Mesoderm Segmentation in the Amphipod Crustacean Parhyale Hawaiensis
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Mesoderm Segmentation in the Amphipod Crustacean Parhyale hawaiensis by Roberta Lydia Farago Hannibal A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Molecular and Cell Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Nipam Patel, Chair Professor Jennifer Fletcher Professor John Gerhart Professor Chelsea Specht Professor David Weisblat Fall 2010 Mesoderm Segmentation in the Amphipod Crustacean Parhyale hawaiensis © 2010 by Roberta Lydia Farago Hannibal Abstract Mesoderm Segmentation in the Amphipod Crustacean Parhyale hawaiensis By Roberta Lydia Farago Hannibal Doctor of Philosophy in Molecular and Cell Biology University of California, Berkeley Professor Nipam Patel, Chair In arthropods, annelids, and chordates, both ectodermal and mesodermal tissues are organized into segments. Although these phyla share the trait of segmentation, a widely accepted phylogenetic hypothesis places them into the three separate clades of the bilatarians. Since each of these clades also contains many unsegmented phyla, it is debatable whether segmentation in arthropods, annelids and chordates is homologous. A limitation of previous studies comparing segmentation between these three groups is that most of the existing data is on mesoderm segmentation in vertebrates and ectoderm segmentation in Drosophila. Homologous mechanisms of segmentation, such as in the mesoderm of vertebrates and arthropods, could be missed in these studies. To further investigate the evolution of segmentation, I have examined mesoderm segmentation in the arthropod Parhyale hawaiensis, an amphipod crustacean. In Parhyale, segments are added progressively from anterior to posterior, similar to most other arthropods. Additionally, Parhyale is an ideal system for experiemental manipulations. For example, lineage tracing, cell ablation, and gene knock-down reagents are easily targeted to the segmental mesoderm and ectoderm. My dissertation focuses on two aspects of Parhyale mesoderm segmentation. First, whether the mesoderm and the ectoderm require signals from each other in order to segment. Second, whether the Parhyale homologs of the snail family of transcriptional repressors play a role in mesoderm segmentation. My first research project tests a hypothesis that mesoderm segmentation is autonomous and induces segmentation in the overlying ectoderm. Using ablations, I show that the ectoderm segments normally without the mesoderm. When I ablate the ectoderm, the mesoteloblasts, the mesodermal stem cells, continue to undergo normal rounds of division. However, they fail to migrate properly. Additionally, the progeny of the mesoteloblasts, the mesoblasts, neither proliferate nor express known markers of mesoderm segmentation. This suggests that the ectoderm provides a permissive signal, allowing mesoblasts to divide, and, possibly, an instructive signal, patterning the mesoblast lineage. My second research project addresses whether Parhyale homologs of the snail family of transcriptional repressors play a role in mesoderm segmentation and/or specification, similar to their role in other species. There are four known homologs of snail in Parhyale. Since one homolog was previously characterized, I characterized the three other homologs. In addition, I performed analysis of all four family members to determine their relationship with each other as well as snail homologs in other species. The snail family has two branches in bilatarians, snail 1 and scratch. I found that there are three snail homologs and one scratch homolog in Parhyale, which I named Ph-snail1, Ph-snail2 and Ph-snail3, and Ph-scratch, respectively. Since snail plays a major role in early mesoderm development in other animals, I expected one or more Parhyale snail homologs to be expressed in early mesoderm. I found that Ph-snail1 is the only Parhyale snail gene expressed in the mesoderm, and is expressed later in development than expected. Ph-scratch is not expressed during early development and germband elongation. Ph- snail2 and Ph-snail3 are expressed in the ectoderm, suggestive of a role in nervous system development. In order to investigate the role of Ph-snail1 (Ph-sna1) in the mesoderm, I knocked down Ph-sna1 transcripton using targeted siRNAs. The expression pattern of Ph-sna1 suggests three potential roles in the mesoteloblasts: specification, migration, and/or cell-cycle progression. By injecting siRNAs against Ph-sna1 into the mesoteloblast precursor cell, I found that mesoteloblasts form but undergo fewer divisions than controls. This is consistent with the hypothesis that Ph-sna1 is involved in mesoteloblast cell cycle progression. In addition, when siRNAs for Ph-sna1 are injected at the one cell stage, the embryo fails to develop after the initiation of germ band formation due to knock-down of Ph-sna1 in the anterior head ectoderm. My thesis work has refined our understanding of the role of the ectoderm and snail homologs in Parhyale mesoderm segmentation. Comparison of segmentation in Parhyale to segementation in the arthropod Drosophila show that, although the role of snail homologs differ in mesoderm segmentation, the interactions between the segmental ectoderm and mesoderm are similar. These interactions differ between arthropods, vertebrates and annelids, though, suggesting that either their common ancestor was not segmented, or that segmentation in these groups has diverged to the point where the germ layer containing the primary information for segmentation has been reversed. 2 Dedication i Table of Contents Abstract 1 Dedication i Table of Contents ii List of Figures iv List of Tables vi List of Abbreviations vii Acknowledgements viii Chapter 1: Introduction 1 Segmentation 1 What is a segment? 1 Evolution of segmentation 5 Parhyale hawaiensis 7 Parhyale hawaiensis 7 Reproduction and early development 8 Mesoderm and ectoderm segmentation 8 Chapter 2: Interplay between mesoderm and ectoderm segmentation 23 Summary 23 Introduction 23 Materials and methods 25 Results 26 Ph-Even-skipped is expressed segmentally in the mesoderm 26 Relationship between the segmental mesoderm and ectoderm 27 Ectoderm, but not mesoblasts, has regional identity 28 Ectoderm segmentation does not require mesoderm 29 Mesoteloblast can divide autonomously, but mesoteloblast migration and mesoblast division require the ectoderm 29 In the absence of ectoderm, mesoblasts do not express markers of segmentation 30 Discussion 30 Ectoderm segmentation does not require the mesoderm, refuting a hypothesis from arthropod literature 30 Development of segmental mesoderm in Parhyale involves an autonomous and an ectoderm-dependent phase 31 Evolution of segmentation 32 Chapter 3: The Parhyale snail superfamily 51 Summary 51 Introduction 51 Materials and methods 53 Results 55 There are three snail and one scratch homologs in Parhyale 55 Common N-terminal sequence motifs in Ph-sna1 55 ii Analysis of Parhyale snail homolog BACs 55 Ph-sna2 and Ph-sna3 are expressed in the ectoderm, suggesting a role in Dorsal-Ventral patterning 56 Lack of Ph-scratch expression in Parhyale embryos 57 Discussion 57 Parhyale snail phylogenetic analysis and nomenclature 57 Lack of Parhayle snail expression during mesoderm specification and gastrulation 58 Subfunctionalization of the Parhyale snail family 58 Chapter 4. Ph-snail1 function 76 Summary 76 Materials and methods 76 Results 78 Knock-down of Ph-sna1 prevents germband development 78 Ablation of anterior head ectoderm phenocopies Ph-sna1 knock-down 79 Mesoteloblast behavior and Ph-sna1 expression 79 Knock-down of Ph-sna1 in the mesoteloblasts 80 Ph-sna1 overexpression and knock-down constructs 81 Discussion 83 Ph-sna1 function in the anterior head ectoderm 83 Ph-sna1 function in the mesoteloblasts 83 Future outlook for Ph-sna1 functional experiments 84 Chapter 5: Future directions and closing remarks 107 Mesoderm segmentation in Parhyale hawaiensis 107 Evolution of Segmentation 107 Appendix A: BAC screening (Mef2, twist, msx1, msx2, vnd, Notch, Delta, Su(h)) 109 Appendix B: cDNA sequences and additional data for the Parhyale snail family 114 Appendix C: DV patterning in the mesoderm 136 Appendix D: Ectopic mesoteloblasts 149 Appendix E: Parhyale FGF receptor (Ph-heartless) 155 References 163 iii List of Figures 1.1. What is a segment? 9 1.2. Phylogenetic relationship among segmented and unsegmented animals 11 1.3. Parhyale development and reproduction 13 1.4. Parhyale fate map 15 1.5. Parhyale fate map for the segmental mesoderm and ectoderm 17 1.6. Ectoderm segmentation 19 1.7. Mesoderm segmentation 21 2.1. Ph-Even-skipped (Ph-Eve) expression in the mesoderm 33 2.2. Coordination of mesoderm and ectoderm segmentation during development 35 2.3. Diagram of coordination of mesoderm and ectoderm segmentation during development 37 2.4. Relationship between the segmenting mesoderm and ectoderm 39 2.5. Ph-Ultrabithorax (Ph-Ubx) is expressed in the ectoderm but not in the underlying mesoblasts 41 2.6. Ectoderm segments autonomously 43 2.7. Mesoteloblast division is autonomous, but mesoteoblast migration and mesoblast division require the ectoderm 45 2.8. Mesoblasts express neither Ph-twi nor Ph-Eve, markers of the segmental mesoderm, without the ectoderm 47 2.9. Models of mesoderm development 49 3.1. Phylogenetic relationship among members of the snail superfamily 60 3.2. Zinc finger alignment of the snail superfamily 62 3.3. Comparison of sequence motifs in the snail superfamily 64 3.4.