Browsing by Author "McClay, David R"

Specification and differentiation of a cell is accomplished by changing its gene expression profiles. These processes require temporally and spatially regulated transcription factors (TFs), to induce the genes that are necessary to a specific cell type. In each cell a set of TFs interact with each other or activate their targets; as development progresses, transcription factors receive regulatory inputs from other TFs and a complex gene regulatory network (GRN) is generated. Adding complexity, each TF can be regulated not only at the transcriptional level, but also by translational, and post-translational mechanisms. Thus, understanding a developmental process requires understanding the interactions between TFs, signaling molecules and target genes which establish the GRN.

In this thesis, two genes, FoxN2/3, a TF and FGFR1, a component of the FGF signaling pathway are investigated. FoxN2/3 and FGFR1 have different mechanisms that function in sea urchin development; FoxN2/3 regulates gene expression and FGFR1 changes phosphorylation of target proteins. However, their ultimate goals are the same: changing the state of an earlier GRN into the next GRN state.

First, we characterize FoxN2/3 in the primary mesenchyme cell (PMC) GRN. Expression of foxN2/3 begins in the descendants of micromeres at the early blastula stage; and then is lost from PMCs at the mesenchyme blastula stage. foxN2/3 expression then shifts to the secondary mesenchyme cells (SMCs) and later to the endoderm. Here we show that, Pmar1, Ets1 and Tbr are necessary for activation of foxN2/3 in the descendants of micromeres. The later endomesoderm expression is independent of the earlier expression of FoxN2/3 in micromeres and independent of signals from PMCs. FoxN2/3 is necessary for several steps in the formation of larval skeleton. A number of proteins are necessary for skeletogenesis, and early expression of at least several of these is dependent on FoxN2/3. Furthermore, knockdown (KD) of FoxN2/3 inhibits normal PMC ingression. PMCs lacking FoxN2/3 protein are unable to join the skeletogenic syncytium and they fail to repress the transfating of SMCs into the skeletogenic lineage. Thus, FoxN2/3 must be present for the PMC GRN to control normal ingression, expression of skeletal matrix genes, prevention of transfating, and control fusion of the PMC syncytium.

Second, we show that the FGF-FGFR1 signaling is required for the oral-aboral axis formation in the sea urchin embryos. Without FGFR1, nodal is induced in all of the cells at the early blastula stage and this ectopic expression of nodal requires active p38 MAP kinase. The loss of oral restriction of nodal expression results in the abnormal organization of PMCs and the larval skeleton; it also induces ectopic expression of oral-specific genes and represses aboral-specific genes. The abnormal oral-aboral axis formation also affected fgf and vegf expression patterns; normally these factors are expressed in two restricted areas of the ectoderm between the oral and the aboral side, but when FGFR1 is knocked down, Nodal expands, and in response the expression of the FGF and VEGF ligands expands, and this in turn affects the abnormal organization of larval skeleton.

Embryonic development occurs with precisely timed morphogenetic cell movements directed by complex gene regulation. In this orchestrated series of events, some epithelial cells undergo extensive changes to become free moving mesenchymal cells. The transformation resulting in an epithelial cell becoming mesenchymal is called an epithelial to mesenchymal transition (EMT), a dramatic cell biological change that occurs throughout development, tissue repair, and disease. Extensive in vitro research has identified many EMT regulators. However, most in vitro studies often reduce the complicated phenotypic change to a binary choice between successful and failed EMT. Research utilizing models has generally been limited to a single aspect of EMT without considering the total transformation. Fully understanding EMT requires experiments that perturb the system via multiple channels and observe several individual components from the series of cellular changes, which together make a successful EMT.

In this study, we have taken a novel approach to understand how the sea urchin embryo coordinates an EMT. We use systems level methods to describe the dynamics of EMT by directly observing phenotypic changes created by shifting transcriptional network states over the course of primary mesenchyme cell (PMC) ingression, a classic example of developmental EMT. We systematically knocked down each transcription factor in the sea urchin's PMC gene regulatory network (GRN). In the first assay, one fluorescently labeled knockdown PMC precursor was transplanted onto an unperturbed host embryo and we observed the resulting phenotype in vivo from before ingression until two hours post ingression using time-lapse fluorescent microscopy. Movies were projected for computational analyses of several phenotypic changes relevant to EMT: apical constriction, apical basal polarity, motility, and de-adhesion.

A separate assay scored each transcription factor for its requirement in basement membrane invasion during EMT. Again, each transcription factor was knocked down one by one and embryos were immuno-stained for laminin, a major component of basement membrane, and scored on the presence or absence of a laminin hole at the presumptive entry site of ingression.

The measured results of both assays were subjected to rigorous unsupervised data analyses: principal component analysis, emergent self-organizing map data mining, and hierarchical clustering. This analytical approach objectively compared the various phenotypes that resulted from each knockdown. In most cases, perturbation of any one transcription factor resulted in a unique phenotype that shared characteristics with its upstream regulators and downstream targets. For example, Erg is a known regulator of both Hex and FoxN2/3 and all three shared a motility phenotype; additionally, Hex and Erg both regulated apical constriction but Hex additionally affected invasion and FoxN2/3 was the lone regulator of cell polarity. Measured phenotypic changes in conjunction with known GRN relationships were used to construct five unique subcircuits of the GRN that described how dynamic regulatory network states control five individual components of EMT: apical constriction, apical basal polarity, motility, de-adhesion, and invasion. The five subcircuits were built on top of the GRN and integrated existing fate specification control with the morphogenetic EMT control.

Early in the EMT study, we discovered one PMC gene, Erg, was alternatively spliced. We identified 22 splice variants of Erg that are expressed during ingression. Our Erg knockdown targeted the 5'UTR, present in all spliceoforms; therefore, the knockdown uniformly perturbed all native Erg transcripts (∑Erg). Specific function was demonstrated for the two most abundant spliceoforms, Erg-0 and Erg-4, by knockdown of ∑Erg and mRNA rescue with a single spliceoform; the mRNA expression constructs contained no 5'UTR and were not affected by the knockdown. Different molecular phenotypes were observed, and both spliceoforms targeted Tbr, Tel, and FoxO, only Erg-0 targeted FoxN2/3 and only Erg-4 targeted Hex. Neither targeted Tgif, which was regulated by ∑Erg knockdown sans rescue. Our results suggest the embryo employs a minimum of three unique roles in the GRN for alternative splicing of Erg.

Overall, these experiments increase the completeness and descriptive power of the GRN with two additional levels of complexity. We uncovered five sub-circuits of EMT control, which integrated into the GRN provide a novel view of how a complex morphogenetic movement is controlled by the embryo. We also described a new functional role for alternative splicing in the GRN where the transcriptional targets for two splice variants of Erg are unique subsets of the total set of ∑Erg targets.

Dorsal closure in Drosophila embryos provides an excellent model system for the analysis of the coordinated cell shape changes and biomechanical processes that drive morphogenesis. During closure, the dorsal surface of the embryo displays an eye–shaped structure consisting of amnioserosa flanked by sheets of lateral epidermis. The canthi are found at the corners of the eye–shaped dorsal opening and are the focus of this dissertation. A synthesis of the four biomechanical processes that contribute to dorsal closure occurs in each canthus. Apical constrictions of amnioserosa cells and contractile actomyosin cables provide forces that favor closure. The two opposing sheets of lateral epidermis that flank the amnioserosa come together in the canthi where they are zipped together. Zipping at the canthi ensures the formation of a continuous epithelium and serves to maintain the curvature of the actomyosin cable necessary to resolve force in a dorsal–ward direction. This dissertation first describes the formation of the canthi, particularly interesting due to the radically different tissue organization for germ band retraction, the preceding stage of development. After canthus ontogeny, I describe dorsal closure stage canthi in three morphologically and mechanically distinct zones. I interrogate each zone by both confocal fluorescent microscopy and laser microsurgery to achieve a thorough visual and mechanical description. Finally, I describe the results of completely removing both canthi — the lateral epidermis leading edges straighten out to become parallel or nearly parallel fronts that move at native or nearly native rates and closure completes at the dorsal midline. Closure, again, proves to be robust and resilient — it can proceed without zipping or much if any leading edge curvature that in control embryos resolves purse string contraction into dorsal–ward forces. In total, the canthus proves to be an excellent source for many avenues of investigation with many more questions left to answer.

The mouse embryonic gonad provides an outstanding model to study the complex mechanisms involved in cell fate specification and maintenance. At the bipotential stage, both XX and XY gonads are capable of becoming testes or ovaries upon specific molecular cues. The specification of the supporting cell lineage (as either Sertoli cells in the male or granulosa cells in the female) initiates the testis or ovary program, leading to male or female fate. However, there are significant gaps in our understanding of how the somatic cells in the gonad arise, are competent to differentiate, and determine and maintain their fates. In this dissertation, we addressed these questions.

We found that NUMB (an antagonist of Notch signaling) serves as competence factor for somatic cell differentiation during early gonadogenesis. The asymmetric allocation of NUMB to the basolateral domain of actively dividing coelomic epithelial (CE) cells is indispensable to (1) maintain the totipotent stem cell-like reservoir at the CE domain, and (2) give rise to progenitor cells that can further differentiate into supporting and interstitial cell lineages. Deletion of Numb; Numbl resulted in disruption of cell polarity in the CE domain as well as a reduction of multiple differentiated cell lineages within XX and XY gonads, including supporting cells and male steroidogenic cells, which were most severely affected. We detected elevated Notch downstream signaling in the Numb; Numbl mutant gonads. Moreover, treatment of DAPT (which blocks Notch signaling) rescued the Numb; Numbl mutant phenotypes, strongly suggesting that upregulation of Notch is responsible.

Previous experiments indicate that when supporting cells commit to the male (Sertoli) fate, they must repress the alternative female (granulosa) cell fate. In another line of experiments, we investigated the hypothesis that the Polycomb repressive complex (PRC1) plays a critical role in repressing the female pathway during male gonad patterning. We found that loss of Ring1B (a component of PRC1) led to the disruption of XY gonad development specific to the posterior region of male gonads. Sry, the upstream driver of the male pathway, was not appropriately expressed in the posterior domain, which contained cells expressing female markers and, in some cases, small aggregates of undifferentiated cells. Using ChIP-Seq, we identified potential targets of PRC during male gonad development. Moreover, a key gene in the male pathway, SOX9, interacts with Ring1B, based on immunoprecipitation results, leading to the hypothesis that it may be involved in the recruitment of PRC to its target sites to execute the repression of female genes in male gonads.

Our findings provide insight into how somatic cell fate is determined and maintained during mammalian sex determination. Our results may be valuable for patients with disorders of sexual development with unidentified genetic contributions.

Multicellular organisms can be made up of hundreds of different cell types, each with their own unique morphology and characteristics to carry out their specific functions. For over 100 years, the sea urchin embryo has been used as a model to examine how cell types are specified during embryonic development. In each cell type of the embryo, transcription factors and signaling molecules must interact to form a gene regulatory network (GRN) which controls cell differentiation. When developmental GRNs are revealed, they provide insights into the stepwise mechanisms of how cells in the embryo differentiate from a multipotent progenitor to a fully differentiated specialized cell type. To understand the evolutionary history of specialized cells, GRNs that control specification of cell types in one species are compared to GRNs of similar cell types in other species. These comparisons provide insights into how ancestral cell types were changed during animal evolution to give rise to specialized cells in extant species. In this thesis, we use a combination of gene expression and perturbation assays to dissect the molecular mechanisms of cell type specification focusing on the sea urchin nervous and digestive system. We then infer evolutionary conclusions about when these cell types and the mechanisms of their differentiation evolved in metazoans.

In Chapter 3, we build a foundation to study how neural cells are specified and evolved in the nervous system by analyzing spatial and temporal gene expression during sea urchin neurogenesis. We report the expression of 23 genes expressed in areas of active neurogenesis in the sea urchin embryo from blastula stage (just before neural progenitors begin their specification sequence) through pluteus larval stage (when much of the nervous system has been patterned and is functional). Though this chapter is largely descriptive, it is essential to better understand what molecules and transcription factors are required for proper neural development in a basal deuterostome, the sea urchin. The expression patterns can be used as a starting point to 1) identify how subtypes of neurons are specified in the embryo 2) build a spatial gene regulatory network for sea urchin neurogenesis, and 3) perform comparative studies with the sea urchin, protostome and vertebrate organisms.

In Chapter 4, we build off the information found in Chapter 3 to examine the molecular mechanisms of neuronal subtype specification in three distinct neural subtypes in the Lytechinus variegatus larva. We show that these subtypes are specified through Delta/Notch signaling and identify a different transcription factor required for the development of each neural subtype. Our results show achaete-scute and neurogenin are proneural for the serotonergic neurons of the apical organ and cholinergic neurons of the ciliary band, respectively. We also show that orthopedia is not proneural but is necessary for the differentiation of the cholinergic/catecholaminergic postoral neurons. Interestingly, these transcription factors are used similarly during vertebrate neurogenesis. We believe the results in this chapter are a starting point for building a neural gene regulatory network in the sea urchin and for finding conserved deuterostome neurogenic mechanisms (Slota and McClay, 2018).

In Chapter 5, we focus on a neuronal cell type found in Chapter 4 to examine the evolutionary origin of the neural crest cell, a transient embryonic stem cell population unique to vertebrates. The mechanism of neural crest evolution has perplexed biologists since its discovery in the 1860s (Huang, 2004). The emergence of this cell type was critical for vertebrate evolution since it gives rise to tissues in the embryo required for complex predatory behaviors such as connective tissues of the head and neck and peripheral sensory neurons (Gans and Northcutt, 1983). In the last decade, two embryonic cell types in the tunicate Ciona intestinalis, have been proposed to be rudimentary neural crest cell types (Abitua et al., 2012; Stolfi et al., 2015). In this chapter, we show that a population of neurons in Lytechinus variegatus, which is a basal deuterostome, shares features with the neural crest-derived spinal neurons and C. intestinalis bipolar tail neurons. Like the neural crest, this cell type arises from the lateral borders of the neuroectoderm, expresses the transcription factor neurogenin and the acid sensing ion channel gene asicl, undergoes migration, requires MAPK signaling for its specification, and gives rise to afferent neurons in the peripheral nervous system. We believe this is an ancient cell type that is homologous to Ciona bipolar tail neurons and therefore the neural crest. We propose that this cell type existed before the split of chordates and the clade that includes sea urchins and acquired a multipotency gene regulatory program in the vertebrate lineage to give rise to the neural crest.

In Chapter 6, we shift focus to cell type specification in the sea urchin digestive system. We find that molecular inputs from tissues outside the gut provide inductive signals that contribute to cell type specification and anterior/posterior patterning of the developing gut. We show that the Wnt signaling ligand, wnt1, which is expressed in a ring of expression surrounding the developing blastopore, provides an inductive signal to the developing endoderm. In Wnt1 knockdown embryos, gastrulation occurs normally but anterior/posterior pattern of gene expression and regionalized cell type specification is lost in the developing mid and hindgut. Wnt1 knockdown results in a loss of transcription factor expression in the hindgut and anus including cdx, foxd, foxi and phb1. Furthermore, wnt1 knockdown results in loss of expression of the pyloric sphincter markers lox and nkx6.1 and the midgut marker gaba transporter (gat). When wnt1 RNA is ectopically expressed, ectoderm is then fated to become endoderm and the embryo becomes a large tripartite gut with an expansion of hindgut and midgut markers. Using a live imaging digestion assay, we then show the consequences to the organism when this gene expression pattern in the gut is lost, namely that larva cannot properly hold food in their gut. We propose that the inductive capabilities of wnt1 is ancient to metazoans and that another signal, possibly a different Wnt ligand, is the activating signal for regional cell type identity in the digestive system in the vertebrate lineage.

The sea urchin embryo has been used for decades for building developmental GRNs that control the separation of germ layers and for specification of cell types in the early embryo. At later developmental stages however, particularly after gastrulation is complete, little work has been done to build GRNs for specialized cell types required for the complex behavior of the larva. Identifying cell types in the sea urchin, understanding the mechanisms that lead to their specification and differentiation and then comparing that to cell types in other species allows us to understand how cells were modified and specialized during animal evolution.

Sea urchins are relegated as background or non-playable characters in Western Science and Western culture on a daily basis. This dissertation shines a spotlight on my non-human relatives and gives readers a chance to learn about the sciences and cultures that sea urchin species play a role in. Along with offering research within the realms of development biology and immunology, there are pieces relevant to Science & Society and general audience blended throughout the chapters. I address two major problems in this work. The first problem is determining how the complex immune system of the Lytechinus variegatus larva develops and reacts to injury. The second, is uncovering what colonial practices have led to the sea urchin research climate today.The first chapter of this dissertation offers a broad Introduction to sea urchins. Moving away from a “cold” or impersonal description, you’ll learn about the diversity of sea urchins that move about the world’s oceans every day. Topics include what urchin’s eat, their etymological origin, their influence on historical and contemporary cultures, what some taste like, and other ranging topics. Parallel to these topics, this chapter contains basic biology on the adult and larval sea urchin, and the development of the embryo’s mesenchyme cell populations, which will be the foci of multiple chapters. Moving into wet lab research, chapter 2 provides the methods used in molecular, cellular, and embryological experiments. The major novel research method is an injury assay where we are able crush or “squish” L. variegatus larvae, causing a reproducible and quick immune cell response. Chapter 3 involves a review and recommendations on how Western Science developmental biology researchers think about and approach studying the larva’s mesenchyme. Using these new practices, we were able to uncover tissue specific candidate markers in silico, by cloning, and through expression data. Chapter 4 is the characterization of the immune-related cytokine family, the macrophage migration inhibitory factors (MIFs) in sea urchins. Utilizing newly published genomes, we were able to provide a fuller description of MIF gene duplications, and confidently clone select MIFs and perturb candidate regulators. The following chapter addresses the conundrum of wound healing and the immune system’s role in the process. By crushing L. variegatus larvae, we have been able to describe and characterize roles of pigment and blastocoelar cells in epithelial and skeletal wound repair. Our conclusions for the respective chapters are, the MIF family has gone through a major gene duplication, and are now able to perform tissue specific and potentially redundant roles in immune cell function; and pigment cells and blastocoelar cell networks are activated quickly in injuries and can remodel tissues in the larvae. The Science & Society, and penultimate chapter of this dissertation takes a deeper look into why Western Scientists study sea urchin, doing so through the lens of colonialism. In outlining personal kinship, obligations, and an interdisciplinary lens, I am able to name the colonial intentions of marine stations and their use of invertebrate species as research materials. For a contemporary example, I name general instances of colonial research methods and propose select ways to promote anti- and decolonial practices in sea urchin science that move beyond metaphors. This project, and the questions it answers and generates moves away from myths of Western Science such as neutrality, and away from a spiritless resource for academic consumption. Along with learning about the immune system, cytokines, and injury response from my non-human, sea urchin relatives, we can also begin to address broad and systemic problems that are faced in sea urchin science and culture. My goal, and what should be thought about early in reading this work, is to learn from, appreciate, reciprocate, respect, and co-create knowledge with sea urchins in a way that doesn’t harm the Land.

During embryogenesis, a single zygote gives rise to a multicellular embryo with distinct spatial territories marked by differential gene expression. How is this patterning process organized? How robust is this function to perturbations? Experiments that examine normal and regulative development will provide direct evidence for reasoning out the answers to these fundamental questions. Recent advances in technology have led to experimental determinations of increasingly complex gene regulatory networks (GRNs) underlying embryonic development. These GRNs offer a window into systems level properties of the developmental process, but at the same time present the challenge of characterizing their behavior. A suitable modeling framework for developmental systems is needed to help gain insights into embryonic development. Such models should contain enough detail to capture features of interest to developmental biologists, while staying simple enough to be computationally tractable and amenable to conceptual analysis. Combining experiments with the complementary modeling framework, we can grasp a systems level understanding of the regulatory program not readily visible by focusing on individual genes or pathways.

This dissertation addresses both modeling and experimental challenges. First, we present the autonomous Boolean network modeling framework and show that it is a suitable approach for developmental regulatory systems. We show that important timing information associated with the regulatory interactions can be faithfully represented in autonomous Boolean models in which binary variables representing expression levels are updated in continuous time, and that such models can provide direct insight into features that are difficult to extract from ordinary differential equation (ODE) models. As an application, we model the experimentally well-studied network controlling fly body segmentation. The Boolean model successfully generates the patterns formed in normal and genetically perturbed fly embryos, permits the derivation of constraints on the time delay parameters, clarifies the logic associated with different ODE parameter sets, and provides a platform for studying connectivity and robustness in parameter space. By elucidating the role of regulatory time delays in pattern formation, the results suggest new types of experimental measurements in early embryonic development. We then use this framework to model the much more complicated sea urchin endomesoderm specification system and describe our recent progress on this long term effort.

Second, we present experimental results on developmental plasticity of the sea urchin embryo. The sea urchin embryo has the remarkable ability to replace surgically removed tissues by reprogramming the presumptive fate of remaining tissues, a process known as transfating, which in turn is a form of regulative development. We show that regulative development requires cellular competence, and that competence is lost early on but can be regained after further differentiation. We demonstrate that regulative replacement of missing tissues can induce distal germ layers to participate in reprogramming, leading to a complete re-patterning in the remainder of the embryo. To understand the molecular mechanism of cell fate reprogramming, we examined micromere depletion induced non-skeletogenic mesoderm (NSM) transfating. We found that the skeletogenic program was greatly temporally compressed in this case, and that akin to another NSM transfating case, the transfating cells went through a hybrid regulatory state where NSM and skeletogenic marker genes were co-expressed.

Gene regulatory networks (GRNs) provide a systems-level orchestration of an organism’s genome encoded anatomy. As biological networks are revealed, they continue to answer many questions including knowledge of how GRNs control morphogenetic movements and how GRNs evolve. Morphogenesis is a complex orchestration of movements by cells that are specified early in development.

The activation of an upstream GRN is crucial in order to orchestrate downstream morphogenetic events. In the sea urchin, activation of the endomesoderm GRN occurs after the asymmetric 4th cleavage. Embryonic asymmetric cell divisions often are accompanied by differential segregation of fate-determinants into one of two daughter cells. That asymmetric cleavage of the sea urchin micromeres leads to a differential animal-vegetal (A/V) nuclear accumulation of cell fate determinants, β-Catenin and SoxB1. Β-Catenin protein is localized into the nuclei of micromeres and activates the endomesoderm gene regulatory network, while SoxB1 is excluded from micromeres and enters the nucleus of the macromeres, the large progeny of the unequal 4th cleavage. Although nuclear localization of β-Catenin and SoxB1 shows dependence on the asymmetric cleavage, the mechanics behind the asymmetrical division has not been demonstrated. In Chapter 3, we show that micromere formation requires the small RhoGTPase, Cdc42 by directing the apical/basal orientation of the mitotic spindle at the apical cortex. By attenuating or augmenting sea urchin Cdc42 function, micromere divisions became defective and failed to correctly localize asymmetrically distributed determinants. As a consequence, cell fates were altered and multiple A/V axes were produced resulting in a “Siamese-twinning” phenotype that occurred with increasing frequency depending on the quantitative level of perturbation. Our findings show that Cdc42 plays a pivotal role in the asymmetric division of the micromeres, endomesoderm fate-determinant segregation, and A/V axis formation.

This dissertation also characterizes, at high resolution, the repertoire of cellular movements contributing to three different morphogenetic processes of sea urchin development: the elongation of gut, the formation of the primary mouth, and the migration of the small micromeres (the presumptive primordial germ cells) in the sea urchin, Lytechinus variegatus. Descriptive studies of the cellular processes during the different morphogenetic movements allow us to begin investigating their molecular control.

In Chapter 4, we dissected the series of complex events that coordinate gut and mouth morphogenesis. Until now, it was thought that lateral rearrangement of endoderm cells by convergent extension was the main contributor to sea urchin archenteron elongation and that cell divisions were minimal during elongation. We performed cell transplantations to live image and analyze a subset of labeled endoderm cells at high-resolution in the optically clear sea urchin embryo. We found that the endomesoderm cells that initially invaginate into the sea urchin blastocoel remained contiguous throughout extension, so that, if convergent extension were present, it was not a major contributor to elongation. We also found a prevalence of cell divisions throughout archenteron elongation that increased the number of cells within the gut linearly over time; however, we showed that the proliferation did not contribute to growth, and their spindle orientations were randomized during divisions and therefore did not selectively contribute to the final gut length. When cell divisions were inhibited, we saw no difference in the ability of the cells within the gut to migrate in order to elongate. Also in Chapter 4, we describe our observations of the cell biological processes underlying primary mouth formation at the end of gastrulation. Using time-lapse microscopy, photo-convertible Kaede, and an assay of the basement membrane remodeling, we describe a sequential orchestration of events that leads to the fusion of the oral ectoderm and the foregut endoderm. Our work characterizes, at higher resolution than previously recorded, the temporal sequence and repertoire of the cellular movements contributing to the length of the sea urchin larval gut and tissue fusion with the larval primary mouth.

In Chapter 5, the migration of the small micromeres to the coelomic pouches in the sea urchin embryo provides an exceptional model for understanding the genomic regulatory control of morphogenesis. An assay using the robust homing potential of these cells reveals a “coherent feed-forward” transcriptional subcircuit composed of Pax6, Six3, Eya, and Dach1 that is responsible for the directed homing mechanism of these multipotent progenitors. The linkages of that circuit are strikingly similar to a circuit involved in retinal specification in Drosophila suggesting that systems-level tasks can be highly conserved even though the tasks drive unrelated processes in different animals.

The sea urchin gene regulatory network (GRN) describes the cell fate specification of the developing embryo; however, the GRN does not describe specific cell biological events driving the three distinct sequences of cell movements. Our ability to connect the GRN to the morphogenetic events of gastrulation, primary mouth formation, and small micromere migration will provide a framework for characterizing these remarkable sequences of cell movements in the simplest of deuterostome models at an unprecedented scale.

This dissertation combines live imaging, embryological manipulations and computational analysis to understand the development of pigmented immunocytes in the sea urchin. Three areas received special attention: the mechanism and timing of the epithelial to mesenchymal transition, the function of pigment cells in larvae, and identification of new candidate molecules that may participate in those activities.

Chapter 2 is a methods chapter combining embryo cut and paste approaches with live imaging. Improvements over earlier methods including development of techniques to better image sea urchin embryos in vivo are described. One of the primary strengths of the sea urchin is its optical clarity and simplistic developmental morphogenetic movements. The embryo develops cilia prior to undergoing morphogenetic movements. Consequently, long-term live imaging is a challenge. This can be overcome through multiple treatments and image processing techniques, resulting in the ability to acquire time-lapse movies for up to 24hrs.

Chapter 3 describes an investigation of pigment cells epithelial-to-mesenchymal transition (EMT) a dynamic cellular process during which cells must change polarity, de-adhere from neighboring cells, breach through the basement membrane and become motile. Building off previous work in the lab, a comparison of the gene regulatory network that directs this process was identified and compared with other cell types undergoing an EMT to learn whether the same, or a different network is deployed in different cell types. I show that most of the TFs deployed in the skeletogenic EMT are not expressed in pigment cells before or during EMT, suggesting that there are different mechanisms controlling EMT in the pigment cells. There is some overlap, however, in that morpholino knock down of the TF twist disrupts EMT in pigment cells as it does in skeletogenic cells.

Chapter 4 is a study of pigment cell biology. A number of questions were addressed. We learned where pigment cells incorporate into the ectoderm, how are they are patterned in the ectoderm and demonstrate several components of their function. Pigment cell incorporation initially occurs into the posterior dorsal ectoderm and the cells then migrate in the dorsal ectoderm to spread into anterior regions of that tissue. Observations were made describing how the cells move with the ectoderm. Functionally, pigment cells are shown to reversibly respond to light exposure. They also are shown to respond to wounding. Further, the immunosurveillance activities are recorded revealing populations of immobile and migratory pigment cells, suggesting that the population may diversify functionally.

Computational and molecular approaches were used in Chapter 5 to identify new candidate molecules, specific to pigment cells, to begin to understand molecular processes underlying pigment cell behaviors. This approach provides the largest expansion to date of previously un-described genes expressed in pigment cells.

Pigment cells serve in a protective role during larval development. This project has advanced an understanding of how those cells work, and it opens new possibilities for further in depth approaches toward molecular explanations of function. The final synthesis addresses unanswered questions in the development, differentiation and function of pigment cells and provides insight into how these activities can now be explored.

My dissertation work integrates comparative transcriptomics and functional analyses to investigate gene expression changes underlying two significant aspects of sea urchin evolution and development: the dramatic developmental changes associated with an ecologically significant shift in life history strategy and the development of the unusual radial body plan of adult sea urchins.

In Chapter 2, I investigate evolutionary changes in gene expression underlying the switch from feeding (planktotrophic) to nonfeeding (lecithotrophic) development in sea urchins. In order to identify these changes, I used Illumina RNA-seq to measure expression dynamics across 7 developmental stages in three sea urchin species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph Heliocidaris tuberculata, and an outgroup planktotroph Lytechinus variegatus. My analyses draw on a well-characterized developmental gene regulatory network (GRN) in sea urchins to understand how the ancestral planktotrophic developmental program was altered during the evolution of lecithotrophic development. My results suggest that changes in gene expression profiles occurred more frequently across the transcriptome during the evolution of lecithotrophy than during the persistence of planktotrophy. These changes were even more pronounced within the GRN than across the transcriptome as a whole, and occurred in each network territory (skeletogenic, endomesoderm and ectoderm). I found evidence for both conservation and divergence of regulatory interactions in the network, as well as significant changes in the expression of genes with known roles in larval skeletogenesis, which is dramatically altered in lecithotrophs. I further explored network dynamics between species using coexpression analyses, which allowed me to identify novel players likely involved in sea urchin neurogenesis and endoderm patterning.

In Chapter 3, I investigate developmental changes in gene expression underlying radial body plan development and metamorphosis in H. erythrogramma. Using Illumina RNA-seq, I measured gene expression profiles across larval, metamorphic, and post-metamorphic life cycle phases. My results present a high-resolution view of gene expression dynamics during the complex transition from pre- to post-metamorphic development and suggest that distinct sets of regulatory and effector proteins are used during different life history phases.

Collectively, my investigations provide an important foundation for future, empirical studies to investigate the functional role of gene expression change in the evolution of developmental differences between species and also for the generation of the unusual radial body plan of sea urchins.

Sea urchin larvae possess a beautiful, intricately patterned, calcium-carbonate skeleton. Formation of this complex structure results from two independent processes within the developing embryo: specification of the mesenchymal cells that produce the skeletal rods, and patterning inputs from the ectoderm, which secretes signals directing the growth and shape of the skeleton. To understand patterning of the skeleton therefore, the specification events behind these two processes must be understood separately, and then connected in order to understand how ectoderm signaling directs skeletal growth. While the former processes of mesenchyme specification and mineralization are under study elsewhere, the means by which ectodermal cues directing skeletal growth are activated and localized is not known. Using molecular genetics, including gene knock downs and mis-expression, as well as microsurgical manipulations of early cleavage embryos, I show how a previously undescribed territory within the ectoderm, the border ectoderm (BE) is specified with short range signaling inputs. Then, experiments show that the BE provides signals that initiate, and contribute to the propagation of skeletogenesis. From this dataset, and from biological experiments I outline a model for how the BE patterns and contributes to the directed growth of the skeleton. I also discuss challenges to this model that need to be addressed in future research. In principle, the mechanism proposed herein depends on the integration of information from both the primary and secondary embryonic axes. It requires both short-range signaling by Wnt5 from the endoderm to establish the BE fate, and TGFß signaling from the oral and aboral ectoderm which subdivides the BE into four territories. These findings demonstrate that the short-range signaling cascade that subdivides the embryo into first mesoderm and then endoderm also specifies ectodermal fates. Ultimately, this research paves the way for understanding how the larval skeleton is patterned during embryogenesis and may provide a paradigm for understanding other, more complex, developmental problems.