Cell Type Specification and Evolution of the Developing Sea Urchin Nervous and Digestive Systems

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.