B-Cell Genes Andrea L. Schraven

Transcriptomic Analysis of the Gray short-tailed opossum (Monodelphis domestica) B-cell Genes

Photo credit: J. Old

Andrea L. Schraven

A thesis presented as fulfilment of the requirements for the degree of Master of Research Western Sydney University, Hawkesbury School of Science and Health Date: December 2019

Table of Contents

Table of Contents ...... 2 Table of Figures ...... 4 Table of Tables ...... 10 Acknowledgements ...... 11 Statement of Authenticity ...... 12 Preface ...... 13 Publications and Prepared Manuscripts...... 14 Conference Presentations ...... 15 Abstract ...... 16 Introduction ...... 18 1.1 Mammalian Evolution ...... 19 1.1.1 Marsupial Reproductive Strategies ...... 20 1.2 Marsupial Immunology ...... 22 1.3 Marsupial Genomics ...... 27 1.3.1 Transcriptomics ...... 29 1.4 Model Marsupial: The gray short-tailed opossum (Monodelphis domestica) ...... 32 1.5 Research Aim ...... 34 1.6 Thesis Outline ...... 35 1.7 Chapter 1 References...... 36 Immunogenetics of Marsupial B-cells ...... 44 2.1 Chapter Outline and Authorship ...... 45 2.2 Introduction ...... 46 2.3 Mammalian Evolution ...... 48 2.4 Marsupial Immunology ...... 49 2.5 B-cells ...... 51 2.5.1 Immunoglobulins ...... 58 2.5.2 Cluster of Differentiation Markers ...... 66 2.5.3 Signal Transduction Molecules ...... 69 2.5.4 Transcriptional Regulators ...... 71 2.6 Conclusions ...... 72 2.7 Conflicts of Interest ...... 72 2.8 Chapter 2 References...... 73 Single-cell transcriptome analysis of the B-cell repertoire reveals the usage of immunoglobulins in the gray short-tailed opossum (Monodelphis domestica) ...... 83

3.1 Chapter Outline and Authorship ...... 84 3.2 Introduction ...... 85 3.3 Materials and Methods ...... 87 3.3.1 Ethics Statement...... 87 3.3.2 Tissue Collection, Cell Sorting and RNA Extraction ...... 87 3.3.3 cDNA Synthesis, Sequencing and Alignment ...... 87 3.3.4 Single-cell Identification and Statistical Analyses ...... 88 3.4 Results ...... 89 3.4.1 Heavy and Light Chain Immunoglobulin Usage ...... 89 3.4.2 Immunoglobulin Variable and Joining Domains ...... 90 3.4.3 VH and VL Gene Segment Pairing ...... 93 3.5 Discussion ...... 95 3.6 Acknowledgements ...... 99 3.7 Conflicts of Interest ...... 99 3.8 Chapter 3 References...... 100 3.9 Chapter 3 Supplementary Figures ...... 103 Single-cell transcriptomic analysis of marsupial B-cells, the gray short-tailed opossum (Monodelphis domestica) ...... 105 4.1 Chapter Outline and Authorship ...... 106 4.2 Introduction ...... 107 4.3 Materials and Methods ...... 109 4.3.1 Ethics Statement...... 109 4.3.4 Transcriptome Annotation and Gene Ontology ...... 110 4.3.5 Statistical Analyses ...... 111 4.3.6 Phylogenetic Analyses and Motif Comparison ...... 111 4.4 Results ...... 112 4.4.1 Overview of the Transcriptome ...... 112 4.4.2 Highly Expressed Genes ...... 114 4.5 Discussion ...... 126 4.6 Acknowledgements ...... 130 4.7 Conflicts of Interest ...... 130 4.8 Chapter 4 References...... 131 Concluding Summary and Future Directions ...... 135 5.1 Concluding Summary ...... 136 5.2 Future Directions ...... 140 5.3 Chapter 5 References...... 142

Table of Figures

Figure 2.1 Phylogenetic tree based on B-cell relationship in vertebrate groups and divergence of marsupials referred to in this review, constructed using the NJ method.

Figure 2.2 Unrooted phylogenetic tree based on amino acid alignments of vertebrate IgD sequences, constructed using the NJ method. Genbank accession numbers: I. punctatus IgD,

ADF56020; S. trutta IgD, AAV28076; E. coioides IgD, AEN71107; M. musculus IgD,

AAB59653; X. tropicalis IgD, ABC75541; P. sinensis IgD, ACU45375; O. anatinus IgD,

ACD31540; C. siamensis IgD, AFZ39208; E. macularius IgD, ABY67439; H. sapiens IgD,

AAA52771; P. troglodytes IgD, PNI88328; S. scrofa IgD, AAP23939; B. taurus IgD,

AAN03673; O. aries IgD, AAN03671.

Figure 2.3 Unrooted phylogenetic tree based on amino acid alignments of marsupial and eutherian IgA and IgG subclasses sequences, constructed using the NJ method. Genbank accession numbers: M. domestica IgG, AAC79675; T. vulpecula IgG, AAD55590; M. domestica IgA, AAD21190; T. vulpecula IgA, AAD41690; N. eugenii IgA, ABV01995; M. musculus IgA, AAB59662; H. sapiens IgA2, AAB59396; H. sapiens IgA1, AAC82528; H. sapiens IgG1, BAC87418; H. sapiens IgG3, CAA27268; H. sapiens IgG2, AAB59393; H. sapiens IgG4, CAC20457; M. musculus IgG1, CAD32497; M. musculus IgG3, ADK11691;

M. musculus IgG2b, ACX70084; M. musculus IgG2a, BAC44883; M. musculus IgG2c,

CAA34864.

Figure 2.4 Genomic organisation of the M. domestica IgH, Igκ, and Igλ loci. V, D (in the

IgH), and J gene segments are clustered, and C gene segments are shown (not to scale).

Direction of transcription is indicated by the Telomeric and Centromeric ends. Dotted lines indicate where there are gaps in MonDom5 genome sequence. For the IgH locus, the C

4 domain is shown below the IgH main line, the presumptive IgM pseudogene is indicated with a psi. H identifies the sequences with an encoding hinge region. Polyadenylation and transmembrane protein domain are designated with Poly A and TM, respectively. Double angled lines indicate where there are large regions of separation. The Igκ shows two large clusters of Vκ gene segments with an 800 Kb region separating them. Circles on the Igκ identify a recombining element, RE, and two kappa enhancers, Ke5’and KE3’P, respectively.

Pairing IgλJ and IgλC genes are designated according to their order.

Figure 2.5 Amino acid alignment of expressed (A) CD79α and (B) CD79α mammalian sequences. Dots indicate identical bases to M. domestica CD79 transcripts. The Ig domain,

TM regions and CYT regions are indicated by arrows below the sequences, the ITAM motifs are identified by a box around the amino acids. Genbank accession numbers: M. domestica

(CD79α, ACZ13436; CD79β, ACZ13438.1), N. eugenii (CD79α, ACZ13437; CD79β,

ACZ13439.1), (O. fraenata CD79α, AGO64767; CD79β, AII23405.1), H. sapiens (CD79α,

AAI13732; CD79β, AAH02975.2).

Figure 3.1 Fraction of cells expressing immunoglobulin heavy (IgM, IgG, IgA, IgE) and light (Igκ and Igλ) chains in both spleen and blood single cells.

Figure 3.2 Fraction of cells expressing V and J domains of the Ig heavy (IgM, IgG, IgA, IgE) chains in both spleen and blood single cells. NA indicates no information.

Figure 3.3 Fraction of cells expression variable and joining domains of the immunoglobulin light (Igκ) chains in both spleen and blood single cells. NA indicates no information.

Figure 3.4 Fraction of cells expression variable and joining domains of the immunoglobulin light (Igλ) chains in both spleen and blood single cells. NA indicates no information.

Figure 3.5 Heat map indicating the frequency of VH/Vκ gene segment pairs expressed in the opossum repertoire.

Supplementary Figure 3.1 Previously annotated pseudogene VH1.10. Identification of ORF on the reverse strand of frame 1, 3’ – 5’. Start codon, 2250; stop codon, 415; length, 612 amino acids. Stop codon is indicated by the asterisks.

Supplementary Figure 3.2 Previously annotated pseudogene Vκ1.17. Identification of ORF on the reverse strand of frame 1, 3’ – 5’. Start codon, 273; stop codon, 37; length, 79 amino acids. Stop codon is indicated by the asterisks.

Supplementary Figure 3.3 Previously annotated pseudogene Vκ7.11. Identification of ORF on the reverse strand of frame 3, 3’ – 5’. Start codon, 990; Stop codon, 235; length, 252 amino acids. Stop codon is indicated by the asterisks.

Figure 3.6 Heat map indicating the frequency of VH/Vλ gene segment pairs expressed in the opossum repertoire.

Figure 4.1 Overall distribution of GO categorical terms in the opossum cell transcriptome.

(A) Molecular function, (B) Biological process, and (C) Cellular components. Percentages are relative of all genes which are categorised as a percentage of all annotated transcripts.

Figure 4.2 Overall distribution of GO categorical terms within the Immune System Process

Category. Percentages are relative of all genes which are categorised as a percentage of all annotated transcripts.

Species designations are abbreviated to the first two letters of their scientific names. M. domestica (Modo), S. harrisii (Saha), P. cinereus (Phci), V. ursinus (Vour), O. anatinus

(Oran), H. sapiens (Hosa), and M. musculus (Mumu). Genbank accession numbers are: Modo

SDC4, XP_007475896.1; Saha SDC4, XP_012395261.1; Phci SDC4, XP_020839750.1;

Vour SDC4, XP_027707442.1; Oran SDC4, XP_028927254.1; Hosa SDC4, NP_002990.2;

Mumu SDC4, NP_035651.1.

Figure 4.4 Two-dimensional non-transformed multidimensional scaling (MDS) plot visually representing the single-cell transcriptomes of the top 10 highly expressed genes in the spleen

(grey upwards triangles) and blood (black downward triangles).

Figure 4.5 Two-dimensional non-transformed multidimensional scaling (MDS) plot visually representing the single-cell transcriptomes of the top 10 highly expressed genes in the IgM

(black downward triangle), IgG (dark grey diamond), IgA (grey squares), and IgE (grey upwards triangle).

Figure 4.6 (A) Multiple sequence alignment of HNRNPK gene products and (B) a

Phylogenetic tree based on the HNRNPK sequences, constructed using the NJ method.

Protein sequences were aligned using the ClustalW algorithm. Dots indicate identical matches to the opossum sequences. Domains of the HNRNPK protein are indicated by a line underneath the sequence. Symbols above sequence are: (1) Inverted triangles mark cysteine residues (2) Bullets mark nucleic acid binding regions, (3) Diamonds G-X-X-G motif.

Species designations are abbreviated to the first two letters of their scientific names. M. domestica (Modo), S. harrisii (Saha), P. cinereus (Phci), O. anatinus (Oran), H. sapiens

(Hosa) and M. musculus (Mumu). Genbank accession numbers are: Modo HNRNPK,

XP_016279557.1; Saha HNRNPK, XP_012398527.1; Phci HNRNPK, XP_020822678.1;

Oran HNRNPK, XP_016083874.1; Hosa HNRNPK, AAH14980.1; Mumu HNRNPK,

ACC69193.1.

Figure 4.7 (A) Multiple sequence alignment of ELMOD1 gene products and (B) a

Phylogenetic tree based on the ELMOD1 sequences, constructed using the NJ method.

Protein sequences were aligned using the ClustalW algorithm. Dots indicate identical matches to the opossum sequences. Domains of the ELMOD1 protein are indicated by a line underneath the sequence. Symbols above sequence are: (1) Inverted triangles mark cysteine residues. Species designations are abbreviated to the first two letters of their scientific names.

M. domestica (Modo), S. harrisii (Saha), P. cinereus (Phci), V. ursinus (Vour), O. anatinus

(Oran), and H. sapiens (Hosa). Genbank accession numbers are: Modo ELMOD1,

XP_007494949.1; Saha ELMOD1, XP_012400499.1; Phci ELMOD1, XP_020851842.1;

Vour ELMOD1, XP_027705369.1; Oran ELMOD1, XP_028904013.1; Hosa ELMOD1,

NP_061182.3.

(Saha), P. cinereus (Phci), V. ursinus (Vour), O. anatinus (Oran), H. sapiens (Hosa), and M. musculus (Mumu). Genbank accession numbers are: Modo GLG1, XP_007477569.1; Saha

GLG1, XP_012395893.2; Phci GLG1, XP_020819407.1; Vour GLG1, XP_027699023.1;

Oran GLG1, XP_028931633.1; Hosa GLG1, AAH60822.1; Mumu GLG1, EDL11491.1.

Figure 4.9 (A) Multiple sequence alignment of HLA-DRA gene products and (B) a

Phylogenetic tree based on the HLA-DRA sequences, constructed using the NJ method.

Protein sequences were aligned using the ClustalW algorithm. Dots indicate identical matches to the opossum sequences. Domains of the HLA-DRA protein are indicated by a line underneath the sequence. Symbols above sequence are: (1) Inverted triangles mark cysteine

8 residues, (2) Bullets mark heterodimer interface residues, (3) Diamonds mark MHC binding domain interface residues. Species designations are abbreviated to the first two letters of their scientific names. M. domestica (Modo), S. harrisii (Saha), P. cinereus (Phci), V. ursinus

(Vour), and H. sapiens (Hosa). Genbank accession numbers are: Modo HLA-DRA,

XP_007483702.1; Saha HLA-DRA, XP_003770700.1; Phci HLA-DRA, XP_020829855.1;

Vour HLA-DRA, XP_027732036.1; Hosa HLA-DRA, ARB08447.1.

Figure 4.10 (A) Multiple sequence alignment of marsupial MHC DAB1 gene products and

(B) a Phylogenetic tree based on the MHC DAB sequences, constructed using the NJ method.

Protein sequences were aligned using the ClustalW algorithm. Dots indicate identical matches to the opossum sequences. Domains of the MHC DAB protein are indicated by a line underneath the sequence. Symbols above sequence are: (1) Inverted triangles mark cysteine residues. Species designations are abbreviated to the first two letters of their scientific names.

M. domestica (Modo), S. harrisii (Saha), P. cinereus (Phci), N. eugenii (Noeu), and T. vulpecula (Trvu). Genbank accession numbers are: Modo DAB1, NP_001028163.1; Saha

DAB, ABV58848.1; Phci DAB, ALX81653.1; Noeu DAB, AAX54675.1; Trvu DAB,

AAK84172.1.

Table of Tables

Table 1.1 Summary marsupial immune tissues, examined in the thymus, bone marrow, lymph nodes, and spleen.

Table 2.1 List of B-cell genes that have either been expressed or predicted in marsupial species, including their Genbank accession Identifications (ID).

Table 3.1 IgL isotypes Igκ:Igλ ratio expression in mammals.

Table 4.1 Top 100 most highly expressed genes in the opossum B-cell transcriptome. Total percentages are calculated from spleen and blood counts.

Acknowledgements

Firstly, I’d like to thank Associate Professor Julie Old, for all the support and encouragement throughout my Masters. Thank you for pushing me outside my comfort zone, especially when it came to presenting at conferences. I’d like to thank my co-supervisors Dr. Hayley Stannard and Dr. Oselyne Ong for reading and providing feedback on my endless drafts, and your words of encouragement when tackling the journal revisions.

Thank you Professor Robert Miller for being a wonderful collaborator and sharing your data with me. Thank you Kimberly Morrisey and Bethaney Fehrenkamp for your assistance in the initial analysis, and Associate Professor Ricky Spencer for your help with the statistical analyses. Special thanks to Danny Douek, Galina Petukhova and especially Victoria Hansen for generating the data used in all analyses.

I’d like to give thanks to the friends I have made along the way, Blaire Vallin, Chandni

Sengupta, Rowan Thorley, Janice Vaz, Corrine Letendre and Kristen Petrov who have all supported me during some of my highest and lowest points throughout my Masters. I am forever grateful and wish you all well in your own degrees and future endeavours.

To my parents, Mark and Linda, whose work ethics have always been an inspiration to me.

Thank you for your support in everything I do and always showing an interest in my research.

Lastly to my sisters, Rebekah and Alicia, thank you for keeping my sanity in check.

Statement of Authenticity

This thesis is submitted to the Western Sydney University in fulfilment of the requirement for

the Degree of Master of Research.

The work presented in this thesis is, to the best of my knowledge, composed of my original

work, and contains no material previously published or written by another person, except where due reference has been made in the text. I have clearly stated the contribution by others

to jointly authored works that I have included in my thesis.

......

Andrea L. Schraven

Preface

This thesis is presented as a series of manuscripts, which describes the gene expression of B-

cell in the gray short-tailed opossum (Monodelphis domestica). Each chapter presented is self-contained, and includes a specific outline and authorship. Of the manuscripts presented here, Chapter 2 has been published in a peer-review journal, Chapter 3 has been submitted to

a peer review journal and is currently under review, chapter 4 has been prepared for

submission to a peer-reviewed journal. An introductory and general conclusion has been

included in this thesis to assist with the continuity throughout. Format of manuscripts and

reference lists will vary according to the guidelines of journals they are submitted to. All

manuscripts are jointly authored, but in each case, I am first author.

Publications and Prepared Manuscripts

Schraven, A. L., Stannard, H. J., Ong, O. T. W., Old, J. M. 2020. Immunogenetics of marsupial B-cells, Molecular Immunology. 117, 1-11. https://doi.org/10.1016/j.molimm.2019.10.024

Schraven, A. L., Hansen, V. L., Morrissey, K. A., Stannard, H. J., Ong, O. T. W., Douek, D.

C., Miller, R. D., Old, J. M. Single-cell transcriptome analysis of the B-cell repertoire exposes the usage of immunoglobulins in the gray short-tailed opossum (Monodelphis domestica). Immunology and Cell Biology. Under Review.

Schraven, A. L., Hansen, V. L., Morrissey, K. A., Stannard, H. J., Ong, O. T. W., Douek, D.

C., Miller, R. D., Old, J. M. Single-cell transcriptomic analysis of marsupial B-cells, the gray short-tailed opossum (Monodelphis domestica). Prepared for Immunology and Cell Biology.

Conference Presentations

Schraven, A. L., Miller, R. D., Stannard, H. J., Ong, O. T. W., Hansen, V. L., Morrissey, K.

A. Old, J. M. Bioinformatics analyses of B-cell repertoire expression in the gray short-tailed opossum (Monodelphis domestica). June, 2019. Presented at the North American

Comparative Immunology Workshop, Waterloo, Canada.

Abstract

Marsupials and eutherians are mammals that differ in their physiological traits, predominately their reproductive and developmental strategies; eutherians give birth to well-developed young, while marsupials are born highly altricial after a much shorter gestation. These developmental traits result in differences in the development of the immune system of eutherian and marsupial species. B-cells are key to humoral immunity, are found in multiple lymphoid organs, and have the unique ability to mediate the production of antigen-specific antibodies in the presence of pathogens. Marsupial B-cell investigations have become increasingly important in understanding an adaptive immune system that develops primarily ex utero. In comparison to eutherians and monotremes, marsupial B-cells have four

Immunoglobulin (Ig) heavy (H) chain isotypes (IgA, IgG, IgM and IgE) and two light (L) chain isotypes; lambda (Igλ) and kappa (Igκ). The gray short-tailed opossum (Monodelphis domestica) is a well-established model marsupial species, with a well annotated genome. The

B-cell transcriptome of an individual opossum was investigated by Next Generation RNA-

Seq techniques at the single-cell level. A total of 273 single-cells and 575,721 contigs were generated, annotation of the transcriptome identified 14,654 unique genes. The first study of this thesis analysed the IgH and IgL usage in the opossum B-cell repertoire. Not surprisingly,

IgM had the highest expression in the repertoire, followed by IgA, IgG, and very few cells expressing IgE. Despite Igκ being the most complex IgL isotype, the ratio of Igκ to Igλ was

35:65. IgL isotypes have been identified to have a greater contribution to antibody diversification than IgH isotypes, due to the complexity and abundance of IgL variable (V) gene segments. The second study of this thesis examined the whole opossum B-cell transcriptome and analysed the most highly expressed genes. The most abundant gene transcripts were Sydnecan-4, making up 0.66% of the entire transcriptome. IgM and IgG cells produced significantly more transcripts of the golgi glycoprotein 1 and ELMO domain-

16 containing protein genes in comparison to IgA. Since IgE expressing cells were very low in number, a definitive comparison could not be made between all IgH cells. Highly expressed genes associated with the marsupial immune system included MHC class II DRα chain and

MHC class II DAβ chain. The diverse array of genes identified in the opossum single-cell transcriptome reveals the importance of marsupial B-cells in producing endogenous antibody responses, and has allowed for a comparative analysis with other mammalian lineages.

CHAPTER 1

Introduction

1.1 Mammalian Evolution

Marsupials, or metatherians, are one of three major mammalian lineages, along with monotremes (Prototherians) and eutherian mammals (Selwood and Coulson, 2006).

Monotremes, the egg-laying mammals, diverged from viviparous mammals over 186 million years ago (MYA) (Bininda-Emonds et al., 2007). There are two distinct living monotreme clades comprising of five species (Rowe et al., 2008); the short-beaked echidna

(Tachyglossus aculeatus) (Griffiths, 2012), three long-beaked echidna species (Zaglossus attenboroughi, Zaglossus bartoni, and Zaglossus bruijni) (Gambaryan and Kuznetsov, 2013), and the platypus (Ornithorhynchus anatinus) (Johansson et al., 2002, Bininda-Emonds et al.,

2007). Eutherians and marsupials shared their last common ancestor more recently, between

130 to 180 MYA (Nilsson et al., 2004, Nilsson et al., 2010), resulting in more physiological similarities than with monotremes, such as giving birth to live young (Tyndale-Biscoe and

Renfree, 1987).

One of the key differences between eutherians and marsupials is their prenatal and postnatal investment in offspring. Marsupials have a relatively short gestational period in comparison to eutherians, resulting in highly altrical young (Block, 1964, Ashman and

Papadimitriou, 1975, Tyndale-Biscoe and Renfree, 1987, Old and Deane, 2000); Block

(1964) described a newborn marsupial to be developmentally equivalent to a nine-week-old human foetus. The major developmental stages which take place during the first few postnatal months in marsupials have been the focus of many reproductive investigations of marsupials, as these events occur outside a sterile uterine environment. (Block, 1964, Yadav et al., 1972) In contrast, the major developmental milestones in eutherians only occur during their prenatal period, and as a result low survival rates occur in underdeveloped individuals

(Hamilton et al., 2010). At birth marsupial neonates have well-developed forelimbs and mouth parts, allowing them to climb towards the mother’s teat and successfully attach to the

19 teat (Edwards and Deakin, 2013). The development of the forelimbs and mouth parts allow the neonate to embark on a lengthy and fixed lactation period , where important physical

(Cooper and Steppan, 2010) and immunological maturation occurs (Old and Deane, 2000). In the past, the advantages of marsupial reproduction have been heavily debated (Kirsch, 1977a,

Kirsch, 1977b, Parker, 1977, Low, 1978, Cockburn, 1989). Specific benefits for short gestation periods include reduced physiological energy cost on the mother, reduced susceptibility to predator attack, and increased foraging capabilities (Kirsch, 1977a, Kirsch,

1977b). Since marsupials are ectothermic at birth and gradually become endothermic during the lactation period (Ikonomopoulou and Rose, 2006), neonates have lower metabolic rates, which increases postnatal parental care, postpartum (Hopson, 1973, Case, 1978).

1.1.1 Marsupial Reproductive Strategies

Protection from environmental risks is afforded to marsupials through the marsupium, or pouch (Edwards and Deakin, 2013). Russell (1982) described six different types of pouches that have varying structural distinction between species. Type one pouches only have skin folds on the underbelly of the mother for the neonate to adhere to (or ‘pouchless’)

(Griffiths and Slater, 1988). Type two has a partial covering of the mammary area; type three, the fold of skin is arranged in a circular pattern with a central opening to the teat. In type four pouches, the mammary area is covered and the mammary glands are located in two pockets on the underbelly of the mother. Type five and six pouches are completely enclosed and deep, the difference being, type five is located on the underbelly (anterior), whereas type six is located on the backside (posterior) of the mother (Russell, 1982). Different pouch types can also influence the parental care given to neonates (Russell, 1982). Species with deep and enclosed pouches will typically have 1-2 neonates per litter (Edwards and Deakin, 2013) in which the neonate will remain in the pouch until a certain stage of development has occurred

20 and the mother can either leave the neonate in a nest, or the neonate has the ability to follow the mother on foot (Russell, 1982). Deep and enclosed pouches are beneficial, as these species have better control of environmental factors, such as humidity and warmth, which allows the neonate to reach the developmental stage where their eyes are open, they have fur, and can thermoregulate before leaving the pouch (Russell, 1982). On the other hand, the

‘pouchless’ marsupials like that of the gray short-tailed opossum (Monodelphis domestica)

(Fadem et al., 1982), have multiple offspring per litter and the mother leaves the young in the nest at an early developmental stage where their eyes are still closed and they have no apparent thermoregulation (Edwards and Deakin, 2013). Although ‘pouchless’ marsupials leave their neonates in a nest at a substantially early developmental stage in comparison to deep and enclosed pouched species, parental care of the neonate is still ongoing (Russell,

1982). When neonates of ‘pouchless’ species are left in the nest, the mother only leaves the nest briefly for foraging purposes, otherwise the mother will remain in the nest keeping the neonates close to her underbelly, allowing the neonates to voluntarily suckle from her mammary glands (Aslin, 1974, Morton, 1978).

In addition to the pouch, marsupials also produce milk for the neonate as another form of protection in the form of antibodies (or immunoglobulins) (Deane and Cooper, 1984). The transfer of milk is multifaceted over four phases associated with different developmental stages. Joss et al. (2009) described these phases in the tammar wallaby (Notamacropus eugenii); phase 1 includes secretions of high levels of antibodies, lipids and proteins

(equivalent to colostrum), and ceases 48 h postpartum. Phase 2a occurs during the first 100 days postpartum, where the neonate adheres permanently to the teat and most immunological development occurs (Nicholas, 1988, Joss et al., 2009). Phase 2b covers days 100-200 and the neonate suckles less frequently, both phase 2a and 2b includes secretions of high levels of carbohydrates and decreased levels of proteins and fats (Nicholas, 1988). At phase 3, the final

21 phase, milk secretions consist of high levels of proteins and fats, and low levels of carbohydrates (Young et al., 1997). During this final phase, the neonate spends less time in the pouch as it is less reliant on the milk (Edwards and Deakin, 2013).

1.2 Marsupial Immunology

It was once believed that the marsupial immune system was ‘primitive’ in comparison to eutherians (Jurd, 1994), however copious studies have since described it to be just as complex as the eutherian immune system (Bell, 1977, Harrison and Wedlock, 2000, Miller and Belov, 2000, Belov et al., 2013, Old, 2016), despite the marsupial immune system developing and maturing in a non-sterile environment (Old and Deane, 1998). The development of marsupial immune tissues have been studied extensively over the past couple of decades (Table 1.1) (Block, 1964, Ashman and Papadimitriou, 1975, Basden et al., 1996,

Basden et al., 1997, Old et al., 2003, Old et al., 2004b, Belov et al., 2013, Borthwick et al.,

2014, Borthwick and Old, 2016). Marsupials, like eutherians, have both primary and secondary immune tissues, which have proven to be comparable in development, appearance, and function (Old et al., 2003). Eutherian primary immune tissues are involved in the production and maturation of lymphocytes (e.g. B- and T-cells), and include the thymus and bone marrow (Block, 1964, Ashman and Papadimitriou, 1975, Old et al., 2004a). The secondary tissues includes, but is not limited to, the spleen, lymph nodes, mucosal-associated lymphoid tissues (MALT), gut-associated lymphoid tissues (GALT), bronchus-associated lymphoid tissues (BALT), tonsils, and Peyer’s patches (Block, 1964, Ashman and

Papadimitriou, 1975, Basden et al., 1996, Cisternas and Armati, 1999). In contrast to eutherians, marsupials are immunologically immature at birth (Block, 1964, Old et al., 2003,

Belov et al., 2013), and therefore the maturation of primary and secondary tissues occurs during ‘pouch life’ (Old and Deane, 2000, Borthwick et al., 2014, Borthwick and Old, 2016).

Table 1.1 Summary marsupial immune tissues, examined in the thymus, bone marrow, lymph nodes, and spleen.

Species Tissue First Observation Maturation Observation Reference Virginian opossum Thymus Day 1 (thoracic) Days 23-32 (Block, 1964, Cutts and (Didelphis virginiana) Bone Marrow Days 6-7 Days 65-100 Krause, 1982) Lymph Nodes Day 4 (mediastiunum), Days 8-9 (caudal) Day 100+ Spleen Day 4 Day 100+ Quokka (Setonix Thymus Day 1 (cervical), Day 7 (thoracic) Days 90-120 (cervical), Day 120 (thoracic) (Ashman and brachyurus) Bone Marrow Day 4 Not observed Papadimitriou, 1975, Lymph Nodes Day 5 (cranial) Days 120-150 Stanley et al., 1972, Spleen Day 7 Not observed Yadav and Eadie, 1972) White-eared opossum Thymus 10 mm CRL* 24 mm CRL* (Coutinho et al., 1995) (Didelphis albiventris) Bone Marrow Not observed Not observed Lymph Nodes 75 mm CRL* Not observed Spleen Not observed 80 mm CRL* Tammar wallaby Thymus Day 2 (cervical), Day 6 (thoracic) Day 38 (cervical), Day 60 (thoracic) (Basden et al., 1996, (Notamacropus eugenii) Bone Marrow Day 4 Not observed Basden et al., 1997, Old Lymph Nodes Day 1 Day 150 and Deane, 2003) Spleen Day 3 Day 120 Northern brown bandicoot Thymus Day 1 (thoracic) Day 60 (thoracic) (Cisternas and Armati, (Isoodon macrourus) Bone Marrow Not observed Not observed 1999, Haynes, 2001) Lymph Nodes Day 1 (deep inferior cervical), Day 4 Day 60 (inferior cervical and axillary) Spleen Day 7 Not observed Common brushtail possum Thymus Day 1 (thoracic) Day 48 (thoracic) (Johnstone, 1898, Baker (Trichosurus vulpecula) Bone Marrow Not observed Not observed et al., 1999) Lymph Nodes Days 48-53 Not observed Spleen Day 48 Day 150+ Stripe-faced dunnart Thymus Day 12 Not observed (Old et al., 2003, Old et (Sminthopsis macroura) Bone Marrow Day 11 Days 40-60 al., 2004a, Old et al., Lymph Nodes Day 15 Day 31 (cervical and mammary) 2004b) Spleen Day 12 Day 74 * CRL = crown-lump length. Measurement of the marsupial neonate, from the head (crown) to the buttocks (rump). Coutinho et al. (1995) utilised neonates at 12, 14, 24, 29, 45, 50, 58, 60, 75, and 80 mm CRL to measure the tissue development of the white-eared opossum.

The thymus is the first immunological tissue to mature in marsupials (Basden et al.,

1997), and due to its important role in cell-mediated immunology, via the production of T- cells, it is the most widely investigated immune tissue in marsupial immunology (Johnstone,

1898, Yadav et al., 1972, Cisternas and Armati, 1999, Wong et al., 2011b). Anatomical location and whether both the cervical and thoracic thymuses are present varies among the marsupial lineage (Old and Deane, 2000). All marsupials (except wombats) have a thoracic thymus, while the Diprotodontia order of marsupials also have a cervical thymus (Yadav et al., 1972, Haynes, 2001). Yadav et al. (1972) reported the variance in presence of a cervical thymus in the quokka (Setonix brachyurus) was due to the small size of the thoracic cavity, whilst Ashman and Papadimitriou (1975) thought it may be due to the microbiological enriched environment of the pouch. During the first few days after birth, the thymus begins to mature rapidly and mature lymphocytes start to appear (Block, 1964, Rowlands et al., 1964,

Stanley et al., 1972, Johnstone, 1898, Baker et al., 1999). Detection of CD3+ T-cells were detected at day 2 postpartum in both the white-eared opossum (Didelphis albiventris)

(Coutinho et al., 1994) and common brushtail possum (Trichosurus vulpecula) (Baker et al.,

1999), however full maturation of the thymus occurred in the possum on day 25 postpartum

(Baker et al., 1999). Whereas in the Virginian opossum (Didelphis virginiana), lymphocytes appeared as early as day 1 postpartum, histological development of the Hassall’s corpuscles began on day 5 and the opossum thymus continued to develop its medullary and cortical areas until day 13-16, being fully matured by day 23 postpartum (Table 1.1) (Block, 1964).

Unlike eutherians, the bone marrow is not actively haematopoietic at birth, this role is taken on by the liver due to the bones of the neonate being solely cartilaginous (Old and

Deane, 2000). During the first two weeks postpartum, haematopoiesis is increasingly observed in the bone marrow of marsupial species and the liver’s involvement starts to decline. In the Virginian opossum, primitive bone marrow appeared in the diaphysis of the

24 endochondral bones and primary bone marrow observed in the membranous bone on day 5 postpartum (Block, 1964). On days 13-16, the bone marrow continued to increase in the endochondral bones and at days 23-32, they were filled with bone marrow (except in the epiphysis). The opossum bone marrow appeared to be similar to that of adult mammals by days 65-100 postpartum (Table 1.1) (Block, 1964). In other marsupial species, such as the quokka (Setonix brachyurus) and tammar wallaby, primary bone marrow started to appear as early as day 4 postpartum (Ashman and Papadimitriou, 1975, Basden et al., 1996), and by the end of the second week postpartum, a diversity of cells including granulocytes, erythroblasts and lymphocytes, had increased their numbers substantially (Ashman and Papadimitriou,

1975, Basden et al., 1996). By the end of the first month in the tammar wallaby, the bone marrow had fully taken over the role of haematopoietic production (Block, 1964, Old et al.,

2004a).

Secondary tissues such as the spleen and lymph nodes start to develop and mature much later than the thymus (Old and Deane, 2000). Characteristically, adult spleen and lymph nodes have follicles and germinal centres throughout their respective tissues which are the major sites for B-cell proliferation and continued development (Belov et al., 2013). White and red pulp have been observed in adult marsupial splenic tissues, similar to eutherians, however studies have reported very small differences in size, and number of sinuses and follicles (Cutts and Krause, 1982, Baker et al., 1999, Old et al., 2004a). Spleen and lymph node development has increasingly been studied in numerous species, including the Virginian opossum (Block, 1964), quokka (Ashman and Papadimitriou, 1975), white-eared opossum

(Coutinho et al., 1995), tammar wallaby (Basden et al., 1996, Basden et al., 1997, Old and

Deane, 2003), northern brown bandicoot (Isoodon macrourus) (Cisternas and Armati, 1999), common brushtail possum (Baker et al., 1999), and stripe-faced dunnart (Sminthopsis macroura) (Old et al., 2004a, Old et al., 2004b). In marsupial neonates, the spleen is very

25 immature at birth. Composed of mesenchymal tissue, the spleen starts to develop as early erythrocytes and megakaryocytes begin to populate the tissue, which is followed on by granulopoietic cells (Cutts and Krause, 1982). The spleen begins to mature with the appearance of red and white pulp, and reaches maturity when germinal centres can be found

(Old, 2016). In the Virginian opossum, large lymphocytes were apparent on day 2, and only rare erythrocytes and megakaryocytes appeared on day 3 (Block, 1964). However, megakaryocytes were more abundant in the tammar wallaby on day 3 (Basden et al., 1996).

Haematopoiesis, erythropoiesis, granulocytopoiesis, and size of the spleen continually increased during the first two weeks postpartum, in the Virginian opossum, and between days

60 and 100, splenic nodules and germinal centres started to appear (Block, 1964, Cutts and

Krause, 1982). By day 60, in the tammar wallaby, the splenic tissue was much more distinct with areas of red and white pulp, with the white pulp continuing to increase in abundance until four months after birth (Basden et al., 1996). As the spleen continues to mature, the germinal centre and follicles are utilised more for B-cell maturation, differentiation, and proliferation (Old, 2016). In comparison to the spleen, rudimentary lymph node tissues are not present until day 4 postpartum, of the Virginian opossum, quokka, and northern brown bandicoot (Block, 1964, Ashman and Papadimitriou, 1975, Cisternas and Armati, 1999).

However, the developmental pattern of the lymph nodes is similar to that of the spleen

(Bryant and Shifrine, 1974), whereby during the first two weeks postpartum, various cell types, lymphocytes, myelocytes and erythroblasts were visible in the Virginian opossum

(Block, 1964), and additional myeloid cells and megakaryocytes were seen in the quokka

(Ashman and Papadimitriou, 1975, Old, 2016).

1.3 Marsupial Genomics

In the past studying the genome of marsupials has been difficult and traditional strategies required an abundance of pre-existing resources (Abts et al., 2015, Sette et al.,

2005). The utilisation of conventional experimental methods that generate primers based on eutherian sequences for reverse-transcriptase polymerase chain reactions (RT-PCR) have limited the practicability of marsupial genomic studies (Harrison and Wedlock, 2000).

Advancements in the field have enabled researchers to utilise sequencing technology, from a whole shotgun sequencing (WGS) approach (Mikkelsen et al., 2007) to now using next generation sequencing (NGS) technology for Sanger sequencing (Murchison et al., 2012,

Deakin, 2012). The marsupial genomes are similar in size to eutherians (Deakin, 2012), although they are packaged into larger and fewer chromosomes. The gray short-tailed opossum was the first marsupial species to have its genome sequenced and is available through the Broad Institute and NCBI Genbank (Mikkelsen et al., 2007). This resource has provided invaluable data to analyse the evolution of developmental biology (Samollow,

2006), immunogenetics (Deakin et al., 2006, Mikkelsen et al., 2007), cancer development, and disease susceptibility (Wong et al., 2006) of the different mammalian lineages. The opossum genome was Sanger-sequenced to ~7x coverage or draft quality, producing a highly usable assembly, reflected by the large contig N50 of 108kb and scaffold N50 of 60mb

(Papenfuss et al., 2010). However, the annotation of the opossum was largely based on human transcripts and protein sequences (Hubbard et al., 2007), due to no large-scale transcriptomic data for the species (Papenfuss et al., 2010). Despite sequencing of large amounts of ‘good quality’ conserved genes in the opossum, the genes predominately involved in immune responses have failed to be identified and/or annotated due to the high rate of divergence (Wong et al., 2011a). Immune genes, for the most part, succumb to intense selective pressure due to the requirements of forever evolving pathogens (Zelus et al., 2000).

As a result, the Ensembl Consortium has missed many immune genes and less than a third of the opossum immune genes previously annotated (Wong et al., 2006, Belov et al., 2006) have been predicted by Ensembl (Curwen et al., 2004). Meanwhile, WGS and Sanger sequencing approaches were used to sequence the genome of the tammar wallaby (Renfree et al., 2011).

From this data for the tammar wallaby, ~2x sequence coverage was submitted to the NCBI archives and the initial assembly was constructed from low coverage Sanger sequences.

Additionally, a 5.9x sequence coverage was generated at Baylor College of Medicine Human

Genome Sequencing Centre and used for performing contig correction, super-scaffolding, and improvements on the initial assembly (Renfree et al., 2011).

Since the initial sequencing of the opossum and tammar wallaby genomes there have been multiple genome sequencing projects. The Tasmanian devil (Sarcophilus harrisii), for example, has provided much needed insights into Devil Facial Tumour Disease (DFTD), the tissue of origin, and the evolution of the tumour (Pyecroft et al., 2007). To understand the responses to DFTD, two male devil genomes were first sequenced, each inhabiting different locations (Miller et al., 2011). Sequencing was undertaken by multiple NGS platforms and the data from each devil combined to produce a genome assembly of a 147.5 kb N50 contig with ~140,000 scaffolds (Miller et al., 2011). The combination of genome information was done with the aim of detecting genetic variation between the two individuals (Deakin, 2012).

At the conclusion of the first sequencing project, a second genome project was initiated, in which a 5-year-old female devil was sequenced and annotated. This project utilised the

Illumina platform to assemble a 1.8 Mb N50 contig with ~35,000 scaffolds (Murchison et al.,

2012). Along with flow cytometry, 99% of the scaffolds were assigned to chromosomes, which was an immense improvement on the initial Tasmanian devil genome project

(Murchison et al., 2012). Furthermore, annotated genes were identified using the Ensembl

Genebuild Pipeline from 12 pooled devil tissues to construct the first marsupial

28 transcriptome. As a result of such efforts, a total of 18,775 protein coding genes were identified, 1,213 of these genes did not have a human or opossum orthologue (Murchison et al., 2012).

Recently, Johnson et al. (2018) assembled the koala (Phascolarctos cinereus) genome by long-read Illumina sequencing. Its genome was sequenced and assembled using 57.3-fold

PacBio long-read coverage. The primary contigs consist of 3.19 Gb with an N50 of 11.6Mb and the alternate contigs totalled 230 Mb with an N50 of 48.8Kb. Additionally, ~30x sequence coverage of Illumina short reads were used to improve the initial assembly

(Johnson et al., 2018). The koala genome project resulted in the highest coverage of coding regions in any marsupial genome project, scoring 95.1% of mammalian single-copy orthologues, as well as identifying 6,124 protein-coding genes from 2,118 gene families. An analysis of the gene families showed 1,089 of these gene families have more members in the koala than in any species investigated to date (Johnson et al., 2018). The quality of the koala genome has initiated research into the genetic diversity of different populations across

Australia, provided a resource into the disease pathogenesis and defence of the Chlamydia pecorum infection, and the characterised specific genes related to taste and smell (Burgess,

2018).

1.3.1 Transcriptomics

The development of NGS technologies has increased the potential to expand our knowledge of genomics and transcriptomics (Abts et al., 2015), where a transcriptome is a complete set of RNA molecules (transcripts) found at a specific stage of development or physiological condition in a cell (Wang et al., 2009a). The aim of transcriptomics is to identify and register all the species of the RNA transcript (coding and non-coding inclusive),

29 determine the transcriptional structure of the gene, and quantify the different expression levels of the transcripts at different developmental stages and/or under numerous physiological conditions (Wang et al., 2009a). Previously, the characterisation of the marsupial genome has largely been based on the use of gene prediction tools developed for the human genome (Papenfuss et al., 2010). Analysis of the transcriptome allows any species genome to be sequenced at a higher quality than any traditional laboratory technique completed previously (Allendorf et al., 2010). A number of marsupial species have since been used for gene expression analyses.

Several transcriptomic studies have been undertaken to show the gene expression profiles in various tissues in the tammar wallaby. Wong et al. (2011b) analysed the transcriptome of the cervical and thoracic thymuses in a 178-day old tammar wallaby neonate, which was the first investigation to compare the gene expression of both thymuses in any mammalian species. Their results saw that T-cell development and basic thymic function of both tammar wallaby cervical and thoracic thymuses were highly similar to eutherian thymuses and found specific genes involved in the differentiation and function of

T-cell in both thymuses (Wong et al., 2011b). A complete transcriptome was sequenced in the koala (Hobbs et al., 2014), whereby a number of different tissues were sampled from both a female (liver, brain, heart, kidney, lung, adrenal gland, spleen, uterus, and pancreas) and male (liver, kidney, spleen, bone marrow, lymph node, salivary gland, and testes) individual.

This study used RNA-sequencing technology and de novo assembly to generate about 100 Gb of sequences per individual and covering ~15,000 koala genes (Hobbs et al., 2014). Recently, the long-nosed bandicoot (Perameles nasuta) also had multiple tissues (liver, heart, kidney, and spleen) sequenced from one male individual (Morris et al., 2018). Immune genes and immune gene families of both the passive and adaptive immune systems were characterised, including immunoglobulins, T-cell receptors, major histocompatibility complex (MHC I and

MHC II classes) receptors, natural killer cells, cytokines, chemokines, and pattern recognition receptors. This survey indicated the bandicoot has a diverse range of expressed immune genes which are similar to those previously identified in the opossum, Tasmanian devil and koala (Morris et al., 2018).

With the aim to create a better understanding of the evolutionary gestational and lactational periods which are unique to marsupials, transcriptomic data has been generated and analysed from the milk and mammary glands of the koala (Morris et al., 2016), milk of the Tasmanian devil (Hewavisenti et al., 2016), uterus of the opossum (Hansen et al., 2016), and the mammary glands and placenta of the tammar wallaby (Lefèvre et al., 2007, Guernsey et al., 2017). Hewavisenti et al. (2016) sequenced, annotated and characterised the genes of the passive immune system of the Tasmanian devil. They successfully identified 233,660 transcripts, of which more than 17,000 were identified as protein-coding genes and 846 were immune genes. They collected milk samples at mid-lactation with the assumption that marsupial neonates have encountered new pathogens as they begin to emerge from the pouch

(Adamski and Demmer, 2000, Daly et al., 2007). The immune compounds in the milk of the devil found highly expressed caseins, β-lactoglobulin, and α-lactoglobulin (Hewavisenti et al., 2016) during the mid-lactation period. β-lactoglobulin was also highly expressed during mid-lactation of the tammar wallaby (Lefèvre et al., 2007) and during the early and late lactation periods in the koala (Morris et al., 2016). In comparison to the tammar wallaby

(Lefèvre et al., 2007), of the devil had a higher expression of early lactation proteins (ELP) and late lactation proteins (LLP) during mid-lactation. Hewavisenti et al. (2016) made the assumption that the discrepancies between expression levels of these genes between the two species are due to either recruitment of different proteins during mid-lactation or ELP and

LLP having additional and unknown functions. A global uterine transcriptomic analysis has been performed on the gray short-tailed opossum using NGS on virgin, late stage pregnant

31 and non-pregnant individuals with the objective to establish what genes are associated with pregnancy in marsupials (Hansen et al., 2016). Hansen et al. (2016) generated paired-end reads using the Illumina HiSeq 2000 platform and then aligned them to the opossum genome

(Mikkelsen et al., 2007). The outcome of this investigation found over 900 opossum genes had significantly higher expression in pregnant individuals compared to non-pregnant individuals, and another 1400 opossum genes expressed more so in non-pregnant individuals.

1.4 Model Marsupial: The gray short-tailed opossum (Monodelphis domestica)

It has been well established that the gray short-tailed opossum is an important, fully developed laboratory-bred marsupial utilised for many comparative biological investigations

(Lucero et al., 1998). The opossum has been bred in captive colonies in North America

(Kraus and Fadem, 1987, Miller et al., 1998, Wang et al., 2009b), Australia (Fadem et al.,

1982), and Europe (Moore, 1996, Zeller and Freyer, 2001) since the 1970s (Samollow, 2008), providing researchers with the unique opportunity to investigate various evolutionary processes that have given rise in modern mammalian lineages (Samollow, 2006).

The gray short-tailed opossum belongs to the family Didelphidae (Szalay, 2006) which is the most abundant of all the marsupial families in the Didelphimorphia order, with opossums being the most species-rich genus within the family (Gardner, 1993). The gray short-tailed opossum is mostly covered in a light grey fur with slight variations (red and/or white) between populations (O'Connell, 2007). Their tails are naked, semi-prehensile, and usually make up about half of their body length. The adult opossum body length ranges between 10-15cm, adult males weigh between 90-155g, whereas adult females weigh between 80-100g, making them one of the smallest didelphids (Gardner, 1993, O'Connell,

2007). The sexual maturity of the opossum is reached at about 18-20 weeks and their mating

32 behaviour is strongly linked to the olfactory system, in which males will mark their surroundings via their sternal gland secretions (Trupin and Fadem, 1982). The opossum has a short 14 day pregnancy, and produces young that are developmentally equivalent to a 40-day- old human embryo (Block, 1964, Wang et al., 2009b, Rousmaniere et al., 2010). Opossums have approximately three litters a year, of up to thirteen (average of eight), hence has established itself as a true “laboratory marsupial” (Samollow, 2008). Unlike laboratory rodents and guinea pigs, opossums are housed separately unless paired up for breeding purposes. At 6-months of age, individuals can be housed together to induce oestrus, which occurs within 8 days (Stone et al., 1996). Since opossums have no enclosed pouch, the neonates are exposed on the underbelly of the mother, allowing for easy observation and manipulation during the early postnatal period (Rousmaniere et al., 2010). The neonates are attached to the teat of the mother for about two weeks, weaned at two months and reach sexual maturity between five and six months of age. Reproductive decline commences between 18 and 30 months of age and age-related death naturally occurs between 36 and 42 months (Stone et al., 1996).

The opossum has been actively used in a variety of research programs over the past

30 years, covering topics from behaviour, reproduction, physiology, immunology, genetics and several human-related health processes. Now with availability of the opossum genome

(Mikkelsen et al., 2007), research areas have successfully progressed into comparative genomics, detection and mapping of specific genes and gene expression. This resource is not just vital for investigating normal verses abnormal physiological states, but also practical uses for animals that are susceptible to disease in the real world (Samollow, 2006, Samollow,

2008).

1.5 Research Aim

To investigate the expression of immune genes in marsupials, a transcriptomic analysis is required. RNA-sequencing (RNA-Seq) will help us to identify and annotate key B- cell transcriptomes, and ultimately address uncertainties about immune function in marsupials and advance the examination of the immunological divergence between mammalian groups (Wang et al., 2009a). RNA-Seq was developed from various novel high- throughput DNA sequencing data methods (Wang et al., 2009a). The RNA-Seq method utilises deep-sequencing technologies as an approach to transcriptomic profiling (Wang et al.,

2009a, Chu and Corey, 2012). The methodology allows for a population of RNA to be converted to a library of cDNA fragments (or primers) with adaptors attached to one or both ends. As most reads are between 30-400 base pairs (bp) long (Toung et al., 2011), single-end or pair-end sequencing (Wang et al., 2009a) is necessary to obtain shortened reads

(Mortazavi et al., 2008). Reads can then be mapped to a reference genome or assembled de novo (Cloonan et al., 2008), and the expression levels for a gene can subsequently be determined by counting the number of reads that align to its exons (Wang et al., 2009a).

B-cells have a unique role in the humoral immune response of mammals. B-cells are able to provide detection of almost any type of foreign molecule, or antigen, through their differentiated cells to produce antibodies and elicit an immune response (Painter et al., 2011).

Therefore, the aims of this thesis are focused on the expression of B-cell genes in the gray short-tailed opossum. The research detailed here for the first time in a marsupial analyses the use of IgH and IgL chains in the B-cell repertoire of single cells isolated from spleen and peripheral blood. Secondly, the single-cell transcriptome of these B-cells was further analysed and the genes most highly expressed compared between the different IgH producers.

These findings will contribute to our understanding of comparative immunology and genomics, and aid in future studies of the opossum and other marsupial species.

1.6 Thesis Outline

This thesis is presented in five chapters, inclusive of this introduction (Chapter 1).

The following three chapters are presented as manuscripts for peer review journals, in which

Chapter 2 is published, Chapter 3 is Under Review, and Chapter 4 is a prepared manuscript.

These chapters are formatted according to the journal specifications, and includes the referencing style for those journals. The final chapter of this thesis (Chapter 5) discusses the overall findings of the research and presents an overall conclusion and future direction for this field of study. The following chapters of this thesis are:

Chapter 2: Immunogenetics of marsupial B-cells

Chapter 3: Single-cell transcriptome analysis of the B-cell repertoire reveals the usage of immunoglobulins in the gray short-tailed opossum (Monodelphis domestica)

Chapter 4: Single-cell transcriptomic analysis of marsupial B-cells, the gray short-tailed opossum (Monodelphis domestica)

Chapter 5: Concluding summary and future directions

1.7 Chapter 1 References

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CHAPTER 2

Immunogenetics of Marsupial B-cells

Andrea L. Schraven1, Hayley J. Stannard2, Oselyne T.W. Ong3, Julie M. Old1

1School of Science and Health, Hawkesbury Campus, Western Sydney University, Locked

bag 1797, Penrith, NSW 2751, Austral00ia

2Charles Sturt University, School of Animal and Veterinary Sciences, Wagga Wagga, NSW

2678, Australia

3QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia

Molecular Immunology; 2020, vol. 117, pp. 1-11

2.1 Chapter Outline and Authorship

Chapter 2 provides a detailed literature review on the knowledge of marsupial B-cell genetics and biology. It discusses the similarities and differences between marsupial and eutherian B- cells, in regard to genetic presence, homology, and developmental stages, as well as highlights the areas requiring further research.

This chapter has been published in Molecular Immunology, and the manuscript is formatted as per journal specifications. The manuscript is jointly authored; I am the primary author. I collected and analysed the data and wrote the manuscript. Hayley Stannard, Oselyne Ong, and Julie Old reviewed the manuscript and provided feedback.

Published version available as:

Schraven, A. L., Stannard, H. J., Ong, O. T. W., Old, J. M. 2020. Immunogenetics of marsupial B-cells. Molecular Immunology. 117, 1-11. https://doi.org/10.1016/j.molimm.2019.10.024

2.2 Introduction

The gray short-tailed opossum (Monodelphis domestica) was the first marsupial species to have its genome sequenced and available through the Broad Institute and NCBI

Genbank (Mikkelsen et al., 2007). After the release of the gray short-tailed opossum genome, the sequencing of other marsupials, in particular Australian marsupials, soon followed, including the tammar wallaby (Notamacropus eugenii) (Renfree et al., 2009), Tasmanian devil (Sarcophilus harrisii) (Murchison et al., 2012) and koala (Phascolartos cinereus)

(Hobbs et al., 2014). The opossum genome was Sanger-sequenced to ˜7x coverage or draft quality, producing a highly usable assembly, reflected by the large contig N50 of 108 kb and scaffold N50 of 60 mb (Papenfuss et al., 2010). However, at the time, the Ensembl

Consortium had no large-scale transcriptomic data available for the annotation of the species

(Papenfuss et al., 2010), and consequently, the annotation was based largely on human

(Homo sapiens) transcripts and protein sequences (Hubbard et al., 2007). Despite the sequencing of large amounts of ‘good quality’ conserved genes, many genes predominately involved in immune responses were not identified and/or annotated due to the high rate of divergence (Wong et al., 2011a). Immune genes, for the most part, succumb to intense selective pressure due to the requirements of forever evolving pathogens (Zelus et al., 2000), and for this reason, the Ensembl Consortium began annotating the immune genes of the gray short-tailed opossum. However, the majority of marsupial genes are yet to be identified through expression and have therefore only been predicted by Ensembl (Curwen et al., 2004,

Wong et al., 2006, Belov et al., 2006). With recent bioinformatic advancements, in particular genome annotation, researchers now have the ability to understand the immunocompetence of different mammalian species through cell-mediated and humoral immune response investigations.

To date, researchers have detected various genes and processes, including immunoglobulins (Ig), T-cell receptors, and MHC genes, suggesting the marsupial immune system is highly similar and just as complex as their eutherian counterparts using traditional molecular techniques (Samollow, 2006). Responses to graft rejection, an example of a cell mediated response have been investigated in the Virginian (Didelphis virginiana) and gray short-tailed opossums, tammar wallaby and quokka (Setonix brachyurus) (Laplante et al.,

1969). In the Virginian opossum, pouch young accept skin grafts up to day 12 postpartum; after this time, the rejection of tissue is apparent and has been suggested to occur as a result of T-cell responses (Laplante et al., 1969). For humoral responses, Igs have been measured in the serum of the Virginian opossum (Rowlands et al., 1964). Rowlands et al. (1964) found that Igs could not be detected in the serum until after exposure to a bacterial antigen at day 8–

15, and Kalmutz (1962) only detected an Ig response at day 17 to viral antigens. Whilst techniques used in immunochemistry have allowed investigators to detect tammar wallaby

CD79b+ lymphocytes at day 7 postpartum (Old and Deane, 2003).

In eutherians, B-cells are important for humoral immunity as their genes related to immunity undergo vigorous developmental, differentiation, regulation, and functional processes and checkpoints to enable them to mediate the production of antigen-specific antibodies in the presence of foreign molecules. In contrast to eutherians, little is known about marsupial B-cells due to the lack of investigation into the marsupial genome. For this reason, the purpose of this paper is to review what is already known in regards to marsupial

B-cells, with a comparison to eutherian studies. Analyses of B-cell genes have the potential to shine light on marsupial humoral immune responses as well as contribute to comparative evolutionary studies of B-cells.

2.3 Mammalian Evolution

Mammals arose ˜200 million years ago (Archibald and Deutschman, 2001, Bininda-

Emonds et al., 2007) and are classified into three lineages; eutherians, marsupials

(metatherians), and monotremes (prototherians). Monotremes were the first to diverge ˜166 million years ago and exhibit both reptilian and mammalian features (Renfree et al., 2009).

Marsupials then separated from eutherians ˜160 million years ago (Kumar and Hedges,

1998). Both eutherians and marsupials give birth to live young, while monotremes are ‘egg laying’ mammals (Warren et al., 2008). The major difference between eutherians and marsupials is while eutherians are born fully developed after a long gestational period (e.g. humans have nine month gestation period), marsupials have a shorter gestation period and are born with no mature lymphoid tissues (Deane and Cooper, 1988, Block, 1964, Tyndale-

Biscoe and Renfree, 1987). The survival of the marsupial is heavily dependent on the young having the ability to obtain passive acquired immunity from its mother. At birth marsupials are considered to be at the same physical and physiological stage of development as a nine- week-old human foetus (Block, 1960). Therefore, while marsupials continue developing externally, eutherians are still undergoing prenatal development at a similar life stage (Wang et al., 2012b). Marsupial pouch young can range from 3 mg (honey possum, Tarsipes rostratus) to 828 mg (western grey kangaroo, Macropus fuliginosus) (Tyndale-Biscoe and

Renfree, 1987) at birth, with no developed hind legs (Graves and Westerman, 2002).

Newborn marsupials only have partially developed forelimbs to aid their upwards climb from the vaginal opening to their mother’s teat, where they undergo a lengthy lactation period to complete their physical and immunological development (Tyndale-Biscoe and Renfree,

1987).

For many decades our understanding of the marsupial immune system has lagged behind that of the eutherian immune system (Morris et al., 2010) although there is evidence

48 that suggests the lymphoid tissues and cells examined in marsupials are highly similar to eutherians (Ashman and Papadimitriou, 1975, Old and Deane, 2000, Wong et al., 2006,

Borthwick and Old, 2016). In the past, it was thought that marsupials (and monotremes) were only evolutionary steps in the progression to eutherians and were therefore inferior (Jurd,

1994). However there is now evidence suggesting that marsupials and eutherians have similar lymphoid tissues and cells (Block, 1964), and their immune system presumably functions in a similar manner (Block, 1964, Deane and Cooper, 1984, Graves and Westerman, 2002, Old,

2016, Wong et al., 2006). In addition, the knowledge that marsupials give birth to underdeveloped young could expand our knowledge on the evolution of developmental immunity (Old and Deane, 2000), including defence mechanisms used via an adaptive immune response (Edwards et al., 2012).

2.4 Marsupial Immunology

Marsupial haematopoiesis commences in the primitive yolk sac in utero, which continues in the liver (postpartum) until the bone marrow and thymus have matured (Old,

2016). This process is already complete in newborn eutherians, where the haematopoietic stem cells (HSCs) undergo development and maturation in the fetal liver and give rise to B- cells prior to moving to the bone marrow in the last stages of foetal development (Doulatov et al., 2012, Hadland and Yoshimoto, 2018). The liver has been identified as the major site for haematopoietic activity for the first few weeks postpartum (Borthwick et al., 2014), and is similar in the pattern of development as that in eutherians despite occurring prenatally in eutherians. After this time, the liver ceases its involvement in haematopoiesis and the bone marrow and thymus take over the role (Borthwick et al., 2014). Different studies have found that both granulocyte and erythrocyte lineages were observed in the liver, day 1 postpartum of the quokka, stripe-faced dunnart (Sminthopsis macroura) and tammar wallaby (Ashman

49 and Papadimitriou, 1975, Basden et al., 1996, Old et al., 2004). On day 3, the quokka’s haematopoietic cells have started to aggregate and form islands throughout the liver (Old et al., 2004), while on day 4, the northern brown bandicoot (Isoodon macrourus) liver has a high abundance of erythrocytes and erythrocyte precursor islands (Cisternas and Armati,

1999). After the first week postpartum, haematopoiesis rapidly decreases in the liver of the

Virginian opossum (Didelphis virginiana) (Cutts et al., 1973) and northern brown bandicoot

(Cisternas and Armati, 1999). Over the next few weeks to months, haematopoiesis continues to decrease, and the tammar wallaby and quokka are observed to have a few sparse haematopoietic islands during development by the end of the third month (Ashman and

Papadimitriou, 1975, Basden et al., 1996).

Lymphoid tissues and cell types develop at different stages in marsupials from the time of birth (Belov et al., 2013). The thymus is the first of the lymphoid tissues to develop and mature in marsupials, and is also the case for humans where the thymus is responsible for the maturation of T-cells (Wong et al., 2011b). The thymus has been identified in all marsupials studied, but not all species investigated have both the thoracic and cervical thymus, most only have the former (Old, 2016). For those marsupials that have both a thoracic and cervical thymus, the cervical thymus develops earlier and is considerably larger in size, however both perform the same function (Stanley et al., 1972, Wong et al., 2011b).

Block (1964) detailed the histological development of the thymus in the Virginian opossum, observing on day 1 postpartum a single sheet of epithelial cells and between day 23 and 32 postpartum a fully functional thymus. The timing of bone marrow maturation varies across different marsupial species (Old, 2016). The tammar wallaby has been observed to develop early formation of erythrocytes and granulocytes on day 4, by day 8 megakaryocytes were apparent, and by day 14, increased numbers and the first stages of diversification of haematopoietic cells were observed (Old, 2016).

The spleen is the last of the lymphoid tissues to mature (Old, 2016), where mesenchymal cells are the only representation of the spleen for the first couple of days postpartum for the Virginian opossum (Cutts and Krause, 1982), tammar wallaby (Basden et al., 1996), and quokka (Ashman and Papadimitriou, 1975). Haematopoietic activity slowly increases over the next few weeks and by the end of the first month, erythroblastic haematopoiesis is prominent in the quokka (Ashman and Papadimitriou, 1975). As for the

Virginian opossum, the appearance of white pulp increases exponentially in contrast to the red pulp (Block, 1964). The spleen continues to undergo erythropoiesis through adulthood and into old age of the Virginian opossum (Cutts and Krause, 1982, Old, 2016).

2.5 B-cells

B-cells are of particular importance in the immune system as they are found in multiple lymphoid organs (Holmes et al., 2008), and are classified as an adaptive humoral immune cell type (MacConmara and Lederer, 2005). They are able to detect foreign molecules that invade the body through the production of antibodies released from their differentiated cells (Painter et al., 2011). In mice (Mus musculus), B-cells are produced from the pluripotent haematopoietic stem cells which are found in the liver during the late stages of foetal development and continues development in the bone marrow after birth (Hardy and

Hayakawa, 2001). The appearance of marsupial B-cell development is similar to eutherians except B-cells are still produced in the liver postpartum until the bone marrow fully matures

(Borthwick et al., 2014, Old, 2016).

Many genes are specifically expressed in B-cells, throughout multiple stages of B-cell development, differentiation, and regulation. Signature genes associated with the B-cell include Igs (heavy and light chains) (Palmer et al., 2006), cell surface markers (e.g. CD79α/β)

(Deneys et al., 2001), signal transduction molecules (e.g. BTK, Lyn, and Syk) (Kurosaki,

2002) and transcriptional regulators (e.g. EBF, E2A and Pax5). These B-cell genes have been extensively studied in eutherian mammals particularly in humans and mice (LeBien, 2000,

Muschen et al., 2002, MacConmara and Lederer, 2005, Sanz et al., 2010). The distribution of

B-cells has been observed throughout the lymphoid tissues of mammals (Figure 2.1) by using various antibodies to track developmental stages of B-cells (Old and Deane, 2003). In eutherians, there are a multitude of genes expressed throughout the different stages of the B- cell’s lifetime, inclusive of development, differentiation, and function.

Figure 2.1 Phylogenetic tree based on B-cell relationship in vertebrate groups and divergence of marsupials referred to in this review, constructed using the NJ method.

In eutherians, developing B-cells are in constant contact with stromal cells that facilitate their development. Stromal cells produce interleukin (IL)-7 to support the proliferative expansion of progenitor (pro-) B-cells (Bagwell et al., 2015). Pro-B-cells are the earliest of the B-cell developmental stages and express the IL-7 receptor, CD43, c-Kit, B220,

BP1, and terminal deoxynucleotide transferase (TdT). During this developmental stage, the Ig heavy chain undergoes a genetic recombination called V(D)J (somatic) recombination where variable (V), joining (J), and sometimes diversity (D) gene segments are rearranged to allow for the Igs to adhere to the antigen, allowing for an adaptive immune response (Li et al.,

2004, Schatz and Swanson, 2011). Igs are glycoproteins constituting four polypeptide chains: two identical light and heavy chains which are linked together by disulphide bonds and noncovalent forces (Painter, 1998), and primarily involved in secondary immune responses.

Early pro-B-cells undergo D–J rearrangement while late pro-B-cell undergoes V-D–J rearrangement. Eutherian B-cell development then transitions from a pro-B-cell to a precursor (pre-) B-cell stage where chances of apoptosis are its highest. At this time, the Ig heavy chain is expressed at the cell surface with the surrogate light chain, known as the pre-

B-cell receptor (pre-BCR), to determine if the Ig heavy chain has undergone a successful recombination (Bagwell et al., 2015).

The maturation of eutherian B-cells is thought to be similar to marsupials and involves several stages (Conley, 2003) defined by the rearrangement of different Igs, expression of cluster of differentiation markers (CD), and location of the cells within the bone marrow, spleen, and/or lymph nodes (Conley and Cooper, 1998). The few B-cell genes that have been examined in marsupials, for example the gray short-tailed opossum, demonstrate that a B-cell dependent immune response can only be initiated after the first postnatal week, as all genes necessary to elicit an adaptive immune response have not yet

53 been expressed (Table 2.1) (Wang et al., 2012b). Marsupial B-cell dependent immune responses are correlated with the appearance of cells expressing mature lymphocyte-specific markers (Old and Deane, 2000, Duncan et al., 2010, Wang et al., 2012c). While developing

B-cells have been detected in late stage prenatal embryos, followed by B-cell maturation, which occurs exclusively postnatal in the opossum (Wang et al., 2012c). Despite having the knowledge of when specific B-cell stages are present, it remains unknown if these Ig repertoire changes in marsupial B-cells are analogous to eutherians.

Table 2.1 List of B-cell genes that have either been expressed or predicted in marsupial species, including their Genbank accession Identifications (ID). Gene Species Expressed/Predicted Genbank Expression Reference accession ID

CD10 M. domestica Predicted XM_007502344.1

P. cinereus Predicted XM_020988925.1

S. harrisii Predicted XM_012546630.2

V. ursinus Predicted XM_027876124.1

CD19 M. domestica Predicted XM_016423695.1

P. cinereus Predicted XM_021006522.1

S. harrisii Predicted XM_023497850.1

V. ursinus Predicted XM_027862685.1

CD20 M. domestica Predicted XM_001379088.3

P. cinereus Predicted XM_020976111.1

S. harrisii Predicted XM_003774020.3

V. ursinus Predicted XM_027858851.1

CD21 M. domestica Predicted XM_007481319.2

P. cinereus Predicted XM_021003160.1

S. harrisii Predicted XM_023504041.1

V. ursinus Predicted XM_027871517.1

CD22 M. domestica Predicted XM_001369696.3

P. cinereus Predicted XM_020977007.1

S. harrisii Predicted XM_012545693.2

V. ursinus Predicted XM_027855546.1

CD32 M. domestica Predicted XM_007480653.2

P. cinereus Predicted XM_020988655.1

S. harrisii Predicted XM_012547618.2

V. ursinus Predicted XM_027873972.1

CD34 M. domestica Predicted XM_007481323.2

P. cinereus Predicted XM_021007822.1

S. harrisii Predicted XM_003767657.3

V. ursinus Predicted XM_027871708.1

CD38 M. domestica Predicted XM_007496701.2

P. cinereus Predicted XM_020992954.1

S. harrisii Predicted XM_012553075.2

V. ursinus Predicted XM_027836551.1

CD72 M. domestica Predicted XM_007498802.1

P. cinereus Predicted XM_020977042.1

S. harrisii Predicted XM_023497642.1

V. ursinus Predicted XM_027839551.1

CD79a M. domestica Expressed ACZ13436.1 (Duncan et al., 2010) (IgA) XM_021004022.1 P. cinereus Predicted XM_023500926.1 S. harrisii Predicted XM_027858134.1 V. ursinus Predicted ACZ13437.1 N. eugenii Expressed AGO64767.1 (Duncan et al., 2010) O. fraenata Expressed (Suthers and Young, 2014)

CD79b M. domestica Expressed ACZ13438.1 (Duncan et al., 2010)

P. cinereus Predicted XM_020997545.1

V. ursinus Predicted XM_027872889.1

N. eugenii Expressed ACZ13439.1 (Duncan et al., 2010)

O. fraenata Expressed AII23405.1 (Suthers and Young, 2014)

IgG M. domestica Expressed AF098643.1 (Wang et al., 2009)

N. eugenii Expressed EF599608.1 (Daly et al., 2007)

T. vulpecula Expressed AF157619.1 (Belov et al., 1999a)

IgM M. domestica Expressed AF012109.2 (Miller et al., 1998)

IgE M. domestica Expressed AF035194.1 (Aveskogh and Hellman, 1998)

N. eugenii Expressed EF599607.1 (Daly et al., 2007)

T. vulpecula Expressed AF157617.1 (Belov et al., 1999b)

IGκ M. domestica Expressed AAF25575.1 (Wang et al., 2012c) ALB75225.1 S. harrisii Expressed ABV02004.1 (Ujvari and Belov, 2015)

N. eugenii Expressed AAL17619.1 (Daly et al., 2007) T. vulpecula Expressed (Belov et al., 2001)

IGλ M. domestica Expressed AAC98628.1 (Lucero et al., 1998) XP_020834443.1 P. cinereus Predicted ABV02011.1 N. eugenii Expressed (Daly et al., 2007) AAL37211.1 T. vulpecula Expressed (Belov et al., 2002b)

Pax5 M. domestica Predicted XM_007497997.2

P. cinereus Predicted XM_021004585.1

S. harrisii Predicted XM_003761344.1

V. ursinus Predicted XM_027859120.1

POU2AF1 M. domestica Predicted XM_007494855.2

P. cinereus Predicted XM_020989802.1

S. harrisii Predicted XM_023501417.1

V. ursinus Predicted XM_027847318.1

SPIB P. cinereus Predicted XM_020971799.1

S. harrisii Predicted XM_023506369.1

V. ursinus Predicted XM_027849772.1

SPI1 M. domestica Predicted XM_001370178.3

P. cinereus Predicted XM_003773728.3

S. harrisii Predicted XM_012552632.2

V. ursinus Predicted XM_027837740.1

Pu.1 M. domestica Predicted XM_001370178.3

P. cinereus Predicted XM_021009704.1

S. harrisii Predicted XM_003773728.3

V. ursinus Predicted XM_027837740.1

E2A M. domestica Predicted XM_016432215.1

P. cinereus Predicted XM_021009149.1

S. harrisii Predicted XM_012542196.2

V. ursinus Predicted XM_027853230.1

EBF1 M. domestica Predicted XM_007473955.2

P. cinereus Predicted XM_020992364.1

S. harrisii Predicted XM_023505848.1

V. ursinus Predicted XM_027841851.1

LYN M. domestica Predicted XM_007487109.2

P. cinereus Predicted XM_021005934.1

S. harrisii Predicted XM_012541316.2

V. ursinus Predicted XM_027877022.1

N. eugenii (isoform a) Expressed KC153239.1 (Suthers and Young, 2013)

N. eugenii (isoform b) Expressed KC153241.1 (Suthers and Young, 2013)

O. fraenata (isoform a) Expressed KC153240.1 (Suthers and Young, 2013)

O. fraenata (isoform b) Expressed KC153242.1 (Suthers and Young, 2013)

Syk M. domestica Predicted XM_016431278.1

P. cinereus Predicted XM_020979707.1

S. harrisii Predicted XM_003762986.3

V. ursinus Predicted XM_027839299.1

BTK M. domestica Predicted XM_001374558.3

P. cinereus Predicted XM_020963393.1

S. harrisii Predicted XM_023507144.1

V. ursinus Predicted XM_027837226.1

BLNK M. domestica Predicted XM_007478713.2

P. cinereus Predicted XM_020997077.1

S. harrisii Predicted XM_023495007.1

V. ursinus Predicted XM_027859783.1

BLK M. domestica Predicted XM_001381609.3

P. cinereus Predicted XM_020985383.1

S. harrisii Predicted XM_012539921.2

V. ursinus Predicted XM_027840386.1

IRF4 M. domestica Predicted XM_001378689.2

P. cinereus Predicted XM_020976679.1

S. harrisii Predicted XM_003760227.3

V. ursinus Predicted XM_027841712.1

IRF8 M. domestica Predicted XM_016427817.1

P. cinereus Predicted XM_020989230.1

S. harrisii Predicted XM_003758467.3

V. ursinus Predicted XM_027847649.1

2.5.1 Immunoglobulins

Early studies of the marsupial immune system included measurements of antibody responses and comparing these levels of response to those of eutherians (Kalmutz, 1962).

Primary immune responses of marsupials are very similar to eutherians, however the secondary immune responses are delayed and less effective than that of eutherian responses

(Kalmutz, 1962, Yadav and Eadie, 1972). In comparison to eutherians and monotremes, marsupial B-cells have four Ig heavy (H) loci isotypes (IgA, IgG, IGM and IgE), defined by their constant regions (Cα, Cγ, Cμ and Cε respectively) (Belov et al., 1999c). So far, multiple

Ig heavy chain genes, IgA, IgG, IgM and IgE inclusive, have been observed in a number of marsupial species including the Virginian opossum (Rowlands and Dudley, 1968), quokka

(Bell et al., 1974b), common brushtail possum (Trichosurus vulpecula) (Ramadass and

Moriarty, 1982), koala (Aveskogh and Hellman, 1998) and the gray short-tailed opossum

(Aveskogh and Hellman, 1998). Interestingly, IgD described in eutherians and the platypus

(Ornithorhynchus anatinus) has not been identified in any marsupial species to date (Figure

2.2) (Aveskogh and Hellman, 1998, Aveskogh et al., 1999). Homologous-based searches in the regions where IgD is expected to be located have been unsuccessful in the gray short- tailed opossum (Wang et al., 2009), despite large duplications and insertions of a non- functional copy of IgM being identified (Wang et al., 2009). The internal duplications of this downregulated IgM may have contributed to the loss of the IgD gene in the gray short-tailed opossum and possibly all modern marsupial species. IgD is still present in most eutherians, monotremes and some non-mammalian species (Figure 2.2) suggesting this Ig isotype is evolutionarily ancient (Ohta and Flajnik, 2006).

Figure 2.2 Unrooted phylogenetic tree based on amino acid alignments of vertebrate IgD sequences, constructed using the NJ method. Genbank accession numbers: I. punctatus IgD, ADF56020; S. trutta IgD, AAV28076; E. coioides IgD, AEN71107; M. musculus IgD, AAB59653; X. tropicalis IgD, ABC75541; P. sinensis IgD, ACU45375; O. anatinus IgD, ACD31540; C. siamensis IgD, AFZ39208; E. macularius IgD, ABY67439; H. sapiens IgD, AAA52771; P. troglodytes IgD, PNI88328; S. scrofa IgD, AAP23939; B. taurus IgD, AAN03673; O. aries IgD, AAN03671.

In all mammals, IgA is passively transferred from the mammary gland epithelium via the colostrum to the young (during suckling), which provides protection in the mucus membranes of both the respiratory and gastrointestinal systems of the newborn (Adamski and

Demmer, 1999). In eutherians, the mammary gland epithelium lymphocytes secrete IgA as a dimer joined by the J chain (Koshland, 1985), in turn IgA is transported across the epithelial

59 cells in the milk by the polymeric Ig receptor (pIgR) and released by proteolytic cleavage of the extracellular domain at the apical membrane (Mostov, 1994). sIgA was first identified in marsupial milk and other secretory sites of the quokka (Bell et al., 1974b). Since then, IgA has been isolated from the colostrum of the Virginian opossum (Hindes and Mizell, 1976) and common brushtail possum (Ramadass and Moriarty, 1982). In the common brushtail possum, Adamski and Demmer (1999) have reportedly cloned and characterised the J chain and pIgR cDNA sequences and found these genes to be highly conserved when compared to those of eutherians. The expression studies found both the J chain and pIgR had increased in expression levels at two distinct time periods throughout the lactation period of the pouch young and would concur with the time of exposure to a potential new pathogen (Adamski and

Demmer, 1999). The first IgA enhancement being at the beginning of lactation is consistent with eutherians, however the second increase occurs at the end of the switch phase (80–120 days of lactation) and is unique to marsupials (Adamski and Demmer, 1999).

IgG makes up 80 % of serum antibody levels (Smith et al., 2019). Like IgA, IgG plays a major role in respiratory protection, however IgG primarily acts to prevent local and systemic infections in the lower rather than the upper respiratory tract (Stokes and

Rajasekaran, 2008). IgG has been isolated from the foetal serum of the tammar wallaby two days prior to birth (Renfree, 1973), and again detected in the blood of pouch young, as well as maternal colostrum and placental extracts (Deane et al., 1990), suggesting it can be transferred transplacentally in this species. It is apparent that separate gene duplication events have occurred in IgA and IgG isotypes in some mammalian species. In eutherians, humans and mice have four functional IgG subclasses; humans have IgG1, IgG2, IgG3, and IgG4

(Vidarsson et al., 2014), whereas mice have IgG1, IgG2a/c, IgG2b, and IgG3 in which they either present IgG2a or IgG2b depending on their strain (Zhang et al., 2012). Humans also have two functional IgA subclasses, IgA1 and IgA2 with a higher preference for IgA1 in

60 lymphoid tissues (Kett et al., 1986). These gene duplications seem to have evolved quite recently and independently within individual mammalian lineages as no IgA nor IgG subclasses have been identified in any marsupial or monotreme (not shown) species, and not all eutherians (Figure 2.3).

IgM was the first of the Igs to appear in vertebrate evolution and is the first Ig to be expressed during development of the immune system (Lawton et al., 1975). Membrane bound

IgM (mIgM) has a monomer structure which resides on the B-cell surface and acts upon the presence of an antigen, whilst serum IgM (sIgM) is a pentamer and aids in the activation of the complement system (Atwell and Marchalonis, 1977). In marsupials, studies of IgM have mostly been limited to its initial characterisation including those in the Virginian opossum

(Rowlands and Dudley, 1968), quokka (Bell et al., 1974a), common brushtail possum

(Ramadass and Moriarty, 1982), koala (Wilkinson et al., 1991), and gray short-tailed opossum (Shearer et al., 1995). Belov et al. (2002a) has also detected transcripts of IgM in the spleen and liver as early as day 10 postpartum of the common brushtail possum which is similarly expressed in humans at the same developmental stage (Cooper, 1987).

IgE is found in much lower concentrations in comparison to the other Ig classes, however it plays a major role in immediate hypersensitivity reactions and defence against parasitic infections (Ishizaka et al., 1966). Aveskogh and Hellman (1998) cloned IgE in the gray short-tailed opossum and observed a single IgE isotype in the species, which has also been identified in eutherians. During evolutionary divergence, where IgG was duplicated into four isotypes, and two duplicates in IgA, IgE was not duplicated and still exists as only one isotype in marsupials (Aveskogh and Hellman, 1998).

Light chain (Igκ and Igλ) genes have been extensively studied in eutherian mammals, however, only a few key marsupials have characterised their respective Igκ (gray short tailed opossum, AAF25575; tammar wallaby, ABV02004; common brushtail possum, AAL17619;

Tasmanian devil, ALB75226; human, ACJ1716; mouse, AAA65945) and Igλ (gray short- tailed opossum, AAC98628; tammar wallaby, AAL37211; human, S25738; mouse

AAO53439) light chains (Lucero et al., 1998, Miller et al., 1999, Deakin et al., 2006). The L loci in the opossum are highly diverse, with at least four Vκ families and three Vλ families

(Miller and Belov, 2000) that collectively contain more than 60 gene segments (Lucero et al.,

1998, Miller et al., 1999). Evidence has shown the IgH and IgL loci of marsupials appears to be evolving and diversifying due to their complexity (Sitnikova and Su, 1998, Miller and

Belov, 2000), which may be due to the IgL loci compensating for the lack of gene segment diversity in the IgH loci (Baker et al., 2005). In the gray short-tailed opossum and common brushtail possum, the IgL loci appears to have a higher rate of complexity than the IgH loci and therefore contributes more to overall antibody diversity (Figure 2.4) (Baker et al., 2005,

Wang et al., 2009). It is not yet known whether marsupials favour one light chain over the other, and in eutherians, different species express Igκ and Igλ at different ratios, for example, the Igκ:Igλ ratio in the mouse (Mus musculus) in 95:5, whereas in the horse (Equus caballus) it is 10:90 (Belov et al., 2001). The gray short-tailed opossum does have two identified conserved kappa enhancers (KE5′ and KE3′D) and one recombining element (RE), however due to gaps the gray short-tailed opossum genome (MonDom5) specific kappa-deleting elements (KDE) have not been detected (Das et al., 2009). The Igλ locus of the gray short- tailed opossum has a confirmed eight J-C duplicated pairs which is also present in eutherians, this proves to be conserved in all mammals, where in avians it only contain a single J-C region in the Igλ locus (Lucero et al., 1998).

Figure 2.4 Genomic organisation of the M. domestica IgH, Igκ, and Igλ loci. V, D (in the IgH), and J gene segments are clustered, and C gene segments are shown (not to scale). Direction of transcription is indicated by the Telomeric and Centromeric ends. Dotted lines indicate where there are gaps in MonDom5 genome sequence. For the IgH locus, the C domain is shown below the IgH main line, the presumptive IgM pseudogene is indicated with a psi. H identifies the sequences with an encoding hinge region. Polyadenylation and transmembrane protein domain are designated with Poly A and TM, respectively. Double angled lines indicate where there are large regions of separation. The Igκ shows two large clusters of Vκ gene segments with an 800 Kb region separating them. Circles on the Igκ identify a recombining element, RE, and two kappa enhancers, Ke5′and KE3′P, respectively. Pairing IgλJ and IgλC genes are designated according to their order.

Of the Ig genes, IgA, IgG, IgE, Igκ and Igλ have all been found to be expressed in the gray short-tailed opossum, tammar wallaby and common brushtail possum (Lucero et al., 1998,

Aveskogh and Hellman, 1998, Belov et al., 1999a, Belov et al., 1999b, Belov et al., 2001,

Duncan et al., 2010, Wang et al., 2009, Suthers and Young, 2014, Ujvari and Belov, 2015), whereas IgM has only been expressed in the gray short-tailed opossum (Miller et al., 1998).

All three mammalian lineages therefore have fundamentally the same heavy (H)

(except IgD) and light (L) loci with respect to their Ig classes (Baker et al., 2005), and two

IgL isotypes kappa (Igκ) and lambda (Igλ) (Lucero et al., 1998, Miller et al., 1999). Antibody diversity and specificity is due to the variation of amino acid residues at the N-terminal ends of the H and L loci and are primarily produced by V(D)J recombination (Figure 2.4) (Baker et al., 2005). Marsupials, unlike other mammals have contrasting diversity between the germline H and L loci. Miller et al. (1998) reported that all but one variable (V) H loci gene segment resided in the VH1 family, with the remaining (closely related) member residing in the VH2 family observed in the gray short-tailed opossum. Wang et al. (2009) analysed the gray short-tailed opossum genomic sequence and uncovered a third subgroup, IGHV3.1. This previously unrecognizable partially germline-joined VH gene appears to be joined at the end of the V and fused to the D segment in the IgH DNA germline of the opossum (Wang and

Miller, 2012). In a typical mammalian V(D)J recombination event, pro B-cells firstly rearrange the DH to the JH, followed by the rearrangement of VH to the D-JH (Schatz and

Swanson, 2011). However, where a VH3.1 is apparent, there is a direct rearrangement of VH to JH as the VH3.1 gene has previously joined the VH to the DH (Wang and Miller, 2012).

Uniquely, IGHV3.1 is significantly longer in length when compared to previously identified mammalian IgH gene segments and shares <80 % of nucleotide identity with other gray short-tailed opossum VH gene segments (Wang and Miller, 2012).

2.5.2 Cluster of Differentiation Markers

In eutherians, the first appearance and expression of CD markers can aid in detecting and analysing the events involved in the development and differentiation of B-cells (Vale and

Schroeder, 2010). Various CD markers, such as CD10, CD19, CD34, CD79α and CD79β can help to identify the stages of B-cell development, differentiation, and responses to antigens in mammals. CD10, also known as neprilysin (Mishra et al., 2016) is a prime example of a marker used to detect early stages of B-cell differentiation (Hystad et al., 2007). CD10 is commonly expressed in early eutherian pro-B-cells (Torlakovic et al., 2005) with an immature phenotype (Bachelard-Cascales et al., 2010), suggesting it plays a role in the development and maturation of immature B-cells (Stamenkovic and Seed, 1988) before ceasing its expression once B-cells mature (Ichii et al., 2010). The high expression levels of

CD10 in a common lymphoid progenitor (CLP) has been observed to aid in commitment towards the development of early B-cells (Ichii et al., 2010). In eutherians, CD19 expression is regulated by the transcription factor Pax5 (Wang et al., 2012a). Its initial expression begins after the initiation of D–J gene rearrangement of the Ig heavy chain locus during the early pro-B-cell stage (Nadler et al., 1983, Tedder, 2009) and then disappears during the terminal plasma cell differentiation stages (Haas and Tedder, 2005, Tedder, 2009). CD34 plays a major role in the developmental pathway from HSCs (Davi et al., 1997) through to the early

B-cell stages where it is highly expressed (LeBien and Tedder, 2008). CD34 and CD10 are both expressed at the CLP stage through to the pro-B-cell stage, however CD19 expression is initiated at the pro-B-cell stage (Sanz et al., 2010). From there B-cells develop into pre-B-cell stages and the expression of CD34 ceases (Fluckiger et al., 1998). CD10, CD19, CD34 and other CD marker genes have previously been described in many eutherians (Furness and

McNagny, 2006), and have now been identified in the gray short-tailed opossum, koala, and

Tasmanian devil (Wong et al., 2011a).

CD79α and CD79β are both essential for the differentiation and functionality of B- cells in eutherians. These genes produce the BCR by forming a non-covalent heterodimer with a sIg (Reth, 1992) and is required for BCR surface expression (Hashimoto et al., 1995).

The BCR is a complex hetero-oligomeric structure (Wienands, 2000), where ligand binding surface Igs; Igα (CD79α) and Igβ (CD79β) are separated into distinct receptor subunits (Chu and Arber, 2001). Both CD79 transcripts are expressed in all stages of a B-cell’s life; immature and mature, as they are key to guiding the B-cell differentiation and antigen response pathways (Mason et al., 1992, Chu and Arber, 2001). CD79α and CD79β have been identified in various marsupials, and reported to have been transcribed and characterised in the gray short-tailed opossum, tammar wallaby (Duncan et al., 2010) and bridled nail-tail wallaby (Onychogalea fraenata) (Suthers and Young, 2014). Duncan et al. (2010) was able to analyse the expression of CD79α and CD79β in pouch young tissues of the tammar wallaby.

At day 10 postpartum, both transcripts were detected in the bone marrow, cervical thymus and lungs. CD79α was also detected at this time point in the spleen and blood, however

CD79β was not detected in these tissues until day 21 (Duncan et al., 2010), thus a tammar wallaby would be unable to mount a specific immune response until at least day 21, when the entire BCR is fully expressed. The genes encoding the CD79α (Figure 2.5A) and CD79β

(Figure 2.5B) protein sequences in these marsupials have shown a high similarity between their highly conserved structural domains, inclusive of, an Ig domain, transmembrane (TM) region, and a cytoplasmic tail (CYT). Their TM region in particular has been presented as highly conserved within mammalian species, with 100 % identity in CD79α (Figure 2.5A) and >80 % identity in CD79β (Figure 2.5B) between marsupials and the human protein sequences. An immunoreceptor tyrosine activation motif (ITAM) was also identified on the

CYT in both CD79α and CD79β protein sequences (Figure 2.5). Reth (1989) first identified

ITAM in eutherians which has now also been characterised in different marsupials (Duncan

et al., 2010, Suthers and Young, 2014). CD79α and CD79β initiates an extracellular signal by producing a specific antigen on the surface of the BCR through the cell membrane to the cytoplasmic tail where it binds to the tyrosine residues within the ITAMs (Kim et al., 1993).

As a result, a rapid increase in protein tyrosine phosphorylation levels occurs (Gold et al.,

1991) and launches a cascade of intracellular signalling within the B-cells (Chu and Arber,

2001, Dal Porto et al., 2004, Radaev et al., 2010). The structural features of this ITAM are highly conserved between marsupial species, even more so than with their eutherian counterparts (Duncan et al., 2010). Transcribed CD79α of the gray short-tailed opossum and tammar wallaby identified the presence of three conserved cysteine residues on the extracellular domain (Duncan et al., 2010). The first two residues (C50 and C101) are able to form an intrachain bond within the fold of the Ig protein, while the third residue (C113) forms an intermolecular dimer between CD79α and CD79β on the BCR (Duncan et al.,

2010), and has also been observed in humans (Yu and Chang, 1992).

Figure 2.5 Amino acid alignment of expressed (A) CD79α and (B) CD79α mammalian sequences. Dots indicate identical bases to M. domestica CD79 transcripts. The Ig domain, TM regions and CYT regions are indicated by arrows below the sequences, the ITAM motifs are identified by a box around the amino acids. Genbank accession numbers: M. domestica (CD79α, ACZ13436; CD79β, ACZ13438.1), N. eugenii (CD79α, ACZ13437; CD79β, ACZ13439.1), (O. fraenata CD79α, AGO64767; CD79β, AII23405.1), H. sapiens (CD79α, AAI13732; CD79β, AAH02975.2).

2.5.3 Signal Transduction Molecules

Signal transduction is fundamental in the cellular processes used for communicating molecular events from the cell surface and the nucleus, altering gene expression and inducing a cellular response (Campbell, 1999). These pathways are activated by an array of external stimuli and mediated by a series of physiological and pathological functions within the cell

(Niiro and Clark, 2002). In the instance where an Ig is expressed on the surface of a B-cell, it results in the formation of a BCR. Upon this formation, the genes which are expressed within the BCR bind to the BCR ligand, facilitating the signal to be sent from the cell surface to the nucleus to elicit an immune response (Ha et al., 1992).

Key transducers involved in intracellular downstream BCR signalling are the tyrosine-protein kinase (Lyn), spleen tyrosine kinase (Syk), and Bruton’s tyrosine kinase

(BTK) (Niiro and Clark, 2002). Lyn is a cytoplasmic non-receptor protein kinase that is encoded by the Scr-family kinase Lyn gene and is associated with the cell surface receptor proteins of the BCR. Syk is a protein kinase (Takata et al., 1994) with a structure that is highly homologous to ZAP-70 which is expressed in T-cells and Natural Killer cells (Takata et al., 1994) of the tammar wallaby (Flenady and Young, 2014). BTK is an intracellular signalling molecule required by B-cells (Fluckiger et al., 1998) to link the signal responses produced by the BCR through activation of various transcript factors (Afar et al., 1996). As of yet, only the isoforms of Lyn tyrosine kinase have been characterised in marsupials

(Suthers and Young, 2013) due to its role in regulating the B-cell response and being the first molecule to be activated in the downstream pathway of the BCR. Suthers and Young (2013) detailed the expression of the Lyn isoforms, LynA and LynB in the spleen, lymph nodes, leucocytes, whole and peripheral blood of marsupials and confirmed that the LynB arises from the same splicing mechanism as humans. The coding domain of Lyn in both the tammar wallaby and bridled nail-tail wallaby appears to have the same six structural regions (SH1, 2,

3, 4, a ‘unique’ domain, and COOH domains) as seen in other mammalian lineages (Suthers and Young, 2013). The tammar wallaby Lyn gene has also been confirmed to have two tyrosine residues (Y399 and Y510) corresponding to the human residues, Y397 and Y508

(Suthers and Young, 2013). These tyrosine residues in eutherians appear to phosphorylate, hence regulates the activation and inhibition of intracellular kinase activity in the BCR (Dal

Porto et al., 2004). Therefore, the identification of Lyn isoforms in marsupials suggests that

Lyn is also utilised in the signalling mechanisms of marsupial B-cell responses.

2.5.4 Transcriptional Regulators

In eutherians, the fate of B-cells in early development and lineage commitment is highly dependent on the activity of the paired box protein 5 (Pax5) as well as other transcription factors including purine box factor 1 (PU.1), E box binding protein 2A (E2A) and early B-cell factor-1 (EBF1) (Singh et al., 2005, Holmes et al., 2008). PU.1 primarily acts in the promotion of lymphoid progenitor formation and the initiation of EBF1 expression

(Medina et al., 2004). Low concentrations of PU.1 are also required to secure the fate of B- cells (Medina et al., 2004). Once these early lymphoid progenitors are formed, EBF1 and

E2A work together to initiate the expression of specific genes within the B-cell (O'Riordan and Grosschedl, 1999). EBF1 is exclusively expressed by eutherian B-cell lineages, and has an atypical helix-loop-helix zinc finger protein structure, while E2A has two basic helix-loop- helix proteins; E12 and E47 (Hagman et al., 1993). E2A and EBF1 work together to initiate the transcription of several B-cell genes (i.e. Vpreb1), which are encoded in the pre-B-cell receptor and the V-D and J DNA rearrangements located in the IgH locus (Pongubala et al.,

2008).

The Pax5 gene belongs to the family of transcription factors which are important for the control of tissue-specific transcription throughout many types of cellular differentiation

(Holmes et al., 2008). It is however the only one (of nine factors) that can be found throughout the haematopoietic system and its expression is confined to that of B-cells in eutherians (Fuxa and Busslinger, 2007). Pax5 expression is initiated in the pro- to pre- B-cell stage and maintains a stable level of expression throughout the life of a B-cell until the downregulation of plasma cell differentiation (Kallies et al., 2007). To date, Pax5 has only been identified in a highly conserved transcriptional module that was generated to compare human and tammar wallaby WNT4 promoter regions and shares 97 % homology between the species (Yu et al., 2009). Other transcriptional regulators have been predicted in genome

databases such as ENSEMBL (Curwen et al., 2004) and Genbank (Benson et al., 2005). A wider investigation into these key molecules in marsupials is required to confirm the developmental and commitment process involved in marsupial B-cells.

2.6 Conclusions

Advances in genomics have and will continue to enable genome-wide investigations of the marsupial immune system and will enable marsupials to be utilised in additional studies in skin cancer, reproduction, evolutionary development (Selwood and Coulson, 2006), new antimicrobial research (Wang et al., 2012c), and no doubt additional areas of research yet to be defined. However, as many marsupial immune genes (equivalent to those identified in eutherians) have yet to be expressed and/or characterised, our understanding is still lagging that of the eutherian immune system. This review has highlighted the major similarities and differences between known marsupial and eutherian B-cell genes, such as, only four of the five Ig heavy isotypes being identified in marsupials. This has left scientists speculating that the IgD isotype was lost in modern day marsupials during mammalian evolution.

Like eutherian B-cells, marsupial B-cells are vital in adaptive humoral immune responses, as they are responsible for producing antibodies, via the expression of specific genes, to be used to defend against the presence of extracellular pathogens and identify them upon repeated exposure (Conley, 2003). Thereby, investigations into marsupial B-cell genes continues to increase in importance, and will aid our understanding of the marsupial adaptive immune system.

2.7 Conflicts of Interest

The authors declare no conflicts of interest.

2.8 Chapter 2 References

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CHAPTER 3

Single-cell transcriptome analysis of the B-cell repertoire reveals the usage of immunoglobulins in the gray short-tailed opossum (Monodelphis domestica)

A. L. Schraven1, V. L. Hansen2, K. A. Morrissey2, H. J. Stannard3, O. T.W. Ong4, D. C.

Douek5, R. D. Miller2, J. M. Old1

1School of Science and Health, Hawkesbury Campus, Western Sydney University, Locked

bag 1797, Penrith, NSW 2751, Australia

2Center for Evolutionary and Theoretical Immunology, Department of Biology, University of

New Mexico Albuquerque, New Mexico, USA

3Charles Sturt University, School of Animal and Veterinary Sciences, Wagga Wagga, NSW

2678, Australia

4QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia

5Human Immunology Section, Vaccine Research Center, National Institute of Allergy and

Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

Manuscript has been submitted to Immunology and Cell Biology and is currently under review.

3.1 Chapter Outline and Authorship

Chapter 3 evaluates the usage of Immunoglobulin Heavy and Light chains in the gray short- tailed opossum (Monodelphis domestica) B-cell repertoire. The B-cell transcriptome was generated by single-cell RNA-Seq technology. Analysis of the opossum B-cell repertoire identified the fraction of cells expressing each heavy chain, as well as the overall light chain isotopic ratio.

This chapter is a prepared manuscript and is jointly authored; I am the primary author, analysed the raw data, identified paired immunoglobulin chains and gene segments, annotated pseudogenes, performed all statistical analysis, and prepared the manuscript.

Victoria Hansen performed Cell sorting, RNA extraction and cDNA synthesis, Daniel Douek performed the sequencing, and Kimberly Morrissey assisted with the initial analysis. Robert

Miller, Hayley Stannard, Oselyne Ong and Julie Old provided advice on data analysis, and have reviewed and provided feedback on the manuscript.

This chapter is a manuscript that has been submitted to the Journal of Immunology and Cell

Biology for publication and is currently under review, and is therefore formatted to the journal specifications.

3.2 Introduction

Typically, Ig molecules comprise of two identical IgH and two identical IgL chains, which are paired together on the surface of the B-cell. Both of these chains have a constant

(C) domain which determines the effector function of the Ig molecule, and a variable (V) domain responsible for binding to an antigen (Alt et al., 1992). Ig isotypes are determined by their CH domain, where four of the five mammalian IgH isotypes have been confirmed in the gray short-tailed opossum (Monodelphis domestica). Previous investigations have isolated

IgM, IgG, IgA, and IgE heavy chain isotypes (Miller et al., 1998, Aveskogh and Hellman,

1998, Belov et al., 1998, Aveskogh et al., 1999, Belov et al., 1999a, Belov et al., 1999b,

Belov et al., 1999c), however, unlike eutherians and monotremes, IgD has not been found in any marsupial species to date. The genomic region predicted to encode the IgD constant domain in the opossum, appears to be replaced with insertions of large duplicated regions of repetitive DNA (Wang et al., 2009). It is not known whether these insertions are typical for all marsupials, or just the opossum. Although, it is clear the absence of IgD is due to a gene loss in the opossum, the presence of IgD in both eutherian and monotreme species (and some non-mammalian vertebrates) is consistent with IgD being ancient in vertebrates (Ohta and

Flajnik, 2006, Wang et al., 2009). Like all mammals, marsupials have two IgL isotypes; kappa (Igκ) and lambda (Igλ) (Lucero et al., 1998). In the opossum Igκ, the V and joining (J) gene segments are present in different clusters, upstream of a single Cκ domain (Miller et al.,

1999), whereas Igλ has multiple Jλ-Cλ pairs (Lucero et al., 1998, Wang et al., 2009).

Antibody diversity and specificity is determined by the V domain located on the N- terminal ends of the IgH and IgL chains (Baker et al., 2005). The abundance and diversity of the VH domain appears to be highly variational within the eutherian lineage, humans and mice have a large set of VH gene segments, whereas other eutherians, such as rabbits and cattle, have a limited VH diversity (Butler, 1997). This pattern of VH diversity seems to have a

coevolving relationship with the diversity of VL, where species with high VH diversity

(humans and mice) also have a high diversity of VL gene segments, and species with low VH diversity (rabbits and cattle) have low VL diversification (Sitnikova and Su, 1998). In the opossum however, this coevolution of VH and VL diversification doesn’t appear to apply, the

VH is both limited in diversity and abundance (Miller et al., 1998, Baker et al., 2005) while

VL has a set of highly diverse gene segments (Lucero et al., 1998, Miller et al., 1999, Wang et al., 2009). This pattern appears true for other marsupial species as well (Baker et al., 2005). A highly complex VL domain seems to compensate for limited VH diversity, and therefore contribute more to overall antibody diversity in marsupials (Lucero et al., 1998, Miller et al.,

1999, Baker et al., 2005, Wang et al., 2009).

The availability of the fully mapped and annotated IgH and IgL chains of the gray short-tailed opossum has facilitated the comparative analysis of mammalian B-cells (Deakin et al., 2006, Wang et al., 2009). The opossum is born highly altrical and like all marsupials,

B-cell competence develops postpartum. IgH transcripts have been detected as early as the first 24 h postpartum, though IgL transcripts have not been detected until day 7 after birth

(Wang et al., 2012). Therefore, the earliest the opossum neonate would be able to generate an endogenous antibody response is at least one week postpartum (Wang et al., 2012), leaving the neonate completely dependent on the antibodies transferred via the maternal milk for the first week (Samples et al., 1986). During the second week after birth opossum antibody diversification increases, seemingly due to the complexity of the IgL chain (Wang et al.,

2012). Here we identify and compare the usage of IgH and IgL isotypes in the B-cell repertoire of the opossum. In doing so, we present the first study to investigate the usage of Ig chains in any marsupial species and compare opossum IgL isotype ratios to other mammalian species.

3.3 Materials and Methods

3.3.1 Ethics Statement

This study was approved under the protocol numbers 16-200407-MC and 15-200334-

B-MC from the University of New Mexico Institutional Animal Care and Use Committee and protocol numbers BIO-17-969 from the Uniformed Services University of the Health

Sciences (USUHS) Animal Care and Use Committee. The opossums used in this study originated from a captive-bred research colony housed at USUHS (Bethesda, MD, USA).

3.3.2 Tissue Collection, Cell Sorting and RNA Extraction

A 10-month old male gray short-tailed opossum was euthanized by carbon dioxide- induced asphyxiation, whole spleen and blood samples extracted, and splenocytes (spleen) and peripheral blood mononuclear cells (blood) isolated by density gradient centrifugation with Ficoll-Paque Plus (GE Healthcare, Chicago, IL). The purified spleen and blood were counted and then stained with LIVE/DEAD Aqua (1:800) (Invitrogen, Waltham, MA) to differentiate viable cells, and then sorted into individual wells on 96-well plates. Single cells were sorted into individual wells of 96-well plates containing 5 L 1% (v/v) 2- mercaptoethanol (Sigma-Aldrich, St. Louis, MO) in TCL buffer (Qiagen, Hildon, Germany) per well. All sample plates were stored at -80C until further use.

3.3.3 cDNA Synthesis, Sequencing and Alignment

The Sciclone G3 Automated Liquid Handler (PerkinElmer, Waltham, MA) was used for most of the liquid handling. RNA was isolated using the Agencourt RNA Clean XP SPRI beads (Beckman Coulter, Brea, CA). First strand Master Mix (Illumina, San Diego, CA) was added to the RNA to conduct first strand cDNA synthesis. Second strand synthesis was

conducted using Second Strand Master Mix (Illumina) in a thermocycler to produce the double-stranded cDNA. Agencourt AMPure XP Beads (Beckman Coulter) and Zephyr G3

NGS workstation (PerkinElmer) were used to isolate the double stranded DNA. The 2100

Bioanalyzer (Agilent, Santa Clara, CA) was used for quality assessment of the PCR products and concentration were calculated using a Qubit 2.0 Fluorometer (Life Technologies, Grand

Island, NY, USA). Nextera indexed adapters were ligated to cDNA libraries according to manufacturer’s instructions (Illumina). Nextera libraries were then quantified by qPCR using

KAPA SYBR FAST Master Mix (Kapa Biosystems, Wilmington, MA) according to manufacturer’s instructions on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad,

Hercules, CA). Up to 96 Nextera libraries were pooled together in 10 nM equimolar concentrations, were by Illumina HiSeq 3000/4000 instrument (Illumina) performed all

Illumina sequencing to create paired end reads which were demultiplexed to generate FASTQ files. FASTQC was used to perform the quality assessment of the read data. Reads were assembled de novo and aligned to the annotated opossum genome. The combined assembled reads of all tissues resulted in 273 single-cells and 575,721 contigs, with a mean contig length of 892 bp, and a transcript sum of 513.7 Mb.

3.3.4 Single-cell Identification and Statistical Analyses

Briefly, the identification of paired IgH and IgL chains in the single-cell transcriptome was performed using BLASTx searched through the NCBI database with an E value cut-off of 10-5. Open reading frames (ORF) were searched for in the ORFfinder (NCBI) using the contig sequences which were previously annotated as pseudogenes in the opossum genome to test whether these sequences were functional in the individual opossum used.

Prism 8.2 (Graphpad) software was used for all statistical analyses and graphing purposes. To determine the Igλ:Igκ ratio for spleen and blood single-cells, Student’s (unpaired) t-tests was

performed using default parameters. Single-factor ANOVA was used for the VH/Vκ and

VH/Vλ pairing analysis.

3.4 Results

3.4.1 Heavy and Light Chain Immunoglobulin Usage

IgH and IgL chain usage in the opossum was compared between spleen and blood single B-cells. Overall, the percentage of cells in the spleen and blood was dominated by

IgM+ B-cells (79% and 70%, respectively) in comparison to other IgH isotypes (Figure 3.1).

IgG and IgE had consistent but low expression levels throughout, whereas IgA had a slight increase of expressed Igλ B-cells in the blood (15%). The expression of the IgL isotypes showed a significant difference between the usage of Igκ and Igλ in the opossum B-cell repertoire (Student’s t-test, p = 0.0421) (Figure 3.1). In the spleen 64% of B-cells were using

Igλ compared to 36% using Igκ, and in the blood 66% used Igλ and only 34% used Igκ, revealing an overall average ratio of opossum Igλ:Igκ in the B-cell repertoire to be 65:35.

Figure 3.1 Fraction of cells expressing immunoglobulin heavy (IgM, IgG, IgA, IgE) and light (Igκ and Igλ) chains in both spleen and blood single cells.

3.4.2 Immunoglobulin Variable and Joining Domains

Annotation of the opossum IgH and IgL chains used in the single-cell repertoire revealed limited functional usage of the IgH variable (VH) gene segment. A total of 27 VH gene segments were used in the opossum B-cell repertoire, in which 6 VH gene segments were originally annotated as being among the unaligned VH in the opossum genome assembly

(Figure 3.2, (Wang et al., 2009)). A VH gene segment, VH1.10, previously annotated as being a pseudogene was found in the repertoire and appears to be functional in the individual opossum used in this study, consistent with it having non-functional alleles rather than being a pseudogene (Supplementary Figure 3.1). As IgM+ B-cells had the highest overall frequency among the individual spleen and blood cells, it was expected that it would utilise the majority of the variable gene segments. Interestingly, a high number of VH1.18 (11%) and VH1.Ue

(14%) gene segments were identified in the blood cells largely due to a high frequency of

+ IgA B-cells; 36% of VH1.18 and 100% of VH1.Ue were utilised by B-cells that had switched to IgA.

The pattern of recombination between VH and JH was also analysed. There are 6 JH in the opossum IgH locus, 4 appear functional and 2 pseudogenes (Wang et al., 2009). Only two, JH1 and JH2, appeared be used in the single-cell repertoires. These are the two functional

JH in closest proximity to the functional C (Wang et al., 2009). JH2 was used in V(D)J recombinations in 78% of splenic B-cells, while JH1 was used in 22% of splenic B-cells.

There was little difference between blood B-cells using JH1 (44%) and JH2 (56%) (Figure

+ + 3.2). The repertoire of both the IgM and IgG B-cells recombined JH2 more so than JH1

(57% and 89%, respectively) in all cells examined, however blood IgA+ B-cells utilised more

JH1 (70%) than JH2 (30%).

Figure 3.2 Fraction of cells expressing V and J domains of the Ig heavy (IgM, IgG, IgA, IgE) chains in both spleen and blood single cells. NA indicates no information.

In the opossum single-cell repertoire 37 Vκ gene segments were found to be utilised out of the 122 Vκ annotated previously in the opossum genome (Figure 3.3, (Wang et al.,

2009)). As was found in the VH repertoire, two V, Vκ1.17 and Vκ7.11, that were annotated as pseudogenes previously (Wang et al., 2009), were used in the B-cell repertoire in the current dataset (Supplementary Figures 3.2 and 3.3). There was no significant bias between which Vκ gene segments were present and the IgH isotype being used by splenic B-cells. In contrast,

+ the Vκ gene segments used in blood B-cells were mostly found in IgM B cells. Gene segments Vκ1.11, Vκ4.2, and Vκ4.3 were being used by B-cells that have switch to IgA and

Vκ1.29 was only found in B-cells that switched to IgG in the blood cells. Vκ3.20 was the only

+ + Vκ gene segment found in the blood cells to have been used by both IgM and IgG B-cells. A high proportion of cells had no expression information for their J gene segments, therefore no comparison could be made between Jκ1 and Jκ2 (Figure 3.3).

Figure 3.3 Fraction of cells expression variable and joining domains of the immunoglobulin light (Igκ) chains in both spleen and blood single cells. NA indicates no information.

The Igλ locus had the highest proportion of Vλ gene segments to be produced by the opossum B-cell repertoire; a total of 43 Vλ gene segments were identified in the Igλ locus, 7 of which were classified as unassembled in the opossum genome (Figure 3.4). As seen in the usage of Vκ, the Vλ gene segments used in splenic B-cells were highly diverse, the usage of the Vλ gene segments were evenly distributed, with each Vλ utilised <10% in the B-cells. In the blood cells however, the usage of Vλ gene segments was less diverse, in which, Vλ1.38 was utilised in 14% of B-cells. In comparison to the Vκ gene segments used in blood B-cells, multiple Vλ gene segments were found paired by more than one IgH isotype. Notably, 14% of

+ + Vλ gene segments used were Vλ1.38 and found in both IgM (1%) and IgA (13 %) B-cells.

The opossum has 8 Jλ gene segments and all were utilised in the spleen and blood cell repertoires (Figure 3.4). Gene segments Jλ1, Jλ6, Jλ7, and Jλ8 all had consistent use in the

+ spleen and blood cells, all of which were dominated by IgM B-cells. The Jλ3 gene segment had an increase of expression in the blood cells associated with the increase of IgA+ B-cells

(Figure 3.4).

Figure 3.4 Fraction of cells expression variable and joining domains of the immunoglobulin light (Igλ) chains in both spleen and blood single cells. NA indicates no information.

3.4.3 VH and VL Gene Segment Pairing

Having single cell-based B-cell repertoires allowed us to analyse whether there was any bias in which VH pair with which VL in the opossum. In those B-cells using Igκ there was no significance between VH/Vκ gene segment pairing (ANOVA, p = 0.167) (Figure 3.5).

However, a significant pairing was identified between the VH/Vλ gene segments (ANOVA, p

+ = 0.008), this was largely due to the high abundance of IgA B-cells using VH1.Ue paired with Vλ1.38, a bias that may be unique to this animal (Figure 3.6).

Figure 3.5 Heat map indicating the frequency of VH/Vκ gene segment pairs expressed in the opossum repertoire.

Figure 3.6 Heat map indicating the frequency of VH/Vλ gene segment pairs expressed in the opossum repertoire.

3.5 Discussion

All three modern mammalian lineages, eutherians, marsupials, and monotremes, have typical Ig molecules consisting of two identical heavy (H) and light (L) chains (Baker et al.,

2005). However, unlike eutherians and monotremes, marsupials only have four of the five

IgH isotypes (IgD has not been identified) (Miller and Belov, 2000), along with both IgL isotypes; Igλ and Igκ (Lucero et al., 1998, Miller et al., 1999). The goal of this study was to analyse the expression of IgH and IgL loci in the single-cell transcriptome of the opossum B- cells and examine their usage of the variable and joining gene segments.

In contrast to other mammals, marsupial IgH constant (C) regions appear to only contain a single functional copy of IgM, IgG, IgA and IgE subclasses (Aveskogh and

Hellman, 1998, Miller et al., 1998). Both the monotremes investigated, the platypus and short-beaked echidna (Tachyglossus aculeatus), each have two IgA and IgG subtypes

(Vernersson et al., 2002, Belov and Hellman, 2003). There is a second IgM locus identified in the gray short-tailed opossum, however, it appears to be a non-functional pseudogene (Wang et al., 2009, Miller, 2010). Analyses found more IgM+ B-cells than other IgH isotypes in the opossum repertoire. This is not surprising given the cells analysed came from a naïve animal and IgM is the first Ig to be expressed during ontogeny of the immune system (Lawton et al.,

1975, Belov et al., 1999c),

The VH domain is encoded by an exon assembled from gene segments: the variable

(V), diversity (D), and joining (J) gene segments, through V(D)J recombination (Alt et al.,

1992, Schatz and Swanson, 2011). In contrast, IgL isotypes use only V and J gene segments to encode the V domain. The limitation in the VH domain diversity appears to have occurred during early marsupial evolution, with previous investigators reporting a limited number of

VH families in marsupials (Miller et al., 1998, Baker et al., 2005, Wang et al., 2009). Three major groups of VH genes are found across vertebrates (Kabat et al., 1992). These groups are

further divided into families (or subgroups) based generally on nucleotide sequence identity and/or cross-hybridization (Brodeur and Riblet, 1984). Most of the VH in the opossum belong to a single family (VH1), and there are two other families (VH2 and VH3) that have a single gene segment in each (Miller et al., 1998, Baker et al., 2005, Wang et al., 2009, Wang and

Miller, 2012). Our study found 21 of the annotated VH segments (VH2.1 included) used in the

B-cell repertoire, one of which, VH1.10, had been previously designated as a pseudogene

(Wang et al., 2009). Our results suggest that VH1.10 is actually functional in the individual animal investigated due to the presence of an ORF identified in the transcript contigs, indicating that VH1.10 can be a functional allele, rather than a pseudogene. Curiously, VH3.1 was not found in our dataset, in spite of it having previously shown to be used in the opossum

IgH repertoire (Wang and Miller, 2012). Only two of the six identified JH gene segments (JH1 and JH2) were found to be used in the repertoire analysed in this dataset. JH gene segments are grouped into two sets of three, in which JH1 and JH2 are directly upstream of the functional

IgM constant region in the IgH genomic organisation (Wang et al., 2009). JH1 and JH2 are also the only JH gene segments to have been previously found in IgH encoding cDNAs isolated from adult opossum spleen (Aveskogh et al., 1999).

Our study is the first to determine the relative usage of Igλ and Igκ in any marsupial species. The opossum has a Igλ:Igκ ratio of 65:35, whereas in eutherian mammals this ratio is highly diverse among species (see table 3.1; reviewed in Nezlin (1998)).

Table 3.2 IgL isotypes Igλ:Igκ ratio expression in mammals. Species Igλ:Igκ References

Gray short-tailed opossum 65:35 This study

Human 40:60 (Butler, 1997)

Mouse 5:95 (Kirschbaum et al., 1996)

Guinea pig 30:70 (Cebra et al., 1977)

Rabbit 10:90 (Waterfield et al., 1973),

Pig 50:50 (Butler and Brown, 1994)

Sheep 20:1 (Reynaud et al., 1995)

Horse 90:10 (Home et al., 1992)

Dog 91:9 (Arun et al., 1996)

Cat 92:8 (Arun et al., 1996)

In contrast to the limited VH diversification, the VL domain appears to have retained a broader and more ancient diversity of V gene segments. Therefore, the IgH and IgL loci of the opossum does not appear to follow common rules which emerged in other vertebrates, including other mammalian species (i.e. humans and mice) that have high IgH and IgL diversification (Kirschbaum et al., 1996, Butler, 1997, Baker et al., 2005). The opossum IgL domain seemingly contributes more to antibody diversity than the IgH domain due the complexity of the VL domain (Miller et al., 1998, Baker et al., 2005, Wang et al., 2009).

Preferential use of mammalian IgL isotypes appears to correlate with the overall complexity

(or number) of IgL V gene segments (Lucero et al., 1998), with horses and sheep appearing to be an exception (Home et al., 1992, Reynaud et al., 1995). The opossum also seems to be an exception, as Igκ, undoubtedly, has the most complex V domain of the two IgL isotypes, consisting of 122 Vκ gene segments (both functional and pseudogenes) separated into seven subgroups (Wang et al., 2009). However Igλ has a total of 64 Vλ gene segments within four subgroups, of which all but six gene segments are functional (Wang et al., 2009). Further

analyses of marsupial IgL isotype expression will be required to determine whether the entire marsupial lineage follows the same expression pattern as the opossum, or like eutherians, whereby a high dissimilarity between species occurs. Only 37 Vκ gene segments were found to be used in the opossum Ig repertoire; 14 from Vκ1, seven from Vκ2 and Vκ3, four from Vκ4 and Vκ5, and three from Vκ7. Previously, Vκ1.17 and Vκ7.11 have both been annotated as pseudogenes in the opossum genome (Mikkelsen et al., 2007, Wang et al., 2009). However, our findings reveal that these gene segments are functional due to an ORF in the assembled sequence contigs, suggesting that there are functional alleles in some individuals. No transcripts were found to express any of the gene segments from the Vκ6 subgroup. In comparison, 35 previously annotated Vλ gene segments were used in the opossum Ig repertoire; 27 from Vλ1, 6 from Vλ2, and 2 from Vλ3. No Vλ4.1. The Vλ4 gene family is only newly identified in the opossum and appears functional (Wang et al., 2009), and it still remains unclear if Vλ4 contributes to antibody diversity. Analysis of the gene segment pairing showed a significant difference between cells using VH/Vλ pairs, which was largely due the

+ high abundance of cells expressing VH1.Ue:Vλ1.38 in IgA B-cells. This may reflect selection that has occurred on the B-cell pool in this opossum that expanded those B-cells using

VH1.Ue:Vλ1.38.

J and C domains vary between opossum IgL isotypes. The opossum Ig locus contains only a single Cκ gene and two Jκ gene segments (Wang et al., 2009). Although the dataset contained full length Ig transcripts, we could not distinguish which Jκ were being used. The opossum has eight Jλ-Cλ pairs that have previously been identified (Lucero et al.,

1998, Wang et al., 2009), all of which were being used in the B-cell repertoire. The presence of multiple Jλ-Cλ gene pairs in the opossum is a feature conserved across mammals

(Kirschbaum et al., 1996), whereas birds only contain one Jλ-Cλ pairing (McCormack et al.,

1989).

It should also be mentioned that a number of “unaligned” gene segments were utilized in the opossum B-cells; six from VH belonging to the VH1 subgroup, and seven from Vλ1 subgroup. These sequences have previously been identified in the opossum genome, but it is still unclear if any of these are alleles of VH annotated in the IgH locus or were unique gene segments that are located in gaps in the assembly (Mikkelsen et al., 2007).

Our study has provided valuable insights into the Ig gene repertoire in opossum B- cells. Until now, no studies have investigated the usage of IgL isotypes, Igλ and Igκ, in any marsupial species at the single-cell level, and this information can now be utilised as a key resource for future analysis of marsupial Ig usage. However, as it has already been seen in the eutherian lineage, the Igλ:Igκ ratio is highly variable between species, therefore, we cannot suppose the same variation occurs in all marsupials.

3.6 Acknowledgements

The authors would like to thank Galina Petukhova (USUHS) for generously making available M. domestica for this project and assistance with animal husbandry and tissue collection. This research was funded in part by a National Science Foundation award IOS-13531232.

Research reported in this publication was also supported in part by the National Institute of General

Medical Sciences of the National Institutes of Health under award P30 GM110907. The content is solely the responsibility of the authors and does not necessarily represent the official views of the

National Institutes of Health.

3.7 Conflicts of Interest

The authors declare no conflicts of interest.

3.8 Chapter 3 References

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duck-billed platypus, Ornithorhynchus anatinus. European Journal of Immunology, 32, 2145-2155. WANG, X. & MILLER, R. D. 2012. Recombination, transcription, and diversity of a partially germline-joined VH in a mammal. Immunogenetics, 64, 713-7. WANG, X., OLP, J. J. & MILLER, R. D. 2009. On the genomics of immunoglobulins in the gray, short-tailed opossum Monodelphis domestica. Immunogenetics, 61, 581-96. WANG, X., SHARP, A. R. & MILLER, R. D. 2012. Early postnatal B cell ontogeny and antibody repertoire maturation in the opossum, Monodelphis domestica. PLoS One, 7, e45931. WATERFIELD, M. D., MORRIS, J. E., HOOD, L. E. & TODD, C. W. 1973. Rabbit immunoglobulin light chains: correlation of variable region sequences with allotypic markers. The Journal of Immunology, 110, 227.

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3.9 Chapter 3 Supplementary Figures

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CHAPTER 4

Single-cell transcriptomic analysis of marsupial B-cells, the gray short-tailed opossum (Monodelphis domestica)

A. L. Schraven1, V. L. Hansen2, H. J. Stannard3, O. T.W. Ong4, D. C. Douek5, R. D. Miller2,

J. M. Old1

1School of Science and Health, Hawkesbury Campus, Western Sydney University, Locked

bag 1797, Penrith, NSW 2751, Australia

2Center for Evolutionary and Theoretical Immunology, Department of Biology, University of

New Mexico Albuquerque, New Mexico, USA

3Charles Sturt University, School of Animal and Veterinary Sciences, Wagga Wagga, NSW

2678, Australia

4QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia

5Human Immunology Section, Vaccine Research Center, National Institute of Allergy and

Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

Manuscript has been prepared for submission to Immunology and Cell Biology

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4.1 Chapter Outline and Authorship

Chapter 4 presents an investigation of the gray short-tailed opossum (Monodelphis domestica) B-cell transcriptome at the single cell level. This chapter annotates all the genes identified in the opossum and analyses the most highly expressed genes, inclusive of those which are associated with marsupial immune system.

This chapter is a prepared manuscript and is jointly authored; I am the primary author, analysed the raw data, identified and annotated the genes, performed structural motif and phylogenetic analysis, and prepared the manuscript. Victoria Hansen performed Cell sorting,

RNA extraction and cDNA synthesis, and Daniel Douek performed the sequencing. Robert

Miller, Hayley Stannard, Oselyne Ong and Julie Old provided advice on data analysis, and have reviewed and provided feedback on the manuscript.

This chapter is a manuscript that has been prepared for submission to Immunology and Cell

Biology, and is therefore formatted to the journal specifications.

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4.2 Introduction

The gray short-tailed opossum (Monodelphis domestica) is arguably a leading marsupial model for evolutionary research (Samollow, 2006), as it has been widely used as a model for comparative and biomedical studies (Vandeberg and Robinson, 1997). Since the sequencing of the opossum genome (Mikkelsen et al., 2007), many components of the marsupial innate and adaptive immune system have been characterised (Wong et al., 2011,

Edwards et al., 2012, Morris et al., 2018), and the opossum Immunoglobulin (Ig), T cell receptor (TCR), and major histocompatibility complex (MHC) genes have been annotated

(Wang et al., 2009). The resulting information suggests that the marsupial immune system is highly similar, and as complex, to that of eutherian mammals.

Marsupials are a unique group of mammals and of great interest in immune system development and functionality studies (Old and Deane, 2000). As with all marsupials, the opossum is born at an early stage of immunological development after a considerably short gestational period of only 13.5 days (Mate et al., 1994). Unlike eutherian mammals, physical and physiological development is completed ex utero while attached to the mother’s teat

(Edwards and Deakin, 2013). During this time, the young will endure a lengthy lactation period and undergo major developmental stages including the maturation of lymphoid tissues and cells.

B-cells are of particular importance in humoral (adaptive) immune responses and are found in multiple lymphoid organs (Holmes et al., 2008). B-cells are unique because of their production of antibodies released from their differentiated cells and are able to detect foreign molecules that invade the (Painter et al., 2011). In mice, B-cells are produced from the pluripotent haematopoietic stem cells, which are found in the liver during the late stages of foetal development and haematopoiesis continues in the bone marrow after birth (Hardy and

Hayakawa, 2001). Several stages are involved in the maturation of eutherian B-cells,

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including the rearrangement of Igs, expression of CD79α+ and CD79β+ cells, and their population in both primary and secondary immune tissues (Conley and Cooper, 1998). In the opossum, detection of Ig rearrangement has only been expressed as early as day 7, postpartum, suggesting marsupial neonates cannot produce an endogenous antibody response until after the first postnatal week (Wang et al., 2012). The earliest time point detected for

+ CD79β cells was identified in the gut of the tammar wallaby (Notamacropus eugenii) at day

7 postpartum (Old and Deane, 2003). By day 10, B-cell receptor CD79α+ and CD79β+ transcripts were detected in cervical thymus and bone marrow, along with CD79α+ in the spleen, gut and blood of the wallaby (Duncan et al., 2010). In the common brushtail possum

(Trichosurus vulpecula) IgM transcripts were detected as early as day 10 postpartum, and only after two months, switched to IgG (Belov et al., 2002). Likewise, this switch to IgG was detected in the opossum during the fifth postnatal week, when permanent detachment of the mother’s teat has ceased (Wang et al., 2012).

There are many genes that are specifically expressed in B-cells, throughout development, differentiation and regulation. Recent advances in genomics have enabled researchers to make comparisons between marsupial and eutherian immune systems

(Samollow, 2008), and several investigations have found many overlapping similarities of the structural and functional components of B-cells in groups of mammals (Stone et al., 1996,

Miller and Belov, 2000, Wang et al., 2012). Here, we identify and annotate the expressed genes of the gray short-tailed opossum B-cell transcriptome at the single cell level.

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4.3 Materials and Methods

4.3.1 Ethics Statement

This study was approved under the protocol numbers 16-200407-MC and 15-200334-B-

MC from the University of New Mexico Institutional Animal Care and Use Committee and protocol numbers BIO-17-969 from the USUHS Animal Care and Use Committee. The opossums used in this study originated from a captive-bred research colony housed at the

Uniformed Services University of the Health Sciences (USUHS; Bethesda, MD, USA).

4.3.2 Tissue Collection, Cell Sorting and RNA Extraction

4.3.3 cDNA Synthesis, Sequencing and Alignment

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double-stranded cDNA. Agencourt AMPure XP Beads (Beckman Coulter) and Zephyr G3

NGS workstation (PerkinElmer) were used to isolate the double stranded DNA. The 2100

Bioanalyzer (Agilent, Santa Clara, CA) was used for quality assessment of the PCR products and concentration were calculated using a Qubit 2.0 Fluorometer (Life Technologies, Grand

Island, NY, USA). Nextera indexed adapters were ligated to cDNA libraries according to manufacturer’s instructions (Illumina). Nextera libraries were then quantified by qPCR using

KAPA SYBR FAST Master Mix (Kapa Biosystems, Wilmington, MA) according to manufacturer’s instructions on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad,

Hercules, CA). Up to 96 Nextera libraries were pooled together in 10 nM equimolar concentrations, were by Illumina HiSeq 3000/4000 instrument (Illumina) performed all

4.3.4 Transcriptome Annotation and Gene Ontology

Functional transcriptome annotation was performed using the Galaxy analysis platform (Afgan et al., 2018) (https://usegalaxy.org/). Briefly, BLASTn searches were performed using the Ensembl database as the target with an E value cut-off of 10-5. The annotated genes matched a total of 14,654 unique Ensembl or Genbank genes. Gene ontology

(GO) analyses of all annotated genes was performed using online Panther tools (Mi et al.,

2019) (http://pantherdb.org/) to gain on overview of the assembled opossum transcriptome.

Genes were mapped to the opossum reference genome and classified under GO-Slim

Molecular Function, Biological Process, and Cellular Component terms. Any “unclassified”

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terms were ignored in all analyses. Prism 8.2 (Graphpad) software was used to graph GO annotation percentages.

4.3.5 Statistical Analyses

For the comparison of the most highly expressed genes in the in the single-cell transcriptome of the opossum B-cells, PRIMER-e (v7) (Clarke et al., 2014) was used.

Analysis of Similarity (ANOSIM) was used to compare the genes between the spleen and blood cells. To compare the genes expressed in the individual transcriptomes, ANOSIM and

Similarity Percentage Analysis (SIMPER) was used, and Ig heavy isotypes were grouped together. Non-transformed multidimensional scaling plots were used to visualise the multivariate differences in gene expression.

4.3.6 Phylogenetic Analyses and Motif Comparison

Geneious Prime 2019.2 (Kearse et al., 2012) (http://www.geneious.com) software was used to analyse the evolutionary relationship between mammalian lineages of the top 10 highly expressed genes and the ribosomal protein family genes. Protein sequences from the gray short-tailed opossum, Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctos cinereus), bare-nosed wombat (Vombatus ursinus), tammar wallaby (Notamacropus eugenii), common brushtail possum (Trichosurus vulpecula), platypus (Ornithorhynchus anatinus), human (Homo sapiens), and mouse (Mus musculus) were used for phylogenetic tree constructions. Sequences were aligned in BioEdit (Hall, 1999)

(http://www.mbio.nscu.edu./BioEdit/bioedit.html) using the ClustalW algorithm (Thompson et al., 1994), and phylogenetic trees were constructed based on the alignments in Geneious

Prime using Blossum62 (Henikoff and Henikoff, 1992) with a global alignment. The unrooted morphological phylogenetic trees were constructed using the neighbour-joining tree

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build method, and the sequences were evolved according to the Jukes-Cantor model using

1000 bootstrap replicates (Jukes and Cantor, 1969). Additionally, sequence motif analyses were conducted to compare the 10 most highly expressed genes in the opossum B-cell transcriptome with other mammals. Protein sequences were entered into MOTIF (Pagni et al.,

2007) (http://www.genome.ad.jp/) using the PROSITE pattern notation. The functionally important residues and sites were labelled and amino acids with >75% identity were highlighted.

4.4 Results

4.4.1 Overview of the Transcriptome

The B-cell transcriptome was assembled and annotated using the opossum spleen and blood samples. Of the 14,654 unique gene hits, 13,376 genes were assigned GO-slim annotation classifications (Figure 4.1). In the molecular function category, the most common

GO terms included ‘binding’ (37.5%) and ‘catalytic activity’ (35.3%). The most common cellular component categories were ‘cell’ (42.0% of genes), ‘organelle’ (30.9%), and ‘protein coding complex’ (13.5%). In the biological process category, the highest abundance of genes came under the ‘cellular process’ category (32.1%), followed by ‘metabolic process’ (32.1%) and ‘biological regulation’ (15.1%).

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Figure 4.1 Overall distribution of GO categorical terms in the opossum cell transcriptome. (A) Molecular function, (B) Biological process, and (C) Cellular components. Percentages are relative of all genes which are categorised as a percentage of all annotated transcripts.

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Within the GO-Slim Biological process category, a total of 340 genes (or 2.5%) were termed ‘immune system process’ and then further categorised into GO-Slim subcategories

(Figure 4.2). Of these genes, ‘immune response’ (2.0%) were the most abundant, followed by

‘leukocyte activation’ (0.7%), ‘immune effector process’ (0.6%), ‘leukocyte migration’

(0.3%), ‘immune system development’ (0.2%), and ‘activation of immune response’ (0.1%).

Figure 4.2 Overall distribution of GO categorical terms within the Immune System Process Category. Percentages are relative of all genes which are categorised as a percentage of all annotated transcripts.

4.4.2 Highly Expressed Genes

The most highly expressed genes in the opossum B-cell transcriptome transcribe an array of protein classes with many of these transcripts coding for immune proteins (Table

4.1).

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Table 4.1 Top 100 most highly expressed genes in the opossum B-cell transcriptome. Total percentages are calculated from spleen and blood counts. Rank ID Gene Description Percentage (%)

1 ENSMODG00000042119 SDC4 Syndecan 4 0.66

2 ENSMODG00000014722 HLA-DRA major histocompatibility 0.45 complex, class II, DR alpha 3 ENSMODG00000016394 UBC ribosomal protein S28 0.36

staphylococcal nuclease domain- 4 ENSMODG00000015182 SND1 0.31 containing protein

5 ENSMODG00000028756 CD74 CD74 molecule 0.31

6 NCBI: 554228 MODO-DAB1 MHC class II beta chain 0.28

7 ENSMODG00000014629 GLG1 golgi glycoprotein 1 0.26

ELMO domain-containing 8 ENSMODG00000014705 ELMOD1 0.20 protein

heterogeneous nuclear 9 ENSMODG00000008402 HNRNPK 0.20 ribonucleoprotein K

10 ENSMODG00000002120 FCMR Fc fragment of IgM Receptor 0.18

glutathione S-transferase omega 11 ENSMODG00000010839 GSTO2 0.17 2

12 ENSMODG00000012499 MED27 mediator complex subunit 27 0.17

13 ENSMODG00000007640 H3F3C H3 histone family member 3c 0.17

eukaryotic elongation factor-2 14 ENSMODG00000007732 EEF2K 0.16 kinase

15 ENSMODG00000014450 MODO-UC MHC class I antigen 0.16

16 ENSMODG00000025091 RPS24 ribosomal protein S4 0.15

17 ENSMODG00000009657 RPL4 ribosomal protein L4 0.15

18 ENSMODG00000012981 RHOA rac homolog family member A 0.14

heterogeneous nuclear 19 ENSMODG00000000603 HNRNPA2B1 0.14 ribonucleoprotein A2/B1

20 ENSMODG00000008731 RPL10 ribosomal protein L10 0.14

rho GDP dissociation inhibitor 21 ENSMODG00000017941 ARHGDIB 0.14 beta

22 ENSMODG00000021572 COI cytochrome c oxidase subunit 1 0.14

eukaryotic translation initiation 23 NCBI: 100012315 EIF1 0.13 factor 1

24 ENSMODG00000009945 LIMD2 LIM domain containing 2 0.13

destrin, actin depolymerizing 25 ENSMODG00000005428 DSTN 0.13 factor

26 NCBI: 3074661 ND1 NADH dehydrogenase subunit 1 0.13

27 ENSMODG00000018533 EEF1A1 elongation factor 1-alpha 0.13

28 ENSMODG00000023940 ACTB actin cytoplasmic 1 0.13

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29 ENSMODG00000006222 PABPC1 polyadenylate-binding protein 0.12

30 ENSMODG00000017520 RPS12 ribosomal protein S12 0.12

31 ENSMODG00000009238 HMGB1 high mobility group protein B1 0.12

32 ENSMODG00000002647 ACTG1 actin gamma 1 0.12

NADH: ubiquinone 33 ENSMODG00000007201 NDUFA13 0.12 oxidoreductase subunit A13

ENSMODG00000003499 ubiquitin A-52 residue ribosomal 34 UBA52 0.12 protein fusion product 1

35 ENSMODG00000009274 IKZF1 IKAROS family zinc finger 1 0.12

36 ENSMODG00000021012 JAM2 junctional adhesion molecule 2 0.11

serine and arginine rich splicing 37 ENSMODG00000011362 SRSF5 0.11 factor 5

38 ENSMODG00000009539 RPL13 ribosomal protein L13 0.11

39 ENSMODG00000011635 RPL5 ribosomal protein L5 0.11

membrane spanning 4-domains 40 ENSMODG00000020324 MS4A1 0.11 A1

NADH-ubiquinone 41 ENSMODG00000021584 NADH4 0.11 oxidoreductase chain 4

42 ENSMODG00000017688 B2M beta-2-microglobulin 0.11

43 ENSMODG00000008857 C5AR1 complement C5a receptor 1 0.11

SH3 domain-binding glutamic 44 ENSMODG00000014189 SH3BGRL2 0.11 acid-rich-like protein

heat shock cognate 71 kDa 45 ENSMODG00000012958 HSPA8 0.11 protein

46 ENSMODG00000000635 RAC2 rac family small GTPase 2 0.11

47 ENSMODG00000015665 CORO1A coronin 0.11

48 ENSMODG00000017253 TPM3 tropomyosin 3 0.11

49 ENSMODG00000005299 PFN1 profilin 0.10

Cullin-associated NEDD8- 50 ENSMODG00000006377 CAND2 0.10 dissociated 2

51 ENSMODG00000003534 RPL17 ribosomal protein L17 0.10

52 ENSMODG00000012693 RPL19 ribosomal protein L19 0.10

53 ENSMODG00000014099 RPL18 ribosomal protein L18 0.10

54 ENSMODG00000002481 UROD uroporphyrinogen decarboxylase 0.10

55 ENSMODG00000012118 EGR1 early growth response protein 1 0.10

56 ENSMODG00000010967 RPSA ribosomal protein SA 0.10

57 ENSMODG00000003743 DDX5 DEAD-box helicase 5 0.10

58 ENSMODG00000013766 RPL10A ribosomal protein L10A 0.10

59 ENSMODG00000004091 RACK1 receptor for activated C kinase 1 0.10

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arf-GAP with coiled-coil, ANK 60 ENSMODG00000006852 ACAP1 0.10 repeat and PH domains 1

61 ENSMODG00000007691 EEF1G elongation factor 1-gamma 0.10

heterogeneous nuclear 62 ENSMODG00000011916 HNRNPDL 0.10 ribonucleoprotein D like

63 ENSMODG00000012580 NME2 nucleoside diphosphate kinase 2 0.10

64 NCBI: 100030473 LOC100030473 myeloblastin 0.10

65 ENSMODG00000015201 RPLP0 ribosomal protein LP0 0.10

66 ENSMODG00000005983 H3F3A histone H3 0.10

67 ENSMODG00000020255 RPS23 ribosomal protein S23 0.10

signal sequence receptor subunit 68 ENSMODG00000008581 SSR4 0.10 4

69 ENSMODG00000007078 RPS13 ribosomal protein S13 0.10

70 ENSMODG00000007582 FTH1 ferritin 0.10

nascent polypeptide associated 71 NCBI: 100030834 NACA 0.09 complex subunit alpha

72 ENSMODG00000015929 PLA2G2C phospholipase A(2) 0.09

73 ENSMODG00000023391 RPL13A ribosomal protein L13A 0.09

peptidyl-prolyl cis-trans 74 ENSMODG00000016089 PPIA 0.09 isomerase

heterogeneous nuclear 75 ENSMODG00000009436 HNRNPH2 0.09 ribonucleoprotein H2

76 ENSMODG00000014210 RPS18 ribosomal protein S18 0.09

77 ENSMODG00000028389 RPS8 ribosomal protein S8 0.09

78 ENSMODG00000011070 RPS4X ribosomal protein S4X 0.09

79 ENSMODG00000000969 RPS3A ribosomal protein S3A 0.09

80 ENSMODG00000010572 CWF19L1 CWF19-like protein 1 0.09

ENSMODG00000003264 leucine rich repeats and 81 LRIG1 0.09 immunoglobulin like domains 1

82 ENSMODG00000012195 TYSND1 serine protease 0.09

83 ENSMODG00000019444 CTSH Pro-cathepsin H 0.09

84 ENSMODG00000010095 RPS30 ribosomal protein S30 0.09

85 ENSMODG00000013434 RPS11 ribosomal protein S11 0.09

86 ENSMODG00000003663 RPL13 ribosomal protein L13 0.09

eukaryotic translation elongation 87 ENSMODG00000016012 EEF1B2 0.09 factor 1 beta 2

88 ENSMODG00000023336 TUBA3E tubulin alpha chain 0.09

89 ENSMODG00000021464 MYL12B myosin light chain 12B 0.09

90 ENSMODG00000000275 RPL28 ribosomal protein L28 0.09

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91 ENSMODG00000001819 RPS27A ribosomal protein S27A 0.09

92 ENSMODG00000003890 RPS28 ribosomal protein S28 0.08

93 ENSMODG00000019167 RPL18A ribosomal protein L18 0.08

94 ENSMODG00000028168 COX5A cytochrome c oxidase subunit 5A 0.08

95 ENSMODG00000003974 RPS9 ribosomal protein S9 0.08

96 ENSMODG00000021591 CYTB cytochrome b 0.08

97 ENSMODG00000025578 RPL36A ribosomal protein L36A 0.08

coiled-coil domain containing 98 ENSMODG00000028326 CCDC184 0.08 184

99 ENSMODG00000004571 RPL12 ribosomal protein L12 0.08

100 ENSMODG00000006682 RPL38 ribosomal protein L38 0.08

Syndecan 4 (SDC4) had the highest expression in the entire B-cell transcriptome, accounting for 0.66% of the total gene expression in the opossum B-cell transcriptome (Table

4.1). Alignment comparison between the opossum SDC4 protein sequence and other mammalian species showed a >90% identity between opossum and other marsupial SDC4 protein sequences, and >75% identity between the opossum and investigated eutherian protein sequences (Figure 4.3).

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Figure 4.3 (A) Multiple sequence alignment of SDC4 gene products and (B) a Phylogenetic tree based on the SDC4 sequences, constructed using the NJ method. Protein sequences were aligned using the ClustalW algorithm. Dots indicate identical matches to the opossum sequences. Domains of the SDC4 protein are indicated by a line underneath the sequence. Species designations are abbreviated to the first two letters of their scientific names. M. domestica (Modo), S. harrisii (Saha), P. cinereus (Phci), V. ursinus (Vour), O. anatinus (Oran), H. sapiens (Hosa), and M. musculus (Mumu). Genbank accession numbers are: Modo SDC4, XP_007475896.1; Saha SDC4, XP_012395261.1; Phci SDC4, XP_020839750.1; Vour SDC4, XP_027707442.1; Oran SDC4, XP_028927254.1; Hosa SDC4, NP_002990.2; Mumu SDC4, NP_035651.1.

The spleen and blood single cells showed significant differences between their expression of highly expressed genes identified in the opossum B-cell transcriptome

(ANOSIM Global R = 0.035, P = 0.046) (Figure 4.4). Average dissimilarity revealed that this significant difference between the spleen and blood was largely due to the proportion of the

HNRNPK gene (heterogeneous nuclear ribonucleoprotein K) in the spleen (55%) compared to the blood (36%). There was no significant difference between the different Ig expressing cells (ANOSIM Global R = 0.063, P = 0.066), as the abundance of IgE cells were too small to compare (Figure 4.4 and Figure 4.5). ANOSIM pairwise tests showed significant differences between the expression of specific genes produced by IgM (R = 0.168, P = 0.002) and IgG (R = 0.138, P = 0.001) cells in comparison to IgA cells (Figure 4.5). SIMPER

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revealed that these significant scores are largely based on the expression of the GLG1 (golgi glycoprotein 1) and ELMOD1 (ELMO domain-containing protein) genes. The proportion of

GLG1 and ELMOD1 in IgM producing cells (74% and 62%, respectively) was significantly higher than in IgA producing cells (21% and 24%, respectively). GLG1 and ELMOD1 were also significantly higher in IgG (61% and 70%, respectively) than in IgA (21% and 24%, respectively) producing cells (Figure 4.5).

Figure 4.4 Two-dimensional non-transformed multidimensional scaling (MDS) plot visually representing the single-cell transcriptomes of the top 10 highly expressed genes in the spleen (grey upwards triangles) and blood (black downward triangles).

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Figure 4.5 Two-dimensional non-transformed multidimensional scaling (MDS) plot visually representing the single-cell transcriptomes of the top 10 highly expressed genes in the IgM (black downward triangle), IgG (dark grey diamond), IgA (grey squares), and IgE (grey upwards triangle).

Analysis of these opossum protein sequences when compared to other mammalian sequences showed a 90% identity between all mammalian HNRNPK protein sequences

(Figure 4.6). The opossum ELMOD1 protein sequence had 90% identity between marsupials and 70% identity between all mammalian ELMOD1 protein sequences (Figure 4.7), and the opossum had 75% identity between all mammalian GLG1 protein sequences (Figure 4.8).

Motif comparison of the GLG1 mammalian protein sequences identified fifteen conserved cysteine rich repeats containing four cysteine residues in each (Figure 4.8).

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Figure 4.6 (A) Multiple sequence alignment of HNRNPK gene products and (B) a Phylogenetic tree based on the HNRNPK sequences, constructed using the NJ method. Protein sequences were aligned using the ClustalW algorithm. Dots indicate identical matches to the opossum sequences. Domains of the HNRNPK protein are indicated by a line underneath the sequence. Symbols above sequence are: (1) Inverted triangles mark cysteine residues (2) Bullets mark nucleic acid binding regions, (3) Diamonds G-X-X-G motif. Species designations are abbreviated to the first two letters of their scientific names. M. domestica (Modo), S. harrisii (Saha), P. cinereus (Phci), O. anatinus (Oran), H. sapiens (Hosa) and M. musculus (Mumu). Genbank accession numbers are: Modo HNRNPK, XP_016279557.1; Saha HNRNPK, XP_012398527.1; Phci HNRNPK, XP_020822678.1; Oran HNRNPK, XP_016083874.1; Hosa HNRNPK, AAH14980.1; Mumu HNRNPK, ACC69193.1.

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Figure 4.7 (A) Multiple sequence alignment of ELMOD1 gene products and (B) a Phylogenetic tree based on the ELMOD1 sequences, constructed using the NJ method. Protein sequences were aligned using the ClustalW algorithm. Dots indicate identical matches to the opossum sequences. Domains of the ELMOD1 protein are indicated by a line underneath the sequence. Symbols above sequence are: (1) Inverted triangles mark cysteine residues. Species designations are abbreviated to the first two letters of their scientific names. M. domestica (Modo), S. harrisii (Saha), P. cinereus (Phci), V. ursinus (Vour), O. anatinus (Oran), and H. sapiens (Hosa). Genbank accession numbers are: Modo ELMOD1, XP_007494949.1; Saha ELMOD1, XP_012400499.1; Phci ELMOD1, XP_020851842.1; Vour ELMOD1, XP_027705369.1; Oran ELMOD1, XP_028904013.1; Hosa ELMOD1, NP_061182.3.

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Figure 4.8 (A) Multiple sequence alignment of GLG1 gene products and (B) a Phylogenetic tree based on the GLG1 sequences, constructed using the NJ method. Protein sequences were aligned using the ClustalW algorithm. Dots indicate identical matches to the opossum sequences. Cysteine rich repeats are indicated by lines underneath the sequence. Symbols above sequence are: (1) Inverted triangles mark cysteine residues. Species designations are abbreviated to the first two letters of their scientific names. M. domestica (Modo), S. harrisii (Saha), P. cinereus (Phci), V. ursinus (Vour), O. anatinus (Oran), H. sapiens (Hosa), and M. musculus (Mumu). Genbank accession numbers are: Modo GLG1, XP_007477569.1; Saha GLG1, XP_012395893.2; Phci GLG1, XP_020819407.1; Vour GLG1, XP_027699023.1; Oran GLG1, XP_028931633.1; Hosa GLG1, AAH60822.1; Mumu GLG1, EDL11491.1.

Immune genes that were highly expressed genes included the MHC class II DRα chain (HLA-DRA) and MHC class II β chain (MODO-DAB1) genes (both ranked in the top

10 most highly expressed genes), and along with SDC4, make up >1.3% of the total expressed genes in the opossum B-cell transcriptome (Table 4.1). Comparison of these protein sequences with other mammals showed HLA-DRA to have a >90% identity between marsupial and eutherian mammals while MODO-DAB1 had a >70% identity with other

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marsupial MHC DAB protein sequences (Figure 4.9 and Figure 4.10). Motif analysis identified an Ig C1-set domain and an α domain on both HLA-DRA and MODO-DAB1 protein sequences. Additionally, HLA-DRA protein sequences included seven heterodimer interface residues and ten MHC binding domain interface residues (Figure 4.9).

Figure 4.9 (A) Multiple sequence alignment of HLA-DRA gene products and (B) a Phylogenetic tree based on the HLA-DRA sequences, constructed using the NJ method. Protein sequences were aligned using the ClustalW algorithm. Dots indicate identical matches to the opossum sequences. Domains of the HLA-DRA protein are indicated by a line underneath the sequence. Symbols above sequence are: (1) Inverted triangles mark cysteine residues, (2) Bullets mark heterodimer interface residues, (3) Diamonds mark MHC binding domain interface residues. Species designations are abbreviated to the first two letters of their scientific names. M. domestica (Modo), S. harrisii (Saha), P. cinereus (Phci), V. ursinus (Vour), and H. sapiens (Hosa). Genbank accession numbers are: Modo HLA-DRA, XP_007483702.1; Saha HLA-DRA, XP_003770700.1; Phci HLA-DRA, XP_020829855.1; Vour HLA-DRA, XP_027732036.1; Hosa HLA-DRA, ARB08447.1.

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Figure 4.10 (A) Multiple sequence alignment of marsupial MHC DAB1 gene products and (B) a Phylogenetic tree based on the MHC DAB sequences, constructed using the NJ method. Protein sequences were aligned using the ClustalW algorithm. Dots indicate identical matches to the opossum sequences. Domains of the MHC DAB protein are indicated by a line underneath the sequence. Symbols above sequence are: (1) Inverted triangles mark cysteine residues. Species designations are abbreviated to the first two letters of their scientific names. M. domestica (Modo), S. harrisii (Saha), P. cinereus (Phci), N. eugenii (Noeu), and T. vulpecula (Trvu). Genbank accession numbers are: Modo DAB1, NP_001028163.1; Saha DAB, ABV58848.1; Phci DAB, ALX81653.1; Noeu DAB, AAX54675.1; Trvu DAB, AAK84172.1.

4.5 Discussion

The release of the opossum genome (Mikkelsen et al., 2007) and several companion articles have both reported structural details of the genome and in depth comparisons between eutherian and marsupial immunological components. Along with this report, the opossum has become the first marsupial to begin analysing the gene expression of B-cells at the single-cell level. Marsupials are born immunologically incompetent, unable to mount an endogenous antibody response until after the first week postpartum (Wang et al., 2009). Initial studies of primary humoral immune response in the opossum utilised sheep red blood cells and haemolytic titration to conclude that humoral immune responses are similar to that of

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eutherians (Croix et al., 1989). Our investigation identified the most highly expressed genes in the opossum’s single-cell transcriptome and analysed these genes to show any preferential production from IgM, IgG, IgA and IgE expressing cells, however since the number of cells expressing IgE were far too small, a definitive comparison with IgE could not be made.

SDC4 had the highest accumulative expression in the single-cell transcriptome, making up 0.66% of the opossum B-cells. Analyses of the SDC4 gene found no preferential variation between the IgH expressing cells. SDC4 is a transmembrane heparin sulphate proteoglycan located on the cell surface (Lee et al., 1998), encoding proteins that play major roles in cell adhesion, signalling and defence (Tkachenko et al., 2005, Elfenbein and Simons,

2013). Although the specific immunological role of SDC4 remains unclear (Endo et al.,

2015), it has previously been expressed in the uterine transcriptome of a pregnant opossum

(Hansen et al., 2016), as well as in the splenic B-cells of mice (Yamashita et al., 1999).

Furthermore a 4.1 m domain (putative band 4.1 homologues’ binding motif) was identified in both eutherian and marsupial SDC4 protein sequences and has been acknowledged for its importance in transmembrane signalling and cell-matrix adhesion (Lee et al., 1998, Wang et al., 2013). Other genes which had a high accumulative expression within the opossum B-cell transcriptome including HNRNPK, ELMOD1, and GLG1. HNRNPK had a significant abundance of transcripts expressed by spleen cells, though no difference was seen in between the single IgH expressing cells. ELMOD1 and GLG1 on the other hand, had significantly higher expression of these transcripts in both IgM and IgG in comparison to IgA. Due to low expression of IgE in the transcriptome, it is still unclear whether these genes are definitively produced by IgE expressing cells. HNRNPK was first identified to facilitate the processing and/or transport of pre-mRNA between the nucleus and cytoplasm of cells (Matunis et al.,

1992). Though analysis of human HNRNPK distribution has shown greater function in signal transduction and gene expression involved in the B-cell receptor signalling pathway

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(Bomsztyk et al., 1997, Jeon et al., 2005). The KH-1 motif identified on the investigated mammalian HNRNPK sequences suggests this gene is highly conserved between mammals and present the similar function in the B-cell receptor (Adinolfi et al., 1999). The ELMOD1 gene encodes for a protein with GTPase-activating properties (Ivanova et al., 2014), regulating actin-mediated processes such as chemotaxis and engulfment in many cell types

(Brzostowski et al., 2009). Previous investigations have expressed the ELMOD1 gene in the uterine transcriptome of the opossum (Hansen et al., 2016), showing a decrease of expression in non-pregnant opossum individuals. ELMOD1 has been identified in number of eukaryotes, suggesting it is highly conserved and has a distinct set of cellular functions which are homologous in eukaryotes (East et al., 2012). Lastly, GLG1 gene has not been fully characterised in mammals (Ahn et al., 2005). Previous investigations of GLG1 have shown significant localisations with basic fibroblast growth factors binding proteins, along with number of cysteine rich repeats identified on the GLG1 mammalian alignments suggests

GLG1 may assist with processing growth factors in numerous cells (Zuber et al., 1997, Köhl et al., 2000).

Specific immune genes that were highly expressed in the in transcriptome included

HLA-DRA (ranked 2nd) and MODO-DAB1 (ranked 6th). Both HLA-DRA and MODO-DAB1 belong to the MHC Class II family, which have extensive involvement in B-cell immunological responses (Cheng et al., 2009). Neither of these genes showed any preferential variation amongst the different IgH isotype expressing cells. Previously, both

HLA-DRA and MODO-DAB1 genes have been identified and characterised in marsupials.

The MHC DAB1 gene has been characterised in several marsupial species; two DAB alleles have been isolated in the red-necked wallaby (Macropus rufogriseus) (one was identified as a pseudogene) (Schneider et al., 1991), six DAB1 alleles have been isolated from the

Tasmanian devil (Siddle et al., 2007), five DAB1 alleles in the common brushtail possum

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(Lam et al., 2001), and only a single DAB1 transcript isolated in the spleen from a male opossum (Stone et al., 1999). The marsupial MHC DAB gene family, along with DBB (Belov et al., 2004) and DCB (Belov et al., 2006), are not present in any eutherian species (Belov et al., 2004). MHC DAB and DBB gene families are considered to have the highest expression in marsupials (Jobbins et al., 2012), with DAB the most polymorphic (Holland et al., 2008), yet this investigation could only identify the expression for DAB1 in the B-cell transcriptome. HLA-DRA has been investigated in both the red-necked wallaby (Slade and

Mayer, 1995), and koala (Abts et al., 2015). Slade and Mayer (1995) showed that the marsupial DRA sequence forms a distinct clade with DRA sequences observed in eutherians and northern blot analysis revealed very high expression of DRA in the spleen of the red- necked wallaby. Unlike DAB1, marsupial DRA is present in eutherians, suggesting some

MHC class II genes of mammals have evolved from different ancestral genes (Schneider et al., 1991).

The investigation of these highly expressed genes expressed in the opossum B-cell transcriptome has shown marsupial B-cells are comparable to eutherians. B-cells are vital for the humoral immune system response which is fundamental in the investigatory studies of mammalian immunity. This report provides a key resource for future transcriptomic studies of the opossum B-cells and assist with comparisons between the immunological divergence of marsupials and eutherians, and will contribute to the understanding of defence mechanisms used by marsupials, particularly the adaptive immune response.

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4.6 Acknowledgements

The authors would like to thank Ricky Spencer (WSU) for his help with the MDS plots, and Galina Petukhova making available M. domestica and her assistance with animal husbandry and tissue collection. This research was funded in part by a National Science

Foundation award IOS-13531232. Research reported in this publication was also supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under award P30 GM110907. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

4.7 Conflicts of Interest

The authors declare no conflicts of interest.

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4.8 Chapter 4 References

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KÖHL, R., ANTOINE, M., OLWIN, B. B., DICKSON, C. & KIEFER, P. 2000. Cysteine- rich fibroblast growth factor receptor alters secretion and intracellular routing of fibroblast growth factor 3. Journal of Biological Chemistry, 275, 15741-15748. LAM, M. K. P., BELOV, K., HARRISON, G. A. & COOPER, D. W. 2001. Cloning of the MHC class II DRB cDNA from the brushtail possum (Trichosurus vulpecula). Immunology Letters, 76, 31-36. LEE, D., OH, E., WOODS, A., COUCHMAN, J. R. & LEE, W. 1998. Solution structure of syndecan-4 cytoplasmic domain and its interation with phosphatidylinositol 4,5- bisphosphate. The Journal Of Biological Chemistry, 273, 13022-13029. MATE, K. E., ROBINSON, E. S., PEDERSEN, R. A. & VANDEBERG, J. L. 1994. Timetable of in vivo embryonic development in the grey short‐tailed opossum (Monodelphis domestica). Molecular Reproduction and Development, 39, 365-374. MATUNIS, M. J., MICHAEL, W. M. & DREYFUSS, G. 1992. Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein. Molecular and Cellular Biology, 12, 164. MI, H., MURUGANUJAN, A., HUANG, X., EBERT, D., MILLS, C., GUO, X. & THOMAS, P. D. 2019. Protocol Update for large-scale genome and gene function analysis with the PANTHER classification system (v.14.0). Nature Protocols, 14, 703-721. MIKKELSEN, T. S., WAKEFIELD, M., AKEN, B., AMEMIYA, C., CHANG, J., DUKE BECKER, S., GARBER, M., GENTLES, A. J., GOODSTADT, L., HEGER, A., JURKA, J., KAMAL, M., MAUCELI, E., SEARLE, S., SHARPE, T., BAKER, M. L., BATZER, M. A., BENOS, P., BELOV, K. & LINDBLAD-TOH, K. 2007. Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences. Nature 447, 167-77. MILLER, R. D. & BELOV, K. 2000. Immunoglobulin genetics of marsupials. Developmental & Comparative Immunology, 24, 485-490. MORRIS, K. M., WEAVER, H. J., O'MEALLY, D., DESCLOZEAUX, M., GILLETT, A. & POLKINGHORNE, A. 2018. Transcriptome sequencing of the long-nosed bandicoot (Perameles nasuta) reveals conservation and innovation of immune genes in the marsupial order peramelemorphia. Immunogenetics, 70, 327-336. OLD, J. M. & DEANE, E. 2003. The detection of mature T- and B-cells during development of the lymphoid tissues of the tammar wallaby (Macropus eugenii). Journal of Anatomy, 203, 123-131. OLD, J. M. & DEANE, E. M. 2000. Development of the immune system and immunological protection in marsupial pouch young. Developmental & Comparative Immunology, 24, 445-454. PAGNI, M., IOANNIDIS, V., CERUTTI, L., ZAHN-ZABAL, M., JONGENEEL, C. V., HAU, J., MARTIN, O., KUZNETSOV, D. & FALQUET, L. 2007. MyHits: improvements to an interactive resource for analyzing protein sequences. Nucleic acids research, 35, W433-W437. PAINTER, M. W., DAVIS, S., HARDY, R. R., MATHIS, D., BENOIST, C. & IMMUNOLOGICAL GENOME PROJECT, C. 2011. Transcriptomes of the B and T lineages compared by multiplatform microarray profiling. Journal of Immunology, 186, 3047-57. SAMOLLOW, P. B. 2006. Status and applications of genomic resources for the gray, short- tailed opossum, Monodelphis domestica, an American marsupial model for comparative biology. Australian Journal of Zoology, 54, 173-196. SAMOLLOW, P. B. 2008. The opossum genome: insights and opportunities from an alternative mammal. Genome Research, 18, 1199-1215.

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SCHNEIDER, S., VINCEK, V., TICHY, H., FIGUEROA, F. & KLEIN, J. 1991. MHC class II genes of a marsupial, the red-necked wallaby (Macropus rufogriseus): identification of new gene families. Molecular Biology and Evolution, 8, 753-766. SIDDLE, H. V., SANDERSON, C. & BELOV, K. 2007. Characterization of major histocompatibility complex class I and class II genes from the Tasmanian devil (Sarcophilus harrisii). Immunogenetics, 59, 753-760. SLADE, R. W. & MAYER, W. E. 1995. The expressed class II alpha-chain genes of the marsupial major histocompatibility complex belong to eutherian mammal gene families. Molecular Biology and Evolution, 12, 441-450. STONE, W., BRUUN, D., MANIS, G., HOLSTE, S., HOFFMAN, E., SPONG, K. & WALUNAS, T. 1996. The immunobiology of the marsupial, Monodelphis domestica. Modulators of Immune Responses, The Evolutionary Trail, Breckenridge Series, 2. STONE, W. H., BRUUN, D. A., FUQUA, C., GLASS, L. C., REEVES, A., HOLSTE, S. & FIGUEROA, F. 1999. Identification and sequence analysis of an MHC class II B gene in a marsupial (Monodelphis domestica). Immunogenetics, 49, 461-463. THOMPSON, J. D., HIGGINS, D. G. & GIBSON, T. J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673-4680. TKACHENKO, E., RHODES, J. M. & SIMONS, M. 2005. Syndecans. Circulation Research, 96, 488-500. VANDEBERG, J. L. & ROBINSON, E. S. 1997. The laboratory opossum (Monodelphis domestica) in laboratory research. The Institute for Laboratory Animal Research Journal, 38, 4-12. WANG, W., TAO, C., ZHOU, P., ZHOU, X., ZHANG, Q. & LIU, B. 2013. Molecular characterization, expression profiles of the porcine SDC2 and HSPG2 genes and their association with hematologic parameters. Molecular Biology Reports, 40, 2549-2556. WANG, X., OLP, J. J. & MILLER, R. D. 2009. On the genomics of immunoglobulins in the gray, short-tailed opossum Monodelphis domestica. Immunogenetics, 61, 581-96. WANG, X., SHARP, A. R. & MILLER, R. D. 2012. Early postnatal B cell ontogeny and antibody repertoire maturation in the opossum, Monodelphis domestica. PLoS One, 7, e45931. WONG, E. S., PAPENFUSS, A. T. & BELOV, K. 2011. Immunome database for marsupials and monotremes. BMC Immunology, 12, 48. YAMASHITA, Y., ORITANI, K., MIYOSHI, E. K., WALL, R., BERNFIELD, M. & KINCADE, P. W. 1999. Syndecan-4 Is expressed by B lineage lymphocytes and can transmit a signal for formation of dendritic processes. The Journal of Immunology, 162, 5940. ZUBER, M. E., ZHOU, Z., BURRUS, L. W. & OLWIN, B. B. 1997. Cysteine‐rich FGF receptor regulates intracellular FGF‐1 and FGF‐2 levels. Journal of cellular physiology, 170, 217-227.

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CHAPTER 5

Concluding Summary and Future Directions

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5.1 Concluding Summary

Marsupials are immunologically comparable to eutherians (Old et al., 2003), and like eutherians, marsupial B-cells are necessary for an adaptive humoral response to occur

(Kalmutz, 1962). Early investigations into marsupial immunology included measuring antibody responses (Kalmutz, 1962). These early antibody response studies showed consistent differences between eutherians and marsupials in terms of their intensity of primary and secondary antibody responses. Sustained primary antibody responses have been investigated in the quokka (Yadav, 1971), opossum (Croix et al., 1989), and common brushtail possum (Deakin et al., 2005), in which elevated IgG responses lasted 26, 37, and 15 weeks respectively. In marsupials secondary antibody responses were reportedly delayed and considerably lower in magnitude compared to eutherian responses, as poor isotype switching of IgM in secondary responses were observed (Yadav et al., 1972, Croix et al., 1989, Shearer et al., 1995). Later studies utilised a variety of traditional immunisation procedures typically used for eutherians to report more comparable responses between the two mammalian lineages (Kitchener et al., 2002, Deakin et al., 2005). Prior to the completion and availability of the opossum genome, comparative studies of eutherian and marsupial immune genes relied heavily on the use of heterologous probes to isolate cDNA sequences from cDNA libraries.

Harrison and Wedlock (2000) designed consensus primers from eutherian gene sequences with similar structure and function to isolate and clone a number of cytokines. However, techniques such as these were limited to isolating only highly conserved genes (Wakefield and Graves, 2005). The value of the opossum genome has been paramount for the comparative analysis of distinctive traits of each mammalian lineage, as well as paving the way for the identification of highly divergent genes (Mikkelsen et al., 2007).

The research presented in this thesis investigated the gene expression of gray short- tailed opossum B-cells. My research has successfully analysed the usage of IgH and IgL

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chain in the opossum B-cell repertoire of single cells, the first report for any marsupial species. Furthermore, my research has investigated the single-cell transcriptome of opossum

B-cells and made a comparative analysis of the genes that were highly expressed.

The first objective of the thesis was to discuss the knowledge of marsupial B-cell genetics in terms of genetic presence, homology and developmental stages in comparison to eutherians. The literature review (Chapter 2) explored these key similarities and differences between the two mammalian lineages and also identified the knowledge gaps and areas of research that still require attention. This chapter gives a comprehensive review of signature genes associated with B-cell development, differentiation and regulation. The maturation of

B-cells seems to be similar in both mammalian lineages, defined by several stages involving the production of cells within the lymphoid tissues, the rearrangement of Igs and expression of CD markers (Conley, 2003). Marsupial B-cells appear to develop differently compared to eutherians, as the majority of development is delayed until after birth, and the production of

B-cells still occurs in the liver as the bone marrow is immature (Borthwick et al., 2014, Old,

2016). My review reports on the abundance and diversity of IgH and IgL isotypes investigated in various marsupial species; four of the five Igs identified in eutherians and monotremes have been reported in marsupials (Aveskogh and Hellman, 1998, Belov et al.,

1998, Aveskogh et al., 1999, Belov et al., 1999a, Belov et al., 1999b, Belov et al., 1999c).

IgD has not been identified in any investigated marsupial species to date, leaving researchers to speculate its loss in all marsupials. The review also discusses the roles of CD markers, signal transduction markers, and transcriptional regulators in a few marsupial species. A number of CD markers have recently been identified in marsupials, CD79α and CD79β have both been transcribed and characterised in the tammar wallaby (Duncan et al., 2010) and bridled nail-tail wallaby (Suthers and Young, 2014). Duncan et al. (2010) investigated the expression of CD79α and CD79β in developing pouch young, reporting the detection of

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CD79α on day 10 postpartum, and CD79β on day 21 postpartum, concluding that the tammar wallaby would only be able to elicit a humoral antibody response after both CD markers are expressed. To date only the isoforms of the signal transduction molecule Lyn have been characterised in marsupials (Suthers and Young, 2013). LynA and LynB in the tammar wallaby and bridled nail-tail wallaby show the genomic organisation of these isoforms to be highly conserved, suggesting their signalling mechanism is alike in all mammals (Suthers and

Young, 2013). Many transcriptional regulators have only been predicted in marsupial genomic regions. My review highlights the importance of key regulators in terms of their role in developmental and commitment processes in B-cells, as many of these key molecules are yet to be identified in marsupials.

The usage of IgH and IgL isotypes has never been investigated in any marsupial species before, which has left a major knowledge gap in the comparison of mammalian immunological evolution. Therefore, the objective of the first study in the thesis (Chapter 3) was to analyse the expression of IgH and IgL isotypes in the opossum B-cell repertoire. This study determined IgM had the overall highest number of expressed cells, not surprisingly as

IgM is the first isotype to be formed during an immune response (Aveskogh et al., 1999). IgA had an increased abundance of expressed blood cells, with a significantly high number of IgA cells pairing with Igλ. IgG and IgE had low numbers of expressed cells in both the spleen and blood. A major discovery in this study was the Igκ:Igλ ratio of expressed cells in the opossum

B-cell repertoire, a question which has been left unanswered for marsupials since the 1990s.

The analysis of the opossum single-cells shows a highly distinct Igκ:Igλ ratio of 35:65.

Previous investigations have shown this ratio to be highly diverse within the eutherian lineage, in human, mice and rabbits expression of the IgL chain is clearly dominated by the

Igκ isotype (Butler, 1997, Kirschbaum et al., 1996, Waterfield et al., 1973). On the other end of the scale, eutherian species dominated by Igλ usage include horses, dogs and cats (Home

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et al., 1992, Arun et al., 1996), whilst pigs have a 50:50 ratio when it comes to IgL isotopic expression (Butler and Brown, 1994). The V domain of the IgH and IgL chains contributes to antibody diversity and establishes its specificity (Baker et al., 2005). My study identified a limited set of VH gene segments in the expressed cells of the opossum in contrast to a much higher variability of cells expressing Vκ and Vλ gene segments. Coevolution of opossum VH and VL diversity does not appear to follow the same pattern of V diversification as seen in other mammals investigated. The eutherian lineage has shown that species with highly diverse VH domains also have a highly diverse VL domain, while eutherian species with low

VH diversity have low VL diversity (Butler, 1997, Sitnikova and Su, 1998).

A further investigation was undertaken to gain insights into the whole opossum B-cell transcriptome (Chapter 4). By analysing the single-cells of paired Igs 14,654 unique genes were identified, of which 13,376 were successfully annotated. Accumulation of all cells found SDC4 was the most highly expressed gene throughout the entire transcriptome, with no preferential expression between the IgH cells. SDC4 has previously been expressed in the uterine transcriptome of a pregnant opossum (Hansen et al., 2016). ANOSIM pairwise and

SIMPER analyses found highly expressed ELMOD1 and GLG1 genes were significantly expressed in IgM and IgG cells when compared to IgA cells. As cells expressing IgE were far too low, no definitive comparison could be made between all marsupial IgH isotypes. MOTIF comparison and phylogenetic analysis of ELMOD1 and GLG1 genes suggest their protein sequences are highly conserved throughout mammalian evolution. Of all the genes annotated, only 2.5% (340 genes) were termed immunological. Of these immune genes, the most abundant were HLA-DRA and MODO-DAB1, both affiliated with the MHC Class II family.

These immune genes have previously been isolated and characterised in a number of marsupial species; red-necked wallaby (Schneider et al., 1991), Tasmanian devil (Siddle et al., 2007), common brushtail possum (Lam et al., 2001) and opossum (Stone et al., 1999).

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Unlike the HLA-DRA gene, MODO-DAB1 has only been identified in marsupials, suggesting that the MHC class II gene family has undergone a further evolutionary event after the divergence of the mammalian lineages has occurred (Schneider et al., 1991).

5.2 Future Directions

The results presented in my thesis confirm the vastness of the opossum B-cell transcriptome, and in some ways has raised more questions about the humoral immunity of marsupials. Prior to this work, studies for IgH and IgL usage in the B-cell repertoire has been limited to eutherian species. The opossum is now the first marsupial to have its ratio of

Igκ:Igλ usage identified in the B-cell repertoire, paving the way for future analysis. In eutherians the IgL isotopic ratio is highly variable, therefore future transcriptomic analyses are necessary to determine whether the marsupial lineage follows a similar pattern of IgL isotopic ratio variability.

The results of this thesis have shown that marsupial VH and VL domain usage may be coevolving in marsupial species. Due to the lack of VH gene segment diversity, IgL isotopic

V domains compensate by utilising a highly diverse pool of gene segments in the opossum B- cell repertoire. The common brushtail possum is the only other marsupial species to be investigated for VH and VL usage (Baker et al., 2005), and along with the opossum, this pattern of VH and VL is contradictory to eutherians. Investigations with humans and mice have demonstrated that IgH chains have a higher contribution to antibody-antigen interactions than IgL chains (Wilson and Stanfield, 1994), leaving some researchers questioning the importance of IgL chains (Pilström, 2002). Hence, further investigations are needed on marsupial IgL isotype contribution to antibody diversity. Immune genes are known to rapidly evolve within the genome and are therefore of great interest in comparative studies of mammalian evolution. My thesis has provided an in depth analysis of the opossum B-cell

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transcriptome, which can now be utilised as a reference point for future comparative studies within and between the mammalian lineages.

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5.3 Chapter 5 References

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