine serum albumin cDNA Complementary deoxyribonucleic acid
CO2 Carbon dioxide ddH2O Double distilled water DNA Deoxyribonucleic acid dNTPs Deoxyribonucleotides dpc Days post coitus EEmbryo EB Elution buffer ECM Extracellular matrix EGF Epithelial growth factor ELP Early lactation protein EST Expressed sequence tag F Cortisol FCS Fetal calf serum FIL Feedback inhibitor of lactation FP Forward primer GAPDH Glyceraldehyde 3-phosphate dehydrogenase
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GIT Gastrointestinal tract h Hour
H2O Water H&E Hematoxylin and eosin IInsulin IGF-1 Insulin-like growth factor 1 LLP-A Late lactation protein A LLP-B Late lactation protein B MMolar ȝJ Micro gram miRNA Micro ribonucleic acid (microRNA) μl Micro litre mM Milli molar ȝ0 Micro molar ml Millilitre mRNA Ribonucleic acid messenger Mya Million years ago ng Nano gram NIH National Institute of Health OD Optical density P Prolactin P1 Phase 1 P2A Phase 2A P2B Phase 2B P3 Phase 3 PAS Periodic acid schiff PBS Phosphate buffered saline
22
PFA Paraformaldehyde PCR Polymerase chain reaction pH Power of hydrogen PY Pouch young qPCR Quantitative Polymerase Chain Reaction RNA Ribonucleic acid RNA-seq RNA Sequencing RP Reverse primer RPY Removal of pouch young RT-PCR Reverse transcription polymerase chain reaction SD Standard deviation SEM Standard error of the mean STAT5 Signal transducer activator of transcription 5 TGF-ß Transforming growth factor beta UUnit WAP Whey acidic protein
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Abstract
All mammals are characterized by the capacity to lactate and feed the young.
Evolutionary pressure on reproductive strategy has classified these mammals into three subclasses: the monotremes, the marsupials and the eutherians. Among these, the marsupials have a short gestation and give birth to an altricial young that is totally dependent upon maternal milk for nutrition, with the progressive changes in milk composition influencing growth and development. This change in milk composition appears to program development of the young. In contrast, eutherians have invested in an extended intrauterine development of the young and produce milk of relatively constant composition, apart from the initial colostrum. In the present study, we have employed marsupial models to better understand the role of milk in regulating stomach development of the pouch young. To examine the regulation of the lactation cycle, a comparison was made between a eutherian model, the mouse (Mus musculus) and a marsupial model, the tammar wallaby (Macropus eugenii). Subsequently, to understand the role of milk in stomach development, the milk of tammar wallaby was examined for a direct effect on mouse in vitro stomach models. In addition, stomach development in another marsupial model, the grey short-tailed opossum (Monodelphis domestica) was examined to determine the potential role of milk on stomach development.
Caveolins (Caveolin 1, 2 and 3) are structural components of the Caveolae, the flask shaped invaginations on the plasma membrane, and play role in diverse cellular functions. Recent studies in the mouse have shown an inverse correlation
24 between expression of the Caveolin 1 and 2 genes and the onset of milk production around parturition. These studies also suggested that Caveolin 1 gene expression was regulated by prolactin and that Caveolin 1 acts subsequently by limiting the response of the mammary gland to prolactin. Our current study using mammary explants from pregnant mice shows that a complex interplay of hormones (I, F, P) is involved in regulating expression of Caveolin 1 and 2 genes which is consistent with that seen for the regulation of milk protein genes. We also confirmed the earlier reports showing the differential expression of Caveolin 1 across lactation with localisation of
Caveolin specific to myoepithelial cells and adipocytes. This probably suggests a cell-cell interaction in regulating milk protein production in the mammary epithelial cells in a paracrine fashion. Comparative studies with the tammar wallaby Caveolin
1 and 2 demonstrated 70-80% homology in the amino acid sequence. However, in contrast to the mouse there was a significantly increased level of expression of
Caveolin 1 and 2 in the mammary gland during lactation as compared to pregnancy and involution. In contrast to the mouse, this differential expression pattern in the tammar wallaby was not regulated by the lactogenic hormones when examined in mammary explant culture. These data suggest that a difference in function of
Caveolins may possibly be related to the difference in reproductive strategy between eutherians and marsupials. It can be presumed that evolutionary pressure on the function of the Caveolin protein may have led to a more hormone responsive regulatory role in mouse lactation.
Previous studies in the tammar wallaby have reported that cross fostering and exposure to later stage milk accelerated the developmental maturation of stomach by
25 regulating the transition of fore-stomach to a cardia glandular phenotype. However, studies examining the role of early and mid-phase milk on stomach development are limited. In the current study, milk collected from early (phase 2A) and mid-phase
(phase 2B) of tammar lactation was examined for effect on stomach proliferation and differentiation using two in vitro stomach models. First, a stomach explant model was developed by culturing mouse embryonic (E14 or E15) whole stomach. Second, a gastric organoid model was developed from the glands of stomach pylorus of adult mice. In the embryonic stomach explant model, milk of day 65 and day 92 lactation resulted in an increased expression level of gastric gene markers for mucus cells
(Trefoil factor 2) and enteroendocrine cells (Somatostatin) respectively. Similarly in the gastric organoid model, day 60 milk resulted in an increased expression of chief cell marker with a parallel down regulation of stem cell marker Lgr5. Furthermore, milk collected from mid-phase lactation resulted in an increased epithelial proliferation with no difference in apoptotic cells when compared to the control treatment in the embryonic explant model. Thus, the current study demonstrated an effect by early phase tammar wallaby milk on differentiation in both embryonic stomach explant and gastric organoid model, whereas mid-phase milk showed a proliferative effect in the embryonic stomach explant model.
The unique reproductive strategy of the marsupials in which the major development of the altricial young occurs after birth provides an opportunity as a comparative model for understanding the developmental events occurring during the embryonic stage in eutherians. The present study demonstrated the timing and sequence of different developmental events in the stomach of grey short-tailed
26 opossum. This marsupial, being an omnivore with a monogastric stomach, would be a better comparison to eutherians like human than the herbivorous tammar wallaby with a digastric stomach. In the opossum, different cell types of the stomach including mucus cells, chief cells and the parietal cells develop during the postnatal period, which might be hypothesised to be regulated by the mother’s milk.
It can be assumed that the signals for the development provided by amniotic fluid in human may be comparable with signals delivered via the marsupial milk of varying composition. To identify human orthologues of amniotic fluid and placental proteins, genes coding for secreted proteins that were highly expressed during the early lactation phase of tammar wallaby was compared with genes/proteins expressed in human amniotic fluid or placenta. Initially, genes coding for secreted proteins expressed during phase 2A tammar wallaby lactation were identified utilising cDNA microarray analysis. The list of candidate genes common to early phase tammar wallaby milk and either human amniotic fluid or placenta included
C1orf160, Calmin, GM2 ganglioside activator, TINAGL1 and Periostin which may have putative role in regulating development of specific organs of the young.
Further, a detailed RNA-seq analysis was performed using RNA extracted from the mammary gland across tammar lactation cycle. This RNA-seq dataset identified the genes coding for secreted proteins expressed during phase 2A tammar wallaby lactation. The list of candidate genes common to early phase tammar wallaby milk and either human amniotic fluid or placenta according to this approach included
Clusterin, Decorin, Insulin-like growth factor-binding protein 7, Laminin subunit alpha-2 precursor and Tenascin which may have putative role in regulating
27 development of specific organs of the young. This is preliminary evidence to show that the proteins provided by amniotic fluid and placenta in the human are also provided by early phase milk in the tammar wallaby. However, detailed investigation is required to examine the role of these individual candidate bioactives.
To conclude, we can propose that the tammar wallaby with an adapted reproductive strategy is increasingly becoming relevant as a legitimate biomedical research model for understanding the regulation of developmental events in mammals including the eutherians.
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CHAPTER 1
General Introduction and Literature Review
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1.1 Introduction Mammals are a subclass of warm blooded vertebrates that have been specifically grouped into the class termed Mammalia by Carl Linnaeus in 1758 based on their capacity to lactate and feed their young (Vorbach et al. 2006). Lactation, the dominant and defining feature of this subclass is the process by which the mother secretes nutrient-rich milk from the mammary gland for the young. In addition to providing nutrition to the young for growth and development, milk also provides protection from infection by the secretion of anti-microbial components. Pond (1984) summarized the process of lactation as a means by which the mother’s food obtained from the surrounding environment is converted into a form suitable for the growth and development of the offspring immediately after birth when the young may face difficulty in finding food. Apart from containing components for the suckled young’s development and protection, it’s now apparent that milk also provides signals which program the development of the mother’s mammary gland (Khalil et al. 2011; Trott et al. 2003; Wanyonyi et al. 2013a).
Based on reproductive strategy, mammals have been classified into eutherians (e.g., human, mice, dog and cow), metatherians (e.g., kangaroo and opossum) and prototherians (e.g., platypus and echidna) (Bininda-Emonds et al. 2007; Hedges and Kumar 2009; Lefèvre et al. 2010). In all these mammals, reproduction has been divided into two phases; the initial gestation phase during which the mammary gland undergoes gradual development preparing itself for the complex process of lactation and the later lactation phase which involves synthesis and secretion of milk after parturition. In eutherians, where lactation has been studied extensively, both gestation and lactation are generally of comparable duration (Hayssen 1993). In these species, the development of the young occurs mainly in utero and the process is driven by the placenta and amniotic fluid (Garnica and Chan 1996; Jansson and Powell 2007; Mulvihill et al. 1985; Tong et al. 2009). The newborn is well-developed and further development occurs during the lactation period, when the young consumes milk of relatively constant composition apart from the initial colostrum (Langer 2009). In contrast to eutherians, marsupials, which
30 represent one of the most refined lactation programs, have a shorter gestation followed by an extended lactation (Tyndale-Biscoe and Renfree 1987). They give birth to an altricial young and the physiological and biochemical development of this young occurs during the extended lactation period and is driven by the milk of varying composition (Tyndale-Biscoe and Janssens 1988). Monotremes, the third and the most ancient group of living mammals, do not give birth to a young, but instead lay parchment eggs which are covered by a leathery shell (Oftedal 2002). Initially, these eggs are incubated outside the mother’s body during which the secreted milk keeps it moist and later the hatchling’s development is completely dependent on milk throughout the period of suckling (Griffiths 1978) which is longer than gestation as in marsupials. Milk contains a wide range of components with diverse roles, but the function of these components remains to be elucidated. Marsupials which have a diverse reproductive strategy present a sophisticated lactation program that provides new opportunities to identify these regulatory molecules that are not readily identified in higher mammals (Sharp et al. 2009). Therefore, in the current study, the adapted reproductive strategy of marsupial animal models like tammar wallaby (Macropus eugenii) and gray short-tailed opossum (Monodelphis domestica)has been exploited to better understand the role of milk (Kuruppath et al. 2012). This review will therefore focus on a comparison between eutherian and marsupial lactation and its regulation, and further the significance of different components of milk on diverse physiological processes. One of the major aims of the present study is to examine the role of milk on stomach development utilising marsupial models and therefore, stomach development in eutherians and marsupials is reviewed. In brief, the areas which require further investigation utilising marsupials as a legitimate model for biomedical research have been highlighted in this review (Tyndale-Biscoe and Janssens 1988).
1.2 Evolution of Mammals
As stated by Capuco and Akers (2009), mammary glands and lactation has been an important characteristic feature in evolution and classification of animal
31 species. During the process of evolution, amniotes (a group of tetrapods with an egg) split into synapsids and sauropsids about 320 Mya. Gradually, by the end of the Triassic period, mammals appeared on the synapsid branch of the evolutionary tree as Mammaliaformes whereas lactation evolved later and was established along the cynodont lineage [Figure 1.1; (Lefèvre et al. 2010)]. Mammals further split into Prototherian and Therian 166-220 Mya (Bininda-Emonds et al. 2007; Hedges and Kumar 2009). Prototherians mainly included the monotremes, with platypus (Ornithorhynchus antinus) and echidna (Tachyglossus and Zaglosus genera) as the only extant families. These are the egg laying mammals considered to exhibit the most ancient lactation characterized by the secretion of complex milk. The Therians split in parallel about 140 Mya into Metatherians and Eutherians (Lefèvre et al. 2010). Metatherians and Eutherians mainly differ in their reproductive strategy in terms of the length of gestation and lactation period and also the composition of milk secreted by the mother. The majority of eutherians have a well-developed invasive placenta that sustained life in utero after which mother gives birth to a developed young (Wildman et al. 2006). Instead in marsupials, even though there is a functional placenta, the major growth and development of the altricial young occurs postnatally, driven by the mother’s milk. During this phase, the teats act in a similar way to an umbilical cord and the conversion of nutrients to body mass is comparable to that observed in utero in eutherians (Renfree 2010).
According to Oftedal (2002, 2012), there is evidence that milk originated as a glandular skin secretion in synapsids which laid parchment-shelled eggs and this occurred even before mammals evolved. Amniotes (Sauropsids- ancestors of living reptiles and birds and Synapsids- ancestors of living mammals) in general laid parchment-shelled eggs; however, sauropsids evolved to develop calcified eggshells resistant to vapour loss (Oftedal 2012). In contrast, synapsids, had a glandular skin and continued to lay the parchment-shelled eggs and prevented the egg from drying under ambient conditions by skin secretions which further evolved into mammary secretions (Oftedal 2002, 2012, 2013). Successively, different types of skin glands evolved including eccrine, apocrine, sebaceous glands, scent glands and mammary glands (Blackburn 1991; Oftedal 2013). Capuco and Akers (2009) proposed that
32 along with the supply of moisture to the egg, other components like anti-microbials were incorporated into this biological fluid helping it to evolve as a better secretion from the mother for the developing young. At present, early mammals which include the extant monotremes (echidna and platypus) that lay parchment-shelled eggs produce these secretions from the mammary gland for the same purpose and may have evolved to provide nutrients (Blackburn et al. 1989). In summary, during the process of evolution, synapsids evolved into mammals wherein the ancestral skin secretions that retained moisture in the skin and eggs evolved in parallel into milk secreted by the mammary gland to act as a source of nutrients for the young (Oftedal 2012). During this gradual process (Blackburn 1993), mammals diverged into 3 subclasses – Monotremes, Marsupials and Eutherians which differ mainly in their reproductive strategies.
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Figure 1.1: Evolution of mammals and lactation (Modified from Lefèvre et al. 2010)
34
1.3 Mammary gland structure and lactation
Mammary gland development termed mammogenesis, begins at the embryonic stage and proceeds beyond parturition (Knight and Peaker 1982). The gland in its mature lactating form consists of the parenchymal region which is a layer of polarised luminal epithelial cells (producing milk) lined by a layer of myoepithelial cells (producing the basement membrane). These form the functional alveoli which along with the ducts are embedded within a complex environment composed of extracellular matrix and various stromal cell types including fibroblasts, adipocytes, blood vessels and immune cells (macrophages and mast cells). During the process of lactation, under the control of different factors, milk is synthesised by the mammary epithelial cells and secreted to the lumen via the ducts by the contractile pressure exerted by the myoepithelial cells (Forsyth and Neville 2009).
1.4 Lactation: comparison between eutherians and marsupials
The majority of studies in mammary gland development and lactation have been concentrated on eutherian species like mouse, rat, cow, rabbit and human. These species have a reproductive strategy with greater maternal investment in an extended gestation period (Renfree 1983). In contrast, in marsupials and monotremes, the lactation cycle is long and complex during which the mammary gland increases in size and milk production changes (Green et al. 1980; Stewart 1984). This varied lactation strategy may provide insight into mammary gland regulatory mechanisms and processes that are present but not readily apparent in eutherian animals (Sharp et al. 2007).
1.4.1 Eutherians
In eutherians such as the mouse, the mammary gland starts growth at the embryonic stage with the invasion of the fat pad by a rudimentary ductal structure (Richert et al. 2000). Ductal growth and early alveolar development occur during sexual maturation and estrous cycle, proliferation and differentiation during pregnancy and lactation and death during involution (Richert et al. 2000).
35
The onset of lactation, the secretion of milk, requires the differentiation of the mammary epithelial cells and is referred to as lactogenesis. It is a two stage process in eutherians (Brisken and Rajaram 2006). Stage 1 of lactogenesis, characterised by presence of lipid droplets in the cells, is initiated at mid pregnancy with an increased H[SUHVVLRQRIPLONSURWHLQVOLNHȕ-casein (CSN2)Į-lactalbumin (LALBA) and whey acidic protein (WAP) in mouse (Brisken and Rajaram 2006; Neville et al. 2002). Stage 2 of lactogenesis, initiated at parturition, is characterised by an additional increase in expression of milk proteins, closure of alveolar tight junctions and movement of cytoplasmic lipid droplets and caseins into the lumen (Neville and Morton 2001). Once lactation is established (galactopoiesis), it is maintained by removal of milk from the mammary gland (Neville et al. 2002).
1.4.2 Marsupials
In marsupials such as the tammar wallaby, all four mammary glands develop equally during gestation showing an increase in alveoli size and number. Immediately after parturition and attachment of the young to one specific gland, the alveolar lumen of this gland enlarges and milk secretion is initiated whereas, the other three glands simultaneously regress to a quiescent state (Findlay 1982). During the whole process of lactation, the gross dimension of gland and alveolar size increases until it attains maximal milk production (Findlay 1982). After weaning, the lactating gland involutes (Findlay 1982).
When compared to eutherians, marsupials have adopted a different reproductive strategy. The lactation cycle is extended and the composition of milk changes progressively to provide timely nourishment to the altricial young in a phase-dependent manner. In tammar wallaby (Figure 1.2), the reproductive cycle has been divided into four different phases (Nicholas et al. 1997; Figure 1.3). It includes phase 1, a short gestation of 26.5 days which involves the preparation of the mammary gland for lactogenesis. Phase 2 is the first 200 days of lactation divided into phase 2A (first 100 days when the PY is continuously attached to the teat) and phase 2B (100-200 days), during which milk is very high in complex carbohydrate
36 and low in fat and proteins (Green and Merchant 1988; Nicholas et al. 1997; Trott et al. 2003). Phase 3 is the final 200-330 days during which herbage is the major component of its diet. At this stage, milk is high in fat and proteins and low in carbohydrates (Green and Merchant 1988). During the extended lactation cycle, milk proteins show differences in expression pattern and based on this, they are allocated to two groups; the first group includes Į-lactalbumin (LALBA), ȕ-lactoglobulin (BLG), Į-casein (CSN1), ȕ-casein (CSN2)andț-casein (CSN3) which are first expressed at parturition and secretion is constant throughout lactation (Nicholas et al. 1997). The second group of milk proteins include early lactation protein (ELP) (Nicholas et al. 1997), whey acidic protein (WAP) (Simpson et al. 2000) and late lactation proteins (LLP-A and LLP-B, Nicholas et al 1987; Trott et al. 2002) which are expressed in phase 2A, 2B and 3 respectively.
37
(a)
(b)
E A B C D
Figure 1.2: Tammar Wallaby (Macropus eugenii) (a) Adult (b) Developmental stages of tammar wallaby (A) Day 6 (B) Day 70 (C) Day 185 (D) Day 220 (E) Adult
38
Figure 1.3: The lactation cycle of tammar wallaby. Phases of the lactation cycle of WDPPDU ZDOODE\ ZLWK PDMRU PLON SURWHLQ JHQHV Į-FDVHLQ ȕ-FDVHLQ Į-ODFWDOEXPLQ ȕ- lactoglobulin expressed throughout lactation and other genes Early lactation protein, Whey acidic protein, Late lactation proteins (A and B) expressed at specific phases of lactation [adapted from Nicholas et al. 1997]
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1.5 Milk and its components
The reproductive strategy, developmental needs of the young and environment are thought to drive variation in milk composition across mammalian species (Nicholas et al. 1995). However, despite the gross compositional variation in milk between species and across lactation in certain species, the major milk components remain the same. In every animal, water is the major requirement for growth and development, and mother’s milk acts as an important source in supplying this to the newborn. Apart from water, there are other factors in the mother’s milk (Goldman 2007; Newburg and Walker 2007) and therefore milk can be defined as a complex mixture containing diverse components including carbohydrates, proteins, fat, vitamins and minerals in addition to water (Oftedal and Iverson 1995).
1.5.1 Carbohydrate
A major component of milk is carbohydrate which is dominated by the sugar lactose, along with a variety of simple and complex sugars. Lactose, the osmole in eutherian milk is responsible for an increased absorption of water from the extracellular fluid. There are also oligosaccharides and protein linked sugars (glyco- proteins) in the milk which provide protective effects from pathological infections (Newburg et al. 1990; Yolken et al. 1992). Milk of marsupial and monotreme species usually have less lactose but include a variety of oligosaccharides in addition to monosaccharides. Broadly, the carbohydrate component is involved in providing energy and most importantly supporting the colonization of beneficial bacteria in the gut (Newburg 1997).
1.5.2 Protein
The two major protein classes in milk are the caseins (insoluble) and whey
VROXEOH 7KH SULPDU\ SURWHLQV IDOOLQJ XQGHU WKHVH WZR FODVVHVDUHĮS1-casein
(CSN1S1), ĮS2-casein (CSN1S2)ȕ-casein (CSN2), ț-casein (CSN3)ȕ-lactoglobulin (BLG) Į-lactalbumin (LALBA), serum albumin (ALB), immunoglobulins (Ig), lactoferrin (LF) and lysozyme (LYZ) (O’Donnell et al. 2004). The protein fraction
40 principally acts as a source of amino acids and nitrogen to the infant. However, research has shown that caseins and whey proteins are important sources of bioactive peptides which may remain inactive in the whole protein form but activity is released by the action of either milk-borne or gut-borne enzymes (Schanbacher et al. 1998). These bioactive peptides are reported to have diverse physiological functions (Korhonen and Pihlanto–Leppälä 2004).
The casein fraction of milk, apart from being a good source of nutrients for the young plays a significant role in preventing the pathological calcification of mammary gland (Holt 1997). However, caseins delivered as bioactive peptides have been shown to have diverse function (Silva and Malcata 2005). Casein phosphopeptide, the major component, was shown to enhance the availability of calcium from milk and promoted absorption in the rat intestine (Erba et al. 2002; Sato et al. 1986). It was also demonstrated to increase the calcification of cultured embryonic rat bone (Gerber and Jost 1986 ). There are other peptides derived from the caseins which showed bioactive properties like opioid activity (Schlimme and Meisel 1995), antihypertensive activity (Maruyama and Suzuki 1982) antithrombotic activity (Fiat AM et al. 1993), anti-microbial activity and immune-modulating activity (Dallas et al. 2013; Lahov and Regelson 1996; Migliore-Samour et al. 1989; Parker et al. 1984).
Whey proteins, which represent a heterogeneous fraction of milk, are well acknowledged for their nutritive value, functional value and biological properties especially in health promotion and prevention of disease (Madureira et al. 2007). These whey proteins have diverse physiological roles both as intact proteins and bioactive peptides. This includes an immunological role (Wong and Watson 1995), anti-microbial effects (Jones et al. 1994; Yamauchi 1990), opioid activity (Tani et al. 1994), anti-tumorigenic and anti-carcinogenic effects (Håkansson et al. 1995; MclNTOSH et al. 1995). Chatterton and co-workers (2006) reviewed the biological DFWLYLWLHVRIĮ-ODFWDOEXPLQDQGȕ-lactoglobulin, both as whole intact proteins and as derived bioactive peptides, emphasising their importance in anti-microbial, anti- carcinogenic, hypocholesterolemic effect, and other metabolic and physiological
41
HIIHFWVĮ-ODFWRUSKLQDQGȕ-ODFWRUSKLQDUHSHSWLGHVGHULYHGIURPĮ-ODFWDOEXPLQDQGȕ- lactoglobulin respectively by gut enzyme hydrolysis. They have potent anti- hypertensive effect due to the angiotensin-I-converting enzyme (ACE) - inhibitory activity (Nurminen et al. 2000; Sipola et al. 2002) in addition to acting as opioid agonists (Antila et al. 1991). ȕ-lDFWRWHQVLQ DQRWKHU SHSWLGH GHULYHG IURP ȕ- lactoglobulin is reported to have ileum contracting and ACE- inhibitory effects (Pihlanto-Leppälä et al. 1997). Albutensin A and Serophin, are bioactive peptides derived from another whey protein, serum albumin, which showed opioid and ACE- inhibiting activity (Nakagomi et al. 2000; Tani et al. 1994). Lactoferrin, an iron binding glycoprotein is another major whey protein with anti-microbial, anti- oxidative, anti-inflammatory, anti-cancer and immune regulatory properties (Lönnerdal and Iyer 1995; Valenti et al. 2004 ). Lactoferricin (Vogel et al. 2002; Yamauchi et al. 1993) and lactoferrampin (Haney et al. 2009; van der Kraan et al. 2004) are bioactive peptides derived from lactoferrin that are proven to have anti- microbial properties.
1.5.3 Fat
Fat, the most variable component of milk, exists mainly in the form of triglycerides. They are present in small globules called milk fat globules (MFG) 0.1- 15μm in size which are enclosed by a tri-layered phospholipid membrane that includes proteins, glycoproteins, enzymes and neutral and polar lipids (Koen Dewettinck et al. 2008). These membranes are derived from the mammary epithelial cells. In recent years, the protein fraction of the milk fat globule membrane (Milk fat globule membrane protein - MFGMP) has gained great importance and accounts for about 1-2% of the total proteins in milk (Riccio 2004). MFGMP has been identified and characterized in different species such as human (Liao et al. 2011), bovine (Reinhardt and Lippolis 2006), sheep (Pisanu et al. 2011) and goat (Cebo et al. 2009). The glycoprotein fraction of human milk fat globule was reported to protect the infant from pathogenic infection (Peterson et al. 1998) whereas rat MFGM conferred protection against colon cancer in rat (Snow et al. 2010). Furthermore, a study by Spertino and co-workers (2012) demonstrated that human, bovine and
42 caprine MFGs had stimulating effects on cell viability of Caco2 cells (human epithelial colorectal adenocarcinoma).
1.5.4 Growth factors
Milk also contains different growth factors like epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF) and granulocyte colony-stimulating factor (G-CSF). These growth factors have physiological significance in mammary gland development and infant development (Donovan and Odle 1994). Klagsbrun (1978) demonstrated a role for milk in promoting in vitro growth of enterocytes and hepatocytes which may be due to the presence of these growth factors or mitogenic factors. There is evidence to prove that growth factors like IGF mediate the action of growth hormone in the mammary gland (Davis et al. 1987), promote gastrointestinal tract (GIT) development and maturation in the young (Corps et al. 1987; Ma and Xu 1997). Similar roles have been also demonstrated for EGF present in milk (Corps et al. 1987)7*)ĮDQGȕDUH two other growth factors present in milk which have a potential role in mammary gland development (Plaut 1993). Milk of many species including human and bovine (Rogers et al. 1996) has been repoUWHGWRFRQWDLQIDFWRUVOLNH7*)ȕZKLFKKDVEHHQ implicated in many processes like epithelial cell growth and differentiation (Heikinheimo et al. 1993; Plaut 1993), carcinogenesis (Buck and Ellenrieder 2006) and immune regulation (Letterio and Roberts 1998).
1.5.4 MicroRNA
Recently, there have been reports showing the presence of a new component in milk comprising immune related microRNA (miRNA) (Kosaka et al. 2010; Zhou et al. 2012). MiRNA are small non-coding RNA of 18-22nt length which negatively regulate the activity of specific mRNA and in turn regulate different physiological processes (Bartel 2004). Earlier reports had already demonstrated the presence of these miRNA in different biological fluids like blood plasma, serum, saliva and urine (Chen et al. 2008; Lawrie et al. 2008; Mitchell et al. 2008). MiRNA’s present in milk are presumed to be carried in specialized structures like the membrane bound
43 vesicles and the exosomes which may help in cell-to-cell transfer (Hata et al. 2010; Lässer et al. 2011; Valadi et al. 2007; Weber et al. 2010; Zhou et al. 2012). These milk miRNA have high stability and withstand harsh conditions such as acidic environments, RNase treatment and freeze thaw cycles (Kosaka et al. 2010). Considering these properties, it can be anticipated that milk miRNA may be involved in mother-infant communication whereby they may cross the gut barrier and enter the infant’s system in turn regulating different physiological processes. Reports by Chen and co-workers (2010) proposed that milk miRNA can be used as biomarkers for determining milk quality.
1.6 Regulation of lactation
There is a long list of signalling components involved in the correct development and differentiation of mammary gland leading to milk production and secretion; factors and processes include hormones, cell-cell interaction from neighbouring cell types, extracellular matrix (ECM) and milk borne factors (Polyak and Kalluri 2010) (Figure 1.4). The significance of such a definite microenvironment involving diverse factors for normal mammary epithelial cell differentiation was supported by a report showing a lack of differentiation of epithelial cells cultured on plastic. In contrast, production of milk proteins was observed in epithelial cells cultured in basement membrane (3D) with lactogenic hormones (Howlett and Bissell 1993; Petersen et al. 1992). These diverse regulatory factors have been studied in detail and are well characterised in a wide range of eutherian species; but not much is known in marsupials. In this section the regulatory factors of both lactogenesis and galactopoiesis have been reviewed and the need for a detailed investigation in marsupials identified.
44
* SECRETORY EPITHELIAL CELLS
MYOEPITHELIAL CELLS
BASEMENT MEMBRANE
EXTRACELLULAR MATRIX
ADIPOCYTES
SECRETED MILK DROPLETS
HORMONES
* ALVEOLAR SPACE
Figure 1.4: Components and regulators of mammary gland differentiation. The arrows represent the different interactions.
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1.6.1 Hormones
Hormones are one of the major factors which regulate the development and function of the mammary gland (Brisken 2002; Brisken and O’Malley 2010; Oakes et al. 2006). Broadly, hormones are divided into two groups (Neville et al. 2002): the reproductive hormones; oestrogen, progesterone, placental lactogen, prolactin and oxytocins, and the metabolic hormones; glucocorticoids, thyroid hormones and insulin which are important for mammary development, differentiation and milk secretion. These hormones either have a direct effect on mammary epithelial cells or may act indirectly by local production of growth factors (Knight et al. 1998). Among all the known hormones; insulin (I), glucocorticoid (F) and prolactin (P) have been reported to play major roles in regulating mammary differentiation. Further, the studies demonstrating the role of these hormones in both eutherians and marsupials have been reviewed. Based on these previous reports, the current study focused on the three lactogenic hormones, I, F and P.
1.6.1.1 Insulin
The role of insulin in mammary gland function in vivo has been difficult to elucidate as prolonged deprivation of this particular hormone results in death. In eutherians, the earlier studies suggested insulin was not required for either lactogenesis 2 or for lactation. This was supported by evidence from studies in rat and goat where there was no inhibition of lactogenesis 2 and little reduction in milk yield with short term decrease in insulin peripheral concentration (Hove 1978; Kyriakou and Kuhn 1973). However, more recent studies have shown that insulin plays a major role in regulation of nutrient flux to the mammary gland in the lactating animal (Neville et al. 2002). It was also demonstrated that the responsiveness of mouse mammary epithelial cells to insulin varied according to physiological state. There was more responsiveness observed during pregnancy and lactation which supports the terminal differentiation of the mammary epithelium (Oka et al. 1974). In addition, insulin is essential for transcription of the casein genes in the mouse (Bolander et al. 1981). Moreover, using murine mammary explants it
46 was demonstrated that insulin was a key regulator of hormone responsiveness during lactogenesis and it plays a crucial role in milk protein synthesis (Menzies et al. 2010). Similarly, in bovine mammary explants insulin plays an important role in stimulating milk protein synthesis at multiple levels in mammary epithelial cells (Menzies et al. 2009b).
1.6.1.2 Glucocorticoid
Adrenal steroids have a role in mammary gland differentiation and lactation which has been demonstrated by organ ablation experiments (Cowie et al. 1980) and organ culture experiments (Brisken 2002; Topper and Freeman 1980). Glucocorticoids are known to be important for maximal ductal growth during mammary gland development and are also known to enhance lobule formation during pregnancy (Topper and Freeman 1980). They play a role in differentiation of mammary epithelial cells by maintenance of ECM during whole organ and explant culture conditions (Casey et al. 2000) and also have a fundamental role in the function and maintenance of cell-cell contact in the mammary epithelia by inducing the formation of tight junctions (Zettl et al. 1992). Glucocorticoids have an indirect effect on lactation because of their role in nutrient flux and in maintaining high levels of glucose during starvation (Neville et al. 2002). These steroids are also reported to inhibit involution and programmed cell death in the mammary gland (Feng et al. 1995). Glucocorticoids act as lactogenic hormones both under in vivo and in vitro conditions and play a role in the formation of rough endoplasmic reticulum, casein mRNA expression and lactogenesis (Topper and Freeman 1980). However in marsupials, apart from triggering parturition (Shaw et al. 1996), the requirement of glucocorticoids in the process of mammary gland function is known to a lesser extent and requires further investigation.
1.6.1.3 Prolactin
Prolactin is an essential hormone secreted by the anterior pituitary under hypothalamic control (Freeman et al. 2000) and has an influence on the development and function of the mammary gland (Topper and Freeman 1980). Moreover, there
47 are reports of prolactin being produced by mammary epithelial cells where it regulates mammary gland function in a paracrine function (Oakes et al. 2006). During pregnancy, prolactin stimulates the secretion of oestrogen and progesterone which regulates mammary gland development (Gallego et al. 2001). At parturition, prolactin plays a dominant role in epithelial growth (Topper and Freeman 1980) and following a decline in progesterone levels it acts as a primary regulator of secretory cell activation (Neville et al. 2002). Also, prolactin plays an essential role in maintenance of milk production and secretion; galactopoiesis was suppressed when prolactin secretion was inhibited by bromocriptine in human and rat (Kulski et al. 1978; Nicholas and Hartmann 1981) but the effect was variable in ruminants (Benedek-Jaszmann and Sternthal 1976). Experiments using mammary explants from pregnant mice have shown that prolactin stabilizes and promotes transcription of the casein gene, stabilises the mRNA, may stimulate synthesis of Į-lactalbumin and increases lipoprotein lipase activity in the mammary gland (Golden and Rillema 1995; Hang and Rillema 1997).
1.6.1.4 Hormonal regulation of milk protein gene expression: comparison between mouse and tammar wallaby
During the 1950’s Elias and colleagues developed an in vitro mammary explant model to investigate the different hormonal regulatory mechanisms of milk protein genes (Elias 1957; Elias 1959; Elias and Rivera 1959). Using this model system to assess control of milk protein gene expression in mouse, it was demonstrated that the expression of casein genes required the three major lactogenic hormones insulin (I), cortisol (F) and prolactin (P) (Juergens et al. 1965; Terry et al. 1977; Turkington et al. 1965). Similarly the synthesis of the whey protein Į- lactalbumin also required I, F and P in the culture media (Ono and Oka 1980a, 1980b).
Meanwhile, studies using tammar wallaby mammary explant culture model have shown that indXFWLRQ RI PLON SURWHLQV OLNH Į-lactalbumin (Nicholas and Tyndale-Biscoe 1985) DQGȕ-lactoglobulin (Collet et al. 1991) was dependent only
48 on the lactogenic hormone prolactin. The milk protein Į-casein induction was dependent on all three lactogenic hormones, insulin, cortisol and prolactin, whereas ȕ-casein induction was dependent on insulin and prolactin (Collet et al. 1992). The tammar WAP gene is a marker for phase 2B lactation, but the gene was expressed in mammary explants from pregnant tammars in the presence of insulin, cortisol, prolactin, oestrogen and thyroid hormone. In contrast, in mammary explants from phase 2B lactation, the endocrine requirement for maintenance of tammar WAP gene expression was limited to insulin, cortisol, and prolactin, indicating that there is a role for oestrogen and/or thyroid hormone for induction of gene expression (Simpson et al. 2000). The late lactation protein (LLP) genes can be down regulated in phase 3 mammary explants and then re-stimulated with insulin, cortisol and prolactin. These reports show the regulation of milk protein expression in tammar wallaby is different to mouse and possibly plays a role in the lactation strategy of the marsupial.
1.6.2 Extracellular Matrix
Extracellular matrix (ECM), a component of the mammary gland that supports morphological development of the tissue during the process of pregnancy and lactation, is a highly complex mixture of collagens, fibronectin, laminin, glycosaminoglycans and others (Knight 1995). It has been known for many years that ECM, the major component of the complex mesenchyme plays an important role in regulating mammary gland function (Bissell et al. 1982; Emerman and Pitelka 1977). The interaction between ECM and secretory cells is bidirectional as the ECM influences the differentiation of epithelial cells and in turn secretory cells are responsible for producing some elements of the basement membrane (Knight 1995) which initiates a signaling mechanism controlling mammary gland development and differentiation. A model of “Dynamic Reciprocity” was proposed by Bissell and co- workers where it was postulated that ECM exerts an influence on milk protein gene expression via transmembrane proteins and cytoskeleton components (Bissell and Aggeler 1987). Cytoskeleton in the mammary epithelial cells is associated with polyribosomes that affect mRNA stability and rates of protein synthesis and with the nuclear matrix that affects mRNA processing and rates of transcription (Bissell et al.
49
1982). This mechanism of regulation occurs at multiple levels including the control of integrin expression by mammary epithelial cells, composition of ECM and presence of secreted matrix proteins (Morrison and Cutler 2010). Recent reports have demonstrated a role of ECM on marsupial mammary gland function (Wanyonyi et al. 2013a, 2013b). The role of ECM specifically in the mouse and tammar is reviewed below.
1.6.2.1 Eutherians
ECM plays a fundamental role in the maintenance of tissue-specific function of mouse mammary epithelial cells in culture (Aggeler et al. 1988; Bissell and Aggeler 1987). The ECM-cell interaction regulates epithelial cell polarity and milk protein gene expression, and this occurs together with the action of lactogenic hormones (Bissell and Aggeler 1987; Topper and Freeman 1980). The importance of the stromal epithelial interaction in mammary differentiation was emphasised by an earlier observation by Cunha and co-workers (1995) where undifferentiated cells acquired mammary epithelial phenotype when cultured on mammary mesenchyma and the proliferative response of the cells to hormones was mediated by stroma (Cunha et al. 1997). In addition, the role of the individual ECM components had been demonstrated in regulating the expression of many eutherian milk protein genes, including PRXVHȕ-casein (Li et al. 1987; Streuli et al. 1991) and whey acidic protein (Lin et al. 1995)ERYLQHȕ-casein (Schmidhauser et al. 1990) DQGUDEELWĮ6- casein (Jolivet et al. 2001).
1.6.2.2 Marsupials
Further understanding of the role of ECM in the local control of lactation exploited the unique lactation strategy of the macropod marsupial tammar wallaby where the ECM composition of the mammary gland varies across different phases of lactation. It was shown that ECM plays a central role in the phase-to-phase transition in tammar lactation resulting in a differential expression pattern of milk protein genes (Wanyonyi et al. 2013a). This study demonstrated a role for ECM in regulating ACL (asynchronous concurrent lactation), where two mammary glands
50 secrete milk of two different compositions at the same time which was as a result of local regulatory mechanisms (Wanyonyi et al. 2013a). The tammar mammary epithelial cells extracted from phase 2B acquired phase 3 phenotype when cultured on phase 3 ECM and phase 2A cells acquired phase 2B phenotype when cultured on phase 2B ECM. This change in phase phenotype was correlated with the phase- specific changes in the ECM composition. Thus, the progressive changes in ECM composition in individual mammary glands provided local regulatory control over milk protein gene expression, in turn assisting the mammary glands to lactate independently. Earlier, the same group reported a role for ECM in cathelicidin gene expression, an immune related milk protein that is produced in the mammary gland, in a phase-dependent manner during lactation in tammar wallaby (Wanyonyi et al. 2013b).
1.6.3 Different cell types
Apart from secretory cells, mammary glands are composed of different cell types such as myoepithelial cells, fibroblasts, adipocytes and immune cells, and cell- cell interactions between these cell types influence gland function and regulate milk protein synthesis (Levine and Stockdale 1984; Levine and Stockdale 1985; Polyak and Kalluri 2010). These different cell types in the mammary gland produce signals to locally regulate epithelia and such regulatory mechanisms fall under paracrine control (Knight 1995).
1.6.3.1 Myoepithelial cells
Myoepithelial cells are one of the major cell types of the mammary gland which are in direct contact with epithelial cells, and are known to influence proliferation, polarity and differentiation of epithelial cells, along with production of basement membrane that forms a physical barrier for the gland (Polyak and Kalluri 2010). The key role of myoepithelial cells in mammary gland function and lactation is to participate in the process of milk ejection by assisting the contraction and release of milk from epithelial cells into the lumen of the alveoli (Sopel 2010). This contractile function is under the control of different factors like oxytocin, myosin
51 light chains, smooth muscle proteins, appropriate cell-ECM interactions and transcription factors (Moumen et al. 2011). Oxytocin released from the pituitary gland stimulates contraction of the myoepithelial cells which leads to milk let down (Moumen et al. 2011). This contraction also requires expression of smooth muscle proteins and appropriate cell-ECM interactions along with regulation by the myosin light chains (Moumen et al. 2011). In addition to these basic functions, myoepithelial cells may have additional roles (Adriance et al. 2005; Barsky and Karlin 2005; Bonnet et al. 2002; Gudjonsson et al. 2002) and much remains to be investigated.
1.6.3.2 Adipocytes and stromal fibroblasts
Other cell types in the mammary gland which influence mammary gland differentiation are adipocytes and stromal fibroblasts. Co-culturing experiments have demonstrated a potent growth response of mammary epithelial cells as a result of interaction with adipocytes and confirmed the hypothesis of cell-cell interactions in mammary gland function (Levine and Stockdale 1984). This co-culture experiment also influenced the sensitivity of epithelial cells to lactogenic hormones resulting in increased mammary differentiation and milk protein production (Levine and Stockdale 1985). In addition, a bidirectional regulatory interaction between epithelial cells and stromal fibroblasts has been reported where co-culturing of these two cell types resulted in an increased responsiveness of epithelial cells to oestrogen, which is important for mammary gland function (Haslam 1988).
1.6.3.3 Caveolins; potential influence on mammary function by a mechanism of cell-cell interaction
Studies during the past decade have shown a correlation between expression levels of the caveolin group of genes and lactation in mouse (Park et al. 2001; Sotgia et al. 2009). Caveolins are the main structural components of flask shaped invaginations found on the plasma membrane of almost all cell types (Palade and Bruns 2004). In mice, studies reported that genetic ablation of the Caveolin 1 (CAV1) gene resulted in enhanced mammary gland development and premature lactation through hyper-activation of STAT5 (Park et al. 2002; Sotgia et al. 2006). The
52 regulation was directed by the caveolins which were localised in myoepithelial cells and adipocytes and not the secretory epithelial cells (Hue-Beauvais et al. 2007). This mechanism can be assumed to be a paracrine regulation of lactation resulting from interaction between secretory epithelial cells, myoepithelial cells and adipocytes which in turn is mediated by lactogenic hormones. However, it was not clear whether the whole process of regulation was controlled by prolactin alone as reported (Park et al. 2001) or by an interaction between the complex group of lactogenic hormones that includes insulin and cortisol together with prolactin. In the present study we have tried to answer this question. In order to have a better understanding about the mechanism, the unique lactation strategy of tammar wallaby was exploited where there is phase-to-phase transition of mammary function during the lactation cycle.
1.6.4 Milk factors
There are numerous factors in the milk which interact with the apical membrane of secretory epithelial cells as long as they remain in the lumen of the gland and thus exert a local control on the process of lactation (Knight et al. 1998). In ruminants, it has been noted that the frequency of milking regulates the rate of milk secretion (Peaker 1995). This regulatory mechanism has been described as a feedback inhibition of milk proteins by FIL (Feedback Inhibitor of Lactation) (Addey et al. 1991a, 1991b; Wilde et al. 1995) which is an autocrine mechanism. FIL has been detected in milk of a number of eutherians species as well as in the marsupial tammar wallaby (Hendry et al. 1998). This local control of mammary gland function may explain the asynchronous concurrent lactation (ACL) observed in tammar wallaby. However, a recent study has demonstrated that a progressive change in the ECM composition in individual mammary glands provided evidence for a local regulatory mechanism for milk protein gene expression thereby enabling the mammary glands to lactate independently (Wanyonyi et al. 2013a).
In addition, there are milk proteins which are known to influence the mammary epithelial cell growth. In mouse, WAP, one of the major milk proteins was proven to play a negative role in cell cycle progression of mammary epithelial cells through an autocrine or paracrine mechanism in vivo (Nukumi et al. 2004).
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Moreover, over expression of the WAP gene significantly inhibited proliferation of mouse mammary epithelial cells in vitro (Nukumi et al. 2004). In contrast, WAP of tammar wallaby and domain III (DIII) of the protein alone stimulated proliferation of mouse mammary epithelial cell line (HC11) and primary cultures of tammar mammary epithelial cells in vitro (Topcic et al. 2009). Another major anti-microbial protein of tammar wallaby, cathelicidin showed differential splicing across lactation and the splice variants MaeuCath1 and MaeuCath7 stimulated proliferation of wallaby primary mammary epithelial cells in culture (Wanyonyi et al. 2011). Expression and secretion of cathelicidin anti-microbial peptides has been observed in the murine mammary glands (Murakami et al. 2005). However, there is no specific report demonstrating effect on proliferation of mammary epithelial cells by this SURWHLQ $QRWKHU PDMRU PLON SURWHLQ Į-lactalbumin demonstrated an effect on mammary gland by initiating apoptosis in mammary epithelial cells (Sharp et al. 2008).
1.7 GIT development and maturation in mammals
Based on the diet of different mammalian orders, herbivores, carnivores and omnivores have evolved different stomach types. Herbivores have a smaller stomach with a long and developed gut and caecum (Hildebrand 1995). In contrast, the carnivores have a lager stomach and a shorter gut. However, classification from a different point of view indicates eutherian mammals like human, rat and mouse have a monogastric stomach (Stevens and Hume 1995) and ruminants to have a digastric stomach (Hofmann 1988; Lyford 1988). Similarly, in marsupials there are both herbivores and omnivores. The macropod marsupial, tammar wallaby which is a herbivore has a digastric stomach with a fore stomach composed of mostly cardia glands acting as the microbial fermentation unit and a hind stomach lined with gastric glands where acids and pepsin are secreted for further digestion of food (Langer et al. 1980; Stevens and Hume 1995). Instead, Monodelphis domestica and Didelphis virginiana are omnivores with a monogastric stomach. Their gut has a small intestine with an enlarged hind gut and has a well-developed caecum (Stevens and Hume 1995).
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Gut development in mammals is initiated during early fetal development when amniotic fluid acts as the major source of nutrition and developmental signals (Mulvihill et al. 1985) and later, the development extends beyond birth during which mother’s milk acts as the major driving force. During embryonic development, by the process of gastrulation, the embryo develops 3 different layers- the ectoderm, mesoderm and the endoderm. Further, the development of digestive tract is initiated as an undifferentiated primitive tube which by subsequent morphogenetic movements is partitioned into esophagus, stomach, small intestine, caecum and the large intestine (Fukuda and Yasugi 2005; Wells and Melton 1999). The digestive tract consists of an epithelial layer originated from the endoderm and the outer mesenchymal layer / muscle layer originated from the mesoderm (Romanoff 1960). The epithelial layer differentiates into specific cell types which vary according to the region of the digestive tract (Karam 1999; Karam et al. 1997) whereas the mesenchymal layer differentiates into muscularis mucosa, sub mucosa and the lamina propria, which remain the same in all parts of the digestive tract (Fukuda and Yasugi 2005).
1.7.1 Stomach development in mammals
Development of the stomach mainly comprises gland formation and differentiation into specific cell types which involves a significant level of epithelial- mesenchymal interaction (Fukuda and Yasugi 2005; Hayashi et al. 1988; Kaestner et al. 1997). This complex programming of the gastric mucosal development and differentiation is mainly controlled by an array of signalling proteins and transcription factors (Khurana and Mills 2010). Four major signaling pathways- the Hedgehog, Wnt, transforming growth factor (TGF) and the receptor tyrosine kinase are involved in the process which determines the different axis of gut development and these in turn co-ordinate with transcription factors determining proper identity for different parts of the digestive tract (Zorn and Wells 2009). Based on the mammalian species, different stomach developmental processes occur at different time points, some occurs prenatal and some postnatal. There is evidence to propose that during prenatal development, placenta and amniotic fluid act as sources of
55 signal (Trahair and Harding 1992) whereas postnatal development is stimulated by the mother’s colostrum/milk which drives development & differentiation of glands, gastric enzyme synthesis and acid secretion (Gama and Alvares 2000; Kverka et al. 2007).
During the process of stomach development, epithelium invaginates to form numerous short pits which are continuous with the glands. Each gland has a pit, isthmus, neck and a base region (Figure 1.5). These regions have diverse types of cells like the mucus secreting pit and neck cells, pepsinogen secreting pre-zymogenic and zymogenic cells, acid secreting parietal cells and enteroendocrine cells (Karam 1999). During the differentiation process, stem cells present in the isthmus region of the stomach (Karam and Leblond 1993; Lee and Leblond 1985) give rise to precursor cells which evolve into respective mature cells via transit cells (Karam 1999; Khurana and Mills 2010). Many of the processes involved in fetal stomach development are also of importance throughout the life of the animal as they help in continuous cellular regeneration and differentiation. The development and differentiation of the cells into the form in which they exist in an adult stomach has been studied in many species including rat (Helander 1969; Kammeraad 1942), human (McIntyre et al. 1965), and rabbit (Hayward 1967; Menzies 1964). The pattern of development in all the reported species remains almost the same except for differences in chronology of appearance of specialized differentiated cells (Deren 1971). The next two sections of the review will summarise the process of stomach development in the eutherian mouse and compared with stomach development in marsupials such as the tammar wallaby and the opossum.
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Figure 1.5: Diagrammatic representation of the gastric gland (Karam 2010)
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1.7.1.1 Stomach development in mouse
1.7.1.1.1 Gastric mucosa development
In mouse, stomach development occurs in four different phases (Karam et al. 1997). The first stage of development (during late fetal phase) involves cyto- differentiation of the endoderm into the epithelial monolayer dominated by stem cells, precursor cells and about 8% differentiated cells. Previous studies by Kataoka and co-workers (1984) had shown that during this initial stage of development, mouse gastric mucosa at E12.5 is lined by a simple epithelial layer which by E14 develops into a pseudo stratified epithelium. The second stage (1-7 days of post natal life) is characterised by a huge increase in the proportion of differentiated pit, neck and enteroendocrine cells. During the third stage (7-15 days of post natal life), there is an increase in total number of cells and during the last stage (post natal 15-28 days) there is formation of distinct regions; pit, isthmus, neck and base.
During stomach development, the epithelial layer contains stem cells concentrated to the proliferative region called the isthmus which give rise to all differentiated epithelial cell types with a turnover time of 3 days (Karam and Leblond 1993). The two immediate descendants of these gastric stem cells are the pre pit cell precursor and the pre neck cell precursor that develop into different cell types at different rates. The pre pit cell precursors, within a time span of 3 days, develop into mucus producing cells which gradually move to the tip of the gastric glands and die. On the other hand, the pre neck precursor cells gradually move to the neck region where they differentiate into the neck cells, and as they continuously move to the base, differentiate into zymogenic cells and die. This whole process takes place within a period of 14 days. The third major cell type is the parietal cell. The precursor of parietal cells, the pre parietal cells undergo differentiation within one day to produce mature cells and the whole process occurs within the isthmus. But later, these parietal cells migrate in both directions i.e., towards pit and base, and death occurs (Karam 2010).
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1.7.1.1.2 Muscle layer development
The muscle layer is another major component of the stomach and it has an inner circular and outer longitudinal layer. Spatial pattern of smooth muscle differentiation in the stomach was studied in detail by Takahashi and co-workers (1998). It was reported that the smooth muscle myoblasts first differentiated and formed the circular muscle layer at 13 days of gestation. The longitudinal muscle layer appeared at 15 days in the forestomach and at neonatal period in the glandular stomach. The lamina muscularis mucosa appeared by the neonatal period.
1.7.1.2 Stomach development in Opossum and Tammar Wallaby
Several earlier studies have examined stomach development in a wide range of marsupial species. For a comparison of the omnivorous and herbivorous stomach, the current review focused mainly on the North American opossum (Didelphis virginiana) and the tammar wallaby. Development of the digestive tract in the North American opossum was studied in detail by Krause and Cutts (1992). In marsupial species such as the opossum, an omnivore with a monogastric stomach, there is limited prenatal stomach development except for a lining of simple columnar epithelium surrounded by a delicate mesenchymal layer (Krause et al. 1976). At birth, mature parietal cells are noticed at the base of the clefts and also in close contact to the gastric lumen, and few enteroendocrine cells at the base of the fundic region. However, in this species and two other related species, red kangaroo (Macropus rufus) (Griffiths and Barton 1966) and Australian native cat (Dasyurus viverrinus) (Hill and Hill 1955), there is no occurrence of a gastric gland at birth but it develops during the extended postnatal period which can be hypothesized to be driven initially by mother’s milk. Postnatally, the fundic region of the gastric mucosa differentiates, resulting in the first subdivisions in the oxyntic gland which becomes definite with parietal cells and enteroendocrine cells by the end of first week. The maximum extent of development of the oxyntic gland was noticed during the time of weaning with the presence of three different cell types – parietal cells, enteroendocrine cells and a few undifferentiated cells. Another remarkable
59 characteristic is that the chief cells appear only post weaning (Krause et al. 1976). This delay may be supporting the transfer of IgG from mother’s milk across the gut in the infant. It is almost at the same time that the stomach pH drops from around 6 to 2-2.5 when there is a shift to a solid diet. The pyloric gland starts differentiating around the 4th week and continues its development throughout adulthood (Acuff et al. 1989). Similarly a cardia gland also starts to appear by the end of the 4th week and is fully established by the end of weaning (Acuff et al. 1989). In brief, it appears that in opossum, parietal cell development occurs before weaning and can be assumed to be driven by the mother’s milk whereas chief cells which are starting to appear by weaning may just need an initial signal from milk and further development occurs based on the diet of the juvenile animal. However, there is no direct evidence for this hypothesis in the North American opossum. In contrast to the omnivorous opossum with a monogastric stomach, the herbivorous marsupial tammar wallaby has a digastric stomach. The tammar wallaby also has no mature gastric gland at the time of birth and parietal cells are found to be distributed in both fore stomach and hind stomach from birth till around 200 days postpartum (Waite et al. 2005). At around 170 days postpartum the gastric gland phenotype emerges in the hind stomach with an increase in the parietal cell number and in the fore stomach, the cardia gland phenotype appears with increased disappearance of parietal cells which may be in preparation to digest milk high in solids including fat and protein from 200 days postpartum (Kwek et al. 2009b; Waite et al. 2005). A similar pattern is reported in red kangaroo (Griffiths and Barton 1966). This change in the stomach phenotype in tammar wallaby is driven by mother’s milk which was demonstrated by a cross fostering experiment by Kwek and co-workers (2009b). However, these studies did not mention the presence and distribution of chief cells in the stomach which is a major functional component of the gastric epithelium. Earlier Davis (1981) examined pepsin activity in both fore and hind stomach of the tammar wallaby which is a representation of functionality of chief cells. It was shown that pepsin activity was comparatively low till around 170 days after which it increased in both parts of the stomach until about 200 days. Thereafter activity was not detected in the forestomach but continued to increase in
60 the hind stomach. Another remarkable development occurring in tammar stomach during the suckling stage is shown by Menzies and co-workers (2009a) and Kwek (2009) who examined the occurrence of ghrelin gene in the stomach of the developing pouch young. Early onset of ghrelin in stomach of the pouch young from about 10 days postpartum was reported by Menzies and co-workers (2009a). Ghrelin was present in forestomach at its gastric like stage, but gradually declined parallel to the disappearance of parietal cells. In contrast, the expression of ghrelin in the hind stomach remained same throughout. Given these few earlier reports of stomach development in opossum and tammar wallaby, one of the specific objectives of the present study was to understand the effect of milk and its components on stomach development in the suckled young of the marsupials where the majority of development occurs after birth.
1.8 Role of milk in GIT development
Apart from being a source of nutrient and providing protection to the immune compromised young, milk plays significant role in regulating mammary gland development (Khalil et al. 2011; Lee et al. 1987; Nukumi et al. 2004; Topcic et al. 2009). Milk contains anti-microbial proteins to protect the mammary gland and the young from infections (Gallois et al. 2007; Wilde et al. 1999) and drives development of different organs of the infant like the gut (Berseth et al. 1983; Yamashiro et al. 1989). The first place for milk to act during suckling is the infant gut. Evidence in the past has shown that breast milk (and not colostrum) play different roles like supporting gut barrier defence function (Leonard et al. 1994), protect against infection, and promotion of growth and development of the gut (Berseth et al. 1983; Yamashiro et al. 1989). Based on these earlier reports and the study by Kwek and co-workers (2009b) which supported a specific role of tammar milk on stomach maturation, the present study aims at identifying the definite role of milk, especially in early stomach development. The approach involved utilisation of the unique lactation strategy of marsupials like tammar wallaby where the role of milk components are more apparent (Kuruppath et al. 2012) due to milk of progressively varying composition (Green et al. 1980) in contrast to eutherian milk
61 of constant composition. The study examined the effect of tammar wallaby milk on stomach development using mouse in vitro models. Therefore, we further reviewed stomach development in mammals with increased emphasis on understanding the developmental differences between mouse and marsupials such as the tammar wallaby and the opossum.
Milk has been proposed to have broad roles in the development of GIT including assisting colonization of beneficial bacteria for enhanced development, immune-protection, and also the most important being driving the development of cells and organs towards particular lineages which involves complex programming (Hanson and Korotkova 2002; Kosiewicz et al. 2011). The different components of milk which may influence the growth and development of GIT include hormones, growth factors, bioactive peptides, oligosaccharides and neuropeptides (Xu 1996).
The leading role of milk is to aid in the colonization of gut flora which helps in the breakdown of nutrients, prevents any sort of neonatal infection (Hanson and Korotkova 2002), promotes gut barrier function (Wang et al. 2006b), helps the development of immune system (Kosiewicz et al. 2011) and cytodifferentiation of the gut (Bates et al. 2006; Bry et al. 1996; Uribe et al. 1994). The components of milk that have the capacity to promote gut colonization are mainly the complex oligosaccharides (Ruhaak and Lebrilla 2012). Studies have shown the difference in colonization of gut between milk fed and formula fed infants (Kapiki et al. 2007; Moreau et al. 1986; Roberts 1986).
Various previous studies have investigated the direct role of milk and milk- derived factors on different aspects of GIT development by employing a wide range of in vitro models including monolayer cell models of cancer origin like AGS, IEC-6 cells and more complex models like Caco2, T84 and HT-29 cells &HQFLþ DQG Langerholc 2010; Chopra et al. 2010; Purup and Nielsen 2012). Studies in a wide range of species like pig (Barneveld and Dunshea 2011; Ma and Xu 1997; Stoddart and Widdowson 1976) and bovine (King et al. 2008; Playford et al. 1999) have shown stimulatory effects for colostrum on growth and development of the GIT due
62 to presence of growth factors such as the TGF and EGF. Similarly, milk of human (Saito et al. 1993) and bovine (Rogers et al. 1996) which contain definite factors such as 7*) ȕ KDve been implicated in epithelial growth and differentiation (Lamprecht et al. 1989), carcinogenesis (Gold 1999) and immune regulation (Letterio and Roberts 1998). Milk also contains different hormones like prolactin isomers which has possible roles in gut development (Bujanover et al. 2002). Apart from having activity in its whole form, milk is rich in proteins that are digested in the infant’s gut and release potential bioactive peptides. There are numerous peptides showing activities like opiate, anti-thrombotic, anti-hypertension role, anti-microbial properties and immune regulation (Ehlers et al. 2011; Korhonen and Pihlanto 2006; Nagpal et al. 2011). However, this field requires further investigation to identify bioactive peptides that may have a role on growth and development of the GIT. There have been reports showing that milk components also determine intestinal permeability in the young (Catassi et al. 1995; Shulman et al. 1998) which is a characteristic feature of GIT development. Therefore, all the different factors in milk can be exploited in such a way that their biological activity is retained in milk-based commercial products to support infant development (Donnet-Hughes et al. 2000). One such proposal was the study by Jantscher-Krenn and co-workers (2012) who reported that a major human milk oligosaccharide disiallyllacto-N-tetraose prevented Necrotising enterocolitis (NEC) using a rat intestinal model. NEC is one of the major fetal intestinal disorders in preterm babies which is often fatal and treatments for this particular disorder are expensive and very limited. It was proposed by Jantscher- Krenn and co-workers (2012) that if the results translate to humans, the oligosaccharide disiallyllacto-N-tetraose can be successfully used to prevent NEC by feeding the infant with mother’s milk or by using it as a component of formula feeds.
Some of the earlier studies reported the role of milk or milk borne factors specifically on stomach development and differentiation. de Andrade Sa and co- workers (2008) demonstrated D GLUHFW HIIHFW RI 7*)ȕ VLJQDOOLQJ RQ LQGXFLQJ apoptosis of gastric epithelial cells and inhibiting proliferation through alteration of cyclic proteins in vivo in suckling rat. This report provided evidence for the potential role of both milk and tissue-H[SUHVVHG7*)ȕLQJDVWULFGHYHORSPHQW(de Andrade
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Sá et al. 2008). Additional studies showed effect of milk on specialized cells of the stomach like parietal cells and chief cells (Andrén and Björck 1986; Morikawa et al. 1979). Morikawa and co-workers (1979) included an accelerated development of parietal cells in the presence of milk in both perinatal rat and premature newborn rats. Similarly, Andren and Bjorck (1986) proved that milk is required for production of prochymosin by the chief cells in the abomasum of the sucking calf. More recent studies in the marsupial tammar wallaby where milk composition progressively changes, the role of milk on stomach development of PY was proven by a cross fostering strategy where the pouch young was transferred to a more advanced stage of lactation (Kwek et al. 2009b). It was demonstrated that later stage milk accelerated stomach development with significant reduction in parietal cell population and expression of gastric glandular marker genes, suggesting down regulation of the gastric glandular phenotype in the fostered PY forestomach which are the characteristic features of development in this species.
In eutherians, major cytodifferentiation of stomach occurs during the fetal stage when the mother’s placenta or amniotic fluid may be acting as the major stimulating factors. In contrast, in marsupials such as tammar wallaby and opossum where the pouch young is born altricial, majority of stomach development is known to occur ex utero after birth and milk of varying composition may play a significant regulatory role. Moreover, there are no reports to verify the role of milk on early stage of stomach development and differentiation. To identify the factors present in milk that may drive early developmental events, we utilised tammar wallaby milk and examined its role on mouse stomach developmental models (in vitro organoid or embryonic models) which are better representatives to examine early stage of stomach proliferation and differentiation.
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1.9 Aim and scope of the study
The process of milk production and secretion is under the control of different factors and the resultant milk secreted plays a role in development of the gland itself and also supports growth of the young. Related studies investigating into the regulation of lactation and the role of milk on development mainly focused on eutherians such as the mouse. However, considerable amount of literature has increasingly proven marsupials to be better comparative models for research. Milk of marsupials such as the tammar wallaby progressively changes in composition across lactation and major development of the young occurs after birth. Therefore it is hypothesised that milk of marsupials may regulate postnatal growth and development of organs such as the stomach.
Following are the research aims of the present project:
¾ Regulation of lactation in tammar wallaby by caveolin group of genes:
The first aim of the project was to examine the role of the caveolin group of genes in regulating tammar wallaby lactation in comparison to the well- established eutherian model, mouse. In vitro mammary explant models were used for the purpose of this study.
¾ Putative role of tammar wallaby milk on stomach development:
The second aim of the project was to understand the role of milk in stomach development of the young. Cross species stomach models; mouse stomach explant and organoid models were developed to examine the role of tammar wallaby milk in stomach development and differentiation.
¾ Comparison of stomach development in gray short-tailed opossum (Monodelphis domestica) to the tammar wallaby:
The third aim of the project was to compare stomach development in the omniovorous marsupial, opossum to the herbiovours marsupial, tammar
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wallaby. This involves understanding the postnatal development of stomach in opossum in a time dependent manner.
¾ A bioinformatic approach to identify putative bioactives of early phase tammar wallaby milk also present in human amniotic fluid and placenta:
The fourth aim of the project was to compare the putative bioactives of early phase tammar wallaby milk that was also present in human amniotic fluid and placenta. This involved a functional genomic analysis of genes expressed in tammar wallaby mammary gland across all stages of lactation in order to identify secreted proteins which may have putative role in stomach development.
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CHAPTER 2
Comparative analysis of caveolins in mouse and tammar wallaby: Role in regulating mammary gland function
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2.1 Introduction
Caveolae are small flask-shaped invaginations found on the plasma membrane (Palade and Bruns 2004) and other organelles like the golgi apparatus (Dupree et al. 1993; Kurzchalia et al. 1992) and caveosomes (Pelkmans et al. 2001). They are mainly involved in regulating different cellular functions like endocytosis (Pelkmans and Helenius 2002), cholesterol homeostasis (Ikonena and Parton 2000; Maxfield and Wüstner 2002), calcium homeostasis (Saliez et al. 2008), vesicular transport and signal transduction (Razani et al. 2002; Smart et al. 1999). They are also reported to be involved in regulating vascular reactivity and blood pressure (Chidlow and Sessa 2010; Yu et al. 2006). The main structural components of these caveolae are caveolins which is a family of three proteins; Caveolin 1, 2 and 3. Caveolin 1 (CAV1) and 2 (CAV2) have higher level of expression in endothelial cells and adipocytes (Kandror et al. 1995; Stan 2005; Thorn et al. 2003) whereas the expression of Caveolin 3 (CAV3) is muscle-specific (Tang et al. 1996; Way and Parton 1995). Reports during the past decade have shown a correlation between caveolin expression in the mammary gland and lactation (Park et al. 2001; Sotgia et al. 2009). In mice, expression of the Caveolin 1 and 2 genes was elevated in the mammary gland before and during early pregnancy, down regulated during lactation and subsequently upregulated with the onset of involution to levels similar to the non- pregnant stage (Park et al. 2001). Subsequent studies reported that genetic ablation of Caveolin 1 in mice resulted in enhanced mammary gland development and premature lactation through hyper-activation of STAT5 (Park et al. 2002; Sotgia et al. 2006). Recently, Sotgia and co-workers (2009) showed that loss of Caveolin 3 expression, a muscle specific protein of the same family, induced lactogenic phenotype in virgin mammary gland. Moreover, implantation of mammary tumor cells in the primary duct of Caveolin 3 null mammary gland resulted in tumor mass reduction which was assumed to be due to the local paracrine effects of lactogenic luminal mammary epithelial cells. Related studies have demonstrated that the caveolin family of proteins play a role in regulating lactation in mice by prolactin via
68 a Ras-p42/44 MAPK-dependent pathway (Park et al. 2002; Park et al. 2001). However, it’s not clear whether this regulation of mammary gland function by caveolins is mediated by a complex interaction of lactogenic hormones that includes insulin and cortisol together with prolactin. The central role of Caveolin 1 in prolactin signaling during lactation in the mouse suggests that these genes may be regulated by complex lactogenic hormones. Parallel to this, there are reports showing the localisation of Caveolin 1 in endothelial and myoepithelial cells in mouse mammary glands (Hue-Beauvais et al. 2007) and not in epithelial cells which may indicate a paracrine regulation of the lactation process. This recent study correlating the caveolin protein family to the process of lactation was focused on the mouse, a eutherian model wherein milk composition and milk production does not change significantly across lactation apart from the initial colostrum produced 24-36 hrs postpartum (Kruse 1983). To further evaluate the role of caveolin in milk synthesis and production, the current study utilised the unique reproductive strategy of tammar wallaby (Macropus eugenii) by examining the expression level of caveolin genes during different phases of lactation. Compared to eutherian mammals (e.g. human, mouse), marsupials such as the tammar wallaby have a different reproductive strategy comprised of a short gestation of 26.5 days followed by a long lactation cycle of about 300-330 days divided into three different phases during which the milk composition and rate of milk production changes progressively (Nicholas et al. 1997; Tyndale-Biscoe and Janssens 1988). The questions of whether these changes in tammar milk composition and production are correlated with the expression profile of caveolin genes would provide new information on the potential role of these genes in regulating a complex lactation cycle. Therefore, the current study investigated the role of three major lactogenic hormones (I, F and P) in regulating the expression of mouse Caveolin 1 and 2 utilising mammary explant models and confirmed the previous reports on localisation of Caveolin 1 in the mammary gland. Further, the genome organisation, protein sequence, expression pattern and regulation of caveolins in the two species, mouse and tammar wallaby were compared.
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2.2 Material and Methods 2.2.1 Animals
BALB/c mice were obtained from Monash Animal Services, Melbourne, Australia. The inguinal and abdominal mammary glands were dissected under sterile conditions from mice at day 12 pregnancy. Tammar wallabies were maintained in the Deakin University Marsupial Facility, Victoria, Australia. Mammary glands were dissected from pregnant - phase 1 (9, 21, 23 and 25 days), lactating - phase 2A (parturition, 1, 2, 3, 5, 10, 22, 37, 40, 62, 80 and 100 days), lactating - phase 2B (110, 114, 133, 135, 150, 151, 171 and 193 days), lactating - phase 3 (216, 240, 260, 266 and 293 days) and involuting (1, 3, 5 and 10 days) tammar wallabies under sterile conditions after the animals were euthanized. The tissues were stored at -80° C and later used for examining the expression profile of different genes. All experiments were approved by the Deakin University Animal Ethics Committee.
Reactivation of pregnancy in the tammar wallaby was performed by removal of pouch young (RPY) and injecting bromocriptine (5 mg/kg body weight) to reinitiate the development of the dormant blastocyst (Gordon et al. 1988; Tyndale 1978). The day of RPY was considered as day 0 pregnancy and the animals were sacrificed at day 21 of pregnancy to collect mammary tissue for explant culture experiments.
2.2.2 Tissue culture
Mammary gland explants were prepared from day 12 pregnant mice and cultured in Medium 199 (11150-059 Invitrogen Gibco, Victoria, Australia) with bovine serum albumin (0.25 mg/ml) (A0281 Sigma Sydney, Australia) (Nicholas and
Tyndale-Biscoe 1985). Explants were incubated at 37°C and 5% CO2 in 5 ml of media per well in 6 well plates. Media was changed on alternate days during the four day experiment. Hormones were added at the following concentration: bovine insulin 1 μg/ml (I; I6634 Sigma Sydney, Australia), cortisol 50 ng/ml (F; H4001 Sigma Sydney, Australia) and ovine prolactin 1 μg/ml (P; National Hormone and Pituitary
70
Program USA). Mammary glands from 4 groups of 3 mice were dissected and pooled and explants were maintained in the indicated hormone combinations for four days. The control explants were maintained in media without addition of exogenous hormones (NH-No Hormone). Explants at different time points were collected and stored at -80°C for RNA extraction.
Tammar mammary glands from day 21 pregnant animals were cut into 1-2 mg sized explants and placed on siliconised lens paper floating on 5 ml of Medium
199 in 6 well plates. These explants were incubated at 37°C and 5% CO2 and media changed every second day. Hormones were added at following concentrations: bovine insulin 1 μg/ml (I), cortisol 50 ng/ml (F) and ovine prolactin 1 μg/ml (P).
2.2.3 RNA extraction and reverse transcription PCR
Mammary tissue was first homogenized in QIAzol lysis reagent (79306 Qiagen, Sydney, Australia) using a polytron kinetamica PT 2100 homogeniser and total RNA was extracted using the RNeasy Lipid Tissue Kit (74804 Qiagen, Sydney, Australia) following manufacturer’s instructions. The cDNA (complementary DNA) was synthesized from total RNA (1 μg) using Superscript III (18080-044 Invitrogen, Australia).
2.2.4 Quantitative polymerase chain reaction
Quantitative polymerase chain reactions (qPCR) were performed on a Bio- Rad CFX Manager using SsoFast Evagreen supermix (172-5200 Bio-Rad Laboratories). The PCR reactions (20 μl) contained 1x SsoFast Evagreen supermix, forward and reverse primers (Table 2.1) and diluted cDNA. The qPCR thermal profile included an initial denaturation (95°C for 3 min) and quantification for 40 cycles (95°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec). All samples were assayed in duplicate. Melting curve analysis was done to confirm the amplification comprised a single PCR product of correct size. 7KH ¨&W PHWKRG ZDV XVHG WR normalize transcripts to housekeeping genes GAPDH for mouse and RPS15 for tammar wallaby (Table 2.1). In both mouse and tammar mammary gland explants,
71 the gene expression levels were represented as fold levels compared to day 0 values of the respective genes
2.2.5 Sequence Analysis
The tammar wallaby caveolin gene sequences were retrieved from the tammar wallaby genome in NCBI and Ensembl using mouse (Mus musculus) caveolin sequence and the blast program (http://www.ncbi.nim.nih.gov/blast). Protein multiple sequence alignment was performed using Clustal W2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
2.2.6 Preparation of mouse mammary gland sections and immunohistochemistry
Mammary glands were collected from pregnant mice and were fixed in 4% paraformaldehyde solution overnight at 4°C, processed and embedded in paraffin. Sections of 5 μm thickness were dewaxed, antigen retrieved by boiling in citric acid buffer (pH = 6) and endogenous peroxidase blocked by incubation in 0.5% H202 for 15 min. Subsequently, the sections were blocked in 5% skimmed milk for 1hr and incubated with primary antibody (1:500, diluted in PBS/1% BSA) (Polyclonal Rabbit Anti-Caveolin, 610059 BD Transduction Laboratories) overnight at 4°C. The slides were washed with PBS and incubated for 1hr in horseradish peroxidase (HRP) - conjugated secondary antibody (Goat Anti-Rabbit HRP, Invitrogen G021234) (1:200, diluted in PBS/1%BSA). All slides were developed with 3,3V- diaminobenzidine tetrahydrochloride (DAB, Dako) and lightly counterstained with haematoxylin prior to mounting in histomount (Zymed). Sections were viewed on a Zeiss imager A1 inverted microscope.
2.2.7 Statistical analysis
All statistical analysis was performed using a two-tailed t-test assuming unequal variances between pairs of means. Quantitative data were presented as
72 means ± standard error of the mean. Statistical significance was determined at either 5 or 1% level.
Table 2.1: Primers used in qPCR.
GENE GENEID PRIMER SEQUENCES AMPLICON
SIZE (bp)
ȕ -Casein CSN2 FP: 5’-AGAGGATGTGCTCCAGGCTA-3’ 230 RP: 5’-TAAGGAGGGGCATCTGTTTG-3’
Glyceraldehyde 3-phosphate GAPDH FP: 5’-AACTTTGGCATTGTGGAAGG-3’ 223 RP: 5’-ACACATTGGGGGTAGGAACA-3’ MOUSE dehydrogenase
Caveolin1 CAV1 FP: 5’-TTGCCATTCTCTCCTTCCTG-3’ 177 RP: 5’-TCTCTTTCTGCGTGCTGATG-3’
Caveolin2 CAV2 FP: 5’-CTCAAGCTAGGCTTCGAGGA-3’ 166 RP: 5’-ACAGGATACCCGCAATGAAG-3’
ȕ lactoglobulin BLG FP: 5’-GCATGTGCCCACTATGTCAG-3’ 154 RP: 5’-AGGGGGATAACTTCGTCTGC-3’
Ribosomal protein S15 RPS15 FP: 5’-AGCAGAAGAAGAAGCGAACG-3’ 160 TAMMAR RP: 5’-TTCAGGAGGGAATGCTGTTT-3’ FP: 5’-ACCCCAAGCATCTCAATGAC-3’ WALLABY Caveolin1 CAV1 243 RP: 5’-TCTTGATGCATGGCACAACT-3’
Caveolin2 CAV2 FP: 5’-ATCTGCAACCACGCTCTCTT-3’ 221 RP: 5’-ACGGGAGCAATGAGAACATC-3’
73
2.3 Results 2.3.1 A time course for hormonal regulation of Caveolin 1 and 2 genes in mouse mammary gland explants ȕ-Casein gene expression was used as a marker to confirm the functional differentiation of epithelial cells in this model. The transcript data normalised using GADPH showed a ȕ-Casein expression pattern as reported earlier (Kanazawa and Kohmoto 2002; Yoshimura and Oka 1990). Based on this level of confidence, Caveolin 1 and 2 expression levels were also determined by normalising with GAPDH.
A basal level of expression of ȕ-Casein was measured in the day 12 pregnant mammary gland in agreement with the previous reports showing differentiation and expression of this milk protein during late pregnancy (Kanazawa and Kohmoto 2002). After incubating explants in the NH control and prolactin alone treatment, this value declined from day 1 onwards whereas maintenance of the milk protein gene expression was noticed in the presence of all three lactogenic hormones (IFP), with values attaining the maximum level at day 4 (Figure 2.1 A). Similarly, the Caveolin 1 and 2 genes showed expression in the day 12 pregnant mammary gland but levels of expression in explants decreased from day 1 and remained low until day 4 of culture in all three treatments. Although Caveolin 1 gene expression declined in explants cultured with both NH and P, the decline was more dramatic in explants cultured in IFP (Figure 2.1 B). Similarly, Caveolin 2 gene expression declined in all treatments but explants cultured with IFP showed comparatively more decline (Figure 2.1 C).
74
A 1.50 ȕ Casein
1.25
1.00 NH 0.75 P 0.50 IFP
0.25
0.00 D0 D1 D2 D3 D4
B 1.50 Caveolin 1 1.25
1.00 NH 0.75 P 0.50 IFP
0.25 Relative Expression
0.00 D0 D1 D2 D3 D4 Relative Fold Expression 1.50 Caveolin 2 C 1.25
1.00 NH 0.75 P 0.50 IFP
0.25
0.00 D0 D1 D2 D3 D4
Days in Culture
Figure 2.1: The levels of ȕ-Casein, Caveolin 1 and Caveolin 2 expression in mouse mammary explants treated with lactogenic hormones. Mammary explants from mid pregnant mice (day 12) were cultured for four days in Media 199 with either no hormone or indicated combinations of insulin (I- 1 μg/ml), cortisol (F- 50 ng/ml) or prolactin (P- 1 μg/ml) for four days. RNA was extracted from explants and the level of gene expression was measured for (A) ȕ-Casein, (B) Caveolin 1 and (C) Caveolin 2 using qPCR. Points represent mean gene expression values normalised to housekeeping gene GAPDH and represented as folds compared to day 0 (D0) levels.
75
The ȕ-Casein gene, the major milk protein gene and the positive control for the experimental system showed a basal level of expression in mammary tissue at day 0 (T0) that was maintained in the explants after 4 days of culture in IFP. This expression declined significantly to very low levels in explants after 4 days of culture in all other treatments (Figure 2.2 A). Caveolin 1 and 2 gene expression in the T0 mammary tissue was maintained after four days of culture except for a significant reduction (<0.01) in Caveolin 1 expression in the treatments with FP and IFP (Figure 2.2 B). Likewise, Caveolin 2 expression was found to be significantly reduced from T0 levels in explants cultured with IF (<0.05), FP (<0.01) and IFP (<0.01) (Figure 2.2 C).
2.3.2 Localisation of Caveolin 1 in mouse mammary gland Mammary gland from pregnant mice is mainly composed of ducts and developing alveoli occupying the fat pad. In day 15 pregnant mammary glands, immunocytological detection of Caveolin 1 was observed in the elongated myoepithelial cells surrounding the epithelial cells and also in the myoepithelial cells lining the ducts whereas none of the epithelial cells showed positive staining (Figure 2.3).
76
A 1.2 ȕ-Casein
1
0.8
0.6 **
0.4
0.2
0 T0 NH I F P IF IP FP IFP
B 2 Caveolin 1
1.5
1
0.5 ** **
Fold Expression 0 T0 NH I F P IF IP FP IFP C 1.2 Caveolin 2
1 Relative Fold Expression Relative 0.8
0.6 * * 0.4 ** 0.2
0 T0 NH I F P IF IP FP IFP
Figure 2.2: The level of expression of genes in mouse mammary explants treated with lactogenic hormones. Mammary explants from mid pregnant mice (day 12) were cultured for four days in Media 199 with either no hormone (NH) or indicated combinations of insulin (I- 1 μg/ml), cortisol (F- 50 ng/ml) or prolactin (P- 1 μg/ml). Expression levels were determined by qPCR for (A) ȕ Casein, (B) Caveolin 1 and (C) Caveolin 2. Bars represent mean ± SEM of gene expression normalised against GAPDH and represented as fold of T0 values. Significant difference compared to T0 (tissue prior to culture) at 5% and 1% level are indicated as * and ** (n = 4).
77
AA BB
CC DD
EE
Figure 2.3: Localisation of Caveolin 1 by immunohistochemistry in mouse mammary gland during pregnancy. Day 15 pregnant mammary gland fragments were fixed and sections were immunolabelled with Caveolin 1 antibody and viewed at (A) 40x and (B) 100x magnification. Arrows show positive staining for Caveolin 1 in myoepithelial cells. The negative control for immunohistochemistry (40x) are represented in (C) and (D). Pictorial representation of the mammary gland (Caffarel et al. 2001) is shown in (E).
78
2.3.3 Comparative genome organisation and protein alignment of Caveolin 1 and 2 of tammar wallaby and mouse The genome organisation of Caveolin 1 and 2 genes from mouse and tammar wallaby (Figure 2.4) showed Caveolin 1 has three exons (Exon 1 = 30bp, Exon 2 = 165bp and Exon 3 = 342bp) which are of similar size in both species. The introns 1 and 2 of tammar wallaby Caveolin 1 are 1668 and 43056 nucleotides respectively, compared to mouse introns 1 and 2 which are of 1401 and 31067 nucleotides respectively. Caveolin 2 consists of exonic and intronic regions of comparable sizes in both the species. Caveolin 2 consists of three exons comprised of 148, 191 and 150 nucleotides in tammar wallaby as compared to 150, 189 and 150 nucleotides in mouse. The intronic regions comprise 382 and 6515 nucleotides in tammar wallaby when compared to 384 and 4814 nucleotides in mouse.
Multiple sequence alignment of Caveolin 1 and 2 amino acid sequences of mouse and tammar wallaby showed 87% similarity between Caveolin 1 orthologues and 70% between Caveolin 2 orthologues (Figure 2.5). The eight amino acid FEDVIAEP motif (caveolin signature motif) towards the amino terminal showed complete conservation in Caveolin 1. In Caveolin 2, there was a single amino acid variation in this domain between the two species with the leucine residue at the 4th position in mouse replaced by valine in tammar wallaby. Caveolin scaffolding domain in Caveolin 1 was conserved between these two species whereas in Caveolin 2 there was variation in three amino acids.
79
30 1668 165 43056 342 tCav 1
30 1401 165 31067 342 mCav 1
148 382 191 6515 150 tCav 2 INTRON 150 384 189 4814 150 mCav 2 EXON
Figure 2.4: Comparative genome organisation of Caveolin 1 and 2 of tammar wallaby and mouse. Tammar wallaby (t) and mouse (m) were used for the comparative analysis. The intronic and exonic organisation is represented by bold lines and rectangular solid boxes respectively as shown. The number above each region represents the respective nucleotide size.
80
A Caveolin 1
MOUSE MSGGKYVDSEGHLYTVPIREQGNIYKPNNKAMADEVTEKQVYDAHTKEIDLVNRDPKHLN 60 WALLABY MSSGKYIDSEGLLYSAPIREQGNIYKPNNKTMTDEMNEKQMYDAHTKEIDLVNRDPKHLN 60 **.***:**** **:.**************:*:**:.***:*******************
MOUSE DDVVKIDFEDVIAEPEGTHSFDGIWKASFTTFTVTKYWFYRLLSTIFGIPMALIWGIYFA 120 WALLABY DDVVKIDFEDVIAEPEGTHSFDGIWKASFTTFTVTKYWFYRLLTVLFGIPMALIWGIYFA 120 *******************************************:.:**************
MOUSE ILSFLHIWAVVPCIKSFLIEIQCISRVYSIYVHTFCDPLFEAIGKIFSNIRISTQKEI 178 WALLABY ILSFLHIWAVVPCIKSYLIEIQCIGRIYSICIHTFCDPLFEAVGKIFNKIRITTQKEI 178 ****************:*******.*:*** :**********:****.:***:*****
B Caveolin 2
MOUSE MGLETEKADVQLFMADDAYSHHSGVDYADPEKYVDSSHDRDPHQLNSHLKLGFEDLIAEP 60 WALLABY MGLETEKADAQIYMDEDSYSRHSGPDYPGPEQVVKSVQDRDPRGLNTHLKLGFEDVIAEP 60 *********.*::* :*:**:*** **..**: *.* :****: **:********:****
MOUSE ETTHSFDKVWICSHALFEISKYVMYKFLTVFLAIPLAFIAGILFATLSCLHIWILMPFVK 120 WALLABY ESAHSFDKVWICNHALFEISKYLIYKVLTVLLAIPLAFVAGIFFATLSCLHIWFVVPFIK 120 *::*********.*********::**.***:*******:***:**********:::**:*
MOUSE TCLMVLPSVQTIWKSVTDVVIGPLCTSVGRSFSSVSMQLSHD 162 WALLABY TCLMVLPSVQTVWKSITDVLIAPVCKSMGLIFSSISLRLSPE 162 ***********:***:***:*.*:*.*:* ***:*::** :
Figure 2.5: Protein sequence alignment of tammar wallaby and mouse Caveolin 1 and 2. Alignment of (A) Caveolin 1 and (B) Caveolin 2 proteins of tammar wallaby (assembled through Ensembl genome browser for Macropus eugenii) and mouse (Caveolin 1, GenBank accession number - AAR16290 and Caveolin 2, GenBank accession number - AAH23095) is shown. Identical amino acids are indicated by the asterisk (*), very similar amino acids are indicated by a colon (:) and more or less similar amino acids are indicated by a dot (.). The caveolin signature motif of the protein family represented by the “FEDVIAEP” stretch has been marked in black boxes and the Caveolin scaffolding domain has been marked in blue boxes.
81
2.3.4 Expression profile of Caveolin 1 and 2 across the tammar wallaby lactation cycle
Mammary tissues from tammar wallaby during phase 1 (pregnancy), 2A, 2B, RI ODFWDWLRQ DQG IURP LQYROXWLQJ SKDVH ZDV DQDO\VHG IRU WKH FDYHROLQV DQG ȕ- Lactoglobulin (BLG) gene expression. The data was normalized with two housekeeping genes RPS15 (data represented) and GAPDH (data not shown), both showing similar profile. Analysis of BLG gene expression (Figure 2.6 A), was used as a positive control for the experimental system and the level of expression of this gene significantly increased post parturition through to phase 3 of lactation when there was maximal milk production (represented as a grey line in the BLG graph; Bird et al. 1994). At involution the level of expression of BLG declined significantly compared to levels in the mammary tissue at phase 3 of lactation. Caveolin 1 gene showed a profile with a higher level of expression at all stages of lactation when compared to phase 1, and at involution decreased to levels comparable to phase 1 (Figure 2.6 B). In contrast, the Caveolin 2 gene showed significantly higher levels of expression only at phase 2B lactation and at all other stages showed level of expression similar to that observed in phase 1 (Figure 2.6 C).
2.3.5 Hormonal regulation of Caveolin 1 and 2 genes in tammar wallaby mammary gland explants The hormonal regulation of Caveolin 1 and 2 tammar wallaby genes was analysed by qPCR in mammary gland explants from tammars at day 21 pregnancy. Explants were cultured for either two or four days in either control media (NH) or media with insulin (I), cortisol (F) and prolactin (P) (Figure 2.7 ȕ-Lactoglobulin (BLG), the major milk protein gene, was used as a positive control for the experimental system and showed a significant level of induction in explants cultured with IFP compared to explants in NH media on both day 2 and day 4 of culture (Figure 2.7 A). In contrast, the level of both Caveolin 1 (Figure 2.7 B) and 2 (Figure 2.7 C) gene expression in explants showed no significant difference between NH control and IFP treatment after two and four days of culture.
82
ɴ-lactoglobulin 1000 * A
800
600 * 400 *
200
0 Phase1 Phase2A Phase2B Phase3 Involution
Caveolin 1
B 0.1 * * 0.08 * 0.06
0.04 Relative Expression 0.02 Relative Expression
0 Phase1 Phase2A Phase2B Phase3 Involution
C 0.01 Caveolin 2
0.008 *
0.006
0.004
0.002
0 Phase1 Phase2A Phase2B Phase3 Involution
Figure 2.6: qPCR analysis of gene expression levels in tammar wallaby mammary gland tissue during the lactation cycle. (A) ȕ-lactoglobulin, (B) Caveolin 1 and (C) Caveolin 2 expression profiles in the mammary gland of the tammar wallaby. Phase 1 included mammary tissue from animals at day 9, 21, 23 and 25 pregnancy. Phase 2A included tissues from animals at parturition, day 1, 2, 3, 5, 10, 22, 37, 40, 62, 80 and 100 days of lactation. Phase 2B included tissues from animals at 110, 114, 133, 135, 150, 151, 171, and 193 days of lactation. Phase 3 included tissues from 216, 240, 260, 266 and 293
83 days of lactation. The involution samples were collected at 1, 3, 5 and 10 days after removal of the pouch young. Bars represent mean ± SEM of gene expression levels normalised to housekeeping gene RPS15. A statistically significant difference in expression from SUHJQDQF\ DW OHYHO ZDV PDUNHG DV ³ ´ 7KH JUH\ OLQH LQ WKH ȕ-lactoglobulin graph represents milk production.
84
A ɴ-lactoglobulin 7 NH 6 * IFP
5
4 **
3
2
1
0 B D2 D4 5 Caveolin 1 NH IFP 4
3
2
Fold Expression Fold 1
0 D2 D4 Relative Fold Expression Relative
C 6 Caveolin 2 NH
5 IFP
4
3
2
1
0 D2 D4
Figure 2.7: qPCR analysis of gene expression levels on day 2 and day 4 in tammar wallaby mammary explants treated with lactogenic hormones. Mammary explants from day 21 pregnant tammar wallabies were cultured in Media 199 with either no hormone (NH) or a combination of insulin (I; 1 μg/ml), cortisol (F; 50 ng/ml) and SURODFWLQ 3 JPO DQG H[DPLQHG IRU $ ȕ ODFWRJOREXOLQ % &DYHROLQ DQG & Caveolin 2 gene expression by qPCR. Bars represent mean ± SEM (n = 4) which is the expression of genes normalised to housekeeping gene RPS15 and represented as fold change
85 compared to T0 values. A statistically significant difference between NH and IFP treatment at 5% and 1% levels are indicated by * and ** respectively.
2.4 Discussion
The caveolins are a family of three proteins (Caveolin 1, 2 and 3) that are markers of the flask-shaped invaginations on the plasma membrane of different cell types called Caveolae (Palade and Bruns 2004) and have recently gained importance in the field of lactation. These proteins are implicated in regulating diverse physiological processes like vesicular transport, signal transduction (Razani et al. 2002; Smart et al. 1999) and endocytosis (Pelkmans and Helenius 2002). Two members of the family, Caveolin 1 (Park et al. 2002; Park et al. 2001; Sotgia et al. 2006) and 3 (Sotgia et al. 2009) were correlated to lactation performance in mice. It was reported that a down regulation of Caveolin 1 was required for milk production and this process was mainly mediated by prolactin via a Jak2/STAT5a pathway (Park et al. 2002; Park et al. 2001; Sotgia et al. 2006) a major signaling pathway promoting milk production (Freeman et al. 2000). Furthermore, Caveolin 1 null mice showed accelerated development of the lobulo-alveolar compartment of mammary gland, premature milk production and hyperphosphorylation of STAT5A (Park et al. 2002). It was demonstrated that when primary mammary epithelial cells from wild- type (WT) and Caveolin 1 null mammary glands were cultured in either a 2D or 3D system, Caveolin 1 deficient mammary epithelial cells displayed the ability to spontaneously generate milk droplets (Sotgia et al. 2006). Likewise, it was reported by Sotgia and co-workers (2009) that Caveolin 3 null virgin mammary glands developed lobulo-alveolar hyperplasia similar to that observed during pregnancy and lactation with an up-regulation of mammary gene transcripts associated with these developmental stages.
Earlier studies have suggested prolactin is the major hormonal mediator for the process of lactation, but more recent studies have provided evidence in different species that lactation is regulated by a complex interplay of different hormones and other factors (Lamote et al. 2004; Neville et al. 2002; Plaut 1993). Murine mammary
86 explant cultures were utilised to show that the maximum induction of milk proteins was possible by prolactin only in co-operation with other lactogenic hormones like insulin and cortisol (Bolander et al. 1981; Menzies et al. 2010; Nagaiah et al. 1981). Since the process of expression of caveolin genes was coupled to milk synthesis in the mouse, we utilised a similar mammary explant culture model to examine whether prolactin alone is the regulator of the Caveolin 1 gene or whether it required a complex regulatory process similar to that of the milk protein genes. To test this hypothesis, we evaluated the in vitro expression of caveolin genes in mouse mammary explants cultured with media and different hormonal combinations. In contrast to the earlier reports, the current study showed that mouse Caveolin 1 gene expression was significantly down regulated from day 0 (T0) by day 4 only when both prolactin and cortisol was present in media. Neither prolactin nor cortisol alone was effective for this response in mammary explant culture in the present study which contrasts with the studies of Park and co-workers (2001). Comparable earlier reports on casein synthesis in mouse mammary explants showed maximal response to prolactin action only in the presence of hydrocortisone and direct evidence for the involvement of gluococorticoids on the modulation of casein gene expression at transcriptional level (Ganguly et al. 1980; Ganguly et al. 1979). This induction of casein accumulation in the mouse mammary explants also required insulin (Bolander et al. 1981; Menzies et al. 2010) in addition to prolactin and hydrocortisone. Previously, prolactin was shown to trigger a dose dependent down regulation of Caveolin 1 in Htert-HME1 cells and a luciferase reporter assay in HC11 cells supported the direct role of prolactin on regulation of increased milk protein gene expression (Park et al. 2001). However, the current study shows that the presence of cortisol in culture media is essential for the down regulation of Caveolin 1 expression. We also studied the regulation of Caveolin 2, the second member of Caveolin family which is colocalised and coexpressed with Caveolin 1 (Scherer et al. 1997). Currently there is no direct evidence of Caveolin 2 having role in regulation of lactation although Park and co-workers (2001) reported a slight decline in the expression level of this gene during peak lactation. In the current study, prolactin was not the only regulator of this gene and a significant decline in levels from T0
87 was observed when cortisol was present in culture together with either insulin or prolactin, or in the presence of all three lactogenic hormones. Therefore, the current study clearly shows that a complex interplay of hormones is involved in regulating expression of caveolin group of genes. This is related to the complex endocrine regulation reported in mouse mammary explants for the synthesis of milk protein genes like caseins (Juergens et al. 1965; Terry et al. 1977; Turkington et al. 1965) and Į-lactalbumin (Ono and Oka 1980a, 1980b) which require the presence of all three hormones insulin, cortisol and prolactin in culture media.
There are conflicting reports on the type of cells in the mammary gland in which caveolin genes are expressed (Amorim et al. 2010; Hue-Beauvais et al. 2007; Hurlstone et al. 1999; Park et al. 2005; Savage et al. 2007). The present study attempted to better understand the localisation of Caveolin 1 in mammary gland and therefore the potential mechanism by which they are involved in signaling the endogenous regulation of lactation. There have been contrary reports about the distribution of caveolins in normal mammary gland in human showing its presence in either epithelial cells or in myoepithelial cells (Hurlstone et al. 1999; Park et al. 2005; Savage et al. 2007). In canine mammary gland (Amorim et al. 2010) caveolins were consistently expressed in myoepithelial cells and not expressed in luminal epithelial cells. In mouse, the eutherian in which caveolins have been negatively correlated with lactation, the proteins were detected in the endothelial and myoepithelial cells while negligible expression was observed in the epithelial cells (Hue-Beauvais et al. 2007). Our current study shows localisation of Caveolin 1 in myoepithelial cells of mouse pregnant mammary gland. This was a confirmation for the previous reports which showed localisation of Caveolin 1 in myoepithelial cells along with endothelial cells and adipocytes of the mammary gland with a minimal level of expression during lactation when compared to pregnancy and involution (Hue-Beauvais et al. 2007). There was no evidence for the presence of Caveolin 1 in epithelial cells. Taken collectively, the current and earlier studies, by Hue-Beauvais and co-workers (2007) and Park and co-workers (2001), probably suggest a cell-cell interaction wherein Caveolin 1 in myoepithelial cells, endothelial cells and adipocytes may be regulating milk production in mammary epithelial cells in a
88 paracrine fashion. There has been an earlier report by Levine and co-workers (1984) confirming cell-cell interaction resulting in potent growth promoting activity of mammary epithelial cells when co-cultured with adipocytes. This co-culture influenced the sensitivity of the epithelial cells to lactogenic hormones leading to increased mammary differentiation in terms of milk protein synthesis (Levine and Stockdale 1984; Levine and Stockdale 1985). However, to confirm the paracrine regulation, a detailed investigation is critical with the co-culture of myoepithelial cells over expressing Caveolin 1 and epithelial cells to examine the effect on milk protein gene expression.
To have a better understanding of how caveolins regulate the process of lactation and milk protein synthesis, we utilised the tammar wallaby, a marsupial in which the composition and production of milk varies both qualitatively and quantitatively throughout lactation (Nicholas et al. 1997). Tammar caveolins were identified and compared with mouse and the exonic-intronic regions of both the genes had comparable sizes with small variations in the size of introns. An alignment of the mouse and tammar wallaby Caveolin 1 and 2 showed 70-80% homology in the protein structural features between species. Earlier, a comparison of Caveolin 1 from two eutherian species, human and mouse had shown 92% homology (Yang et al. 2005). This included an 8-amino acid motif ‘FEDVIAEP’ identified as the caveolin signature motif conserved across species which is an important structural characteristic of the protein family (Razani et al. 2002; Scherer et al. 1996; Williams and Lisanti 2004). However, the functional significance of this motif is not known. The caveolin scaffolding domain, which is necessary for membrane attachment (Schlegel et al. 1999) showed complete identity between mouse and tammar wallaby Caveolin 1 whereas there was variation in the motif in Caveolin 2. This domain is required for Caveolin to bind to its interacting partners (Couet et al. 1997) during the scaffolding function of caveolae. These similarities between mouse and tammar wallaby caveolins in terms of the known domains are probably consistent with its role in lactation. However, the 70-80% similarity between mouse and tammar wallaby caveolins may also reveal some functional difference.
89
Peak lactation in the tammar wallaby occurs in phase 3 of lactation. To evaluate whether this is correlated with a decline in Caveolin 1 as reported in mouse, the current study examined the correlation between Caveolin 1 gene expression and milk production in tammar wallaby. However, in contrast to the mouse there was a significantly increased level of expression of Caveolin 1 in the mammary gland during lactation as compared to pregnancy, with no difference between different phases 2A, 2B and 3 of lactation. Indeed, maximal level of Caveolin 1 and 2 were observed at phase 2B when copious milk production is increased (Trott et al. 2003). This pattern of caveolin gene expression initiated a study of whether these caveolin genes were under the control of hormones known to regulate milk protein gene expression in the tammar. To examine this, we utilised tammar mammary explants from pregnant animals and confirmed the induction of the ȕ-Lactoglobulin gene, major milk protein gene, in the presence of insulin, cortisol and prolactin (Nicholas et al. 1991). However, in contrast there was no significant difference in the expression level of the caveolin genes in explants cultured either in the absence of hormones or in the presence of the three lactogenic hormones, suggesting no endocrine role in regulating caveolin gene expression.
To understand the significance of Caveolin in tammar wallaby lactation, the principal question to be addressed would be the localisation of caveolins in the mammary gland. However, this was not addressed in the current study because antibodies for tammar caveolins are not available. It is possible that the higher expression level of caveolins during lactation may be attributed to different immune cell types like macrophages (Kiss and Geuze 1997; Kiss and Kittel 1995), mast cells (Shin et al. 2000) and dendritic cells (Werling et al. 1999) . Caveolin 1 is known as an important immunomodulatory effector molecule that plays a role in innate immune defence by regulating cytokine production and signaling during pathogen invasion (Gadjeva et al. 2010; Medina et al. 2006; Wang et al. 2006a). The increased expression of caveolins during tammar wallaby lactation may suggest a role in either protecting the mammary gland which undergoes complex developmental changes (Findlay 1982) or protecting the immune naïve young from infection (Trott et al. 2003).
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Earlier, Topcic and co-workers (2009) reported that the tammar whey acidic protein (tWAP) had a specific functional domain III in contrast to the eutherian WAP which had only domain I and II. Domain III showed a unique and specific signalling of proliferation of mammary epithelial cells. This is evidence for a protein with structural difference between marsupials and eutherians resulting in an improved function which can be correlated to the difference in reproductive strategies (Kuruppath et al. 2012). In contrast, the caveolin group of genes represents a family of proteins with a high amino acid similarity between tammar wallaby and mice, however, and therefore any role in the process of lactation in the tammar remains to be elucidated.
To conclude, this study demonstrates that
x In mouse, a complex interplay of hormones is involved in regulating expression of caveolin group of genes. x The expression of Caveolin 1 was localised to the myoepithelial cells and adipocytes in the mouse mammary gland, and the epithelial cells lacked expression. x A 70-80% similarity was observed between mouse and tammar wallaby caveolins. x In contrast to the mouse, in tammar wallaby, Caveolin 1 and 2 expressions were high during lactation when compared to pregnancy and involution.
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CHAPTER 3 Effect of tammar wallaby milk on in vitro stomach models
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3.1 Introduction
The marsupial tammar wallaby (Macropus eugenii) has a reproductive strategy that is different from eutherians (Tyndale-Biscoe and Janssens 1988). It is characterized by a short gestation of 26.5 days followed by a long lactation cycle of approximately 330 days with progressive changes in milk production and composition (Nicholas et al. 1997; Tyndale-Biscoe and Janssens 1988). The growth and development of the immature young is regulated by the mother’s milk during the extended lactation period. This reproductive strategy of the tammar wallaby can be utilized in identifying the bioactives present in milk that may play a role in the development of different organs of the infant, including the stomach (Kuruppath et al. 2012; Kwek et al. 2009b; Waite et al. 2005). In the tammar wallaby, lactation is divided into four phases (Nicholas et al. 1997; Nicholas et al. 1995); phase 1 is the first 26.5 days of gestation followed by a 300-330 days lactation divided into phase 2A, 2B and 3 consisting of approximately 100 days each. As reported by Green and Merchant (1988), early phase milk is low in protein and lipid and rich in carbohydrates. In phase 3 of lactation, milk switches to a composition of increased fat and protein and low levels of carbohydrates. The majority of milk proteins in marsupials are found predominantly in the whey fraction (Green and Renfree 1982; Tyndale-Biscoe and Janssens 1988) whereas in many eutherians, the caseins are the predominant proteins (Thompson and Farrell 1974). Milk proteins in tammar wallaby are comprised of two groups; the first group includes ȕ-&DVHLQĮ-&DVHLQȕ- Lactoglobulin anG Į-Lactalbumin and are induced during parturition, and their secretion in milk remains high throughout lactation. The second group is expressed during specific stages of lactation. Each phase of lactation is characterized by a set of major milk proteins; Early lactation protein (ELP) during phase 2A, Whey acidic protein (WAP) during phase 2B and late lactation proteins (LLP-A and LLP-B) during phase 3 (Trott et al. 2003). Additionally, more genes that are differentially expressed across lactation were recently identified, including 75 genes coding for secreted proteins (Sharp et al. 2009). Thirty proteins from this group had shown bioactivity in at least one of the cell-based assays for immune modulation,
93 inflammatory responses, growth and differentiative effects or altered development of mammospheres. This raises the possibility of milk proteins having a role in regulating growth and function of a range of tissues. Moreover, the significant role played by these milk proteins in development of the newborn in this species is now emerging following a fostering experiment wherein pouch young of an early stage of development showed accelerated growth and stomach maturation when transferred to a mother producing milk at a later stage of lactation (Kwek et al. 2009b; Trott et al. 2003; Waite et al. 2005). Therefore, this model provides new opportunities to identify bioactives in the milk that stimulate the development of specific tissues in the neonate (Sharp et al. 2009).
Milk in general has an important role in supporting early development and providing immune protection to the young (Grosvenor et al. 1993; Schanbacher et al. 1997). Studies have proved the presence of different factors in milk which act as mediators for infant development (Donovan and Odle 1994) highlighting the role of either colostrum or milk specifically in development of gastrointestinal tract (GIT) in a wide range of species (Blum and Baumrucker 2008; Xu 1996). It has been demonstrated that milk promotes GIT development in terms of epithelial cell proliferation, villus height, enzyme activity in bovine species (Blättler et al. 2001; King et al. 2008; Playford et al. 1999; Roffler et al. 2003), increased protein synthesis in the swine intestine (Burrin et al. 1992; Ma and Xu 1997; Simmen et al. 1990; Stoddart and Widdowson 1976) and intestinal epithelial growth and permeability in response to breast milk (Cummins and Thompson 2002; Goldman 2000). Furthermore, some of the earlier studies emphasized the role of milk or milk- borne factors on development of stomach of the young. In the suckling rat where a hyper proliferative response was induced in the stomach epithelia by fasting, there was induction of gastric epithelial cell apoptosis and inhibition of cell proliferation through alteration of cyclic proteins in vivo by 7*)ȕ(de Andrade Sá et al. 2008). This is evidence for milk-ERUQH7*)ȕLQUHJXODWLQJFHOOSUROLIHUDWLRQDQGDSRSWRVLV which are important events in gastric development. Studies by Morikawa and co- workers (1979) DQG $QGUqQ DQG %MĘUFN (1986) showed the effect of milk on specialized cells of the stomach including parietal and chief cells. Cross fostering of
94 tammar pouch young demonstrated that milk of later stage lactation accelerated development of the young (Kwek et al. 2009a; Trott et al. 2003; Waite et al. 2005). Employing a similar cross fostering strategy, it was demonstrated by Kwek and co- workers (2009b), that later stage milk leads to significant reduction in parietal cell population and expression of gastric glandular marker genes in the forestomach suggesting down regulation of the gastric glandular phenotype in the fostered PY which is a characteristic feature of stomach development in this species. Therefore, the evidence of tammar wallaby milk having a role in stomach development, coupled with the progressively changing composition of milk, presents a novel opportunity to identify potential factors in milk that specifically stimulate stomach development.
Previous studies investigating the role of milk and milk-derived factors on GIT development has employed a wide range of in vitro models including monolayer cell models of cancer origin like AGS, IEC-6 cells and more complex models like Caco2, T84 and HT-29 cells &HQFLþ DQG /DQJHUKROF &KRSUD HW DO Purup and Nielsen 2012). The choice of the correct model will be dependent on whether the aim of the study is to identify an effect on either cell proliferation and differentiation or permeability. In addition, the effect may vary depending on the origin and nature of the cell model used as the physiological/developmental stage (differentiated or non-differentiated) represented by each model will be different (Purup and Nielsen 2012). Furthermore, each model may not reflect the complete in vivo condition where signaling and interaction from other cell types or organs play an important role (Purup and Nielsen 2012). The aim of the current study was to examine the effect of tammar wallaby milk on gastric cell proliferation and differentiation and therefore, two different models were developed; (a) the embryonic stomach explant model (Abud et al. 2008; Abud et al. 2004; Hearn et al. 1999) and (b) the gastric organoid model (Barker et al. 2010).
The embryonic stomach explant model required culture of mouse embryonic stomach in suspension by attachment to filter papers similar to the techniques employed previously for intestine (Abud et al. 2008). In the intestinal explant culture, there was maintenance of overall three dimensional architecture of the organ
95 closely mimicking the in vivo system (Abud et al. 2008) with effective development and differentiation in culture in terms of mucin production (PAS staining for goblet cells), desmin, and cKit expression (Abud et al. 2005; Hearn et al. 1999). The long term gastric organoid culture, considered as a “mini-stomach” was developed from the gastric glands of stomach pylorus of adult mice utilizing the Lgr5+ stem cell population. These cells undergo asymmetric division to form a multi-potent stem cell pool that is responsible for the regular self-renewal of the gland and they also differentiate into epithelial cells of all lineages in vivo (Bjerknes and Cheng 2002). This stem cell pool is concentrated in the pyloric region of the stomach in adult mice and is present minimally in the corpus region (Barker et al. 2010).
The adapted reproductive strategy in tammar wallaby provides timely nourishment and/or signaling for the development of the altricial young. This includes the development of different organs like the stomach which is an obvious target for milk to act upon as it lies at the interface between nutrients in the luminal environment and the internal environment of the host &HQFLþand Langerholc 2010). Among the three different phases of lactation in tammar wallaby, the first 100 days of lactation (phase 2A) can be assumed as the major period controlling postnatal development of the altricial young (Nicholas et al. 1995) which may include preparing the stomach to digest the maternal milk immediately after birth and gradually adapt to the external environment to digest solid diet/herbage. Studies have demonstrated the ability of later stage milk to accelerate stomach development but there has been minimal investigation on the role of early stage milk. However, the presence of numerous milk proteins in early tammar wallaby milk that impact on transport, nutrition and immune function has been reported using proteomic analysis techniques comprising two-dimensional electrophoresis, comparative image analysis, matrix assisted laser desorption ionisation mass spectrometry (MALDIMS), de novo peptide sequencing and cross species protein matching (Joss et al. 2007; Joss et al. 2009; Sharp et al. 2009). In addition, there are many unannotated proteins secreted in milk in early lactation that may play roles in growth and development of the GIT (Joss et al. 2009), including early stomach development. To investigate this, the current study involved the development of two different in vitro stomach models; the
96 gastric organoid model and the embryonic stomach explant model. Analysis of the expression of gene markers for muscle development, stem cells, epithelial cells and other specialised cells (parietal cells, chief cells and enteroendocrine cells) by quantitative RT-PCR in both the models provided an opportunity to examine the effect of tammar wallaby milk on development and differentiation of the stomach.
3.2 Material and methods
3.2.1. Animal ethics and sample collection
Wild type C57BL/6 mice were obtained from Monash Animal Services, Clayton, Australia. Tammar wallabies were maintained in the Deakin University Marsupial Facility, Victoria, Australia. All sample collection and experiments were approved by the Deakin University Animal Ethics Committee.
3.2.2 Milk collection and processing
The tammar wallaby mothers were caught, separated from their young for 4 h, anaesthetised by intravenous injection of 0.2 ml (4 mg/kg body weight) of Zoletil 100® (Virbac, NSW, Australia) and administered an intramuscular injection of 1IU of Oxytocin-S® (Intervet, Boxmeer, The Netherlands) for milk let down. Milk was obtained by applying gentle pressure on the mammary gland with an average volume of 0.1 ml of phase 2A milk and 0.5 ml of phase 2B milk rapidly collected from each animal. Milk samples were immediately stored at -80°C until future use.
Phase 2A tammar wallaby milk was thawed on ice, centrifuged at 2000g for 10 min and the skim milk removed from beneath the fat layer and above the cell pellet, and pooled. This was further filtered using Corning Costar Spin-X centrifuge tube filters (CLS8160-96EA Sigma Sydney, Australia) before analysing for bioactivity. The total protein content of milk was estimated using a nanodrop spectrophotometer and an equivalent protein control was prepared for organoid culture using a pure fraction of bovine serum albumin (BSA). Phase 2B tammar
97 wallaby milk was processed similarly. The skimmed fraction was either injected into the lumen of embryonic stomach explants or added to the culture media.
3.2.3 Analysis of gene expression by RT-PCR
Tissue samples were collected at different time points and RNA extracted using a Qiagen RNeasy microkit following the manufacturer’s protocol. The cDNA (complementary DNA) was synthesized from total RNA (1 μg) using Superscript III (18080-044 Invitrogen, Australia). Quantitative polymerase chain reactions (qPCR) were performed on an Agilent Mx3000P qPCR system using Brilliant II SYBR Green qPCR master mix (600828 Agilent technologies). The PCR reaction (18 μl) contained 1x master mix, 10 μM of forward and reverse primers (Table 3.1) and diluted cDNA. The qPCR thermal profile included a denaturation (95°C for 10 min) and quantification for 40 cycles (95°C for 30 sec, 60°C for 30 sec). All samples were assayed in triplicate, and included no RT control and no template control. Melting curve analysis was performed to confirm the amplification comprised a single PCR product of correct size. 7KH ¨&W PHWKRG ZDV XVHG WR QRUPDOL]H WUDQVFULSW WR WKH housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) or beta-2 microglobulin (B2M). The housekeeping genes were selected based on previously reported gene expression studies in mouse stomach (Barker et al 2010).
3.2.4 Imaging and microscopy
A Leica inverted microscope was used for analysis of the gastric organoid culture and images captured using DFC425C camera. A Zeiss imager A1 inverted microscope was used for histological analysis of embryonic stomach explants.
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Table 3.1: Primer sequences used for qPCR.
GENES GENE ID PRIMER SEQUENCE (5' to 3' DIRECTION) PRODUCT SIZE (bp) Leucine-rich repeat containing G FP- CCTTGGCCCTGAACAAAATA protein-coupled receptor 5 LGR5 RP- ATTTCTTTCCCAGGGAGTGG 111 FP- CTTGGCCCTGACCTGTATGT Gastric intrinsic factor GIF RP- TAGGTTGCTCAGGTGTCACG 194 FP- CTGCTGTTGCTGCTCTTCAG Mucin 6 MUC6 RP- ATACTCATGGCCGTCAAAGG 162 FP- CCAACCTGTGGGTGTCTTCT Pepsinogen C PGC RP- TTAGGGACCTGGATGCTTTG 187 FP- TGCTTTGATCTTGGATGCTG Trefoil factor family 2 TFF2 RP- GGAAAAGCAGCAGTTTCGAC 174 FP- CCATCTGCAGTTTGCTGCTA Ghrelin GHRL RP- GCTTGTCCTCTGTCCTCTGG 178 FP- ACCAATGAGGACCTGGAACA Gastrin GAST RP- ATCCATCCGTAGGCCTCTTC 158 FP- CCCAGACTCCGTCAGTTTCT Somatostatin SST RP- GAAGTTCTTGCAGCCAGCTT 240 FP- TCCCCACTGCAGCATCCAGTTC Chromogranin CHG RP- CCTTCAGACGGCAGAGCTTCGG 133 FP- TATGTGAAGTTTGCCCGTCA H+K+ATPase ATP4A RP- TGGCGATGATATTTGTGCTC 214 FP- GTGAAGATGGCCTTGGATGT Desmin DES RP- CGGGTCTCAATGGTCTTGAT 182 FP- GATCTGCTCTGCGTCCTGTT cKIT KIT RP- GACAAAGTCGGGATCAATGC 168 Hypoxanthine FP- CGTCGTGATTAGCGATGATG phosphoribosyltransferase HPRT RP- ACAGAGGGCCACAATGTGAT 178
3.2.5 Effect of phase 2B tammar wallaby milk on embryonic stomach explant culture
3.2.5.1 Embryonic stomach explant culture
Black Millipore ILOWHUSDSHU ȝP%ODFNJULGGHG0LOOLSRUH ZDVFXWLQWR square pieces 3 mm in size and a “V” shaped notch cut on one side of the filter papers. These were sterilized in 70% ethanol and transferred to sterile PBS before use. Mouse embryos of either E14 or E15 days post coitus (dpc) were obtained from timed mated, pregnant C57BL/6 mice. Pregnant mice were sacrificed by cervical dislocation and the embryos were removed. The stomach from these embryos were removed using a dissecting microscope, placed across the “V” shaped notch cut on the filter paper by slight pressure and cultured in individual wells of a 60-well terasaki plate containing 20 ȝORIJURZWKPHGLD7KHPHGLDFUHDWHVDODUJHPHQLVFXV
99 which fully encloses the piece of tissue on the filter paper. This explant culture was maintained in a 5% CO2 incubator at 37°C. Media was changed twice daily. The whole protocol is represented pictorially in Figure 3.1.
To examine the growth and establishment of explants in culture, E14 embryonic stomach was cultured in four different media (A) Dulbecco's Modified Eagle Medium (DMEM), 10% Fetal Calf Serum (FCS), Glutamax, Penicillin/Streptomycin (Pen/Strep) (B) DMEM, 5% FCS, Glutamax, Pen/Strep (C) DMEM, 2% FCS, Glutamax, Pen/Strep (D) DMEM, 0.5% FCS, Glutamax, Pen/Strep. The explants grown in all four different media were collected on day 0 and day 3 of culture for histological analysis and from growth media with 10% FCS to determine the gastric gene marker expression by qPCR. The experimental approach is summarized in the flowchart depicted in Figure 3.2.
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STOMACH EXPLANT CULTURE MOUSE EMBRYO MEDIUM
FILTER SUPPORT
STOMACH
TERASAKI WELL
STOMACH FILTER
STOMACH EXPLANT
Figure 3.1: Protocol for culturing of mouse embryonic stomach. Pregnant mice were sacrificed and embryos removed. The embryonic stomach dissected, placed across the “V” shaped notch cut on the filter paper and cultured in growth media in a 60-well Terasaki plate.
Pregnant mice were sacrificed by cervical dislocation and the embryos were removed
Embryonic stomach was dissected using dissecting microscope
Placed across the “V” shaped notch cut on the filter paper and cultured in growth media in a 60-well Terasaki plate
Explant culture was maintained in a 5% CO2 incubator at 37°C
Media was changed twice everyday and samples collected according to aim of the study
Figure 3.2: Experimental design for stomach explant culture model.
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3.2.5.2 Histology
Stomach explants along with the attached filter paper were removed from the terasaki plate and immersed in 4% paraformaldehyde (PFA) in PBS overnight at 4°C. The fixed stomach samples were then removed from the attached filter paper and embedded in 1% low melting point agarose (AMERSCO) and embedded in paraffin. Sections were cut at 4 μm and stained with haematoxylin (Amber Scientific Pty Ltd) & eosin (Amber Scientific Pty Ltd) using standard techniques (Bancroft and Stevens 1982).
3.2.5.3 Analysis of phase 2B tammar wallaby milk on gastric gene marker expression and stomach epithelial cell proliferation
Embryonic stomach at E14 dpc was dissected and cultured in 20 ȝORIJURZWK media (DMEM, 10% or 2% FCS, Glutamax, Pen/Strep) with or without phase 2B tammar wallaby milk (2 ȝO; pool of day 122, 141, 148, 154, 167) and incubated in
5% CO2 at 37°C for three days. The explants were collected, RNA extracted and analysed by qPCR to define the expression of different markers as described earlier.
Embryonic stomach at E15 dpc was dissected and cultured similarly in 20 ȝO of growth media (DMEM, 2% FCS, Glutamax, Pen/Strep) with or without phase 2B tammar wallaby milk (pool of day 122, 141, 148, 154, 167) and maintained in 5%
CO2 incubator at 37°C for 24 h. The explants were analysed for epithelial cell proliferation (BrdU) and apoptosis (Caspase 3).
3.2.5.3.1 BrdU incorporation assay
The media from E15 embryonic stomach grown in culture for 24 h was replaced with media containing Cell Proliferation labelling reagent (RPN201, Amersham Biosciences), an aqueous solution of 5-fluoro-2V-deoxyuridine and 5- bromo-2V-deoxyuridine (BrdU, 3 mg/ml) at a dilution of 1:1000. The explants were then incubated in this medium for 90 min in a 5% CO2 incubator at 37°C. The tissues were washed in PBS and fixed in 4% PFA for further processing for immunohistochemistry.
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3.2.5.3.2 Immunohistochemistry Freshly dissected embryonic stomach and cultured stomach explants were fixed in 4% PFA either for 1 KDWURRPWHPSHUDWXUHRURYHUQLJKWDWÛ&7LVVXHVZHUH first embedded in low melting point agarose, dehydrated and embedded in paraffin. Sections were cut at 4 μm thickness and collected onto 3-aminopropyltriethoxy- silane-coated slides. Immunohistochemical analysis was performed following previously reported protocol with slight modifications (Abud et al 2005). Sections were dewaxed and antigen retrieved by heating in 10 mM citric acid buffer at pH 6.0 for 10 min in a pressure cooker. Slides washed and endogenous peroxidases blocked by incubation in 0.5% H202 in PBS for 15 min. Sections for BrdU immunohistochemistry were blocked for 1 h in CAS block (00-8120, Invitrogen). After further washing, sections were incubated with mouse anti-BrdU IgG (BD- 555627 Pharmingen), 1:200 dilution (diluted in 1% %6$ RYHUQLJKW DW Û& DQG incubated in horseradish peroxidase (HRP) - conjugated secondary antibodies (goat anti-mouse HRP, G21040 Invitrogen) 1:200 dilution (diluted in 1% BSA) for 1 h. Sections for anti-active Caspase 3 immunohistochemistry were blocked for 1 h in 5% skimmed milk. After further washing, sections were incubated in rabbit anti-active Caspase 3 IgG (550480 Pharmingen), 1:200 dilution (diluted in 1% BSA) overnight DW Û& DQG LQFXEDWHG LQ KRUVHUDGLVK SHUR[LGDVH +53 - conjugated secondary antibodies (goat anti-rabbit HRP, G021234 Invitrogen) 1:200 dilution (diluted in 1% BSA) for 1 h. All slides were developed with 3,3V-diaminobenzidine tetrahydrochloride (DAB, Dako) and lightly counterstained with haematoxylin prior to mounting in histomount (Zymed). Sections were viewed on a Zeiss imager A1 inverted microscope. To quantify the apoptotic cells in the embryonic stomach epithelium, the image analysis software Image-Pro Plus was used. The images captured after active Caspase 3 staining were utilised and a total of atleast 6 images were captured from each slide with a minimum average of at least six segmented regions from each slide considered for counting. The nuclei positively and negatively stained for anti-active Caspase 3 were counted separately and the average percentage of apoptotic cells was determined.
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3.2.5.4 Analysis of tammar wallaby 2A milk on gastric gene markers
Embryonic stomach at E15 dpc was dissected and cultured in 20 ȝORIJURZWK media (DMEM, 2% FCS, Glutamax, Pen/Strep) containing either BSA (control) or phase 2A tammar wallaby milk and maintained in 5% CO2 incubator at 37°C for 48 h. The explants were collected, RNA extracted and qPCR performed to analyse the expression of different gastric gene markers as described earlier.
3.2.6 Analysis of phase 2A tammar wallaby milk on gastric organoid model
3.2.6.1 Gastric gland unit isolation and culture conditions
Gastric glands were isolated from the pyloric region of stomach of mice 6-8 weeks of age according to the method of Barker and co-workers (2010). The stomach was opened and washed thoroughly with cold PBS and the pylorus separated. The epithelium was separated from the muscle layer, divided into pieces of approximately 5 mm size and incubated in PBS with 10 mM EDTA for 4 h at 4°C. After removing the chelating agent, the stomach fragments were thoroughly suspended in the buffered solution using a 10 ml pipette and a gastric gland enriched fraction was obtained by sedimentation and centrifugation.
The gastric gland enriched fraction was mixed with 50 μl of matrigel (356231, BD Biosciences) and plated in 24-well plates. After matrigel polymerization, 500 μl of culture medium (Advanced DMEM/F12 containing Glutamax, B27, N2, 1 μM N-acetylcysteine, 10 nM Gastrin, 50 ng/ml EGF, 100 ng/ml Noggin, 100 ng/ml FGF-10, R-Spondin 1 conditioned media and Wnt3a conditioned media) was added to each well of the 24-well plate. Media was changed every alternate day. The growth of the organoids was observed for a maximum of seven days without any passaging. The whole protocol has been pictorially represented in Figure 3.3.
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The stomach organoid culture was initiated as described above and organoids grown for a period of four days in complete culture media. On day 4, the media was changed and treatments initiated by adding either BSA control or phase 2A milk (day 64 and 65) in complete culture media. The organoids were grown for another two days and analysed for gastric gene expression levels. The experimental approach is represented as a flowchart (Figure 3.4).
3.2.7 Statistical analysis
Statistical analysis was performed using a two-tailed t-test assuming unequal variances between pairs of means. Quantitative data were presented as mean ± standard error of the mean. Statistical significance was determined at the either 5 or 1% level.
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GASTRIC GLANDS
GROWTH
STOMACH
Figure 3.3: Protocol for isolation of gastric glands for stomach organoid culture. Gastric glands were isolated from the pylorus of mouse stomach and growth was initiated in complete culture media in 24-well plates.
Day 0: Gastric glands were isolated from the mouse stomach pylorus and growth was initiated in complete culture media
Day 1: Culture media was changed to remove any cell debris
Day 4: Fresh culture media was added along with initiation of treatments Control – BSA + Complete culture media Treatment – Phase 2A milk + Complete culture media
Day 5: Organoid growth was observed, images acquired and samples collected for RNA extraction
Day 6: Organoid growth was observed, images acquired and samples collected for RNA extraction
Figure 3.4: Experimental design for gastric organoid model.
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3.3 Results
3.3.1 Effect of tammar wallaby milk on embryonic stomach explants
3.3.1.1 Establishment of embryonic stomach explant culture
In the stomach from embryo at E14 dpc cultured in media containing different concentrations of serum ranging from 0.5% to 10%, a pattern of cell death based on cell morphology and cell shrinkage was noticed around the lumen of the stomach after three days in culture (Figure 3.5). Although less cell death was noticed in the explants cultured in DMEM with 10% FCS (Figure 3.5 A), the pattern of cell death became more distinct with decreased concentration of serum in the growth media with almost no epithelial maintenance in explants cultured in DMEM with 0.5% FCS.
Based on the above observation, stomach from embryos at E14 dpc were cultured for three days in media supplemented with 10% FCS. The smooth muscle marker Desmin (DES) and gastric epithelial marker Mucin 6 (MUC6) showed a significant increase in expression at day 3 as compared day 0 levels (Figure 3.6). In contrast, other gene markers including cKit (KIT), Gastrin (GAST), Trefoil factor 2 (TFF2) and Pepsinogen C (PGC) showed no significant change in expression after three days of culture.
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DMEM + 10% FCS DMEM + 5% FCS A B
DMEM + 2% FCS DMEM + 0.5% FCS C D
Figure 3.5: E14 embryonic stomach explants cultured for three days in media containing 0.5% to 10% FCS. Embryonic stomach from pregnant mice at E14 dpc was cultured for three days in different culture media, fixed in 4% PFA, processed, sectioned and stained with H&E. (A) DMEM with 10% FCS (B) DMEM with 5% FCS (C) DMEM with 2% FCS (D) DMEM with 0.5% FCS. Black arrow marks represent cells undergoing death.
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A B
3.0 40 2.5 * 30 * 2.0 1.5 20 1.0 10 0.5 0.0 0
Relative Fold Expression D0 D3 D0 D3 D0 D3 D0 D3 D0 D3 D0 D3 DES KIT GAST MUC6 TFF2 PGC
Figure 3.6: Gene expression analysis by qPCR in day 0 and day 3 embryonic stomach explants. Expression levels of different marker genes were analysed in the embryonic stomach explants at day 3 (D3) in culture and compared with day 0 (D0). Bars represent mean ± SEM (n = 4) of the expression levels normalized to housekeeping gene HPRT and represented as folds relative to day 0. Similar profiles were observed when normalized to the housekeeping gene B2M. A significant difference in gene expression between D0 and D3 at 5% and 1% level is indicated as * and **.
3.3.1.2 Effect of phase 2B tammar wallaby milk on stomach explant culture
3.3.1.2.1 Effect of phase 2B tammar wallaby milk on gastric gene markers
Based on an optimisation from the above experiments, embryonic stomach was further cultured in media supplemented with 2% FCS, serum at an intermediate concentration that allowed better explant maintenance and less interference with tammar milk assay. There was no significant difference between control and phase 2B treatment in the expression of smooth muscle marker DES (Figure 3.7 A; p=0.58 for 10% serum and p=0.04 for 2% serum) and gastric epithelial marker MUC6 (Figure 3.7 B; p=0.45 for 10% serum and p=0.48 for 2% serum) in the stomach from embryos at E14 dpc after three days in culture. Other gene markers also showed no difference (data not shown).
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DesminDES MucMUC6 6
Figure 3.7: Gene expression in E14 embryonic stomach explants at day 3 following treatment with phase 2B milk. Expression levels of Desmin (A) and Mucin 6 (B) were analysed in the embryonic stomach explants at day 3 of culture with and without phase 2B milk. Bars represent mean ± SEM of the relative fold expression levels normalized to housekeeping gene HPRT as compared to day 0 levels (n=3).
3.3.1.2.2 Effect of phase 2B tammar wallaby milk on pattern of proliferation and apoptosis in embryonic stomach explants
The day 0 control explants showed proliferating cells (BrdU, brown) throughout the section (Figure 3.8 A). After 24 h of culture in media supplemented with 2% serum, control explants and explants treated with phase 2B tammar wallaby milk were compared. There was less maintenance of epithelial cells in both tissues (control and phase 2B milk) after treatment for 24 h when compared to day 0 control samples. However, explants injected with phase 2B milk showed extensive staining (Figure 3.8 C) across the section when compared to control explants at 24 h (Figure 3.8 B). The extent of apoptosis in the explants was determined using Caspase 3 staining and counting of cells using Image-Pro Plus (Figure 3.9). There was no significant difference in the number of apoptotic cells between control and phase 2B milk treated explants. However, an average of 17% of the epithelial cells showed Caspase 3 staining under both culture conditions.
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Figure 3.8: Effect of phase 2B tammar wallaby milk on pattern of proliferation in E15 embryonic stomach explants at 24 h. Embryonic stomach from E15 pregnant mice cultured under control conditions or with phase 2B milk in the lumen for 24 h was incubated with Cell Proliferation labelling reagent for 90 min and stained for BrdU (brown) to determine cellular proliferation. (A) E15 embryonic stomach at day 0 (before culture) (B) E15 embryonic stomach after 24 h of culture under control conditions (C) E15 embryonic stomach after 24 h of culture with phase 2B milk injected into the lumen (D) Negative control for staining
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Cell Death Cell Cell Death (Fold levels)
Figure 3.9: Effect of phase 2B tammar wallaby milk on epithelial cell death in E15 embryonic stomach explants at 24 h. Embryonic stomachs from E15 pregnant mice cultured for 24 h under control conditions or with phase 2B milk in the lumen were examined for cell death represented by Caspase 3 staining. A total of 6 images were captured from each slide/section (randomly chosen) with a minimum average of at least 6 segmented regions from each image considered for counting. The nuclei positively and negatively stained for anti-active Caspase 3 were counted separately and the average percentage of epithelial cells stained for active-Caspase 3 was determined and expressed as fold levels compared to control.
3.3.1.3 Effect of phase 2A tammar wallaby milk on stomach explant culture
Stomach from embryos at E15 dpc cultured for two days in culture media (DMEM with 2% FCS) with different treatments; control (media alone), BSA, D13 (pool of D13 and D16), D29, D65 and D92 tammar wallaby skim milk fraction, was examined for expression of gastric gene markers (Figure 3.10). The expression of all gastric gene markers were normalized using housekeeping genes HPRT and B2M (observed similar profile and figure represented is data normalized to HPRT). The smooth muscle marker DES showed no significant increase in expression on day 2 of culture over day 0. MUC6, the gastric epithelial marker showed no significant increase on day 2 in any of the treatments except for BSA treatment showing a
112 significant increase in the level as compared to day 0 levels. TFF2, mucus neck cell marker showed significantly increased level of expression on day 2 in the presence of D65 tammar wallaby milk whereas a significant increase in expression of Enteroendocrine marker SST was observed on day 2 with D92 milk treatment. Parietal cell marker ATP4A, chief cell marker PGC and cell proliferation marker KIT showed no significant difference in expression.
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B A DesminDES cKITKIT 2 2
1.5 1.5
1 1
0.5 0.5
0 0
Muc6 Tff2 C MUC6 D TFF2 20 * 60 * 15 40 10 20 5
0 0 Relative Fold Relative Expression
E SomstnSST F PGCPepCG HKATPaseATP4A
4 4 12
3 3 9 * 2 2 6
1 1 3
0 0 0
Figure 3.10: Gene expression analysis by qPCR in embryonic stomach explants at day 0 and day 2 of treatment with tammar wallaby milk. Embryonic stomach explants (E15) were cultured for two days under control conditions (no treatment) or with BSA, D13, D29, D65 or D92 tammar wallaby milk. The expression levels of different gastric gene marker were analysed in these explants at day 2 in culture and compared with day 0. Bars represent mean ± SEM (n = 3) of the expression levels normalized to housekeeping gene HPRT and represented as fold change relative to day 0 values. A significant difference in gene expression between D0 and D2 at 5% level is indicated as * .Similar profiles were observed when normalized to the housekeeping gene B2M (n=3). The gene markers analysed were (A) Desmin (DES), (B) cKit (KIT), (C) Mucin 6 (MUC6), (D) Trefoil factor 2 (TFF2), (E) Somatostatin (SST), (F) Pepsinogen C (PGC) and (G) H+K+ATPase (ATP4A).
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3.3.2 Effect of phase 2A tammar wallaby milk on gastric organoid culture system
3.3.2.1 Development of gastric organoid culture system
The initial part of the current study shows the growth pattern of the gastric organoids in culture during a period of seven days (Figure 3.11) as reported earlier by Barker and co-workers (2010). The growth of organoids was observed on a daily basis and images captured using an inverted microscope showed that the initial three days of growth involved the formation of spherical structures. These structures gradually increased in size and after day 3 began to show budding which become progressively extensive by day 7. The organoids progressed to death in culture after day 7 if not passaged. The experiment was repeated three times to confirm reproducibility.
3.3.2.2 Gene expression analysis in gastric organoids
The analysis of expression of different gastric marker genes was analysed in the organoids at day 4 and day 7 (Figure 3.12 a). All values were normalized using the housekeeping gene HPRT (normalisation with B2M showed similar profiles) and day 7 expression profiles were represented as fold change compared to day 4. The stem cell marker Leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) showed a two fold increase in expression from day 4 to day 7. The other marker genes; Mucin 6 (MUC6 - gastric epithelial marker, ~5 fold), Pepsinogen C (PGC - Chief cell marker, ~18 fold), Gastric Intrinsic Factor (GIF – gastric epithelial marker, ~85 fold) and Trefoil factor 2 (TFF2 – Mucus neck cell marker, ~8 fold) also showed an increased level of expression by day 7 as compared to day 4. In sharp contrast, other markers like H+K+ATPase (ATP4A - Parietal cell marker), Ghrelin (GHRL - A like cell marker), Gastrin (GAST - G cell marker), Somatostatin (SST – Delta or D cell marker) and Chromogranin (CHG – Enterochromaffin-like or ECL cell marker) showed reduced expression at day 7 as compared to day 4. A similar change in expression was noticed for all the genes when the day 5 expression levels were compared with day 4 levels in the second experiment (Figure 3.12 b).
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D1 D2 D3 D4
D5 D6 D7
Figure 3.11: Growth of gastric organoids. Gastric glands were isolated from mouse stomach pylorus and cultured in matrigel in advanced DMEM/F12 containing Glutamax, B27, N2, 1 μM N-acetylcysteine, 10 nM Gastrin, 50 ng/ml EGF, 100 ng/ml of Noggin, 100 ng/ml FGF-10, R-Spondin 1 conditioned media and Wnt3a conditioned media. Growth was observed and images acquired for a period of seven days. Scale bars (100 μm) are as represented.
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EXPERIMENT 1 a
LGR5 MUC6 PGC GIF TFF2
ATP4A GHRL GAST SST CHG
EXPERIMENT 2
b AB Fold Expression Fold Expression Fold
LGR5 MUC6 PGC GIF TFF2
C Fold Expression
ATP4A GHRL GAST SST CHG
Figure 3.12: Gene expression analysis in gastric organoids by qPCR. [Two separate experiments - a and b] (a) Expression levels of different marker genes were analysed in the gastric organoids at day 4 and day 7. (b) Expression levels of different marker genes were analysed in the gastric organoids at day 4 and day 5. Bars represent the expression levels normalized to housekeeping gene HPRT and represented as folds relative to D4 levels. Similar profiles were observed when normalized to the housekeeping gene B2M.
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3.3.2.3 Effect of phase 2A tammar wallaby milk on gene expression in gastric organoids
The data represents the expression level of genes normalized using the housekeeping gene HPRT and represented as fold changes relative to their value at day 4 (Figure 3.13). The stem cell marker Leucine-rich repeat containing G protein- coupled receptor 5 (LGR5) showed a two-fold increased expression level on both day 5 and day 6 as compared to day 4 whereas, with phase 2A milk treatment there was a gradual decline in expression from day 5 to day 6 when compared to the BSA control. The gastric epithelial marker MUC6 showed a gradual increase in expression by day 6 with no difference between the BSA control and phase 2A milk treatment at both time points (Figure 3.13 A). Figure 3.13 B represents the expression of chief cell marker PGC, epithelial marker GIF and mucus neck cell marker TFF2. PGC showed a gradual increase in level of expression from day 5 to day 6 with phase 2A milk treatment showing increased level as compared to the BSA control. The gastric epithelial marker GIF showed an increase in expression by day 5 and higher levels by day 6 as compared to day 4. Treatment with phase 2A milk showed reduced increase as compared to the BSA control. TFF2 gene showed no difference in expression with phase 2A milk either on day 5 or day 6. Figure 3.13 C represents the expression of gastric gene markers including ATP4A, GHRL, GAST, SST and CHG. None of these showed a difference in expression levels between BSA control and phase 2A milk treatment at both day 5 and day 6 except for GHRL showing a decline in expression level at day 6 alone with milk treatment as compared to BSA control. All these markers showed a decline in their expression by day 5 and day 6 as compared to day 4 of culture.
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A B 2.5 30
25 2.0
20 1.5 15 BSA BSA 1.0 2A MILK 10 2A MILK
0.5 5 Relative Fold Relative Expression Fold Relative Expression 0.0 0 D5 D6 D5 D6 D5 D6 D5 D6 D5 D6 LGR5Lgr5MUC6 Muc6 PepCPGC GIF GIF Tff2TFF2
0.5 C
0.4
0.3
BSA 0.2 2A MILK
0.1 Relative Fold Relative Expression
0.0 D5 D6 D5 D6 D5 D6 D5 D6 D5 D6 HKATPase Ghrelin Gastrin Somstn Chromgn ATP4A GHRL GAST SST CHG
Figure 3.13: Effect of phase 2A tammar wallaby milk on gene expression levels in gastric organoids. Gastric organoids were treated with either BSA (control) or phase 2A tammar wallaby milk after four days of growth in culture and gene expression levels at day 5 and day 6 determined by qPCR. Data shown are the expression levels normalized to housekeeping gene HPRT and represented as fold changes relative to day 4. Similar profiles were obtained when normalized to housekeeping gene B2M (data not shown).
3.4 Discussion
Lactation in the tammar wallaby has been adapted to accommodate the nutritional requirement and developmental changes of the altricial young (Kwek et al. 2009a; Trott et al. 2003; Waite et al. 2005; Wanyonyi et al. 2011; Watt et al. 2012). This reproductive strategy gives a better opportunity as a comparative model to understand the role of milk and milk-borne factors that may signal development of specific organs (Kuruppath et al. 2012; Sharp et al. 2009). It has already been demonstrated by Waite and co-workers (2005) and Kwek and co-workers (2009a)
119 using cross fostering experiments that tammar wallaby milk triggered accelerated stomach development. It was shown that the acid secreting parietal cells present in both forestomach and hind stomach of tammar wallaby PY for the first 200 days postpartum disappears from the forestomach by phase 3 and thereafter is detected only in the hind stomach. This developmental maturation in the PY stomach was driven by later phase tammar wallaby milk. However, the current study is the first to investigate the role of early phase tammar wallaby milk on stomach development using an in vitro model. Newborn tammar wallaby pouch young lacks gastric pit/glands (Setiati 1986) and the development of complex gastric glands is initiated immediately after birth with evidence for mature gastric glands by phase 2B. (Waite 2003). The specialised cells, including parietal cells and mucus cells, are present in stomach from eight days postpartum and gradually increase in number whereas the first evidence for chief cells was towards the end of phase 2B (Waite 2003). This is in contrast to the majority of eutherians including human where the development of gastric glands with the appearance of specialised cells of the stomach were observed at the embryonic phase. This includes the endocrine cells which are present at 9 weeks and the parietal cells at 11-12 weeks, whereas mucous neck cells and zymogen cells were present from the 12th week of gestation (Montella and Dessi 1991; Montella and Dessì 1990). In brief, eutherian gut development and maturation is a gradual process which is initiated in the foetus and continues after birth making it adept to digest all types of nutrition (Khurana and Mills 2010; Weaver 2000). This cascade of events occurs mainly during fetal development and is regulated by the amniotic fluid, and continues during infancy during which milk regulates the process (Wagner et al. 2008). In contrast, in marsupials including the tammar wallaby, stomach and other organs like the lymphoid tissue (Basden et al. 1997), brain (Reynolds et al. 1985) and lung (tissue proliferation and septal development leading to subdivisions in the terminal air sacs) (Runciman et al. 1996) have been reported to undergo development mostly after birth during phase 2A. Therefore, it can be assumed that in tammar wallaby where the major development of the young occurs after birth, different factors present in the mammary gland secretion may play role in signaling these developmental changes in contrast to amniotic fluid in eutherians.
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This may include phase 2A milk assisting the adaptation of the young’s stomach from an intrauterine to extrauterine environment and to drive gut maturation. Additionally, phase 2B milk most likely supports changes in the stomach to cope with a more complex milk rich in proteins and lipids, and also a solid diet/herbage.
The gastrointestinal tract including the stomach is a complex organ that has many interacting cell types and understanding the development of this organ has relied on using in vitro (Corps and Brown 1987; Ma and Xu 1997; Takeda et al. 2004) and in vivo models (Zhang et al. 2001). The current study is the first attempt to examine the effect of early phase tammar wallaby milk (phase 2A and 2B) on stomach development and differentiation in an in vitro model. With this aim, the study initially focused on developing in vitro stomach models, the embryonic stomach explant model and gastric organoid model. Further, the effect of tammar wallaby phase 2A and phase 2B milk on stomach development was examined using these in vitro models.
To investigate the role of tammar wallaby milk using an in vitro model, the embryonic stomach explant culture was utilised. In general, the development of the gastrointestinal tract in all mammals consists of four predominant steps – formation and specification of endoderm during gastrulation, formation of simple gut tube, organ bud formation and the organ-specific cytodifferentiation (Wells and Melton 1999). In mouse, by E10.5 the hind gut and mid gut initially appear and during the next week organize into specialized parts of the GIT. By E12.5, the gastric mucosa is comprised of a simple undifferentiated epithelial monolayer that becomes thicker by E14, and by E18 the epithelium organizes into primordial buds which mainly contain stem cells and a few pre-differentiated/precursor cells (Karam et al. 1997; Kataoka et al. 1984). By the embryonic stage (E18) the differentiated pit, neck and parietal cells start appearing whereas the zymogenic cells appear 21 days postnatally (Karam et al. 1997). Based on these developmental features of the mouse GIT (Karam et al. 1997) and earlier experiments using in vitro culture of embryonic intestine (Hearn et al. 1999), the current study cultured embryonic whole stomach. This retained the 3D architecture which aided the addition of external factors like milk into the growth
121 media and the lumen of the stomach to examine the effect on development. Previously, a catenary culture of mouse gut showed maintenance of gastric functional organization in terms of morphogenesis and differentiation (Hearn et al. 1999). In the current study, initially E14 embryonic stomach was cultured in media containing serum ranging from 0.5% to 10%. After 3 days in culture, there was less maintenance of the epithelium towards the lumen with decrease in serum in the media which was probably due to limited supply of nutrients into the lumen of the stomach. Similarly, Abud and Heath (2004) reported extensive cell death in intestinal culture when there was low serum (0.5%) in culture media. Further, the embryonic stomach grown with 10% serum that showed maximal maintenance of the epithelial layer was examined for growth and differentiation. During the three day culture, the smooth muscle marker DES and the epithelial marker MUC6 showed significant increase in expression level when compared to day 0. This was supported by the earlier study of Hearn and co-workers (1999) wherein intestine from E11.5 embryos (that lack Desmin expression) after 3 days of culture showed immunoreactivity to Desmin which initiates only at E15 under in vivo condition (Torihashi et al. 1997). Thus, this model revealed successful development of GIT smooth muscle in culture. Additionally, there was evidence for progress in intestinal epithelial development in terms of polarisation of the cells and mucin secretion in PAS staining (Hearn et al. 1999). However, lack of significant difference in the other marker genes in tissue during culture may be due to limited differentiation into epithelial cell lineages in the E14 embryonic stomach even after 3 days of culture. Correspondingly, Abud and co- workers (2005) had observed lesser epithelial growth and differentiation after 3 days of culturing of E13.5 intestine. However, progressive growth and differentiation was observed by day 7 as shown by microvilli, junction complex formation and mucin droplets. Furthermore, epithelial proliferation increased slightly in the presence of EGF in culture media and decreased in the presence of an EGF receptor kinase inhibitor (Abud et al. 2005). This evidence was supporting that growth and differentiation in the catenary culture can be manipulated by addition of external factors. Based on these observations, we examined the effect of tammar wallaby phase 2B milk on proliferation and differentiation assuming the putative factors in
122 the tammar milk may accelerate stomach development as reported earlier using an in vivo approach (Kwek et al. 2009b; Waite et al. 2005). However, our results demonstrated no significant effect on differentiation in terms of the markers examined in the current model. Earlier Kuntz and co-workers (2008) had demonstrated a similar reduced differentiation in Caco2 cells in the presence of milk oligosaccharides as this cell type represents an already differentiated phenotype. In brief, the effect of any external signal including milk may vary based on the nature of cell model as the developmental stage represented by each cell type will differ (Purup and Nielsen 2012). Another possibility is the species difference whereby the tammar milk proteins may not bind or activate the orthologous receptors present in the mouse resulting in lack of bioactivity in such cross-species assays. However, previously Khalil (2007) had proven bioactivity utilising bioassays wherein the effect of tammar milk proteins was examined on eutherian cell models. Moreover, in the study by Topcic and co-workers (2009), tammar WAP showed proliferative effect on both wallaby mammary epithelial cells and mouse mammary epithelial cell line (HC11). This suggests the possibility of using cross-species assays is relevant and furthermore, the activity of milk proteins is not restricted to tammar wallaby.
We then conducted a study in a slightly more developed embryonic stomach, E15 to investigate the effect of milk on cell proliferation and apoptosis. To address this question, we included serum in culture media at an intermediate concentration where there was no extensive proliferation with minimal apoptosis (Abud and Heath 2004) and less intereference with tammar milk assay. The apoptosis observed in the culture may be due to an insufficient supply of nutrients for growth and to some extent, a step in endoderm remodelling into epithelial layer as reported by Abud and co-workers (Abud et al. 2005; Saunders 1966). Our current study reports that in the presence of tammar wallaby phase 2B milk there were more proliferating cells in the epithelium as compared to cells in the control media. In addition, the number of apoptotic cells remained the same in tissue cultured in both treatments. Taken together, we can assume that tammar wallaby milk stimulates extensive proliferation of cells while it maintains a basal level of apoptosis necessary for endoderm remodelling (Abud et al. 2005). Experiments using the intestinal catenary culture had
123 emphasized the significance of EGF signaling for the maintenance of the epithelial layer (Abud et al. 2005). Additionally, Takeda and co-workers (2004) showed proliferation of fetal small intestinal cells in the presence of human milk which was higher than the levels observed in the presence of EGF alone and it was demonstrated that this activity was mediated by a unique tyrosine kinase pathway. Many other studies in the human have shown that milk stimulates gastrointestinal mucosal proliferation (Corps and Brown 1987; Ichiba et al. 1992) and maturation (Takeda et al. 2004; Wagner et al. 2008) and the formation of the mucosal barrier (Wagner et al. 1996) due to the presence of various milk-borne factors. Similarly, different growth factors like EGF and IGF-1 are present in tammar wallaby milk and their concentration changes differentially during lactation (Ballard et al. 1995). Therefore, the proliferative role of tammar wallaby milk may be attributed to a cumulative effect of different components in the milk including EGF. There are other components in tammar wallaby milk like lactoferrin protein (Green and Renfree 1982) which has the property of inducing intestinal cell proliferation in humans at higher concentration leading to cell differentiation and inhibition of cell growth at a lower concentration (Buccigrossi et al. 2007). Previously, tammar milk proteins like Whey acidic protein (Topcic et al. 2009) and Cathelicidin (Wanyonyi et al. 2011) have been shown to have a proliferative effect on mammary epithelial cells and the later stage milk caused maturation of the pouch young’s stomach (Kwek et al. 2009b). Likewise, tammar wallaby phase 2B milk may contain components like growth factors, bioactives or other unknown factors (Joss et al. 2009) which may be responsible for the proliferative effect on stomach.
Under similar conditions using E15 embryonic stomach, we examined the effect of phase 2A milk on stomach differentiation in terms of different gastric gene markers. The experiment investigated the effect of milk from different time points spanning early lactation (phase 2A) in the tammar wallaby. There was an increased expression of TFF2 in the presence of D65 tammar wallaby milk. The cells expressing TFF2 represent progenitors for mucus neck, parietal and zymogenic cells in the oxyntic gastric mucosa (Quante et al. 2010). There was also an increased level of expression of SST in the presence of D92 tammar milk. SST LVH[SUHVVHGE\WKHį-
124 cells (endocrine cells) in the stomach which impose a paracrine control over the exo- and endocrine functions of the gastrointestinal tract (Brass et al. 2010). These levels are usually regulated by different factors, including nutrients and hormones like insulin (Schusdziarra 1983). Nutrients including protein in the form of casein hydrolysate have been reported to induce release of Somatostatin peptide in dog (Schusdziarra et al. 1978). Based on the current results, it can be assumed that the milk from later stages of phase 2A contain factors which influence stomach development in terms of TFF2 and SST expression. However, at this stage of growth there is no drastic change in stomach development in the tammar PY except that it adopts an adult spiral groove appearance at around day 60 postpartum which progressively gives rise to the saccariform region (Waite et al. 2005). The gastric pits with simple columnar cells and parietal cells which are seen throughout the mucosa from birth continue development at this stage and finally mature by 170 days postpartum (Waite et al. 2005). Nevertheless, a wide range of whey proteins with diverse bioactivity have been identified from milk of tammar wallaby (Joss et al. 2007; Joss et al. 2009). This includes milk proteins that are differentially expressed across lactation (Sharp et al. 2009) regulating the development of the pouch young in a phase dependent manner (Trott et al. 2002; Wanyonyi et al. 2011; Watt et al. 2012). Moreover, during early stages of lactation (Joss et al. 2009) Alpha-fetoprotein like protein was detected which may regulate early brain development in tammar wallaby (Dziegielewska et al. 1986) and Clusterin assisting the differentiation and morphogenesis of epithelia during embryogenesis (French et al. 1993). Compiling the results of the current study and the earlier reports showing the presence of unknown factors in milk during early phases of lactation, it can be presumed that tammar wallaby phase 2A milk contains putative components that may influence stomach development and differentiation. Despite this evidence for the role of milk- borne factors on stomach development, the individual factors responsible for this function need to be identified either by a genomic or proteomic approach.
Further, to investigate the role of tammar wallaby milk on gastric stem cell maintenance and differentiation into different lineages, a preliminary investigation was undertaken using the gastric organoid model. The gastric organoid model was
125 previously developed by Barker and co-workers (2010) as a long term in vitro culture system from single Lgr5+ cells from the pyloric region of the stomach of adult mice. It utilized the property of multi-potent stem cells to divide and differentiate into various epithelial cell lineages as they move bidirectional towards the pit and gland (Bjerknes and Cheng 2002). In the current experiments, the model was developed as a short term culture examining the expression level of different gastric gene markers. We demonstrated that during 7-9 days culture, the gastric organoids showed extensive budding with a lumen towards the center which is an indication of stem cell division and differentiation (Sato et al. 2009). There was an elevation in the expression of the LGR5 gene in culture over time which indicated that Lgr5+ stem cells that are capable of developing into mature glandular epithelium were maintained and multiplied in culture. Expression of MUC6 and GIF genes as markers for epithelial layer development and PGC as marker for chief cells increased during the 7 days of culture. However, there was no evidence of differentiation of parietal cells or any of the enteroendocrine cells, indicated by low levels of ATP4A, GHRL, GAST, SST and CHG. In the study by Barker and co-workers (2010), a similar differentiation pattern of gastric cell lineages was noticed, and expression of the enteroendocrine markers GAST, GHRL, SST and CHG required lowering of Wnt3a in the media. The expression of TFF2 gene, a mucus neck cell marker, was elevated. This contrasted with the previous study where differentiation into this lineage also occurred only by lowering of the Wnt3a concentration in media during the 10 day culture.
Earlier in vitro experiments have shown the response of Lgr5+ stem cells to external signals whereby addition of FGF10 in media caused an increased number of budding organoids and long term culture maintenance (Barker et al. 2010). Another study utilising the LGR5 expressing stem cells of murine cochlea showed an enhanced capacity to form neurospheres and a capacity to differentiate into hair cells in in vitro culture (Shi et al. 2012) in response to Wnt signals. Based on this evidence for the response of stem cells to external signals in culture, we utilized the organoid model to address the effect of tammar wallaby phase 2A milk on gastric stem cell proliferation and differentiation. A 48 h treatment with phase 2A milk resulted in a
126 decline in the LGR5 expression as compared to control conditions which may be due to the differentiation of stem cells into more differentiated cell lineages when treated with milk. A similar decrease in LGR5 expression upon differentiation was reported by Kemper and co-workers (2012) in primary colorectal cancer derived spheroid cultures. Parallel to this decrease in LGR5 expression, PGC, a marker for chief cells was high in the presence of phase 2A milk. Exposure to bovine milk has previously been demonstrated to be important for the production of prochymosin which is indicative of the function of chief cells and mucosal neck cells (Andrén and Björck 1986). There was also evidence for the role of different factors in milk like oligosaccharides (Kuntz et al. 2008) and lactoferrin (Buccigrossi et al. 2007) in intestinal cell differentiation using in vitro models. Taken together, it is conceivable that in the presence of tammar wallaby phase 2A milk, stem cells in the gastric organoids are differentiated into lineages that include the chief cell population.
To conclude, the current study is the first to demonstrate
x A role for phase 2A tammar wallaby milk on stomach differentiation under in vitro conditions x A role for phase 2B tammar wallaby milk on stomach epithelial proliferation under in vitro conditions.
However, it has not yet been established whether these properties of the tammar milk are phase specific. Nevertheless, the unique reproductive strategy of the tammar wallaby may help identifying putative bioactive components of the milk and their role which to elucidate in eutherians can be difficult (Sharp et al. 2009).
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CHAPTER 4
Putative role of milk in stomach development of gray short-tailed opossum (Monodelphis domestica)
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4.1 Introduction
The lactation strategy in mammals has evolved among species essentially with the idea of meeting the nutritional needs of the offspring. This physiological adaptation was dependent on the metabolic rate of the mother and the energy and growth requirements of the newborn (Peaker 2002). Eutherians, the major class of mammals, have a long gestation relative to the lactation cycle and give birth to a comparatively well-developed young. In contrast, marsupials have a short gestation and give birth to an altricial young whose development is regulated during the extended lactation cycle (Tyndale-Biscoe and Renfree 1987). Of all the marsupials, the tammar wallaby (Macropus eugenii), a small macropod native to Australia, has the most well characterised lactation strategy. Another marsupial that is well established as a laboratory animal for basic and biomedical research is the gray short-tailed opossum (Monodelphis domestica), a small South American member of the Didelphidae family. The major feature that distinguishes these mammals from most other marsupial species is the lack of a marsupium or pouch. It has a wide range of distribution across Eastern and Central Brazil, Bolivia, Paraguay, and North Argentina (Gardner 2007). The reproductive strategy of opossum is characterised by a short gestation of 14.5 days followed by a long lactation of 60-75 days (Figure 4.1). The newborns usually have well-developed forelimbs and jaws which help them to crawl to the teats immediately after birth and to suck milk from the mammary gland teat of the mother. The initial phase of lactation which comprises the first 14 days is characterised by the young continuously attached and hanging from the teat (VandeBerg 1990). After this phase, the young are released from the teat and suck at will (Green et al. 1991) during which time they are carried on the mother’s back (VandeBerg 1990). In contrast to the tammar wallaby, the gray short- tailed opossum is a non-seasonal breeder (VandeBerg 1990). They reach sexual maturity by 4-6 months of age and can produce 3-4 litters per year with each litter size ranging from 13-14 (VandeBerg 1990). These two major characteristics offer an advantage of the gray short-tailed opossum over tammar wallaby as an improved model for scientific research. Similar to other marsupial species, milk composition also varies across the lactation cycle in the gray short-tailed opossum. The early
129 phase of lactation in the gray short-tailed opossum is characterised by dilute milk low in solids, which during the next 20-25 days increase rapidly and remain the same for the remaining 40 days of lactation (Figure 4.2 A) (Green et al. 1991). Milk during this early stage is low in carbohydrates which increase rapidly reaching a high concentration at day 50 and immediately show a rapid decline with lower levels maintained in the milk for the rest of lactation (Figure 4.2 B) (Crisp et al. 1989; Green et al. 1991). These milk carbohydrates are present in milk mainly in the form of monosaccharides like glucose and galactose in addition to free lactose during the first two weeks which later changes to milk composed of predominantly higher oligosaccharides (Crisp et al. 1989). A similar compositional change was observed for milk proteins (Figure 4.2 A) with initial low levels that increased to maximum level by approximately day 25 and continued at the same level for the remainder of lactation (Green et al. 1991). In contrast, lipid concentration in milk was low during the first 55 days and comparatively higher during the later stages (Green et al. 1991).
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(A) CONCEPTION
0 15
0 14 56
BIRTH YOUNG WEANED
DURATION OF:
GESTATION TEAT ATTACHMENT NEST PHASE OF PHASE OF LACTATION LACTATION
(B)
Figure 4.1: Reproductive cycle and developmental stages of gray short-tailed opossum (A) Reproductive cycle represented in days (Harder et al. 1993) (B) Developmental stages (a) Pups 7 days after birth, (b) Pups 20 days after birth and (c) Pups 60 days after birth on mother’s back (http://www.public.iastate.edu/~zoogen/opposum.html).
A B
% of indicated parameters
Figure 4.2: Milk composition of gray short-tailed opossum (A) Changes in % solids (w/w) and % protein (w/v) (B) Change in hexose (w/v) concentration (Green et al. 1991).
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4.1.1 Stomach development in mammals; Monodelphis domestica as a new model Typically, a mammalian digestive tract is divided into fore-, mid- and hindgut of which the foregut consists of mouth, pharynx, oesophagus and stomach; the midgut is the small intestine and the hindgut is the large intestine (Young 1970; Figure 4.3). Development of the digestive tract in vertebrates begins as an undifferentiated tube which is further partitioned into these specialised organs (Fukuda and Yasugi 2005). The wall of each of these digestive tract organs consists of different layers including mucosa, sub mucosa, muscularis externa and serosa. The mucosal layer is composed of specific differentiated cell types which vary according to the region of digestive tract (Karam 1999; Karam et al. 1997). Mammalian orders consume different diets and consequently, herbivores, carnivores and omnivores have evolved different types of stomachs (Stevens and Hume 1995). A large amount of information is available on the structural and functional development of eutherian digestive tracts (Henning and Kretchmer 1973; Koldovský 1969; Langer 2003). However, data for marsupials is limited and further complicated by the complexity of herbivorous, carnivorous and omnivorous species. The tammar wallaby represents a well-studied herbivorous marsupial whereas the dasyurids and didelphids are the most studied carnivorous marsupials. A few earlier reports have examined stomach development in the tammar wallaby (Kwek 2009; Setiati 1986; Waite 2003), which has a digastric stomach composed of both forestomach and hind stomach (Langer et al. 1980). The forestomach is the microbial fermentation chamber which helps digestion of complex carbohydrates from ingested grass and the hind stomach is involved in secretion of gastric acid required for further digestion of food (Hume 1999). At birth, the altricial young has a stomach mainly composed of columnar epithelium which includes mucous secreting cells and pre-parietal cells (Setiati 1986). No dramatic change has been reported in the mucosal layer until about 170 days postpartum (Waite et al. 2005). Initially, the stomach has an absorptive role with parietal cells present in both forestomach and hind stomach, but later the parietal cells completely disappear from the forestomach mucosa. By 200-260 days of pouch life, the forestomach adopts a fermentation
132 function. In the hind stomach, however, the parietal cells increase in number adopting a glandular phenotype (Kwek et al. 2009a; Waite et al. 2005). In the tammar wallaby forestomach, the immature gastric glandular phenotype which is prevalent during the first 200 days of lactation changes to a cardia glandular phenotype by a reduction in parietal cell population accompanied by a down regulation of the gastric glandular phenotype marker genes (Kwek et al. 2009b). Moreover, these developmental events were proven to be regulated by changes in the mother’s milk (Kwek et al. 2009b). It is expected that different milk factors including carbohydrates regulate the microbial colonisation of the pouch young stomach which, in turn, influences development. Moreover, microflora may be acquired through ingested herbage eaten during later stages which may also act as prime regulators of stomach development (Kwek et al. 2009b). The gray short-tailed opossum has been previously established as the most common marsupial model for developmental research as it is a convenient laboratory animal and is the first marsupial whose genome has been sequenced (VandeBerg 1983; 1990). Moreover, the unique reproductive strategy of these marsupials in which the major development of the altricial young occurs after birth provides an opportunity, as a comparative model, for understanding the developmental events occurring during the embryonic stages in eutherians. The present histological study demonstrates the timing and sequence of different developmental events in gray short-tailed opossum stomach. This marsupial, being an omnivore with a monogastric stomach, would be a better comparison to eutherians like human than the herbivorous tammar wallaby with a digastric stomach. Stomach development in the North American opossum (Didelphis virginiana) another omnivorous marsupial with a monogastric stomach has been examined earlier (Krause et al. 1976). Nevertheless, gray short-tailed opossum will most likely be a better model for research as the developmental cycle is shorter than in North American opossum (Harder et al. 1993), and young can be produced economically in larger numbers (VandeBerg 1990). Thus in the current study, we have used the gray short-tailed opossum to examine the development of stomach after birth and correlate these developmental changes with changes in milk composition.
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Figure 4.3: Gastrointestinal tract of gray short-tailed opossum (A) Schematic diagram of the GIT highlighting different regions (Hume 1982) (B) Photograph of GIT (a) Stomach (b) Oesophagus (c) Small Intestine (d) Caecum (e) Colon.
4.2 Material and methods 4.2.1 Animal Ethics Animals were obtained from the University of Melbourne Medical Sciences animal house facility, Melbourne, Australia. All experimentation was approved by the University of Melbourne Animal Ethics Committee and conformed to the National Health and Medical Research Council guidelines. Opossum pups across the lactation cycle (day 8, 14, 29, 35, 45, 61, 75) and adult animals (n§3) were acquired as they became available from the colony.
4.2.2 Stomach tissue collection
Animals were euthanized by CO2 inhalation and the entire gastrointestinal tract was exposed through mid-ventral incision and removed from the body. The stomach was dissected, immediately flushed and washed with sterile PBS. Tissues
134 likely from the body (corpus) of the stomach were fixed overnight in 10% neutral buffered formalin and VWRUHGLQDOFRKRODWÛ& for future use.
4.2.3 Histochemistry The specimens were processed by dehydration in graded ethanol solutions (70% to absolute ethanol), cleared in histolene (Grale Scientific Pty Ltd) and embedded in paraffin. Transverse paraffin sections (5-7 ȝP RI stomach were prepared and processed for staining with a variety of histochemical stains to examine the morphology and differentiation of different cell types of the stomach including mucus cells, parietal cells and chief cells. The histological stains used were hematoxylin and eosin (H&E; Amber Scientific Pty Ltd) and periodic acid schiff (PAS; Amber Scientific Pty Ltd). All staining methods were performed as per standard protocol (Lee and Luna 1968). H&E, the standard stain, stains the nuclei blue and cytoplasm pink, while PAS stains the neutral mucins in the stomach mucosa a dense magenta colour.
4.3 Results The lumen of the stomach of an eight day old opossum is lined by a mucosa consisting of a simple vacuolated epithelial layer (Figure 4.4). The mucosa is loosely folded with evidence of gastric pit formation (Figure 4.4 A and B) and also occasional putative gastric glands that contain mucus which show magenta staining with PAS (Figure 4.4 B). Below the mucosa, the lamina propria blends with the submucosa as there is no obvious muscularis mucosa present at this stage (Figure 4.4 C). The muscularis externa is present as a single undifferentiated layer. In the 14 day old opossum, the stomach mucosa is thicker and there is clear evidence of gastric gland formation (Figure 4.5). Vacuolation of mucosal epithelium is much reduced and is restricted to cells on the luminal surface. A little mucus is present on the surface of the mucosa and is also present at the bottom of some glands (Figure 4.5); while there is no evidence of a muscularis mucosa, the muscularis externa continues to develop.
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By 29 days, vacuolation of the mucosal epithelium has completely disappeared and there is some indication for the presence of chief cells within the mucosa. The muscularis mucosa is also seen for the first time (data not shown).
There is further folding of the mucosa by 35 days and gastric pits and glands are now prominent (Figure 4.6 A, 4.6 B and 4.7 A). Surface mucus cells and mucus neck cells are also apparent (Figure 4.7 A and B), and there is the first clear evidence of chief cells with their characteristic granular cytoplasm (Figure 4.8). Parietal cells, mostly at the base of gastric glands, are now also visible in the mucosa. At this stage, the stomach wall can be clearly differentiated into the layers that are characteristic of the adult stomach i.e., mucosa, lamina propria, muscularis mucosa, submucosa, muscularis externa, and serosa (Figure 4.7 B).
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Figure 4.4: Transverse section of an eight day old opossum stomach (A) H and E stain (B) PAS stain [Black solid arrow indicates gastric pit formations and black arrow indicates vacuolation].
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Figure 4.5: Transverse section of a 14 day old opossum stomach (PAS stain) [White arrows indicate gastric gland formations].
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Figure 4.6: Transverse section of a 35 day old opossum stomach (lower magnification) (A) H and E stain and (B) PAS stain [Gastric pits and gland are prominent].
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Figure 4.7: Transverse section of a 35 day old opossum stomach (higher magnification) (A) PAS stain [Black arrows- gastric pits, yellow arrows- surface mucus cells and white arrows-mucus neck cells] (B) H and E stain [1-serosa, 2-muscularis externa, 3-sub mucosa, 4-muscularis mucosa and 5-lamina propria].
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Figure 4.8: Transverse section of a 35 day old opossum stomach (H and E stain) [Black arrows indicate chief cells].
At 45 days (Figure 4.9), parietal cells are more common and can be seen along the length of the gastric glands, while chief cells are distributed at the base of glands (Figure 4.10). By this stage, there is some evidence that the muscularis externa is beginning to differentiate into inner circular and outer longitudinal layers (Figure 4.11).
When the opossum is weaned at 61 days of age (Figure 4.12 A and 4.12 B), the mucosa is characterised by numerous parietal cells, with chief cells again being more prominent at the base of gastric glands. Although the chief cells do not stain well with PAS stain, their granules are characteristic. Both chief and parietal cells become increasingly prolific by 75 days and by this stage mucus is present both on the surface of the mucosa, and deep within the glands (Figure 4.13 A, 4.13 B and 4.14).
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In the adult opossum, the mucosa is extensively folded with deep gastric pits extending into gastric glands (Figure 4.15 A and 4.15 B). Mucus neck cells and parietal cells are distributed along the glands and chief cells are located towards the base of the mucosa (Figure 4.16 A and 4.16 B). The muscularis mucosa defines the border between the lamina propria and the submucosa, and the muscularis externa has differentiated into two clear layers, the inner circular and outer longitudinal layers (Figure 4.15 A).
(A) (B)
Figure 4.9: Transverse section of a 45 day old opossum stomach (A) H and E stain (B) PAS stain.
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Figure 4.10: Transverse section of a 45 day old opossum stomach (PAS stain) [Black arrows- parietal cells and red arrows- chief cells].
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Figure 4.11: Transverse section of a 45 day old opossum stomach (H and E stain) [1-serosa, 2-muscularis externa, 3-sub mucosa and 4-muscularis mucosa].
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Figure 4.12: Transverse section of a 61 day old opossum stomach (A) H and E stain and (B) PAS stain [Numerous parietal cells, with chief cells more prominent at the base of gastric glands].
144
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Figure 4.13: Transverse section of a 75 day old opossum stomach (A) H and E stain and (B) PAS stain [Mucus is present both on the surface of the mucosa, and deep within the glands].
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Figure 4.14: Transverse section of a 75 day old opossum stomach (PAS stain) [Black bold arrow- mucus surface cells, white arrow- mucus neck cells, red arrows- parietal cells and yellow arrows- chief cells].
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Figure 4.15: Transverse section of stomach of an adult opossum (A) H and E stain and (B) PAS stain [1-serosa, 2-muscularis externa, 3-sub mucosa, 4-muscularis mucosa, 5- mucosa and 6- lamina propria].
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(A)
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Figure 4.16: Transverse section of stomach of an adult opossum (PAS stain) (A) Red arrows- mucus surface secreting cell, yellow arrows- mucus neck cell and asterisk- gastric pit (B) Red arrows- parietal cells and yellow arrows- chief cells.
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4.4 Discussion 4.4.1 Stomach development in gray short-tailed opossum In the current study the morphological development of the stomach of gray short-tailed opossum has now been described, and it can be compared with that of the North American opossum (Didelphis virginiana) (Krause et al. 1976). Development of the stomach is similar in the two species, although there are some differences that can be identified by the use of Epon sections as in the earlier study (allowing greater histological resolution and therefore earlier detection of some cell- types).
The stomach of newborn gray short-tailed opossum has a slightly folded mucosa and at eight days of age the mucosal epithelium is composed of a simple layer of columnar epithelial cells, and there is little development of gastric glands. This is similar to the stomach of newborn North American opossum (Krause et al. 1976) and other Australian marsupials like red kangaroo (Griffiths and Barton 1966b), native cat (Hill and Hill 1955) and tammar wallaby (Setiati 1986). At eight days, the mucosal epithelium is also highly vacuolated. The vacuoles are likely to be lipid droplets as demonstrated by the earlier study in North American opossum (Krause et al. 1976), but in the current study, no special stains were used to confirm this hypothesis. By day 35, the mucosal layer is devoid of these droplets. In North American opossum (Krause et al. 1976) and rat (Helander and Olivecrona 1970) there is evidence that the cells lining the surface of the gastric mucosa have an absorptive function during the early stage of development. Therefore, lipid absorption was probably from the mother’s milk as it is the major source of nutrient at this stage of development. Mucin, which may be secreted from the gastric epithelium (Wabinga 1996) is observed on the surface of the gastric epithelial lining from birth. By day 14, there is increased folding of the mucosa and gastric glands begin to appear. Gradually by day 35, the specialised mucus surface cells develop further and mucus neck cells become apparent during the development of deep gastric pits. Mucin secretion and development of mucus cells are found in didelphis after birth, with the gastric pits
149 and glands showing progressive development from day 9 postpartum (Krause et al. 1976). In the herbivorous tammar wallaby (Figure 4.17), fewer shallow pits were noticed in the cardiac and fundic region of the newborn stomach whereas in the pyloric region there was extensive mucosal folding with deeper pits (Setiati 1986). Further, the pits/glands showed continued development until they formed mature glands consisting of the isthmus, neck and the base by 170 days of age (Waite 2003). However, in humans, well developed gastric pits are noticed by the 9th week of gestational age and mucus neck cells are recognised by 12th week (Montella and Dessi 1991; Montella and Dessì 1990). Thus the gastric glandular development in the opossum after birth is comparable to many eutherians like human wherein major development of the gland occurs during the embryonic period (Salenius 1962). At day 35 postpartum, chief cells become visible in the gray short-tailed opossum stomach, although they may be present as early as 29 days. By day 35 parietal cells may also begin developing at the base of glands, but by 45 days parietal cells are clearly present in association with the developing gastric glands. Concomitant with these changes is an increase in the number of chief cells mainly restricted to the basal region of the glands. By the time of weaning (day 61 postpartum) the gastric mucosa is composed of all specialised cells including mucus cells, parietal cells and chief cells attaining a pattern similar to an adult stomach. In brief, chief cells in gray short-tailed opossum appear a little earlier than the parietal cells. Similarly, in North American opossum the chief cells are observed by 75 days postpartum, just before weaning (Krause et al. 1976). In contrast to the current observation in gray short-tailed opossum, in the majority of species (including the North American opossum and the tammar wallaby), the parietal cells appear first followed by chief cells (Krause et al. 1976; Setiati 1986; Waite 2003). This may simply be explained by the use of high resolution Epon sections in earlier studies which allowed earlier detection of parietal cells. In the tammar wallaby, the pattern of appearance of specialised cells such as the parietal and mucus cells were observed in both cardiac and fundic region of the stomach soon after birth (eight day old pouch young). Parietal cells gradually mature with characteristic internal canaliculi and apical tubulovesicular systems that become prominent by day 40 postpartum.
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However, the chief cells were observed in the stomach only at 170 days postpartum (Setiati 1986; Waite 2003). Nevertheless, there are other macropod species in which the timing of occurrence of the specialised cells is different. These include the Thylogale thetis in which parietal cells were seen at 156 days and not at 146 days (Langer 1979). Similarly in Macropus rufus, there was no evidence of parietal cells during very early stages of development (Griffiths and Barton 1966a). Further, the arrangement of specialised cells in gray short-tailed opossum is similar to that in tammar wallaby (Setiati 1986) and North American opossum (Krause et al. 1976) with the mucus cells towards the surface, parietal cells towards the neck region and the chief cells concentrated more at the basal region of the gland. The muscle layers are extremely thin initially and develop progressively during the suckling phase when the young is continuously attached to the teat. By the time the nest phase is initiated, well-developed and differentiated layers of muscle are noticed representing an adult stomach. Similarly, in North American opossum the muscularis externa is extremely thin at birth and gradually develops with all other layers getting established gradually across the postnatal period (Krause et al. 1976). The tammar wallaby newborn also has a stomach with an incomplete muscular layer which develops during the suckling phase (Setiati 1986). In brief, the muscle layer of the stomach in these marsupial species is immature at birth and develops postnatally.
4.4.2 Possible role of milk in stomach development The current study has shown the progressive development of stomach of gray short-tailed opossum occurs during initial postnatal period when milk is the major source of nutrients and developmental signals. By correlating these events with the changes in the milk composition and production, we can speculate on a role for milk in regulating the process. In gray short-tailed opossum, the development of stomach occurs gradually throughout the suckling period with a major developmental change at 5-6 weeks of age when there is appearance of chief cells followed by parietal cells. This is slightly later than the gradual shift in dilute milk of early stage to a milk rich in solids and protein (Green et al. 1991), and prior to when the young starts feeding on a solid diet
151 in addition to the mother’s milk. The increased protein intake and solid intake would demand an increased capacity for digestion in the stomach of gray short-tailed opossum (Hume 1999; Waite et al. 2005). Similarly, in the North American opossum, the appearance of chief cells in the young stomach occurs prior to the shift to solid diet (Krause et al. 1976). In contrast, in the tammar wallaby, dramatic changes occur in the stomach when the pouch young starts feeding on herbage/solid diet. The specialised cells of the immature gastric glands are lost from the forestomach mucosa without altering the gross morphology of this tissue and the hind stomach mucosa of the pouch young begins to dramatically transform into one with very long thin glands, typical of an adult gastric glandular mucosa (Waite et al. 2005). In tammar wallaby pouch young, the forestomach matures 200 days after parturition by a transition from an immature gastric glandular phenotype to a cardia glandular phenotype (Figure 4.17; Kwek et al. 2009b). It was proven that milk from later stages of development along with the solid diet plays a role in regulating this developmental change in tammar wallaby which might be due to the different factors in the milk and the pattern of gut colonisation occurring under the influence of these milk factors including oligosaccharides (Kwek et al. 2009b). Evidence from studies using a wide range of eutherian species has revealed that milk factors such as oligosaccharides determine gut colonisation (Fuhrer et al. 2010; Jeong et al. 2012; Weiss and Hennet 2012; Zivkovic and Barile 2011) which in turn regulates gut development (Langhendries 2006). Other dietary factors such as lipids and proteins also determine the equilibrium of the gut flora (Corthier et al. 2000). Moreover, previous studies in other species have shown the importance of milk components in gut development (Berseth 1987; Berseth et al. 1983; Xu 1996; Yamashiro et al. 1989) including parietal cell development (Morikawa et al. 1979) and chief cell development (Andrén and Björck 1986). Based on this evidence from eutherians and marsupials, it can be proposed that certain milk born factors along with solid diet may be driving the developmental change observed in the gray short-tailed opossum stomach. These developmental events in the stomach of the omnivore gray short-tailed opossum are different from the stomach of the herbivore tammar wallaby and may be
152 correlated to the changes in the milk composition and secretion pattern between these two species. In gray short-tailed opossum, the Na/K ratio of milk is less during early lactation and more during late lactation (Green et al. 1991). An increase in Na/K ratio is observed as a result of the leakiness of tight junctions which may partially indicate a reduction in milk secretion (Stelwagen et al. 1999). Around 5-6 weeks of lactation the lowest level of potassium is attained in the gray short-tailed opossum milk beyond which it remains constant while the sodium remains constant throughout lactation (Green et al. 1991). This is in contrast to what is observed in other marsupials such as the tammar wallaby wherein during the early stage of lactation the Na/K ratio is high and gradually declines with progress in lactation (Green et al. 1980). Early stage gray short-tailed opossum milk contained carbohydrates in the form of lactose, glucose and galactose (Crisp et al. 1989). Free lactose was prominent in milk until 2 weeks postpartum after which it was oligosaccharides and this shift was at the same time as the pup detaches from teat (Vandeberg 1983). In contrast, in the tammar wallaby milk lactose is the predominant carbohydrate only for the first 4-5 days and subsequently is replaced by complex oligosaccharides (Messer et al. 1984). There are also differences in the oligosaccharides between the opossum milk and the tammar wallaby milk (Crisp et al. 1989). Based on this difference in type of carbohydrates in the diet, gut colonisation may vary (Corthier et al. 2000; Weig et al. 1999) in turn influencing gut developmental events such as mucin composition (Fernandez et al. 2000). Earlier reports have proven that in vertebrates, the host diet influence the diversity of gut flora and this diversity in gut flora showed an increasing trend from omnivory to carnivory to herbivory (Ley et al. 2008a; Ley et al. 2008b). To conclude, in the current study we have described the progressive changes in the monogastric stomach of the marsupial omnivore gray short-tailed opossum. These developmental events occur postnatally and are most likely driven by the mother’s milk. This process may be more comparable to the embryonic phase in human wherein the specialised cells of the stomach including mucus secreting cells, chief cells and parietal cells appear before birth (Montella and Dessi 1991; Montella and Dessì 1990). Moreover, previous reports have shown that different factors like
153 hepatocyte growth factor in human amniotic fluid (Kong et al. 1998) regulate fetal gastrointestinal development (Mulvihill et al. 1989; Mulvihill et al. 1986). However, in marsupials the milk composition changes across lactation cycle (Green et al. 1980; Green et al. 1991) in contrast to a constant composition in human milk (Allen et al. 1991) and this may therefore accommodate the developmental need of the altricial young. Based on this evidence, it can be assumed that signals for development provided by amniotic fluid in human may be comparable with signals delivered via marsupial milk of varying composition. This hypothesis has been addressed in detail in the subsequent chapter. Thus, marsupials like the gray short-tailed opossum with an adapted reproductive strategy are increasingly becoming relevant as unique models to understand developmental events of different organs such as stomach. The marsupial young is assessable and it is relatively simple to examine the milk for bioactives regulating gut development rather than examining gut development in eutherians in which development occurs in utero. Thus, the current study validates the option of exploiting the comparative biology of marsupial species with extreme adaptation to lactation for a better understanding of the regulation of developmental events in eutherians like human.
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Figure 4.17: Stomach development in tammar wallaby (Kwek et al. 2009a; Kwek et al. 2009b; Setiati 1986; Waite et al. 2005).
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CHAPTER 5
Regulation of gut development; a bioinformatic approach to identify putative bioactives of early phase tammar wallaby milk also present in human amniotic fluid and placenta
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5.1 Introduction
Marsupials such as the tammar wallaby (Macropus eugenii) have a unique reproductive strategy characterised by a short gestation of 26.5 days followed by a long lactation cycle of 300-330 days divided into phase 2A, phase 2B and phase 3 (Nicholas et al. 1997; Tyndale-Biscoe and Janssens 1988). One of the most important characteristics that distinguish the marsupials from other mammals is the immaturity of their young at birth (Tyndale-Biscoe and Janssens 1988). The major development of the altricial young’s tissues occurs during the extended lactation cycle (Tyndale-Biscoe and Renfree 1987). Different organs including the brain (Reynolds et al. 1985; Tyndale-Biscoe and Janssens 1988), the gastrointestinal tract (Setiati 1986; Waite 2003) and the lung (Runciman et al. 1996) develop during the initial phase of lactation (phase 2A). Despite this general extreme immaturity, there are certain tissues that are relatively well-developed including the locomotor system, sensory system and muscular system (tongue) (Tyndale-Biscoe and Janssens 1988). This pattern of development of an altricial young in marsupials is supported by a dramatic change in composition of the milk throughout lactation (Green et al. 1980). Early phase tammar wallaby milk is different from milk towards the end of lactation (Nicholas et al. 1997) and there is total dependence of the young on mother’s milk during this phase (Tyndale-Biscoe and Janssens 1988). These changes in milk composition, particularly the milk proteins, are correlated with the changing needs of the pouch young for growth and development (Trott et al. 2003; Wanyonyi et al. 2011; Watt et al. 2012). Cross fostering pouch young to a mother at a more advanced stage of lactation showed that milk with a different composition accelerated growth of young (Trott et al. 2003; Waite et al. 2005) and development of the pouch young’s stomach (Kwek et al. 2009a). Additionally, milk proteins showing differential secretion across the tammar wallaby lactation cycle have demonstrated bioactivity in diverse cell based assays (Khalil 2007; Sharp et al. 2009). Even though the specific relationship between these bioactives and the development of the pouch young remains to be established, their specific timing of delivery during lactation signifies a likely role in pouch young development.
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In contrast to the marsupials, the eutherian young are born well-developed. The development of the young begins during the early embryonic phase in utero and is mostly completed in the early days after parturition. This limited postnatal development is correlated with minimal change in milk composition during lactation (Hayssen 1993). Previous studies in human have shown that the growth of different organs such as the brain (Stiles and Jernigan 2010), lung (functional at birth but matures after birth) (Burri 1984) and gastrointestinal tract (Otani et al. 1993) are initiated and progress during the embryonic period and are functional at birth. During this embryonic phase, fetal growth is regulated by amniotic fluid and placenta which act as the major sources of nutrient and signals for growth and development of the young. Amniotic fluid contributes 15% of the fetal nutrient requirement (Mulvihill et al. 1985) and is a complex fluid composed of different components like insulin, human growth hormone, prolactin, epidermal growth factor, hepatocyte growth factor and insulin-like growth factor (Barka T et al. 1978 ; Chochinov et al. 1976; Delmis et al. 1992). The presence of these factors is consistent with the significant roles of human amniotic fluid on cell proliferation and growth promoting activity (Barka T et al. 1978 ; Scott et al. 1989) leading to the development of different organs such as the gastrointestinal tract (Hirai et al. 2002; Keel et al. 1999; Mulvihill et al. 1989; Mulvihill et al. 1986). Moreover, reports have confirmed the presence of different proteins in the amniotic fluid (Cho et al. 2007; Crettaz et al. 2007). These proteins in amniotic fluid have been matched to major functions including cellular development, growth and proliferation, cell-to-cell signalling and interaction, tissue development and immune response (Cho et al. 2007), all regulating fetal development. The placenta also plays a major role in the developing foetus in terms of oxygen supply, nutrients, antibodies and hormones (Garnica and Chan 1996; Sood et al. 2006). The genes expressed by this organ code for proteins shown to have roles in different processes like signal transduction, immune regulation and growth metabolism (Sood et al. 2006). Recently, Mushahary and co-workers (2013) identified diverse proteins in the human placenta. These different proteins were grouped into different functional classes such as cytoskeletal proteins, transport
158 proteins, cell cycle and growth, signal transduction and metabolism (Mushahary et al. 2013) that may play role in regulating fetal growth and development.
Thus, it is presumed that the development of the foetus in eutherians (Burri 1984; Otani et al. 1993; Stiles and Jernigan 2010) may be equivalent to the first phase of postnatal development in marsupials like the tammar wallaby (Reynolds et al. 1985; Runciman et al. 1996; Setiati 1986) which is consistent with the concept that this animal is a unique model for developmental research (Selwood and Coulson 2006; Sharp et al. 2009). Therefore, it is possible that the signals provided in early phase milk in the tammar wallaby include potent bioactives provided either by human amniotic fluid or placenta during development of the foetus. To address this question, the current study aims at comparing the genes encoding secreted proteins expressed during phase 2A tammar wallaby lactation with the transcriptome and proteome databases for human amniotic fluid and placenta. Initially, genes coding for secreted proteins expressed during phase 2A tammar wallaby lactation were identified utilising the earlier cDNA microarray analysis by Khalil (2007). Subsequently, RNA-seq using RNA from tammar wallaby mammary gland collected across the lactation cycle was performed in the current study and the data analysed to identify the genes coding for secreted proteins expressed during phase 2A of lactation.
RNA-seq is a recently developed approach of transcriptome profiling that uses deep sequencing technologies. In this method, the cDNA generated from the RNA of interest are directly sequenced in a high-throughput manner to obtain short sequences from either one end (single-end sequencing) or both ends (pair-end sequencing), and the reads obtained are aligned to a reference genome in order to produce a whole-genome transcriptome map (Nagalakshmi et al. 2010; Wang et al. 2009). Several platforms are available for RNA-seq such as the Illumina, Applied Biosystems SOLiD and Roche 454 Life Science. Of these, Illumina platform was used in the current study. RNA-seq has a wide range of advantages over microarray based transcriptome profiling methods. This technique uses massively parallel sequencing to allow transcriptome analyses of genomes at a far higher resolution
159 than is available with Sanger sequencing and microarray-based methods (Nagalakshmi et al. 2010). This provides a more precise measurement of levels of transcripts and isoforms than other methods (Nagalakshmi et al. 2010; Wang et al. 2009). RNA-seq does not require probes and hybridisation and allows numerical quantitation of individual genes thereby significantly eliminating bias and background signals (Febbo and Kantoff 2006) which are major advantages when compared to DNA and RNA microarrays. Unlike these hybridisation-based approaches, RNA-seq is not limited to detecting transcripts that correspond to existing genome sequence which makes it attractive for organisms with genome sequences yet to be determined (Oshlack et al. 2010; Wang et al. 2009). RNA-seq also has a large dynamic range of expression levels over which transcripts can be detected in contrast to DNA microarrays which lack sensitivity for genes expressed either at low or very high levels and therefore have a much smaller dynamic range (Wang et al. 2009). Considering all these benefits, RNA-seq which allows the entire transcriptome to be surveyed in a very high-throughput and quantitative manner was performed in the current study. Therefore, a comparison between the two transcriptome profiling approaches, the microarray based technique and RNA-seq, was carried out in identifying putative bioactives of tammar wallaby early phase milk.
5.2 Material and Methods
5.2.1 Microarray based transcriptome profiling of tammar wallaby mammary gland across lactation
5.2.1.1 Identification of secreted proteins of phase 2A tammar wallaby lactation
Previously, a custom tammar wallaby mammary EST (Expressed Sequence Tag) array was printed with 10,000 cDNAs representing genes expressed across the lactation cycle to transcript profile the mammary gland at all major stages of pregnancy and lactation (Khalil 2007). This experiment included mammary glands
160 from virgin, pregnant - phase 1 (5, 22 and 25 days), lactating - phase 2A (1, 5 and 80 days), lactating - phase 2B (130, 168 and 180 days), lactating - phase 3 (213, 220 and 260 days) and involuting (1, 2 and 3 days) animals. A database was established earlier from this dataset by Khalil (2007) to provide sequence analysis and sequence assembly, protein and peptide prediction. The microarray dataset was used to transcript profile approximately 5000 genes and identified approximately 326 known genes with annotation that encode differentially secreted proteins (Khalil 2007). This dataset was utilised to identify the genes coding for secreted proteins that were either specifically or highly expressed during phase 2A of lactation. (http://mamsap.it.deakin.edu.au/~clefevre/VBC/public_html/Wallaby/index.html)
5.2.2 RNA-seq based transcriptome profiling of tammar wallaby mammary gland across lactation
5.2.2.1 Mammary gland collection and RNA extraction
Tammar wallabies were maintained in the Deakin University Marsupial Facility, Victoria, Australia. Mammary glands were dissected from pregnant - phase 1 (17, 18 and 21 days), lactating - phase 2A (2, 3, 4, 5, 6, 10, 22, 40, 62, 70, 80 and 87 days), lactating - phase 2B (133, 151 and 171 days) and lactating - phase 3 (243, 260 and 266 days) tammar wallabies under sterile conditions after the animals were euthanized. The tissues were stored at -80° C and later used for RNA extraction. All experiments were approved by the Deakin University Animal Ethics Committee.
Mammary tissue was first homogenized in QIAzol lysis reagent using a polytron kinetamica PT 2100 homogenizer and total RNA was extracted using the RNeasy Lipid Tissue Kit (74804 Qiagen, Sydney, Australia) following manufacturer’s instructions. The quality and integrity of RNA was assessed by determining the RIN values and 260/280 ratios of the samples using an Agilent bioanalyser.
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5.2.2.2 RNA-seq
RNA samples were sent to Beijing Genomics Institute for sequencing. The total RNA was converted into a library of template molecules suitable for high throughput mRNA sequencing using the Illumina sample preparation guide (Cat # RS-930-1001). This involves purification of the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads and fragmenting into small pieces using divalent cations under elevated temperature. These cleaved RNA fragments were reverse transcribed into first strand cDNA using reverse transcriptase and random primers. The RNA template removed and a replacement strand generating double- stranded cDNA synthesised, the overhangs converted into blunt ends using T4 DNA polymerase and Klenow DNA polymerase. This involved addition of an ‘A’ base to the 3' end of the blunt phosphorylated DNA fragments using the polymerase activity of Klenow fragment (3' to 5' exo minus). This prepares the DNA fragments for ligation to the adapters, which have a single ‘T’ base overhang at their 3' end. Further, the adapters were ligated to the ends of the DNA fragments, preparing them to be hybridized to a single read flow cell. Then, the products of the ligation reaction were purified on gel to select a size range of templates and amplified by PCR. The amplified library was sequenced by Illumina RNA-seq technology.
The details of the experiment were as follows:
Sequencing strategy: single end sequencing for 21P, 3L, 4L, 5L, 6L, 10L, 22L, 40L, 62L, 70L, 80L, 87L, 133L, 151L, 171L, 243L, 260L samples and paired-end sequencing for 17P, 18P, 2L, 266L samples.
Read length: 40 nucleotides for single end sequencing and 90 nucleotides for paired end sequencing.
5.2.2.3 RNA-seq data analysis
The sequence output in FastQ format (Cock et al. 2010) was screened for quality using FastQC software provided by Babraham Bioinformatics
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(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Genomes of both M. eugenii [Meug_1.0 (GCA_000004035.1)] and Monodelphis domestica [BROADO5 (GCF_000002295.2)] were used as reference. The alignment of RNA sequences to M. eugenii genome was for annotation of sequence reads and for validating the dataset by examining the expression profiles of major milk proteins. The alignment to M. domestica genome gave better annotation. The pipeline for the sequence analysis followed the Tuxedo protocol (Trapnell et al. 2012). The sequence reads were mapped to the reference genome using the Bowtie 2 function of TopHat 2.0.5 (Kim and Salzberg 2011; Langmead and Salzberg 2012) and transcripts assembled using Cufflinks (Trapnell et al. 2010). The final transcriptome assembly was performed by merging assembled transcripts using Cuffmerge and SAM tools (Li et al. 2009a), and the differential expression of the mapped reads were determined using Cuffdiff. Seqmonk (version 0.24.1) was used to visualise the data and confirm the location of alignment of the reads to the genome.
Detailed analysis of the dataset aligned to M. domestica genome was performed using Seqmonk by filtering and quantitating the data based on different criteria. Read count quantitation, the simplest kind of quantitation, was performed by counting all reads, corrected to total read per counts and probe length and represented per million reads. To identify the major differentially expressed genes between different phases, the dataset was filtered in Seqmonk to list all genes having DQ)3.0IRUDWOHDVWRQHRIWKHVDPSOHV)XUWKHUEDVHGRQWKHH[SUHVVLRQ during different phases of lactation the genes were grouped into different categories; genes expressed in a phase specific manner and genes expressed across lactation.
5.2.2.4 Identification of genes encoding secreted proteins expressed during phase 2A tammar wallaby lactation
To identify the genes encoding secreted proteins expressed during phase 2A of tammar wallaby lactation, the searchable databases UniProt Knowledgebase (http://www.uniprot.org/help/uniprotkb), GeneCards (http://www.genecards.org/) and LOCATE (http://locate.imb.uq.edu.au/cgi-bin/search.cgi) were utilised. The
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UniProt Knowledgebase (UniProtKB) has a collection of functional information on proteins with accurate, consistent and rich annotation. It includes manually-annotated records extracted from the literature and curator-evaluated computation analysis and a section with computationally analysed records. The GeneCards human gene database has been integrated by gene related transcriptomic, genetic, proteomic, functional and disease information from diverse relevant sources. LOCATE is a curated database that describes the sub-cellular localization of proteins from the RIKEN FANTOM4 mouse and human protein sequence set determined by a high- throughput, immunofluorescence-based assay and by manually reviewing peer- reviewed publications. All the three databases use standard nomenclature and approved gene symbols. In addition, the secreted proteins identified in Khalil’s (2007) study were also used for comparison.
5.2.3 Proteome and transcriptome datasets of human amniotic fluid and placenta The web based body fluid proteome database Sys-BodyFluid which includes list of proteins expressed in amniotic fluid was used as the reference database (Li et al. 2009b). This allows retrieval of proteome data based on protein name, accession number and sequence similarity (http://www.biosino.org/bodyfluid/). It is a summary of different proteome based datasets previously published (Cho et al. 2007; Michaels et al. 2007; Nilsson et al. 2004). For the human placenta proteome list, the expression profiling and compilation of proteins reported by Mushahary and co- workers (2013) was used as the reference dataset. A microarray data analysis was performed by Larrabee and co-workers (2005) using pooled cell free samples of human amniotic fluid across different gestational ages. This gene expression data of human amniotic fluid was used to compare with the expression profile in tammar wallaby mammary gland. In addition, for gene expression data of human placenta, the microarray data available online based on the experimental study comparing smoking and non-smoking mothers was used (http://biogps.org/dataset/870/full-term-placenta-smokers-and-non-smokers/).
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5.2.4 Venn diagram – Comparison of tammar wallaby and human bioactives To represent the comparative relationship between the number of bioactives of human amniotic fluid or placenta and tammar wallaby phase 2A milk, the Gliffy Venn diagram display was utilised (http://www.gliffy.com/uses/venn-diagram- software-template/).
5.3 Results 5.3.1 Microarray based transcriptome profiling of tammar wallaby mammary gland across lactation
5.3.1.1 Genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland identified by microarray transcriptome profiling
The list of genes predicted to encode differentially secreted proteins across tammar wallaby lactation cycle previously identified by Khalil (2007) was examined to select those which are highly or specifically expressed during phase 2A lactation when compared to other phases. From the 326 genes that were differentially expressed during lactation in the tammar wallaby mammary gland and predicted to code for secreted proteins, 23 were highly expressed during phase 2A lactation (Table 5.1).
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Table 5.1: Genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland identified by microarray transcriptome profiling.
CLONE ID ANNOTATION ACPL2 Acid phosphatase-like 2 AHCY S-adenosylhomocysteine hydrolase AOX1 Aldehyde oxidase 1 APE N-acylaminoacyl-peptide hydrolase C1orf160 Chromosome 1 open reading frame 160 C7orf30 Chromosome 7 open reading frame 30 C9orf100 Chromosome 9 open reading frame 100 CATHL2 Cathelicidin 2 CCDC102A Coiled-coil domain containing 102A CCDC80 Coiled-coil domain containing 80 CCL28 Chemokine (C-C motif) ligand 28 CLMN Calmin (calponin-like, transmembrane) CLU Clusterin DNAJC13 DnaJ (Hsp40) homolog, subfamily C, member 13 GM2A GM2 ganglioside activator GNB2 Guanine nucleotide binding protein (G protein), beta polypeptide 2 JARID1B Jumonji, AT rich interactive domain 1B POSTN Periostin, osteoblast specific factor RPS10 Ribosomal protein S10 SCD Stearoyl-CoA desaturase (delta-9-desaturase) SCP2 Sterol carrier protein 2 SLC25A1 Solute carrier family 25 (mitochondrial carrier; citrate transporter), member 1 TINAGL Tubulointerstitial nephritis antigen-like 1
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5.3.1.2 Venn diagram depicting the number of bioactives of tammar wallaby phase 2A lactation and/or human amniotic fluid/ placenta
In total, 754 proteins were identified from human amniotic fluid using the Sys-BodyFluid proteome database which was compiled from previously published reports. This was compared with the 23 genes encoding secreted proteins expressed during phase 2A lactation in tammar wallaby. It was observed that 3 out of the 754 proteins of human amniotic fluid were found to be expressed during phase 2A lactation in tammar wallaby mammary gland (Figure 5.1 A). Similarly, the human amniotic fluid transcriptome dataset was compared with tammar wallaby microarray dataset. From the 1165 genes in the human amniotic fluid database, 9 were found to be expressed during phase 2A lactation (Figure 5.1 B).
Previously published reports have identified 116 proteins in the human placenta of which, one was expressed in tammar wallaby mammary gland during phase 2A lactation (Figure 5.2 A). From the 1182 genes identified in the human placenta, 13 were expressed during phase 2A lactation (Figure 5.2 B).
5.3.1.3 Comparison of genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland with genes/proteins of human amniotic fluid/placenta
The list of secreted proteins of phase 2A tammar wallaby lactation compared with transcriptome and proteome databases of human amniotic fluid and placenta is represented in Table 5.2. Among the 23 secreted proteins highly expressed during phase 2A, 10 were present in both human amniotic fluid and placenta whereas another 3 were present in human placenta alone and none in amniotic fluid alone.
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A
HUMAN TAMMAR WALLABY AMNIOTIC FLUID 3 PHASE 2A MILK 751 20
B
HUMAN TAMMAR WALLABY AMNIOTIC FLUID 9 PHASE 2A MILK 1156 14
Figure 5.1: Venn diagram depicting the common proteins in human amniotic fluid (proteome and transcriptome list) and tammar wallaby phase 2A milk (microarray transcriptome list). (A) The proteome list of human amniotic fluid compared with microarray based transcriptome list of tammar wallaby phase 2A milk (B) The transcriptome list of human amniotic fluid compared with microarray transcriptome list of tammar wallaby phase 2A milk.
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A
HUMAN TAMMAR WALLABY PLACENTA 1 PHASE 2A MILK 115 22
B
HUMAN TAMMAR WALLABY PLACENTA 13 PHASE 2A MILK 1169 10
Figure 5.2: Venn diagram depicting the proteins common to the human placenta (proteome and transcriptome list) and tammar wallaby phase 2A milk (microarray transcriptome list). (A) The proteome list of human placenta compared with microarray transcriptome list of tammar wallaby phase 2A milk (B) The transcriptome list of human placenta compared with microarray transcriptome list of tammar wallaby phase 2A milk.
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Table 5.2: Comparison of genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland with genes/proteins of human amniotic fluid/placenta. (The genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland that showed expression in either human amniotic fluid/placenta are marked with an asterisk sign *).
HUMAN CLONE ID ANNOTATION AMNITOIC FLUID HUMAN PLACENTA ACPL2 Acid phosphatase-like 2 - - AHCY S-adenosylhomocysteine hydrolase ** AOX1 Aldehyde oxidase 1 - * APE N-acylaminoacyl-peptide hydrolase - - C1orf160 Chromosome 1 open reading frame 160 ** C7orf30 Chromosome 7 open reading frame 30 ** C9orf100 Chromosome 9 open reading frame 100 ** CATHL2 Cathelicidin 2 - - CCDC102A Coiled-coil domain containing 102A - - CCDC80 Coiled-coil domain containing 80 - - CCL28 Chemokine (C-C motif) ligand 28 - - CLMN Calmin (calponin-like, transmembrane) - * CLU Clusterin - - DNAJC13 DnaJ (Hsp40) homolog, subfamily C, member 13 ** GM2A GM2 ganglioside activator ** GNB2 Guanine nucleotide binding protein (G protein), beta polypeptide 2 ** JARID1B Jumonji, AT rich interactive domain 1B ** POSTN Periostin, osteoblast specific factor ** RPS10 Ribosomal protein S10 - - SCD Stearoyl-CoA desaturase (delta-9-desaturase) - - SCP2 Sterol carrier protein 2 ** SLC25A1 Solute carrier family 25 (mitochondrial carrier; citrate transporter), member 1 - - TINAGL Tubulointerstitial nephritis antigen-like 1 - *
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5.3.2 RNA-seq based transcriptome profiling of tammar wallaby mammary gland across lactation
5.3.2.1 Milk protein gene expression
To determine the validity of the RNA-seq dataset, the data aligned to the Macropus eugenii genome was examined for the abundance and expression profiles of major milk protein genes (Figure 5.3). This included beta-casein and the major whey proteins expressed throughout lactation cycle such as beta-lactoglobulin and alpha-lactalbumin, and the milk protein genes expressed in a phase-specific manner such as early lactation protein (Phase 2A), whey acidic protein (Phase 2B), late lactation protein-a and late lactation protein-b (Phase 3). These milk protein genes showed a high level of expression in terms of higher FPKM values and expression pattern across lactation cycle as reported earlier (Brennan et al. 2007; Simpson et al. 1998b; Simpson et al. 2000; Trott et al. 2002). Two samples of the dataset (18P and 2L) are not represented because of a computer based problem in aligning the reads of these two samples to the M. eugenii genome.
5.3.2.2 Genes expressed in tammar wallaby mammary gland during the lactation cycle
The dataset aligned to the M. domestica genome was utilised for further analysis as it gave better annotation when compared to the dataset aligned to M. eugenii genome. The datastore summary report provides a brief overview of the data comparing different samples (Table 5.3).
Read count quantitation was carried out using the criteria of counting all reads, corrected to total read per counts and probe length, represented per million reads. This generated a total of 253929 probes. A total of 17775 probes had a FPKM greater than 1.0. In order to identify the highly expressed genes during tammar wallaby lactation, the dataset was filtered in Seqmonk to give a list of genes having a FPKM greater than 1000 for at least one of the 21 samples. Excluding the non- annotated probes, a total of 1889 genes were identified. Among these, 199 genes
171 were either specific or highly expressed during phase 2A lactation (Table 5.4). In addition, 7 genes were identified in phase 2B (Table 5.5) and 20 genes in phase 3 (Table 5.6). A 2D- area graph representing the expression profile of genes expressed in the mammary gland during three different phases of lactation is shown for phase 2A (Figure 5.4), phase 2B (Figure 5.5) and phase 3 (Figure 5.6).
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A
B
C
D
E
F
G
Figure 5.3: Milk protein gene expression level. Milk protein gene expression across lactation cycle determined from the RNA-seq dataset (day of pregnancy, P or lactation, L; x- axis) is represented in fragments per kilobase of exon per million fragments mapped (FPKM; y-axis). This was determined using the dataset aligned to M. eugenii genome. The graph shows the expression profiles of (A) Beta-Casein (B) Beta-lactoglobulin (C) Alpha- lactalbumin (D) Early lactation protein (E) Whey acidic protein (F) Late lactation protein-A and (G) Late lactation protein-B.
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Table 5.3: Data store report of data aligned to Monodelphis domestica genome.
Total Read Mean Read Total Read Fold Samples Count Length Length Coverage Total Quantitation 17P 3328365 68 227795555 0.065040029 7839699.286 18P 3267037 69 227242796 0.064882206 7360776.089 21P 1680601 41 69599345 0.019871957 11742987.223 2L 2580051 69 178404943 0.050938056 7981585.895 3L 1978957 43 86292554 0.02463819 10543068.420 4L 2293944 42 97477740 0.027831777 10516250.494 5L 2610951 41 108147242 0.030878126 11592230.656 6L 2352376 43 101716203 0.02904194 10536948.709 10L 2151767 43 93304409 0.02664021 10271663.622 22L 2433048 42 104223979 0.029757958 10686394.747 40L 1748602 41 72757029 0.020773537 10971394.054 62L 1005887 40 40637262 0.011602723 12578196.696 70L 1471840 40 60280606 0.017211277 11605925.697 80L 1893272 43 83085467 0.023722505 10113563.138 87L 1773401 40 72329215 0.020651387 12338041.889 133L 832063 39 32885611 0.009389477 13454178.381 151L 1224391 40 49157654 0.014035459 12886399.331 171L 1295686 40 52181827 0.014898919 12615234.546 243L 794582 40 32230908 0.009202547 13289705.235 260L 464582 39 18125384 0.005175147 14269111.198 266L 931556 72 67111348 0.019161586 7638933.912
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Table 5.4: Genes expressed in the tammar wallaby mammary gland during phase 2A lactation identified by RNA-seq data analysis.
CLONED ID ANNOTATION ACTA1 Actin, alpha skeletal muscle (Alpha-actin-1) ACTC1 Actin, alpha cardiac muscle 1 (Alpha-cardiac actin) ADAMTS-10 precursor (A disintegrin and metalloproteinase with thrombospondin ADAMTS10 motifs 10)
ADSS Adenylosuccinate synthetase isozyme 2 (Adenylosuccinate synthetase, acidic isozyme) AHI1 Jouberin (Abelson helper integration site 1 protein homolog) (AHI-1) AHNAK Neuroblast differentiation-associated protein AHNAK (Desmoyokin) AKR1B1 Aldose reductase (AR) (Aldehyde reductase) AKT1S1 Proline-rich AKT1 substrate 1 (40 kDa proline-rich AKT substrate) ANKS3 Ankyrin repeat and SAM domain-containing protein 3 Tether containing UBX domain for GLUT4 (Alveolar soft part sarcoma chromosomal ASPSCR1 region candidate gene 1 protein) ATP1A2 Sodium/potassium-transporting ATPase subunit alpha-2 precursor Sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (Calcium pump 1) (SERCA1) ATP2A1 (SR Ca(2+)-ATPase 1) ATP5F1 ATP synthase subunit b, mitochondrial precursor BAT1 Spliceosome RNA helicase BAT1 (DEAD box protein UAP56) BLVRB Flavin reductase (FR) (NADPH-dependent diaphorase) BNC2 Zinc finger protein basonuclin-2 C1orf93 Uncharacterized protein C1orf93 CA3 Carbonic anhydrase 3 CACNA1B Voltage-dependent N-type calcium channel subunit alpha-1B CEP164 Centrosomal protein of 164 kDa (Cep164) CEP192 Centrosomal protein of 192 kDa (Cep192) CEP27 Centrosomal protein of 27 kDa (Cep27) Centrosome-associated protein 350 (Cep350) (Centrosome-associated protein of CEP350 350 kDa) Glycosyltransferase 25 family member 3 precursor (Cerebral endothelial cell adhesion CERCAM molecule) Cold-inducible RNA-binding protein (Glycine-rich RNA-binding protein CIRP) (A18 CIRBP hnRNP) CLK3 Dual specificity protein kinase CLK3 (CDC-like kinase 3) CLU Clusterin precursor (Complement-associated protein SP-40,40) CNTN5 Contactin-5 precursor (Neural recognition molecule NB-2) (hNB-2) CPXM1 Probable carboxypeptidase X1 precursor (Metallocarboxypeptidase CPX-1) Cysteine-rich protein 1 (Cysteine-rich intestinal protein) (CRIP) (Cysteine-rich heart CRIP1 protein) (hCRHP) CRYAB Alpha- crystallin B chain (Alpha(B)- crystallin) CSGALNACT1 Chondroitin beta-1,4-N-acetylgalactosaminyltransferase 1 Cysteine and glycine-rich protein 3 (Cysteine-rich protein 3) (CRP3) (LIM domain CSRP3 protein, cardiac) (Muscle LIM protein) DCN Decorin precursor (Bone proteoglycan II) (PG-S2) (PG40) DDC Aromatic-L-amino-acid decarboxylase (AADC) (DOPA decarboxylase) (DDC) DDX31 Probable ATP-dependent RNA helicase DDX31 DENND4A C-myc promoter-binding protein (DENN domain-containing protein 4A)
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DES Desmin DGCR2 Integral membrane protein DGCR2/IDD precursor Death-inducer obliterator 1 (DIO-1) (Death-associated transcription factor 1) (DATF- DIDO1 1) (hDido1) DOK5 Docking protein 5 (Downstream of tyrosine kinase 5) (IRS6) (Protein dok-5) DST Bullous pemphigoid antigen 1, isoforms 6/9/10 (Trabeculin-beta) Transcription factor E4F1(Putative E3 ubiquitin-protein ligase E4F1) (E4F E4F1 transcription factor 1) (Transcription factor E4F) Transcription factor COE4 (Early B-cell factor 4) (EBF-4) (Olf-1/EBF- like 4) (OE- EBF4 4) (O/E-4) ECHDC2 Enoyl-CoA hydratase domain-containing protein 2, mitochondrial precursor EEF1B2 Elongation factor 1-beta (EF-1-beta) EEF1D Elongation factor 1-delta (EF-1-delta) (Antigen NY-CO-4) Eukaryotic translation initiation factor 3 subunit B (Eukaryotic translation initiation EIF3S9 factor 3 subunit 9) (hPrt1) EML1 Echinoderm microtubule-associated protein-like 1 (EMAP-1) (HuEMAP-1) Ephrin type-A receptor 2 precursor (Tyrosine-protein kinase receptor ECK) EPHA2 (Epithelial cell kinase) Ephrin type-A receptor 3 precursor (Tyrosine-protein kinase receptor ETK1) (HEK) EPHA3 (HEK4) Ephrin type-B receptor 6 precursor (Tyrosine-protein kinase-defective receptor EPH- EPHB6 6) (HEP) ETS translocation variant 3 (ETS domain transcriptional repressor PE1) (PE-1) ETV3 (Mitogenic Ets transcriptional suppressor) FAM45B Protein FAM45A FAM48A Protein FAM48A (p38-interacting protein) (p38IP) FAU Ubiquitin-like protein FUBI FGGY FGGY carbohydrate kinase domain-containing protein FH1/FH2 domain-containing protein 3 (Formin homolog overexpressed in spleen 2) FHOD3 (hFHOS2) (Formactin-2) GRB2-associated-binding protein 1 (Growth factor receptor bound protein 2- GAB1 associated protein 1) (GRB2-associated binder 1) GABRP Gamma-aminobutyric acid receptor subunit pi precursor GCN5L2 General control of amino acid synthesis protein 5-like 2 GLRA3 Glycine receptor subunit alpha-3 precursor Glutaminase kidney isoform, mitochondrial precursor (GLS) (L-glutamine GLS amidohydrolase) (K- glutaminase) GMFG Glia maturation factor gamma (GMF- gamma) GNL1 Guanine nucleotide-binding protein-like 1 (GTP-binding protein HSR1) GTF3A Transcription factor IIIA (Factor A) (TFIIIA) HDAC3 Histone deacetylase 3 HOXD9 Homeobox protein Hox-D9 (Hox-4C) (Hox-5.2) Heparan sulfate glucosamine 3-O-sulfotransferase 6 (Heparan sulfate D-glucosaminyl HS3ST6 3-O-sulfotransferase 6) Insulin-like growth factor-binding protein 7 precursor (IGFBP-7) (IBP- 7) (IGF- IGFBP7 binding protein 7) IL11RA Interleukin-11 receptor alpha chain precursor (IL-11R-alpha)
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T- cell immunomodulatory protein precursor (Protein TIP) (Integrin- alpha FG- GAP ITFG1 repeat-containing protein 1) JARID2 Protein Jumonji (Jumonji/ARID domain-containing protein 2) KIAA0146 Uncharacterized protein KIAA0146 KIAA0564 Novel protein KLF12 Krueppel-like factor 12 (Transcriptional repressor AP-2rep) KNTC1 Kinetochore-associated protein 1 (Rough deal homolog) KRT15 Keratin, type I cytoskeletal 15 (Cytokeratin-15) (CK-15) (Keratin-15) (K15) Lethal(3)malignant brain tumor-like protein (L(3)mbt-like) (L(3)mbt protein homolog) L3MBTL (H-l(3)mbt protein) (H-L(3)MBT) (L3MBTL1) Lethal(3)malignant brain tumor-like 3 protein (L(3)mbt-like 3 protein) (H-l(3)mbt-like L3MBTL3 protein) LAMA2 Laminin subunit alpha-2 precursor (Laminin M chain) (Merosin heavy chain) LIM homeobox transcription factor 1 alpha (LIM/homeobox protein LMX1A) LMX1A (LIM/homeobox protein 1.1) (LMX-1.1) MAP3K11 Mitogen-activated protein kinase kinase kinase 11 (Mixed lineage kinase 3) MCOLN1 Mucolipin- 1 (Mucolipidin) (MG- 2) Mediator of RNA polymerase II transcription subunit 27 (Mediator complex subunit MED27 27) (CRSP complex subunit 8) MGEA5 Bifunctional protein NCOAT Mirror-image polydactyly gene 1 protein MLLT1 Protein ENL (YEATS domain-containing protein 1) MRPL52 39S ribosomal protein L52, mitochondrial precursor (L52mt) (MRP-L52) MXI1 MAX-interacting protein 1 Myosin-13 (Myosin heavy chain 13) (Myosin heavy chain, skeletal muscle, MYH13 extraocular) (MyHC-eo) Myosin-4 (Myosin heavy chain 4) (Myosin heavy chain 2b) (MyHC-2b) (Myosin MYH4 heavy chain, skeletal muscle, fetal) Myosin light chain 1, skeletal muscle isoform (MLC1F) (A1 catalytic) (Alkali myosin MYL1 light chain 1) Myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC-2) (MLC- MYL2 2v) MYOT Myotilin (Titin immunoglobulin domain protein) NEB Nebulin NFATC4 Nuclear factor of activated T-cells, cytoplasmic 4 NGFR Tumor necrosis factor receptor superfamily member 16 precursor NISCH Nischarin NLGN2 Neuroligin-2 precursor NOLC1 Nucleolar phosphoprotein p130 Neurogenic locus notch homolog protein 1 precursor (Notch 1) (hN1) (Translocation- NOTCH1 associated notch protein TAN-1) NOXO1 NADPH oxidase organizer 1 (Nox organizer 1) NP_001028141.1 DNA (cytosine-5-)-methyltransferase NRAP Nebulin-related-anchoring protein (N-RAP) NRG2 Pro-neuregulin-2, membrane-bound isoform precursor (Pro-NRG2) NSMCE4A Non-SMC element 4 homolog A
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OXA1L Inner membrane protein OXA1L, mitochondrial precursor PAM Peptidyl-glycine alpha-amidating monooxygenase precursor PARD3 Partitioning- defective 3 homolog PDSS2 Decaprenyl-diphosphate synthase subunit 2 PKNOX1 Homeobox protein PKNOX1 PKD2 Polycystin-2 (Polycystic kidney disease 2 protein) PLCG1 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-1 PMS1 PMS1 protein homolog 1 PPAP2B Lipid phosphate phosphohydrolase 3 PRDM8 PR domain zinc finger protein 8 PRDX2 Peroxiredoxin-2 PREP Prolyl endopeptidase PRKG1 cGMP-dependent protein kinase 1, beta isozyme PRKRA Interferon-inducible double stranded RNA-dependent protein kinase activator A PRR12 Proline-rich protein 12 PRMT3 Protein arginine N-methyltransferase 3 PSMA5 Proteasome subunit alpha type-5 PSMB10 Proteasome subunit beta type-10 precursor PSMB9 Proteasome subunit beta type-9 precursor PSMC3 26S protease regulatory subunit 6A PSMC5 26S protease regulatory subunit 8 PSME2 Proteasome activator complex subunit 2 PSMF1 Proteasome inhibitor PI31 subunit (hPI31) PTGDS Prostaglandin-H2 D-isomerase precursor QKI Protein quaking (HqkI) (Hqk) R3HDM1 R3H domain-containing protein 1 RAB6IP1 Rab6-interacting protein 1 RAC1 Ras-related C3 botulinum toxin substrate 1 precursor RALGPS1 Ras-specific guanine nucleotide-releasing factor RalGPS1 RCL1 RNA 3'-terminal phosphate cyclase-like protein RGS12 Regulator of G-protein signaling 12 (RGS12) RIPK5 Receptor-interacting serine/threonine-protein kinase 5 RIT2 GTP-binding protein Rit2 RPL11 60S ribosomal protein L11 RPL26 60S ribosomal protein L26 RPL3 60S ribosomal protein L3 RPL35A 60S ribosomal protein L35a RPL4 60S ribosomal protein L4 (L1) RPS15 40S ribosomal protein S15 RPS21 40S ribosomal protein S21 RPS9 40S ribosomal protein S9 RTN1 Reticulon-1 RXFP4 Relaxin-3 receptor 2 S100A11 Protein S100-A11 (S100 calcium-binding protein A11) S100A4 Protein S100-A4 (S100 calcium-binding protein A4) SEL1L Protein sel-1 homolog 1 precursor (Suppressor of lin-12-like protein 1)
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SETDB2 Histone-lysine N-methyltransferase SETDB2 SH3D19 SH3 domain-containing protein 19 SLC6A9 Sodium- and chloride-dependent glycine transporter 1 SNF8 Vacuolar-sorting protein SNF8 SNX7 Sorting nexin-7 SOD1 Superoxide dismutase SRRM2 Serine/arginine repetitive matrix protein 2 STK11IP Serine/threonine kinase 11-interacting protein STX4 Syntaxin-4 STXBP5L Syntaxin-binding protein 5-like SYN1 Synapsin-1 (Synapsin I) (Brain protein 4.1) SYN3 Synapsin-3 SYNE1 Nesprin-1 SYT7 Synaptotagmin-17 TACC3 Transforming acidic coiled- coil- containing protein 3 TBC1D21 TBC1 domain family member 21 TBC1D22A TBC1 domain family member 22A TBRG4 Protein TBRG4 (Transforming growth factor beta regulator 4) Major facilitator superfamily domain- containing protein 10 (Tetracycline transporter- TETRAN like protein) TFAP2D Transcription factor AP-2 delta THBS3 Thrombospondin-3 precursor TIGD5 Tigger transposable element-derived protein 5 TIMP3 Metalloproteinase inhibitor 3 precursor TNC Tenascin precursor TNNC1 Troponin C, slow skeletal and cardiac muscles TNNC2 Troponin C, skeletal muscle TNNI1 Troponin I, slow skeletal muscle TOP2B DNA topoisomerase 2-beta TP53BP1 Tumor suppressor p53-binding protein 1 TP73 Tumor protein p73 (p53-like transcription factor) TPM3 Tropomyosin alpha-3 chain (Tropomyosin-3) TRMT1 N(2),N(2)-dimethylguanosine tRNA methyltransferase TTC17 Tetratricopeptide repeat protein 17 TTC27 Tetratricopeptide repeat protein 27 TUBA8 Tubulin alpha-8 chain (Alpha-tubulin 8) TUBB Tubulin beta chain (Tubulin beta-5 chain) TUBGCP4 Gamma- tubulin complex component 4 USP6NL USP6 N- terminal- like protein VAT1 Synaptic vesicle membrane protein VAT-1 homolog WDR61 WD repeat-containing protein 61 XPR1 Xenotropic and polytropic retrovirus receptor 1 ZMAT4 Zinc finger matrin-type protein 4 ZNF263 Zinc finger protein 263 ZNF362 Zinc finger protein 362 ZNF384 Zinc finger protein 384 ZNF513 Zinc finger protein 513
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ACTA1 ACTC1 ADAMTS10 ADSS AHI1 AHNAK AKR1B1 AKT1S1 ANKS3 ASPSCR1 ATP1A2 ATP2A1 ATP5F1 BAT1 BLVRB BNC2 C1orf93 CA3 CACNA1B CEP164 CEP192 CEP27 CEP350 CERCAM CIRBP CLK3 CLU CNTN5 CPXM1 CRIP1 CRYAB CSGALNACT1 CSRP3 DCN DDC DDX31 DENND4A DES DGCR2 DIDO1 DOK5 DST E4F1 EBF4 ECHDC2 EEF1B2 EEF1D EIF3S9 EML1 EPHA2 EPHA3 EPHB6 ETV3 FAM45B FAM48A FAU FGGY FHOD3 FTO GAB1 GABRP GCN5L2 GLRA3 GLS GMFG GNL1 GTF3A HDAC3 HOXD9 HS3ST6 IGFBP7 IL11RA ITFG1 JARID2 KIAA0146 KIAA0564 KLF12 KNTC1 KRT15 L3MBTL L3MBTL3 LAMA2 LMX1A MAP3K11 MBTPS2 MCOLN1 MED27 MGEA5 MIPOL1 MLLT1 MRPL52 MXI1 MYH13 MYH4 MYL1 MYL2 MYOT NEB NFATC4 NGFR NISCH NLGN2 NOLC1 NOTCH1 NOXO1 NP_001028141.1 NRAP NRG2 NSMCE4A OBSCN PAM PARD3 PDSS2 PKD2 PKNOX1 PLCG1 PMS1 PPAP2B PRDM8 PRDX2 PREP PRKG1 PRKRA PRMT3 PRR12 PSMA5 PSMB10 PSMB9 PSMC3 PSMC5 PSME2 PSMF1 PTGDS QKI R3HDM1 RAB6IP1 RAC1 RALGPS1 RCL1 RGS12 RIPK5 RIT2 RPL11 RPL26 RPL3 RPL35A RPL4 RPS15 RPS21 RPS9 RTN1 RXFP4 S100A11 S100A4 SEL1L SETDB2 SH3D19 SLC6A9 SNF8 SNX7 SOD1 SRRM2 STK11IP STX4 STXBP5L SYN1 SYN3 SYNE1 SYT7 TACC3 TBC1D21 TBC1D22A TBRG4 TETRAN TFAP2D THBS3 TIGD5 TIMP3 TNC TNNC1 TNNC2 TNNI1 TOP2B TP53BP1 TP73 TPM3 TRMT1 TTC17 TTC27 TUBA8 TUBB TUBGCP4 USP6NL VAT1 WDR61 XPR1 ZMAT4 ZNF263 ZNF362 ZNF384 ZNF513
Figure 5.4: A 2D-area graph representing the expression profile of genes expressed in the tammar wallaby mammary gland during phase 2A lactation. (Day of pregnancy, P or lactation, L; x-axis and FPKM; y-axis).
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Table 5.5: Genes expressed in the tammar wallaby mammary gland during phase 2B lactation identified by RNA-seq data analysis.
CLONED ID ANNOTATION ACSL4 Long-chain-fatty-acid--CoA ligase 4 ADAMTS6 ADAMTS-6 precursor (A disintegrin and metalloproteinase with thrombospondin motifs 6) CASR Extracellular calcium-sensing receptor precursor CMAS N-acylneuraminate cytidylyltransferase (CMP-N- acetylneuraminic acid synthetase) MYO5B Myosin-Vb OSBPL6 Oxysterol-binding protein-related protein 6 (OSBP-related protein 6) SPOCK2 Testican-2 precursor (SPARC/osteonectin, CWCV, and Kazal-like domains proteoglycan 2)
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ACSL4 ADAMTS6 CASR CMAS MYO5B OSBPL6 SPOCK2
Figure 5.5: A 2D-area graph representing the expression profile of genes expressed in the tammar wallaby mammary gland during phase 2B lactation. (Day of pregnancy, P or lactation, L; x-axis and FPKM; y-axis).
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Table 5.6: Genes expressed in the tammar wallaby mammary gland during phase 3 lactation identified by RNA-seq data analysis.
CLONED ID ANNOTATION ABCG2 ATP- binding cassette sub- family G member 2 (Placenta- specific ATP- binding cassette transporter) ACSL1 Long-chain-fatty-acid--CoA ligase 1 (Long-chain acyl-CoA synthetase 1) ACSS2 Acetyl-coenzyme A synthetase, cytoplasmic (Acetate--CoA ligase) AGPAT1 1-acyl-sn-glycerol-3-phosphate acyltransferase alpha (1- AGP acyltransferase 1) AGXT2L1 Alanine- - glyoxylate aminotransferase 2- like 1 ALPL Alkaline phosphatase, tissue-nonspecific isozyme precursor ATR Serine/threonine-protein kinase ATR DGKI Diacylglycerol kinase iota (EC 2.7.1.107) (Diglyceride kinase iota) (DGK-iota) (DAG kinase iota) FAM46A Protein FAM46A (HBV X-transactivated gene 11 protein) GLB1 Beta-galactosidase precursor (Lactase) (Acid beta- galactosidase) (Elastin receptor 1) LPL Lipoprotein lipase precursor (LPL) LPO Lactoperoxidase precursor (LPO) (Salivary peroxidase) (SPO) PCTK3 Serine/threonine-protein kinase PCTAIRE-3 (PCTAIRE- motif protein kinase 3) PDCD2L Programmed cell death protein 2-like PIP5K1B Phosphatidylinositol-4-phosphate 5-kinase type-1 beta PROX1 Homeobox prospero-like protein PROX1 RHOBTB2 Rho-related BTB domain-containing protein 2 (Deleted in breast cancer 2 gene protein) (p83) SCD Acyl-CoA desaturase (Stearoyl-CoA desaturase) (Fatty acid desaturase) (Delta(9)-desaturase) SGK1 Serine/threonine-protein kinase Sgk1 (Serum/glucocorticoid-regulated kinase 1) SNX9 Sorting nexin-9 (SH3 and PX domain-containing protein 1)
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ABCG2 ACSL1 ACSS2 AGPAT1 AGXT2L1 ALPL ATR DGKI FAM46A GLB1 LPL LPO PCTK3 PDCD2L PIP5K1B PROX1 RHOBTB2 SCD SGK1 SNX9
Figure 5.6: A 2D-area graph representing the expression profile of genes expressed in the tammar wallaby mammary gland during phase 3 lactation. (Day of pregnancy, P or lactation, L; x-axis and FPKM; y-axis).
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5.3.2.3 Genes encoding secreted proteins expressed during tammar wallaby phase 2A lactation
The 199 genes showing higher or specific expression during phase 2A lactation were examined in detail. These genes were grouped into different classes based on their expression pattern; those showing higher level of expression during early phase 2A, those showing higher level of expression during late phase 2A and those showing same level of expression throughout phase 2A lactation. These genes were identified in UniProt, Genecards and LOCATE database to determine the sub- cellular localisation and identify genes encoding secreted proteins. Among the 199 genes, 28 were genes encoding secreted proteins (Table 5.7). The expression profile of these 28 genes encoding secreted proteins showing specific or higher level of expression pattern during phase 2A lactation is represented (Appendix 1).
In addition, two of the genes identified in phase 2B (ADAMTS6 and SPOCK2) and two of the genes identified in phase 3 (LPL and LPO) code for secreted proteins (Appendix 2 and 3).
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Table 5.7: Genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland identified by RNA-seq transcriptome profiling.
CLONED ID ANNOTATION ADAMTS-10 precursor (A disintegrin and metalloproteinase with thrombospondin ADAMTS10 motifs 10) AKT1S1 Proline-rich AKT1 substrate 1 (40 kDa proline-rich AKT substrate) Tether containing UBX domain for GLUT4 (Alveolar soft part sarcoma ASPSCR1 chromosomal region candidate gene 1 protein) Glycosyltransferase 25 family member 3 precursor (Cerebral endothelial cell adhesion CERCAM molecule) CLU Clusterin precursor (Complement-associated protein SP-40,40) CNTN5 Contactin-5 precursor (Neural recognition molecule NB-2) (hNB-2) CPXM1 Probable carboxypeptidase X1 precursor (Metallocarboxypeptidase CPX-1) Cysteine-rich protein 1 (Cysteine-rich intestinal protein) (CRIP) (Cysteine-rich heart CRIP1 protein) (hCRHP) DCN Decorin precursor (Bone proteoglycan II) (PG-S2) (PG40) Ephrin type-A receptor 3 precursor (Tyrosine-protein kinase receptor ETK1) (HEK) EPHA3 (HEK4) Ephrin type-B receptor 6 precursor (Tyrosine-protein kinase-defective receptor EPH- EPHB6 6) (HEP) HOXD9 Homeobox protein Hox-D9 (Hox-4C) (Hox-5.2) Insulin-like growth factor-binding protein 7 precursor (IGFBP-7) (IBP- 7) (IGF- IGFBP7 binding protein 7) T-cell immunomodulatory protein precursor (Protein TIP) (Integrin- alpha FG-GAP ITFG1 repeat-containing protein 1) KIAA0564 Novel protein Lethal(3)malignant brain tumor-like protein (L(3)mbt-like) (L(3)mbt protein homolog) L3MBTL (H-l(3)mbt protein) (H-L(3)MBT) (L3MBTL1) LAMA2 Laminin subunit alpha-2 precursor (Laminin M chain) (Merosin heavy chain) NOLC1 Nucleolar phosphoprotein p130 NRG2 Pro-neuregulin-2, membrane-bound isoform precursor (Pro-NRG2) PAM Peptidyl-glycine alpha-amidating monooxygenase precursor PTGDS Prostaglandin-H2 D-isomerase precursor SEL1L Protein sel-1 homolog 1 precursor (Suppressor of lin-12-like protein 1) SRRM2 Serine/arginine repetitive matrix protein 2 Major facilitator superfamily domain-containing protein 10 (Tetracycline transporter- TETRAN like protein) THBS3 Thrombospondin-3 precursor TIMP3 Metalloproteinase inhibitor 3 precursor TNC Tenascin precursor TTC17 Tetratricopeptide repeat protein 17
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5.3.2.4 Venn diagram depicting the number of bioactives of tammar wallaby phase 2A lactation and/or human amniotic fluid/ placenta
The 754 proteins identified from human amniotic fluid was compared with the 28 genes encoding secreted proteins highly expressed during phase 2A lactation identified by the current RNA-seq analysis. It was observed that 8 out of the 754 proteins of human amniotic fluid were found to be expressed during phase 2A lactation in tammar wallaby mammary gland (Figure 5.7 A). The 1165 genes in the human amniotic fluid transcriptome dataset were compared with the tammar wallaby RNA-seq dataset and 12 were found to be expressed during phase 2A lactation (Figure 5.7 B).
Among the 116 proteins identified in the human placenta, none was common to the tammar wallaby mammary gland phase 2A genes encoding secreted proteins (Figure 5.8 A). However, from the 1182 genes identified in the human placenta, 19 were expressed during phase 2A tammar wallaby lactation (Figure 5.8 B).
5.3.2.5 Comparison of genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland with genes/proteins of human amniotic fluid/placenta
The list of proteins secreted in phase 2A tammar wallaby lactation identified by the RNA-seq analysis and compared with transcriptome and proteome databases of human amniotic fluid and placenta is represented in Table 5.8. The candidate genes of phase 2A tammar wallaby lactation that showed expression in the human are marked by an asterisk sign (*). Among the 28 secreted proteins highly expressed during phase 2A, 15 were present in both human amniotic fluid and placenta whereas another 2 were present in human amniotic fluid alone and 4 were present in placenta alone.
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A
HUMAN TAMMAR WALLABY AMNIOTIC FLUID 8 PHASE 2A MILK 746 20
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HUMAN TAMMAR WALLABY AMNIOTIC FLUID 12 PHASE 2A MILK 1153 16
Figure 5.7: Venn diagram depicting the number of common proteins between human amniotic fluid (proteome and transcriptome list) and tammar wallaby phase 2A milk (RNA-seq based transcriptome list). (A) The proteins listed in the proteome of human amniotic fluid compared with predicted secreted proteins from the RNA- seq based transcriptome of tammar wallaby phase 2A milk (B) The transcriptome of human amniotic fluid compared with RNA-seq based transcriptome of tammar wallaby phase 2A milk.
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A
HUMAN TAMMAR WALLABY PLACENTA 0 PHASE 2A MILK 116 28
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HUMAN TAMMAR WALLABY PLACENTA 19 PHASE 2A MILK 1163 9
Figure 5.8: Venn diagram depicting the number of common proteins between human placenta (proteome and transcriptome list) and tammar wallaby phase 2A milk (RNA-seq based transcriptome list). (A) The proteins listed in the proteome of human placenta compared with RNA-seq based transcriptome list of tammar wallaby phase 2A milk (B) The transcriptome of human placenta compared with RNA-seq based transcriptome of tammar wallaby phase 2A milk.
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Table 5.8: Comparison of genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland with genes/proteins of human amniotic fluid/placenta. (The genes encoding secreted proteins expressed in phase 2A tammar wallaby mammary gland that showed expression in either human amniotic fluid/placenta are marked with an asterisk sign *).
HUMAN HUMAN CLONED ID ANNOTATION AMNIOTIC FLUID PLACENTA ADAMTS10 A disintegrin and metalloproteinase with thrombospondin motifs 10 * AKT1S1 Proline-rich AKT1 substrate 1 (40 kDa proline-rich AKT substrate) ** ASPSCR1 Alveolar soft part sarcoma chromosomal region candidate gene 1 protein * CERCAM Cerebral endothelial cell adhesion molecule CLU Clusterin precursor * CNTN5 Contactin-5 precursor CPXM1 Probable carboxypeptidase X1 precursor CRIP1 Cysteine-rich protein 1 ** DCN Decorin precursor ** EPHA3 Ephrin type-A receptor 3 precursor EPHB6 Ephrin type-B receptor 6 precursor * HOXD9 Homeobox protein Hox-D9 IGFBP7 Insulin-like growth factor-binding protein 7 precursor ** ITFG1 T-cell immunomodulatory protein precursor ** KIAA0564 Novel protein L3MBTL Lethal(3)malignant brain tumor-like protein (L(3)mbt-like) ** LAMA2 Laminin subunit alpha-2 precursor ** NOLC1 Nucleolar phosphoprotein p130 ** NRG2 Pro-neuregulin-2, membrane-bound isoform precursor PAM Peptidyl-glycine alpha-amidating monooxygenase precursor ** PTGDS Prostaglandin-H2 D-isomerase precursor ** SEL1L Protein sel-1 homolog 1 precursor ** SRRM2 Serine/arginine repetitive matrix protein 2 ** TETRAN Major facilitator superfamily domain- containing protein 10 ** THBS3 Thrombospondin-3 precursor ** TIMP3 Metalloproteinase inhibitor 3 precursor * TNC Tenascin precursor * TTC17 Tetratricopeptide repeat protein 17 **
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5.4 Discussion
All mammals have a reproductive strategy divided into two phases; the gestation phase and the lactation phase. However, when compared to eutherians the marsupials have evolved a reproductive strategy with a sophisticated lactation program (Tyndale-Biscoe and Renfree 1987). The composition of milk changes progressively across the extended lactation period thus supporting the development of the altricial newborn young (Tyndale-Biscoe and Janssens 1988). In contrast, the eutherians have a gestation and lactation cycle of comparable length (Hayssen 1993) and a well-developed young is born after significant development has occurred in utero (deMello and Reid 2000; Huang et al. 2009; Otani et al. 1993). This foetal development is regulated by the amniotic fluid and placenta (Jansson and Powell 2007; Mulvihill et al. 1985). Further postnatal development is regulated by the mother’s milk, although it retains a relatively constant composition apart from the initial colostrum (Langer 2009). Thus, it can be assumed that the developmental events occurring in marsupials during the early phase of lactation may be equivalent to the in utero embryonic stage in eutherians like the human. Given this scenario, it is presumed that the developmental signals provided by the human amniotic fluid and placenta may be comparable with those provided by early phase milk in marsupials. To investigate this, we compared the genes coding for candidate bioactives expressed in tammar wallaby mammary gland during early phase of lactation with those present in human amniotic fluid and placenta.
The Tammar wallaby, one of the most-studied marsupials, has a refined lactation strategy in which the mother regulates milk composition and rate of milk production in turn determining the rate of growth of the pouch young (Trott et al. 2003). This involves two temporally different patterns of milk protein gene expression during the lactation cycle; one group which is expressed throughout lactation and the other group which is expressed in a phase-dependent manner, both to meet the timely needs of either the mother or the developing pouch young (Nicholas et al. 1997). There is evidence to suggest that tammar wallaby milk of specific composition has a role in regulating development of different organs of the
189 pouch young such as the stomach (Kwek et al. 2009b; Waite et al. 2005). Moreover, recent reports have shown that specific milk proteins like CAMP (Wanyonyi et al. 2011) and WFDC2 (Watt et al. 2012) are differentially expressed in a phase dependent manner to meet the requirements of the developing altricial young. The study by Wanyonyi and co-workers (2011) showed a temporal regulation for the tammar milk antimicrobial protein CAMP where alternate splicing was demonstrated for MaeuCath1 in a lactation-phase specific manner. This temporal regulation provides protection for both the pouch young and mother’s mammary gland at the time of increased risk of infection in addition to an increased proliferation of the mammary gland during mid-lactation when increased growth of the mammary gland is observed (Wanyonyi et al. 2011). Similarly, an antibacterial tammar milk protein WFDC2 showed a lactation phase-dependent expression pattern which suggested a role to either reduce mastitis in the mother’s mammary gland or protect the gut of pouch young when it is not immune-competent (Watt et al. 2012). Additional secreted proteins that showed a differential secretion across tammar wallaby lactation and demonstrated bioactivity in cell based assays for immune modulation, inflammatory responses, proliferative and differentiative effects were also identified (Khalil 2007; Sharp et al. 2009). Furthermore, identification of many unknown proteins in the milk of tammar wallaby during the early phase of lactation by Joss and co-workers (2007) may also support the hypothesis of milk having role in early development of the pouch young.
5.4.1 Gene transcriptome profiling to identify putative bioactives in tammar wallaby phase 2A milk
To investigate the putative protein bioactives secreted in early phase milk, we employed two approaches, a microarray based approach and RNA-seq. Initially, the previously identified 326 differentially expressed genes of the tammar wallaby mammary gland that were predicted to code for secreted proteins (Khalil 2007) were examined. The current study focused on the 23 secreted proteins which are highly expressed during early lactation (phase 2A). For comparison, the genes or proteins expressed in the human amniotic fluid (Cho et al. 2007; Crettaz et al. 2007) and
190 placenta (Mushahary et al. 2013; Sood et al. 2006) were retrieved. It was observed that ~56% (13 out of 23) of the secreted proteins of early phase tammar wallaby milk identified by microarray based transcriptome profiling were present in either human amniotic fluid and/or placenta.
Further, RNA-seq data which allows gene transcriptome analysis at a far higher resolution was analysed (Nagalakshmi et al. 2010). Earlier studies examining gene expression profile in the mammary gland (Khalil 2007) of the tammar wallaby have used a microarray based approach. The current study analysing the tammar wallaby mammary gland is the first deep-sequencing approach employed in a marsupial species. To address the limitation of gene annotation in this species, the RNA-seq reads were aligned to both the M. eugenii genome and the M. domestica genome. Aligning the data to the M. eugenii genome showed the expression profile of the major milk protein genes like CSN2, BLG and A-LAC were expressed throughout lactation whereas ELP, WAP, LLP-A and LLP-B genes were expressed at specific phases (Brennan et al. 2007; Simpson et al. 1998b). However, there were minor differences in expression pattern of these genes when analysed by RNA-seq and compared to earlier reports. For example, ELP is detected from early days of phase 2A in the current study whereas in the previous report expression was demonstrated in the mammary gland from later phase 2A (Simpson et al. 1998b). Phase 2B specific milk protein gene WAP showed expression in the mammary gland collected from three stages of phase 2B lactation in the current study. This supports the earlier SDS and northern blot analysis by Simpson and co-workers (2000) who reported the phase 2B expression of WAP. Late lactation protein-A (LLP-A), phase 3 specific milk protein gene, showed an increased level of expression in the phase 3 mammary gland samples. This was demonstrated earlier by Nicholas and co-workers (1987) by SDS/polyacrylamide-gel electrophoresis where LLP-A expression was observed in milk from 26 weeks onwards (182 days) with the secretion increasing and reaching higher levels by 36 weeks (252 days). The second major milk protein gene specific to phase 3, LLP-B, was also detected at higher levels in the three mammary gland samples of phase 3 in the current study. This is in agreement with the earlier report, where northern blot analysis of LLP-B gene expression was
191 detected in the mammary gland after 200 days of lactation (Trott et al. 2002). Therefore, there is a good correlation between the earlier studies reporting expression of major milk protein genes during the tammar wallaby lactation cycle and the profiles observed in the current RNA-seq analysis.
Subsequently the data was aligned to the M. domestica genome which had better gene annotation. This dataset had a total of 253,929 probes of which 17,775 had an FPKM of more than 1.0. This is in contrast to the 14,837 express sequence tags identified by the earlier microarray dataset (Khalil 2007; Lefèvre et al. 2007). The low number in the microarray approach revealed the limitation of shallow library sequencing and identified only the transcripts with high abundance in the mammary gland (Lefèvre et al. 2007). This justifies the major benefit of the deep sequencing approach which covers genome profiling at a higher resolution (Nagalakshmi et al. 2010). Moreover, in contrast to the microarray based transcriptome profiling which is limited to the probes on the array, RNA-seq analysis considers all the genes expressed in the mammary gland (Febbo and Kantoff 2006). However, for detailed investigation, the current study focused only on the genes with FPKM value greater than 1000 for at least one of the 21 samples of the dataset. This approach assisted in examining the major genes expressed in the mammary gland during the lactation cycle, and identifying the expression pattern of these genes permitted grouping them into different classes such as genes which are phase specific and genes which are expressed throughout lactation. Since the main focus of the current study was to identify the putative bioactives of tammar wallaby phase 2A milk, the genes expressed specifically in phase 2B and 3 have not been discussed in detail. Genes could be grouped into different classes; genes which showed higher expression during early phase 2A, late phase 2A and those with same expression across phase 2A. This is consistent with the requirement of phase 2A milk to play a significant role in regulating the early development of tammar pouch young and also in programming the mother’s mammary gland for the complex process of lactation (Nicholas et al. 1997). The increased information available as a result of the current transcriptome profiling of the tammar wallaby mammary gland was achieved because of the precise measurement of levels of transcripts possible by RNA-seq
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(Nagalakshmi et al. 2010; Wang et al. 2009) and due to better coverage of mammary tissues of phase 2A lactation in the current RNA-seq analysis when compared to the earlier study by Khalil (2007).
Furthermore, by analysing the 199 highly expressed genes in the phase 2A dataset, 28 genes were predicted to code for secreted proteins. This is in contrast to the 23 secreted proteins of phase 2A identified in total by microarray based analysis. This clearly indicates the advantage of RNA-seq having better coverage when compared to microarray based techniques (Nagalakshmi et al. 2010). In comparison with the human placenta and amniotic fluid genes or proteins (Cho et al. 2007; Crettaz et al. 2007; Mushahary et al. 2013; Sood et al. 2006), it was observed that ~75% of the proteins (21 out of 28) secreted specifically in the early phase tammar wallaby milk identified by RNA-seq based transcriptome profiling were present in either human amniotic fluid and/or placenta. This is higher than what was observed utilising the microarray data wherein 56% of the proteins secreted specifically in the early phase tammar wallaby milk was found common to either human amniotic fluid and/or placenta. It can be emphasised that the RNA-seq dataset generated in the current study would have higher confidence especially for studies to understand phase 2A profiles as the experiment covered 12 samples of phase 2A in contrast to the 3 samples analysed in the earlier microarray based profiling (Khalil 2007). Moreover, RNA-seq detects transcripts at a large dynamic range of expression levels in contrast to the microarrays which lack sensitivity for genes expressed either at low or very high levels and therefore have a much smaller dynamic range (Wang et al. 2009). This may partially explain why there was not much similarity in the list of genes encoding for secreted proteins identified by the two different approaches.
In brief, this study is the first transcriptome profiling of a marsupial mammary gland using RNA-seq approach and the first evidence to demonstrate that putative bioactives of early phase tammar wallaby milk are also present in either human amniotic fluid or placenta. The putative candidates identified from both microarray and RNA-seq datasets are discussed below with a focus on a potential role in gut development. They have been grouped into two major categories; (a)
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Putative bioactive proteins with plausible role in regulating gut development and (b) Putative bioactive proteins with no known role in regulating gut development.
5.4.2 Putative bioactive proteins with plausible role in regulating gut development
S-adenosylhomocysteine hydrolase
S-adenosylhomocysteine hydrolase (SAHH) is an enzyme that regulates the level of S-adenosylhomocysteine which is an end product and strong physiological inhibitor of S-adenosyl-methionine-mediated transmethylation reactions (Cantoni et al. 1979). This enzyme is required for myc-induced cap methylation which in turn helps in promoting the cell proliferative property of the myc gene (Fernandez- Sanchez et al. 2009). It’s reported that, for stimulating epithelial cell renewal in the gut, c-myc expression is important which is mediated by an activation of Wnt3a signaling (Liu et al. 2012a). One probability for the increased expression of SAHH in milk may be to support this c-myc induced epithelial proliferation in the pouch young’s gut. However to confirm this hypothesis, it may be required to examine the effect of SAHH on an in vitro gastric epithelial cell model and determine the regulation over c-myc and related genes involved in proliferation. Moreover, a nuclear accumulation of this particular protein has been demonstrated in transcriptionally active cells during development of Xenopus laevis (Radomski et al. 1999).
Chromosome 1 Open Reading Frame 160
C1orf160, the transmembrane protein 222 showed increased level of expression during phase 2A lactation. Earlier Khalil (2007) demonstrated that this protein stimulated cell differentiation by inducing loss of pluripotency in embryonic mouse stem cells. This property associated with an increased secretion during early phase lactation may be consistent with a role in tissue and organ differentiation of the pouch young which undergoes major development at this stage (Setiati 1986; Waite et al. 2005). One possibility is that it may influence the differentiation of stem
194 cells in the stomach of tammar pouch young which is the first organ with which milk interacts. Moreover, in Chapter 3 we have demonstrated that phase 2A tammar wallaby milk had an effect on stomach differentiation in both in vitro models which to some extend may be attributed to the increased expression of C1orf160. To address and confirm this hypothesis, the preliminary approach would be to directly examine the effect of purified C1orf160 protein on in vitro stomach models; embryonic stomach explant model and gastric organoid model that has been developed and demonstrated in Chapter 3.
Periostin
Periostin (POSTN) is a secreted ECM protein that can interact with other (&0SURWHLQVDQGLQWHJULQVDQGLVLQGXFHGE\WUDQVIRUPLQJJURZWKIDFWRU7*)ȕDQG BMPs in tissues undergoing remodeling or active stress (Conway et al. 2011). Its expression has been demonstrated in the amnion of developing embryos, bone, heart (Conway et al. 2011; Dobreva et al. 2012; Rios et al. 2005) and also reported to localize to the endocardial cushions suggestive of a role in valvulogenesis and valvular disease (Li et al. 2010a). In general, POSTN influences cell behaviour as well as collagen fibrillogenesis, and therefore exerts control over the structural and functional properties of connective tissues in both health and disease (Hamilton 2008).
While considering the development of gut in the altricial young, among the different factors regulating the process, components of extracellular matrix play a crucial role in morphogenesis and epithelial cell differentiation (Tømmerås et al. 2000; Tremblay and Ménard 1996 ). It was demonstrated that as the gastric epithelia develops from a multilayer cell towards a single cell layer forming glandular structures, ECM proteins like collagen show increased expression (Tømmerås et al. 2000). It is possible that POSTN secreted via the mother’s milk may either directly act on the gastric epithelium or interact with the ECM of the immature gut of tammar pouch young thus regulating early phase of gut development involving differentiation of glandular structures (Setiati 1986; Waite 2003).
195
DnaJ (Hsp40) homolog, subfamily C, member 13
DnaJ, the heat shock proteins in general are involved in the folding of proteins that regulate different cellular processes (Puvirajesinghe et al. 2012). They also regulate the expression of different other heat shock proteins (Straus et al. 1990) and provide protection from pathogen infection (Cui et al. 2011). One direct role for this protein may be to provide pathogen protection to the immune naïve tammar young. Moreover, heat shock proteins play a role in regulating stress response in different organs such as the gut (Ren et al. 2001). It is possible that DnaJ may regulate the stress responses in the immature gut of tammar pouch young which is prone to infection at this early phase of development.
Tubulointerstitial nephritis antigen-like 1
The secretory protein Tubulointerstitial nephritis antigen-like 1 (TINAGL1) is a novel matricellular protein that interacts with both structural matrix proteins and cell surface receptors (Li et al. 2007). It interacts with laminin 1 in the extra embryonic tissue and plays a physical and physiological supportive role in embryo development at post implantation (Igarashi et al. 2009). It has been demonstrated earlier that in the stomach a synergism exists between growth factors and ECM proteins like laminin which regulates the cell polarity and gastric glandular epithelium (Basque et al. 2002). Moreover, gastric basement membrane components like laminin that show a differential expression may play a role in regulating gastric developmental functions like repair (Virtanen et al. 1995). It can be predicted that the secreted protein TINAGL1 may interact with the laminin of pouch young’s stomach during early immature phase thus regulating stomach epithelial polarity and glandular development.
A disintegrin and metalloproteinase with thrombospondin motifs 10
A disintegrin and metalloproteinase with thrombospondin motifs 10 (ADAMTS10) belongs to ADAMTS family of zinc-dependent proteases. ADAMTS10 has been reported to interact with Fibrillin 1 (Kutz et al. 2011), a major component of the ECM and of the microfibril (Sakai et al. 1986). These microfibrils play a
196 structural role in maintaining the ocular lens in its appropriate position in the eye via the zonule of Zinn (the suspensory ligament of the lens), which comprises primarily fibrillin-1 microfibrils (Cain et al. 2006; Ramirez et al. 2004). It is possible that this secreted protein may assist in eye development of the altricial young of the tammar wallaby. A temporo-spatial pattern of distribution of Fibrillin 1 has been reported in different organs emphasising a role in the developmental process (Quondamatteo et al. 2002). Therefore ADAMTS10, having a role in interacting with Fibrillin 1, may regulate tissue remodelling of the developing organs such as the gut of the tammar pouch young.
Clusterin precursor
The glycoprotein Clusterin (CLU) that matures by limited proteolysis forms lipoprotein complexes and is known to be secreted by a number of cell types (Jenne and Tschopp 1992; Wilson and Easterbrook-Smith 2000). It has different physiological roles such as immune defense, tissue remodelling and transporting biologically active peptides (Jenne and Tschopp 1992). It has been identified as an important mediator of neuronal survival and differentiation (Cordero-Llana et al. 2011). This protein has no effect on cell proliferation but decreases apoptotic nuclei and causes a sustained increase in phosphorylated extracellular signal-regulated kinase thus playing a role in cell survival and differentiation (Cordero-Llana et al. 2011). Based on these properties, it can be hypothesised that CLU may assist in tissue remodelling or differentiation of the developing gut or protect the altricial young from infection.
Ephrin type-B receptor 6 precursor
Eph receptors represent the largest subfamily of receptor tyrosine kinases. In general, they are important in embryonic development and in physiological and pathophysiological conditions in adults (Luo et al. 2012). Most reported functions of Ephs occur in the central nervous system (O'Leary and Wilkinson 1999). They are essential in the development of neuronal connections, circuit plasticity, and repair
197
(O'Leary and Wilkinson 1999). Ephs particularly their B family members are expressed in thymocytes and T cells thus regulating T-cell responses and survival (Wu and Luo 2005). In addition, it has been shown that Ephs are involved in intestinal epithelium self-renewal. The EphB receptors are Wnt signaling target genes that control cell compartmentalization along the crypt axis (Clevers and Batlle 2006). Moreover, ȕ-Catenin and TCF signaling in the small intestine determines cell positioning in the intestinal crypt by controlling the EphB genes (Batlle et al. 2002). Therefore, in the current scenario, secreted EPHB6 in the tammar wallaby phase 2A milk may regulate intestinal development and is a high priority for further investigation.
Insulin-like growth factor-binding protein 7 precursor
Insulin-like growth factor-binding protein 7 precursor (IGFBP7) belongs to IGFBP super family, but unlike other members it exhibits low affinity with IGF. It binds to the unoccupied IGF1R, blocks its activation by insulin growth factors and suppresses downstream signalling, thereby inhibiting protein synthesis, cell growth, and survival (Evdokimova et al. 2012). Recombinant IGFBP7 was found to inhibit keratinocyte (KC) proliferation and enhanced their apoptosis thus acting as a regulator of KC proliferation and differentiation (Nousbeck et al. 2010). Similarly, inhibiting proliferation of gastric cells of the tammar pouch young may play a role in regulating differentiation and development. IGFBP7 has also been demonstrated to modulate estrogen induced trophoblast proliferation (Liu et al. 2012b). During pregnancy and immediately after implantation, a series of cell transformation and differentiation steps occurs, such as the transformation of embryonic stem cells into decidual cells for uterine receptivity in mouse (Liu et al. 2012c). Based on this effect on embryonic stem cells, it’s worth investigating whether IGFBP7 has any role on differentiation of the gastric stem cells of the altricial tammar young thus regulating gut development.
198
Laminin subunit alpha-2 precursor
Laminin subunit alpha-2 precursor (LAMA2) belongs to a family of extracellular heterotrimeric glycoproteins that contain alpha, beta and gamma chains. During development, different cell types express different types of laminins in a specific and temporal pattern (Miner 2008). Laminins impart special structural and signalling properties to the basement membrane (Miner 2008). Binding to cells via a high affinity receptor, laminin is thought to mediate the attachment, migration and organization of cells into tissues during embryonic development by interacting with other extracellular matrix components (Miner 2008). It was demonstrated that secreted laminin gets exposed to cell surfaces and plays a crucial role in organizing extracellular matrix, receptor and intracellular proteins at those surfaces (Huang et al. 2003). This property of laminins may plausibly regulate development of immature tissues such as the gut by interacting with the extracellular matrix components.
Protein sel-1 homolog 1 precursor
The role of Protein sel-1 homolog 1 precursor (SEL1L1) in regulating development of vertebrates is not well established. However, experiments have shown it is essential for embryonic survival and the protein impaired protein secretion in mammals (Bernasconi et al. 2012; Francisco et al. 2010). Based on these results, it was concluded that SEL1L plays an important role in regulating embryonic development (Francisco et al. 2010). In addition, it was shown that SEL1L is essential for the growth and differentiation of endoderm-derived pancreatic epithelial cells during mouse embryonic development (Li et al. 2010b). SEL1L acts as a negative regulator for Notch signalling while regulating pancreatic organogenesis (Li et al. 2010b) and Notch signalling regulates glandular differentiation in stomach of vertebrates (Matsuda et al. 2005). Based on this it can be assumed that SEL1L secreted in tammar wallaby milk may play role in regulating Notch signalling controlling stomach epithelial differentiation of pouch young.
199
Metalloproteinase inhibitor 3 precursor
ECM is a component that provides mechanical support to cells and regulates signals reaching the cell that govern cell localization, differentiation, proliferation, and apoptosis (Iozzo 1998). Among the different tissue inhibitors of metalloproteinases, metalloproteinase inhibitor 3 precursor (TIMP-3) is distinguished for its ability to bind to ECM (Yu et al. 2000) and is implicated in the cellular regulation of activity of matrix metalloproteinases for remodelling of ECM during development (Birkedal-Hansen 1995). In addition, TIMP-3 has been reported to be expressed in different fetal cells and also in cells of an adult (Apte et al. 1994). TIMP-3 has been demonstrated to have role in remodelling processes of the extracellular matrix and in human fetal development (Airola et al. 1998). Based on these reports, it can be assumed that TIMP-3 may bind to the ECM of the altricial young gut thus regulating the development and differentiation.
Tenascin precursor
Tenascin (TNC) is a glycoprotein of the ECM produced by both epithelial and myoepithelial cells (Chiquet-Ehrismann et al. 1986; Orend and Chiquet- Ehrismann 2006). During embryogenesis, this protein is expressed in areas of tissue remodelling and regulates proliferation and differentiation (Besser et al. 2012; Ishii et al. 2008). It is also known to control immune response against infection by regulation of TLR-mediated inflammation (Piccinini and Midwood 2012). Considering these properties of TNC, it is possible that the immune regulatory mechanism of the protein may help protect the developing gut of the altricial young from infection.
200
5.4.3 Putative bioactive proteins with no known role in regulating gut development
Calmin
Calmin (CLMN) is a retinoic acid-responsive gene which has a role in promoting cell cycle exit and neuronal differentiation (Marzinke and Clagett-Dame 2012). Its expression is observed early during embryonic development in different regions of the brain (Marzinke et al. 2010). In the tammar wallaby, the spinal cord is immature at birth and gradually develops a mature appearance by 7 weeks of age (Harrison and Porter 1992; Ho and Stirling 1998). Similarly, the tammar brain is also immature at birth and develops during the extended lactation cycle (Renfree et al. 1982) with developmental events including the neocortical differentiation occurring during phase 2A (Reynolds et al. 1985). Thus an increased expression level of Calmin milk protein might possibly support the early functional development of the neuronal system including brain and spinal cord that occurs postnatally in the tammar pouch young (Harrison and Porter 1992; Ho and Stirling 1998; Renfree et al. 1982; Reynolds et al. 1985). However, limited research has been done to investigate the mechanisms of action of this protein in regulating neuronal differentiation and its relevance in gut development remains to be investigated. Interestingly, in gray short- tailed opossum it was reported that immature spinal cord of younger pups has greater capacity for repair after spinal injury and can develop considerable functionality by adulthood whereas such a recovery is limited in older pups (Noor et al. 2011; Wheaton et al. 2011). Therefore, a Calmin protein secreted in the early milk of opossum, another marsupial, may assist the recovery from spinal injury in younger pups.
GM2 ganglioside activator
The GM2 ganglioside activator (GM2A) is a protein that is necessary for the QRUPDO IXQFWLRQLQJ RI WKH HQ]\PH ȕ-hexosaminidase-A (Meier et al. 1991) which assists the degradation of GM2 ganglioside. During the course of brain development, accumulation of GM2 ganglioside (Ngamukote et al. 2007) may lead to neuronal cell
201 death (Huang et al. 1997). The GM2 activator protein helps the degradation of the GM2 ganglioside and further supports normal brain functioning (Beccari et al. 1994). This protein also has a role in intercalated cells regulating kidney functioning (Mundel 2001; Mundel et al. 1999). It may also regulate brain development which occurs during early phases in the tammar pouch young (Renfree et al. 1982). However, this can be confirmed only after an investigation to suggest that proteins such as the GM2 ganglioside activator secreted in tammar phase 2A milk crosses the gut barrier of the altricial young.
Jumonji AT rich interactive domain 1B
Jumonji AT rich interactive domain 1B (JARID1B) also known as Lysine- specific demethylase 5B is mainly expressed in the mammary gland promoting cell proliferation (Catchpole et al. 2011). An increased expression of this protein was demonstrated in epiblast and the outer part of the ectoplacental cone during early embryonic development in mouse (Frankenberg et al. 2007). It plays a developmental role in cell fate decisions through its ability to directly regulate genes that control cell cycle, cell differentiation, and cell lineage during embryoid body formation and neural differentiation, thus increasing the proliferating progenitors and reducing terminally differentiated cells in embryonic stem cells (Dey et al. 2008). Moreover, this protein is reported to mediate a transcriptional control during ESC differentiation (Schmitz et al. 2011). Based on these previous reports, JARID1B can be predicted to play a role in cell differentiation regulating development of different organs in the pouch young. However similar to C1orf160, to address the role in stomach development, it would be reasonable to examine whether JARID1B determines cell fate during stem cell differentiation in stomach using in vitro organoid or explant models.
Sterol carrier protein-2
Sterol carrier protein-2 (SCP2) plays an important role in cholesterol, fatty acid and phospholipid trafficking and metabolism in mammalian cells (Gallegos et al. 2001; Schroeder et al. 2001). The significance for a high expression level of this
202 protein is not well understood. However, its role as a trafficking protein may assist at the gut barrier of the developing pouch young.
Aldehyde oxidase 1 and Guanine nucleotide binding protein (G protein), beta polypeptide 2
Aldehyde oxidases (AOX) are structurally conserved proteins belonging to the family of molybdoflavoenzymes (Garattini et al. 2003). This enzyme showed a developmentally regulated expression in the liver (Ventura and Dachtler 1980) and was also reported to interact with ABC transporter-1 thus regulating the hepatocyte function (Sigruener et al. 2007). However, any milk protein to have a regulatory role on organs such as the liver would need to cross the gut. Guanine nucleotide-binding proteins (G proteins) in general are involved as a modulator or transducer in various transmembrane signaling systems (Gene cards). They have alpha, beta and gamma subunits and are involved in integrating signals between receptors and effector proteins. Detailed investigation is required to understand the significance of both these proteins for the developing pouch young.
Cysteine-rich protein 1 Cysteine-rich protein 1 (CRIP1) is a small LIM-domain containing protein LQYROYHG LQ VPRRWK PXVFOH GLIIHUHQWLDWLRQ DQG UHJXODWHG E\ 7*)ȕ (Järvinen et al. 2012). It’s also known to play a role in immune cell activation or differentiation or in processes associated with cellular repair (Hallquist et al. 1996). Based on the property to regulate smooth muscle differentiation and regulate cell repair, it can be assumed that CRIP1 may regulate gut development in the altricial young. CRIP1 also confers metal-binding properties that are important for zinc transport and/or functions of this micronutrient (Hempe and Cousins 1991). Zinc is important for the formation of biomembranes and zinc finger motifs found in DNA transcription factors and has catalytic function in metalloenzymes that may regulate growth and development (Carol and Semrad 1999).
203
Decorin precursor Decorin (DCN) is a small leucine-rich proteoglycan (SLRP) involved in VHYHUDOLPSRUWDQWELRORJLFDOSURFHVVHVLQFOXGLQJFROODJHQ¿EULOORJHQHVLVDQGPDWUL[ assembly (Goldoni and Iozzo 2008; Goldoni et al. 2008). It regulates assembly of collagen fibrils and acquisition of biomechanical properties during tendon development (Zhang et al. 2006). This study indicates that DCN is a key regulatory molecule and the temporal switch from biglycan to decorin is an important event in the coordinate regulation of fibrillogenesis and tendon development. However, it needs to be investigated whether this regulation of fibrillogenes and matrix assembly is involved in development of other organs of the altricial young of tammar wallaby such as the gut which matures during the post natal period.
Serine/arginine repetitive matrix protein 2
There are not many reports suggesting a role of Serine/arginine repetitive matrix protein 2 (SRRM2) in regulating developmental processes in mammals. However, it is a component of the splicesome and may influence several steps of gene expression. In addition, an orthologue of SRRM2 was shown as a multifunctional protein whose role in transcription influenced C. elegans development (Fontrodona et al. 2013).
Thrombospondin-3 precursor
Thrombospondins mainly TSP-1 and TSP-2 are regulators of angiogenesis (Bornstein 2009). However, TSP-3 lacks this property. However, there is evidence suggesting TSP-3 has a role in regulating skeletal maturation in mice (Hankenson et al. 2005).
Other bioactives In addition to the candidate bioactives of tammar wallaby phase 2A milk discussed above, there are other proteins such as the T-cell immunomodulatory protein precursor, Lethal(3)malignant brain tumor-like protein, Nucleolar
204 phosphoprotein p130, Major facilitator superfamily domain-containing protein 10, Tetratricopeptide repeat protein 17, Proline-rich AKT1 substrate 1, Alveolar soft part sarcoma chromosomal region candidate gene 1 protein, Peptidyl-glycine alpha- amidating monooxygenase precursor and Prostaglandin-H2 D-isomerase precursor which have been identified by RNA-seq in the current study which have not been discussed in detail. Other candidates of phase 2A tammar wallaby milk identified by microarray based approach, like C7orf30 and C9orf90, were present in human amniotic fluid and placenta. The clipped sequence of these genes were retrieved from the database and blasted in NCBI. However, the function of these proteins is not well characterised. Nevertheless, the high level of expression during phase 2A may signify some role which needs to be elucidated.
5.4.4 Conclusion
In the current study we have examined the secreted proteins that are highly expressed during phase 2A tammar wallaby lactation and compared then with the genes/proteins expressed in human amniotic fluid and placenta. Subsequently, candidates were identified (listed or discussed above) that might be of significance in regulating the development of the young, including stomach development. However, detailed investigation examining the effect of individual candidates on in vitro models will demonstrate their bioactivity and help to correlate with the developmental needs of the tammar pouch young. This can be initiated by over expressing these putative bioactive proteins in cell lines such as HEK-293 (Bisana et al. 2013) and examining the effect of specific protein conditioned media on established in vitro stomach models including embryonic stomach explant culture model and gastric organoid model. The earlier study by Khalil (2007) had screened a diverse range of tammar milk proteins for bioactivity using a variety of eutherian cell models proving the conservation of marsupial and eutherian proteins for screening of the candidates for bioactivity. However, the annotation utilised in the current study to identify phase 2A mammary gland genes predicted to code for secreted proteins does not necessarily indicate that these proteins will demonstrate specific bioactivity
205 in a range of bioassays and possible novel roles may be identified. Recently, experiments in our lab have observed a role for tammar wallaby milk proteins of phase 2A in regulating branching development of the lung of the altricial young (Modepalli and Nicholas, Unpublished). Therefore, in addition to examining the role in regulating gut development, the candidate bioactives may regulate development of diverse organs such as the lung, kidney and mammary gland. Furthermore, the major question to be addressed would be whether these candidate putative bioactives will cross the gut of the altricial young. An additional question is whether the bioactivity demonstrated by the whole protein is also present in digested peptides after being hydrolysed by gut borne-proteases of the tammar pouch young. To conclude, the present study increases the likelihood of defining the role of proteins provided by human amniotic fluid or placenta during fetal development by utilising tammar wallaby as an improved model where milk acts as the source of these developmental signals.
206
CHAPTER 6
General Discussion
207
The adapted reproductive strategy of marsupials such as the tammar wallaby (Macropus eugenii) is comprised of a short gestation cycle followed by a long lactation with varying milk composition and production (Sharp et al. 2009). This provides a unique opportunity to understand the role of different milk components in supporting either the development of the young or functioning of the mammary gland. However, regulation of mammary gland function for the sophisticated lactation program in marsupials has limited understanding and may be different to the eutherians. Therefore, the current study initially focused on understanding the regulation of mammary gland function in the tammar wallaby. Despite earlier reports on the role of tammar wallaby milk on stomach development, the current study subsequently investigated the effect of early and mid-phase milk on stomach development using in vitro models. In addition, we examined stomach development in an alternative marsupial model, the gray short-tailed opossum (Monodelphis domestica). This marsupial is an omnivore and so this study helped to correlate the progressive events of stomach development with varying milk composition to propose a putative role for milk in stomach development in this species. Moreover, transcriptome profiling of mammary tissue collected across the lactation cycle used microarray and deep sequencing technology to identify secreted proteins that might have bioactivity. Genes coding for secreted proteins that were highly expressed during the early lactation phase of tammar wallaby were compared with genes/proteins expressed in human amniotic fluid and placenta to identify human orthologues of amniotic fluid and placental proteins. Using this approach, the relevance of marsupials like the tammar wallaby with extreme adaption to reproduction has been increasingly proven as a unique model for research. This approach helps in understanding the genetic regulation of diverse physiological processes by correlating the time of secretion of different milk proteins with potential roles in either regulating mammary gland function or development of the young.
208
6.1 Regulation of mammary gland function in tammar wallaby
In order to examine the regulation of mammary gland function in the tammar wallaby, the current project utilised a comparative approach in understanding the role of the caveolin group of genes in marsupials and eutherians. In mouse, where milk composition and production does not change significantly across lactation, expression of caveolin genes was correlated to the process of lactation. During peak lactation, expression of Caveolin 1 and 2 genes were down regulated and this process was reported to be regulated by prolactin (Park et al. 2002; Park et al. 2001). Conversely, Caveolin 1 and 2 genes were shown to be regulated by a complex interplay of lactogenic hormones including insulin, cortisol and prolactin when examined in mouse mammary explants in the present study. This is similar to the endocrine regulation of major milk protein genes in mouse (Juergens et al. 1965; Ono and Oka 1980a, 1980b; Turkington et al. 1965). However, both earlier (Hue- Beauvais et al. 2007) and current studies showed that mouse mammary epithelial cells lacked expression of the Caveolin 1 gene whilst it was localised to myoepithelial cells and adipocytes. This provides interesting evidence for cell-cell interaction leading to a paracrine control by caveolins over the process of milk secretion by mammary epithelial cells. However, to confirm this, a detailed investigation is critical involving co-culturing of Caveolin 1 transfected myoepithelial cells and adipocytes with epithelial cells to examine the effect on milk protein gene expression. To address this hypothesis, the primary requirement is the availability of pure mammary epithelial cells, myoepithelial cells and adipocytes. An approach using fluorescent sorting of cells demonstrated previously by Smalley and co-workers (1998) can be utilised to separate epithelial and myoepithelial cell types from mouse mammary gland. An alternative approach could utilise mammary epithelial (Zavizion et al. 1996a) and myoepithelial cell lines (Zavizion et al. 1996b) already developed and established from bovine mammary gland. Similarly, adipocytes of the mammary gland also need to be isolated possibly utilising an approach reported by Quirk and co-workers (1990) for rat mammary gland. Further, the myoepithelial cells and the adipocytes need to be transfected with the Caveolin 1
209 gene and co-cultured with mammary epithelial cells. This will address how the Caveolin 1 transfected myoepithelial cells and adipocytes regulate the process of milk protein synthesis by mammary epithelial cells. Earlier, Levine and Stockdale (1985) had utilised a similar co-culturing approach wherein mammary epithelial cells from mid-pregnant mice were plated on 3T3-L1 cells, a sub-clone of the Swiss 3T3 cell line that differentiates into adipocytes in monolayer culture. Milk protein casein was synthesised by mammary epithelium in the presence of co-cultured cells and lactogenic hormones (I, F and P). This co-culturing study showed that the cell-cell interaction between mammary epithelium and other specific non-epithelial cells influenced the acquisition of hormone sensitivity of the epithelium and hormone- dependent differentiation (Levine and Stockdale 1985). In the current scenario, mammary epithelial cells can be co-cultured with normal or Caveolin 1 over- expressing myoepithelial cells and adipocytes, and examined for milk protein gene expression in the presence of lactogenic hormones. This will address how Caveolin 1 localised in the myoepithelial cells and adipocytes influences acquisition of hormone sensitivity of the mammary epithelial cells and regulates milk protein synthesis.
Further, we utilised the unique marsupial model tammar wallaby to investigate the regulation of the caveolin group of genes and to examine whether they play a potential role in regulating mammary gland function in marsupials. Firstly, the gene structural organisation and protein sequence of Caveolin 1 and 2 of both mouse and tammar wallaby showed high similarity. With high homology (70- 80%) observed between the two species, it could be hypothesised that the negative correlation of caveolin gene expression with lactation observed in mice would be similar in tammar wallaby. At phase 3 of lactation in the tammar wallaby when milk production is at its maximum (Cork and Dove 1989; Dove and Cork 1989; Trott et al. 2003), down regulation in the expression of Caveolin 1 and 2 genes was expected. In contrast, both genes showed significantly higher expression level during lactation when compared to pregnancy and involution. This pattern of expression necessitated an investigation to examine whether tammar caveolins are under the control of lactogenic hormones; insulin, cortisol and prolactin. For this purpose, explants from pregnant tammar mammary gland were cultured in the presence of lactogenic
210 hormones to iQGXFH KLJK H[SUHVVLRQ OHYHO RI ȕ-lactoglobulin milk protein gene (Nicholas et al. 1991). The three lactogenic hormones showed no effect in regulating the expression of tammar caveolins and the significance of an increased expression of caveolins during lactation in the tammar remains unknown. However, the role of other factors like extracellular matrix has not been investigated in the current study. An in vitro study by Wanyonyi and co-workers (2013a) showed the role of ECM in tammar wallaby lactation was regulating the switch in phenotype of mammary epithelial cells from phase 2A to 2B and finally to phase 3 that correlated with changes in composition of ECM. It was demonstrated that when wallaby mammary epithelial cells extracted from phase 2B were cultured on phase 3 ECM, the cells acquired phase 3 phenotype. Similarly, phase 2A cells acquired phase 2B phenotype when cultured on phase 2B ECM. Moreover, the differential expressions of specific milk protein genes like cathelicidin were regulated by ECM (Wanyonyi et al. 2013b). A splice variant of the cathelicidins, MaeuCath1a was highly expressed on phase 2A ECM and poorly expressed on phase 2B ECM (Wanyonyi et al. 2011). However, when phase 2B cells were cultured on phase 2A ECM; there was no expression of MaeuCath1a. Similarly, phase 2A cells retained their phenotype when they were cultured on phase 1 ECM. This inferred that ECM regulates cyto- differentiation of mammary epithelial cells in the tammar wallaby, however in a directional manner wherein cells which have acquired a later phase phenotype cannot be programmed backwards to an earlier phase by ECM (Wanyonyi et al. 2013b). These studies demonstrating the regulation of Cathelicidin gene expression by ECM would emphasise the need to investigate the role of other factors, especially ECM, in regulating caveolin gene expression.
Myoepithelial cells, one of the major components of the mammary gland have been demonstrated to play a major role in milk let down during the process of milk secretion and act as a barrier between milk-producing luminal cells and surrounding stroma (Adriance et al. 2005). However, this particular cell type also SOD\V DGGLWLRQDO VLJQLILFDQW UROHV VXFK DV DFWLQJ DV WKH RQO\ VRXUFH RI ĮFKDLQ RI Laminin-1 in the breast to induce polarity (Gudjonsson et al. 2002) and desmosomal proteins to regulate luminal function (Adriance et al. 2005). Much remains to be
211 learned about the physiological role of these cells in mammary gland function and whether they play a role in imposing a paracrine effect over the synthesis of milk proteins by epithelial cells. Determining the localisation of caveolins in the tammar mammary gland would assist this hypothesis and in addition, determine the function of this particular protein. Due to lack of availability of a suitable antibody against tammar caveolins, the current study did not investigate whether caveolins are localised to myoepithelial cells in the tammar mammary gland. However, the more crucial question to be address is why the caveolin group of genes did not show the same expression pattern in the tammar wallaby as observed in the eutherian mouse.
Earlier, a new function was reported for whey acidic protein (WAP)in marsupials and was correlated with the difference in reproductive strategies (Topcic et al. 2009). WAP, a family of 4-DSC (four disulphide core) domain proteins is secreted in milk of various marsupials including the tammar wallaby where the expression is specific to mid-lactation. In the tammar wallaby, WAP is characterized by the presence of 3 domains (I, II, III) (Simpson et al. 2000) in which domain III is specific to this species and specifically signalled increased proliferation of mammary epithelial cells (MEC) (Topcic et al. 2009). This protein has also been identified in milk throughout lactation in a number of eutherians including mice, rat, (Campbell et al. 1984), rabbit (Devinoy et al. 1998), camel (Beg et al. 1986) and pig (Simpson et al. 1998a). However, there is loss of WAP functionality in human and ruminants like cow, goat and ewe where there is a pseudogene due to a frame shift mutation in ruminants (Hajjoubi et al. 2006) and a coding mutation in human (Rival-Gervier et al. 2003). This loss of domain III in the WAP is correlated with the evolution of marsupials to eutherians and a reproductive strategy that requires minimal changes in mammary function during galactopoiesis. In contrast, the structural organisation of Caveolin genes and the encoded proteins remains conserved in eutherian mouse and marsupial tammar wallaby. Even though the structure is conserved in the tammar wallaby, the increased expression of caveolins in tammar wallaby may not result in a putative response by the mammary epithelial cells similar to mouse, consistent with an adaptation in lactation strategies. Further investigation as discussed above is
212 required to understand the significance of caveolins in mammary gland function in the marsupial tammar wallaby.
6.2 Role of tammar wallaby milk in stomach development
The tammar wallaby pouch young is born altricial and major development occurs postnatally during the extended lactation period (Tyndale-Biscoe and Janssens 1988). A progressive change in milk composition and production (Green et al. 1980; Nicholas et al. 1997; Sharp et al. 2009) may regulate the growth and development of the pouch young (Kwek et al. 2009b; Trott et al. 2003; Waite et al. 2005). At birth, the tammar wallaby pouch young has an immature stomach (Setiati 1986) and major development including formation of gastric glands and appearance of differentiated cell types occurs postnatally (Setiati 1986; Waite et al. 2005; Waite 2003). Previously, employing a cross fostering strategy in tammar wallaby, it was shown that milk resulted in enhanced growth of the pouch young (Trott et al. 2003; Waite et al. 2005) and an exposure to later stage milk accelerated stomach maturation (Kwek et al. 2009a). It was observed that later stage milk resulted in a significant reduction in the parietal cell population and expression of gastric glandular marker genes in the fore-stomach suggesting down regulation of the gastric glandular phenotype in the fostered PY which is a characteristic feature of stomach development in this species (Kwek et al. 2009b). Despite these studies, the role of early and mid-phase tammar wallaby milk has not been explored. In Chapter 3, the effect of tammar wallaby milk was demonstrated on stomach proliferation and differentiation using in vitro stomach models developed from mouse. The stomach explant model developed from embryonic mouse at E14 or 15 days post coitus (dpc) represented an early phase of development with limited differentiated cell types (Karam and Leblond 1993; Karam et al. 1997). In this model, tammar wallaby phase 2B milk showed a proliferative effect on gastric epithelial cells. In addition, examination of phase 2A milk demonstrated that milk collected from day 65 and day 92 lactation resulted in an increased expression of a mucus cell marker and an enteroendocrine marker respectively. Similarly, in the gastric organoid model representing a mini-stomach, day 60 milk resulted in an increased expression of a
213 chief cell marker with a concomitant down regulation of stem cell marker indicating epithelial differentiation (Kemper et al. 2012). This organoid model represents a gastric stem cell population which is capable of differentiating into different cell lineages in the presence of external signals (Barker et al. 2010).
Nevertheless, the discrete factor/s responsible for these effects were not identified in the current study. It is possible that the growth factors present in tammar wallaby milk may be responsible for the proliferative effect. Earlier reports have demonstrated a growth promoting activity on cultured myoblasts by tammar milk- borne EGF and IGF-1 (Ballard et al. 1995). The tammar wallaby milk proteins are composed of two types; a group of proteins expressed throughout the lactation cycle and a second group expressed during specific phases of lactation (Nicholas et al. 1997). However, the current study did not perform a comparison of the effects of milk from all three phases of lactation on stomach development to conclude that the effects are either due to phase specific milk-borne factors or factors that are present across lactation.
A phase-specific effect on proliferation may be attributed to proteins like the tammar whey acidic protein (tWAP) and cathelicidin that demonstrate differential expression across lactation (Simpson et al. 2000; Wanyonyi et al. 2011). Earlier, Topcic and co-workers (2009) had demonstrated that tammar whey acidic protein (tWAP) specific to phase 2B of lactation, stimulated proliferation of primary tammar mammary epithelial cells and mouse mammary epithelial cell line (HC11) whereas there was no effect on human embryonic kidney cells (HEK293) and human gastric AGS cells (Topcic 2010). However, the response may vary between cell lines and primary cells as it is dependent on the origin and nature of cell models (Purup and Nielsen 2012). Tammar cathelicidins demonstrate a phase specific differential splicing and showed proliferative effect on mammary epithelial cells (Wanyonyi et al. 2011). Similar proliferative effect on epithelial cells of the skin (Heilborn et al. 2003), intestine (Otte et al. 2009) and lung (Shaykhiev et al. 2005) has been demonstrated by cathelicidins of other species. Therefore, it is possible that specific
214 milk proteins may influence gastric epithelial proliferation to regulate stomach development.
In addition to the known major milk proteins, 326 genes that showed differential expression across lactation and coded for secreted proteins in tammar wallaby have been identified by a genomics approach (Sharp et al. 2009). The 326 proteins were expressed in vitro by Khalil (2007) and at least 30 proteins had bioactivity in one of the cell based assays for immune modulation, inflammatory response, growth and differentiative effects. This included specific assays like ERK1/2 activation determining regulators of cell proliferation and differentiation; cell differentiation assay determining factors inducing loss of pluripotency of mouse embryonic stem cells and a third assay identifying factors inducing trefoil production by mucin secreting epithelial cells that line the gastrointestinal tract. The secreted proteins that revealed a positive response in these assays may be responsible for the proliferative and differentiative effects of tammar wallaby milk demonstrated in the in vitro stomach models in the current study. This group of genes coding for secreted proteins include Cathelicidin, Emopamil binding protein, C1orf160, Transmembrane protein 163, Interferon induced transmembrane protein 3 and a hypothetical protein MGC14327. In addition, Joss and co-workers (2007) used 2D gel electrophoresis, MALDI MS/MS and chemical derivatisation protocols, to identify more proteins that were expressed during early lactation in tammar wallaby. Analysis of milk across the lactation cycle showed that the level of whey proteins varied according to the developmental stage of the animal, with higher number of proteins expressed during early and late phase (Joss et al. 2009). Analysis of these proteins revealed that the majority were of immunological significance which included Transferrin, ȕ2 microglobulin and Cathelicidin, and the timing of their expression correlated with development and growth of the pouch young. This included Į fetoprotein-like protein and clusterin that were expressed during early phase of lactation, in addition to two unknown proteins. The Į fetoprotein-like protein may support brain development (Dziegielewska et al. 1986) of the altricial young whereas clusterin regulates epithelial differentiation (French et al. 1993). To confirm the hypothesis that these milk proteins play a proliferative or differentiative role, individual genes
215 coding for secreted proteins should be expressed in vitro (Khalil 2007) and examined on stomach models to analyse their bioactivity in inducing gastric epithelial cell proliferation and differentiation.
Another important factor in milk that may regulate stomach development is the recently identified microRNAs (miRNA) which regulate diverse developmental processes by targeting mRNA (Lytle et al. 2007). Recently, studies involving high throughput sequencing have identified large numbers of miRNA in milk of a wide range of species (Chen et al. 2010; Kosaka et al. 2010; Weber et al. 2010; Zhou et al. 2012) which were characterised mainly for immune related function. Moreover, a new hypothesis proposed a role for miRNA in inter-cellular communication through horizontal transfer that may also suggest these secreted miRNAs are signalling molecules (Chen et al. 2012). Preliminary experiments from our laboratories indicated the presence of differentially regulated miRNA in tammar wallaby milk (unpublished data). This significant and differential expression of miRNA in milk demands detailed investigation into their potential role in regulating different processes including development of the young. It is possible that secreted miRNAs are delivered to the recipient young in turn regulating target gene expression and cell function. This is supported by a recent study showing exogenous miRNA consumed from plant foods crossed the mammalian intestinal mucosa and reached the plasma (Zhang et al. 2012) suggesting miRNA may plausibly regulate function in the recipient. In addition, the study by Kim and co-workers (2011) indicated that miRNAs (miR-7a and miR-203) are required for digestive tract organogenesis in fetal mice and this mechanism proceeded by regulating the expression of stomach homeotic regulator Barx1. However, this regulation on organogenesis in fetal mice examined miRNA expressed in the stomach. Future studies should examine whether the miRNA delivered by mother’s milk plays a regulatory role in the development of infant stomach.
To determine the individual factor/s that might be either expressed throughout lactation or differentially expressed across tammar wallaby lactation and are involved in regulating stomach development, the use of technology platforms
216 like genomics, proteomics and bioinformatics are required. The approach using a genomics platform has already been initiated in the current study (Chapter 5). This approach involved deep sequencing (RNA-seq) using mammary gland tissue collected across the extended lactation cycle and identified genes coding for secreted proteins that may play a plausible role in stomach development and differentiation. However, individual proteins need to be expressed in vitro and further examined for specific effect on stomach models to confirm their significance on development of the young.
6.3 Gray short-tailed opossum: An alternative marsupial model to understand the putative role of milk in stomach development
Previous studies have focused on stomach development in the tammar wallaby (Setiati 1986; Waite 2003). However, the omnivorous marsupial gray short- tailed opossum (Monodelphis domestica) with a monogastric stomach would be a more appropriate model for comparing the developmental events observed in eutherians such as humans. Therefore, the current study was undertaken to describe the progressive development of stomach in gray short-tailed opossum and has demonstrated (Chapter 4) that timing of development was different from the tammar wallaby. In the tammar wallaby, the digastric stomach has almost a uniform mucosal morphology during the initial 200 days postpartum. The specialised cells including the parietal cells and mucus cells were observed from birth and these cells gradually mature and multiply during development, whereas chief cells were observed after 170 days postpartum (Setiati 1986; Waite et al. 2005). The fore-stomach undergoes a transition from a gastric glandular phenotype to the cardia glandular phenotype post 200 days (Kwek 2009; Waite 2003). In gray short-tailed opossum, the stomach of newborn has an immature mucosa with no evidence for differentiated cell types throughout teat attachment phase (14 days). At around 35 days postpartum there is clear evidence for chief cells followed by parietal cells, and the stomach attains a mature phenotype similar to an adult stomach by the time of weaning. This pattern of appearance of two major cells types of the stomach, chief cells and parietal cells, is
217 different from that observed in the tammar pouch young stomach and the North American opossum, another omnivore marsupial (Krause et al. 1976).
Appearance of both chief cells and parietal cells in the gray short-tailed opossum stomach observed in the current study occurs slightly later than the gradual shift in dilute milk of early stage to milk rich in solids and protein (Green et al. 1991). This transition occurs prior to when the young starts feeding on a solid diet. Similarly, in the tammar wallaby, there is a dramatic change observed in the stomach when there is a compositional change in milk from high carbohydrate to high protein and lipid levels along with the shift to consumption of herbage (Kwek et al. 2009a). However, the direct effect of milk on stomach development, the individual factor/s of milk that regulates the process and whether the regulation is the same in all marsupial species have not yet been confirmed.
One of the major components of milk that may regulate gut development is the carbohydrates. Studies have shown the role of oligosaccharides in influencing gut colonisation (Fuhrer et al. 2010; Jeong et al. 2012; Weiss and Hennet 2012; Zivkovic and Barile 2011) which in turn regulate gut development (Langhendries 2006). In gray short-tailed opossum, which exhibits a lactation cycle of 60-75 days, milk is dilute during the initial five days with low levels of hexose that gradually increases to higher levels by day 50 lactation and then further decreases (Green et al. 1991). In contrast, in tammar wallaby, with a 300-330 day lactation cycle, milk is initially high in carbohydrates and further reduces to very low level post 200 days (Nicholas et al. 1997). Milk from the gray short-tailed opossum is composed of free monosaccharides like glucose and galactose for first five days and monosaccharides and lactose for the first 2 weeks. Subsequently, the milk is composed of higher oligosaccharides (Crisp et al. 1989). This is different from tammar wallaby milk which includes lactose for the first four days, followed by secretion of complex oligosaccharides (Messer et al. 1984). Among the variety of oligosaccharides of gray short-tailed opossum milk, few are identical with the tri- to penta-saccharides (Gal1-
3Lac) of the tammar wallaby milk (Crisp et al. 1989). However, it is unlikely that the
218 nature of milk carbohydrates of these two marsupial species is sufficient to explain the changes in stomach development.
It is important to have a better understanding of the oligosaccharide composition and its temporal variation in gray short-tailed opossum milk and compare with tammar milk in an attempt to correlate with the difference in stomach development between these species. Various earlier studies have shown that milk oligosaccharides control the type of gut colonisation (Newburg 1997; Newburg et al. 1990; Newburg and Walker 2007) and in turn regulate gut differentiation (Bates et al. 2006; Uribe et al. 1994). An abundance of complex oligosaccharides in human milk, apparently indigestible by the developing young is largely known to influence the infant’s gut colonisation (Ruhaak and Lebrilla 2012; Zivkovic et al. 2011). In addition, these components also imply a defensive approach whereby glycans prevent the binding of pathogens to gut epithelial cells there by providing protection (Newburg et al. 2005). Taking advantage of the advanced MS-based analytical techniques, detailed glycoprofiling and structural understanding of the oligosaccharides of human milk has been performed (Zivkovic et al. 2011). Based on the structure of these complex oligosaccharides, a direct systematic link between specific oligosaccharides and bacterial growth especially bifidobacterial growth has been established (LoCascio et al. 2007; Locascio et al. 2009; Sela et al. 2008).
In tammar wallaby, the high levels of oligosaccharides found in milk during the initial 200 days of lactation was hypothesised to inhibit gut colonisation by specific bacteria thus regulating stomach development (Kwek et al. 2009a). However, there is little information available about the specific complex oligosaccharides of marsupial milk and its correlation with gut colonisation. Thin layer chromatography techniques were previously utilised for the basic characterisation of marsupial milk (Crisp et al. 1989; Messer and Green 1979; Messer et al. 1984; Messer and Mossop 1977). More recent approaches like high performance liquid chromatography (Chaturvedi et al. 1997), mass spectrophotometry (MS) (Ninonuevo et al. 2006), MALDI-TOF-MS (Stahl et al. 1994) can be utilised for compositional profiling and characterisation of
219 oligosaccharides. MS is evolving as one of the best analytical method of determining milk oligosaccharide composition due to high sensitivity of the technique with improved reproducibility providing structural information (Zivkovic et al. 2011). Previously, Wickramasinghe and co-workers preformed a chromatographic analysis of the bovine milk oligosaccharides together with a transcriptome profiling of the glycosylation related genes involved in oligosaccharide metabolism using bovine milk somatic cells (2011). Both these analyses together with an extensive literature search provided an in-depth knowledge about the oligosaccharide metabolism with focus on sialic acid and fucose metabolism in bovine milk. Similarly, transcriptome profiling together with milk oligosaccharide characterisation by improved analytical techniques like MS in the marsupial species, the gray short-tailed opossum and the tammar wallaby is recommended and this may provide detailed understanding about the genes regulating milk oligosaccharide composition and its temporal variation (Zivkovic et al. 2011). Deep sequencing of tammar mammary gland RNA across the lactation cycle has already been completed and analysis involving understanding of the genes coding for enzymes related to oligosaccharide metabolism is in progress. This comparative approach may help to predict the relevance and difference of gut flora between the herbivorous (tammar wallaby) and omnivorous (gray short-tailed opossum) marsupials.
Interestingly, there is a major difference in milk carbohydrate component between mammals wherein the eutherians have lactose as the chief component and in monotreme and marsupial milk, oligosaccharides are the major components with either a virtual absence or very low concentration of free lactose (Messer and Green 1979; Messer and Mossop 1977; Urashima et al. 2012). In eutherians especially the human, this dietary lactose is digested by the intestinal lactase (neutral ȕ- galactosidase) located in the membrane of the microvilli of brush border of the small intestine splitting into galactose and glucose. These products are further transported to the enterocytes and thus enter the blood circulation. However, simple oligosaccharides such as the fucosyllactose and sialylactose cannot be digested by this enzyme (Urashima et al. 2001). Marsupial milk is mainly composed of lactose- based oligosaccharides (Messer and Green 1979; Messer et al. 1984) and the
220 intestinal lactase is completely absent from the brush border of the small intestine villi of the suckling young indicating a different digestive mechanism for lactose (Messer et al. 1989b; Walcott and Messer 1980). Instead, acidic ȕ-galactosidase of the enterocytes digest both the lactose and the ȕ1ĺ3 linked galactosides that are present in tammar wallaby milk, in contrast to the neutral brush border lactase of eutherians (Crisp et al. 1987). Thus, it is hypothesised that in the suckled tammar, the milk oligosaccharides might be transported intact into the absorptive cells of the small intestine probably by endocytosis and hydrolysed to monosaccharides by the lysosomal acid ȕ-galactosidase (Messer et al. 1989a).
Furthermore, the oligosaccharide component of the milk has evolved across mammalian classes (German et al. 2008). Human and the primate milk oligosaccharides are similar; however in the primates they are present at lower concentrations with absence of larger oligosaccharides that are present in human milk (Newburg et al. 1999). These complex oligosaccharides of the human milk are composed of a lactose unit at the reducing end and repetitive attachment of galactose and N-acetylglucosamine in ȕ-glycosidic linkage to lactose (Boehm and Stahl 2003). This is mainly composed of the fucosyl neutral oligosaccharides involved in the generation of anti-inflammatory mediators (Eiwegger et al. 2004) and the sialyl acidic oligosaccharides involved in preventing pathogenic adhesion to the intestinal epithelial surface (Boehm and Stahl 2003; Guggenbichler et al. 1997). In monotreme milk, the oligosaccharides are mainly fucosyl based with echidna milk containing sialyllactose (Urashima et al. 2001) whereas in marsupials such as the tammar wallaby, carbohydrates include the major neutral oligosaccharides such as the trisaccharide ȕ1ĺ3galactosyllactose and a series of tetra and heptasaccharides.
To conclude, it is worth considering that the difference in milk carbohydrate may be linked to a difference in gut colonisation which may regulate gut development (Bates et al. 2006; Uribe et al. 1994). Moreover, this difference can probably be linked to the difference in reproductive strategies. This is supported by Messer and co-workers (1989a) argument that the osmotic pressure exerted by a certain mass of oligosaccharides in milk will be lower than that exerted by an equal
221 amount of lactose. The lack of lactose in marsupial milk may support greater concentration of electrolytes and saccharides without it becoming hyper osmotic and further supporting the increased developmental requirement of the altricial young. But, the presence of lactose during the first four days in the tammar wallaby milk and first two weeks in the gray short-tailed opossum milk is intriguing. This differential secretion pattern of lactose in the two marsupials when compared to the eutherians may address a difference in gut colonisation, gut morphology and development or any other significance which needs to be investigated in detail.
6.4 A bioinformatic approach to identity milk bioactives of tammar wallaby
In the tammar wallaby where the young are born altricial, different developmental events such as the opening of eyes (Wye-Dvorak 1984), growth and maturation of different organs like the brain (Reynolds et al. 1985), lung (Runciman et al. 1996) and gastrointestinal tract (Setiati 1986; Waite 2003) occurs during post natal life. This maturation process of different organs occurring during the suckling period in marsupials is similar to that observed in the human foetus in utero. Therefore it is likely that components regulating these developmental events are provided by phase 2A milk in tammar wallaby and by amniotic fluid or placenta in the human. In the present study (Chapter 5) using a bioinformatic platform, the previously established microarray datasets (Khalil 2007; Sharp et al. 2009) and RNA-seq performed in the current study were utilised to identify human orthologues of amniotic fluid and placental proteins that are most likely present in the milk during phase 2A lactation in tammar wallaby. Based on a detailed literature review, we have proposed a putative role for these genes/proteins in regulating different physiological processes. However, a direct correlation between their expression and the development of tammar pouch young has not yet been established.
Furthermore, the transcriptome profiling performed using RNA deep sequencing allows genome analysis at a higher resolution and helps precise quantification of gene expression levels across lactation as compared to the previous
222 microarray dataset. Recently, a RNA deep sequencing was performed using bovine milk somatic cells (Wickramasinghe et al. 2011; Wickramasinghe et al. 2012). This study helped to identify the diverse genes expressed during different stages of bovine lactation (Wickramasinghe et al. 2012) and gave a better understanding of the genes involved in diverse metabolic pathways including milk oligosaccharide metabolism (Wickramasinghe et al. 2011). Similarly, using our tammar wallaby dataset, we can identify the genes expressed at different stages of lactation including genes that are specifically expressed during phase 2A of lactation and therefore provide an improved understanding of the potential regulation of initial development of the altricial young. Thus, both datasets will act as a huge repository for identifying tammar milk bioactives of significance to the developing young and has implication in mammary gland function.
With this information and data, the next approach would be to express the identified genes in vitro and examine for bioactivity on diverse models. In order to determine the role of these identified genes in regulating stomach proliferation and differentiation, the individual proteins expressed in vitro can be examined for bioactivity using models such as the embryonic stomach explant model and gastric organoid model developed and demonstrated in Chapter 3. Nevertheless, it cannot be ruled out that these candidates may have a role in other physiological processes like mammary gland function, immune protection or regulating development of other organs in the pouch young (Khalil 2007; Sharp et al. 2009). To address the broader role in regulating these physiological processes, the proteins need to be examined for bioactivity in specific in vitro models. An earlier study by Khalil (2007) had utilised eutherian cell-based models in which the tammar milk proteins demonstrated stimulative effects. These studies provided evidence that eutherian cell models respond to marsupial signalling proteins.
6.5 Tammar wallaby as a legitimate biomedical research model
Among all the marsupials, the reproductive cycle of the tammar wallaby is the most studied and well characterised including detailed analysis of milk composition and production across lactation. This lactation strategy provides a
223 unique opportunity for examining mammary gland function compared with other eutherian mammals to better understand the evolutionary divergence between mammalian classes with different reproductive strategies. Moreover, the temporal secretion pattern of different milk components correlated with specific phases of development of the young and/or the function of the mammary gland provides new opportunities to address their function. Thus, marsupials with an adapted reproductive strategy are increasingly becoming relevant as a legitimate biomedical research model for understanding the regulation of diverse physiological processes in mammals including eutherians such as stomach development as justified by the current study. Additionally, availability of sequenced public databases along with our RNA-seq results from tammar wallaby provides the increased prospect to identify eutherian orthologues by utilising a comparative genomics and bioinformatics platform (Sharp et al. 2009).
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APPENDICES
274
Appendix 1: Expression profiles of secreted proteins of phase 2A. The expression profile from the RNA-seq dataset across tammar wallaby lactation cycle (Day of pregnancy P or lactation L, x-axis) is represented in fragments per kilobase of exon per million fragments mapped (FPKM, y-axis).
275
276
277
278
279
280
281
282
283
284
285
286
287
288
Appendix 2: Expression profiles of phase 2B specific genes. The expression profile from the RNA-seq dataset across tammar wallaby lactation cycle (Day of pregnancy P or lactation L, x-axis) is represented in fragments per kilobase of exon per million fragments mapped (FPKM, y-axis).
ACSL4 Long-chain-fatty-acid--CoA ligase 4 1500
1200
900
600
300
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
ADAMTS6 ADAMTS-6 precursor 2000
1500
1000
500
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
289
CASR Extracellular calcium-sensing receptor precursor 3000
2000
1000
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
CMAS N-acylneuraminate cytidylyltransferase 1800 1500 1200 900 600 300 0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
290
MYO5B Myosin-Vb 2000
1500
1000
500
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
OSBPL6 Oxysterol-binding protein-related protein 6 2000
1500
1000
500
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
291
SPOCK2 Testican-2 precursor 3000
2000
1000
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
292
Appendix 3: Expression profiles of phase 3 specific genes. The expression profile from the RNA-seq dataset across tammar wallaby lactation cycle (Day of pregnancy P or lactation L, x-axis) is represented in fragments per kilobase of exon per million fragments mapped (FPKM, y-axis).
ABCG2 ATP-binding cassette sub-family G member 2 3000
2000
1000
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
ACSL1 Long-chain-fatty-acid--CoA ligase 1 2500
2000
1500
1000
500
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
293
ACSS2 Acetyl-coenzyme A synthetase, cytoplasmic 1500
1200
900
600
300
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
AGPAT1 1-acyl-sn-glycerol-3-phosphate acyltransferase alpha 3000
2000
1000
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
294
AGXT2L1 Alanine--glyoxylate aminotransferase 2-like 1 1600 1400 1200 1000 800 600 400 200 0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
ALPL Alkaline phosphatase, tissue-nonspecific isozyme precursor 4000
3000
2000
1000
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
295
ATR Serine/threonine-protein kinase ATR 2000
1600
1200
800
400
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
DGKI Diacylglycerol kinase iota 1500
1200
900
600
300
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
296
FAM46A Protein (HBV X-transactivatedgene 11 protein) 2500
2000
1500
1000
500
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
GLB1 Beta-galactosidase precursor 15000
12000
9000
6000
3000
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
297
LPL Lipoprotein lipase precursor 6000
4500
3000
1500
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
LPO Lactoperoxidase precursor 1600
1200
800
400
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
298
PCTK3 Serine/threonine-protein kinase 1800 1500 1200 900 600 300 0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
PDCD2L Programmed cell death protein 2-like 1200
900
600
300
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
299
PIP5K1B Phosphatidylinositol-4-phosphate 5-kinase type-1 beta 1000
800
600
400
200
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
PROX1 Homeobox prospero-like protein 2000
1600
1200
800
400
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
300
RHOBTB2 Rho-related BTB domain-containing protein 2 1600
1200
800
400
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
SCD Acyl-CoA desaturase 8000
6000
4000
2000
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
301
SGK1 Serine/threonine-protein kinase 2000
1500
1000
500
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
SNX9 Sorting nexin-9 2500
2000
1500
1000
500
0 2L 3L 4L 5L 6L 10L 22L 40L 67L 70L 87L 17P 18P 21P 80L 133L 151L 171L 243L 260L 266L
302