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It is a challenge to understand how the information encoded in DNA is used to build a three-dimensional structure. To explore how this works the assembly of a relatively simple skeleton has been examined at multiple control levels. The skeleton of the sea urchin embryo consists of a number of calcite rods produced by 64 skeletogenic cells. The ectoderm supplies spatial cues for patterning, essentially telling the skeletogenic cells where to position themselves and providing the factors for skeletal growth. Here, we describe the information known about how this works. First the ectoderm must be patterned so that the signaling cues are released from precise positions. The skeletogenic cells respond by initiating skeletogenesis immediately beneath two regions (one on the right and the other on the left side). Growth of the skeletal rods requires additional signaling from defined ectodermal locations, and the skeletogenic cells respond to produce a membrane-bound template in which the calcite crystal grows. Important in this process are three signals, fibroblast growth factor, vascular endothelial growth factor, and Wnt5. Each is necessary for explicit tasks in skeleton production.

Gastrulation in the sea urchin begins with ingression of the primary mesenchyme cells (PMCs) at the vegetal pole of the embryo. After entering the blastocoel the PMCs migrate, form a syncitium, and synthesize the skeleton of the embryo. Several hours after the PMCs ingress the vegetal plate buckles to initiate invagination of the archenteron. That morphogenetic process occurs in several steps. The nonskeletogenic cells produce the initial inbending of the vegetal plate. Endoderm cells then rearrange and extend the length of the gut across the blastocoel to a target near the animal pole. Finally, cells that will form part of the midgut and hindgut are added to complete gastrulation. Later, the stomodeum invaginates from the oral ectoderm and fuses with the foregut to complete the archenteron. In advance of, and during these morphogenetic events, an increasingly complex input of transcription factors controls the specification and the cell biological events that conduct the gastrulation movements.

Development of the body plan is controlled by large networks of regulatory genes. A gene regulatory network that controls the specification of endoderm and mesoderm in the sea urchin embryo is summarized here. The network was derived from large-scale perturbation analyses, in combination with computational methodologies, genomic data, cis-regulatory analysis, and molecular embryology. The network contains over 40 genes at present, and each node can be directly verified at the DNA sequence level by cis-regulatory analysis. Its architecture reveals specific and general aspects of development, such as how given cells generate their ordained fates in the embryo and why the process moves inexorably forward in developmental time.

Dissertation, 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 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.

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.

Epithelial-mesenchymal transitions (EMTs) are fundamental and indispensable to embryonic morphogenesis throughout the animal kingdom. At the onset of gastrulation in the sea urchin embryo, micromere-derived primary mesenchyme cells (PMCs) undergo an EMT process to ingress into the blastocoel, and these cells later become the larval skeleton. Much has been learned about PMC specification in sea urchin embryos. However, much less is known about how states of the sequentially progressing PMC gene regulatory network (GRN) controls the EMT process during PMC ingression. Transcriptional regulators such as Snail and Twist have emerged as important molecules for controlling EMTs in many model systems. Sea urchin snail and twist genes were cloned from Lytechinus variegates, and each has been experimentally connected to the PMC regulatory network; these experiments demonstrate several requirements for PMC ingression, and in doing so, begin to illustrate how a gene regulatory network state controls morphogenesis. Functional knockdown analyses of Snail with morpholino-substituted antisense oligonucleotides (MASO) in whole embryos and chimeras demonstrated that Snail is required in micromeres for PMC ingression. Investigations also show that Snail downregulates cadherin expression as an evolutionarily conserved mechanism, and Snail positively regulates a required endocytic clearance of epithelial membrane molecules during EMT. Perturbation experiments indicate that Twist has accessory roles in regulating PMC ingression, and possibly plays a maintenance role in PMC specification network state. In addition, Twist also functions in the post-EMT network state, particularly in directing PMC differentiation and skeletogenesis. The recently annotated sea urchin genome accelerates the discovery of new genes and holds strong promise of mapping out a complete canvas of the micromere-PMC gene regulatory network. Using the genome resources we successfully cloned several newly identified PMC genes, and found most of them to be expressed in micromeres just prior to ingression of the nascent PMCs. Current experiments focus on the roles of these genes in preparing for, executing, and/or controlling the mesenchymal behavior following PMC ingression. The functions and inter-relationships of these genes will greatly augment our understanding of how a gene regulatory network state controls a crucial morphogenetic event.