Embryos develop in dynamic environments and are subject to myriad stressors – both biotic and abiotic. Among those stressors are encounters with pathogens and toxic molecules. The systems for sensing and responding to the environment include a robust suite of molecular machinery that protects the cell, and a heterogeneous population of immune cells that mount coordinated responses. Establishing a whole-animal echinoderm embryo model to address embryo-environment interactions and how stressors influence such systems is the focus of this dissertation work.

Accomplishing this task began with identifying a need for a sea urchin model species that grew more rapidly that other commonly used echinoderms, and establishing a comparable developmental staging scheme and culturing practices for rearing animals in the lab. The painted white urchin, Lytechinus pictus, was a clear choice for the optimal model.

Generating molecular tools was also essential, and yielded a fully sequenced genome with chromosomal resolution and transcriptomic tools pooled across developmental stages. To assess the quality of these molecular resources I annotated the suite of ATP-binding cassette transporters (ABCs). ABCs are expressed in, and play important roles in handling of toxic molecules as well as in the differentiation and migration of, immune cells.

In addition to the ABCs, I combed the genome for other components involved in immunity – including cytokines, transcription factors, enzymes, effectors, and signal mediators. This suite of annotations establishes a foundation of potential targets of exposure in the immune system. From there, I challenged sea urchin larvae with bacterial infection and characterized the first phenotypic responses of an echinoderm model of immunosuppression.

The outcome of this research is a series of tools and resources that open doors for future investigation. With a reliable, dose-dependent model of immunosuppression and complimentary molecular tools in a rapidly developing echinoderm species, it is now possible to address the influence of toxic molecules on whole-animal immune responses. It is also possible to investigate how early life exposures alter later life health or the health of subsequent generations. Finally, we can begin to tease apart the cross-talk of developmental and protective systems in the embryo contributing to environmental sensing and signaling.

This thesis concerns aspects of the development of the sea urchin Lytechinus pictus, a species that can be cultured from egg to breeding adult in six months. The first experimental portion of this thesis (Chapter 2) examines the developmental role of the enzyme phosphatidic acid phosphatase 2B (Pap2b). Fluorescent in situ hybridization demonstrated that Pap2b is expressed ubiquitously in the larval skeletogenic mesenchyme, and not in migrating primordial germ cells where expression was initially expected by analogy with studies on flies and zebrafish. The specific inhibitor Propranolol was used to reduce the function of Pap2b. At a low dose (0.5 µM) the body rods and ventral transverse rods of the larval skeleton failed to fuse normally; higher concentrations resulted in stunted embryos with fragmented, unpatterned skeletal elements. The second major experimental part of this thesis (Chapter 3) concerns the adoption of CRISPR-Cas9 gene editing in L. pictus. The majority of sea urchin CRISPR studies have been done in species that cannot be easily cultured, and have therefore relied on analysis of mosaic F0 embryos. I used CRISPR to create a homozygous knockout sea urchin line, an accomplishment so far attained by only one other laboratory in the world. I mutated the ATP binding cassette (ABC) transporter ABCB1, a small molecule transporter involved in cell signaling, environmental chemical uptake, and immune biology. L. pictus embryos were injected with two guide RNAs against Lp-ABCB1, and genotyping of post-metamorphic juveniles confirmed their activity. Subsequently, non-lethal somatic genotyping was used to identify mutant post-metamorphic individuals. F0 mosaic animals were outbred to wild-type animals to generate F1 heterozygotes, and these were subsequently inbred to produce homozygous F2 mutants. These results provide a foundation for further studies on the function of ABCB1, and more generally for the broader adoption of genetic engineering in sea urchin research. Finally, Chapter 4 of this thesis presents several topics, generally on insertion of transgenes using gene editing, which do not merit a whole chapter individually. These observations may serve as a starting point for future researchers interested in expanding the repertoire of gene editing available in L. pictus.

A number of molecular mechanisms have been implicated in biological tolerance of marine environmental stress. Among the best characterized of these is the heat shock response, which has been shown to play an important role in determining the distribution of marine invertebrates along natural gradients of stress (eg. thermal stress in the intertidal). The heat shock response also appears to play a critical role in determining the susceptibility of invertebrates to anthropogenic stress. In a parallel context, it has been proposed that multidrug resistance may play an important role in determining the survival of invertebrates along natural and anthropogenic gradients of toxic organic compounds.

This study characterized the role of both of these systems in the tolerance of natural stressors in model marine invertebrate systems. ln the first section of the dissertation I demonstrated that, although Pacific oysters are capable of significant adaptive regulation of heat shock protein (HSP) expression, adaptive plasticity occurs at a physiologic cost to inducible stress tolerance. Moreover. although adaptive regulation of HSPs is via a transcriptional mechanism during conditions of acute thermal stress. the response to chronic stress may not require continual transcriptional activation of HSP genes. ln the second section of the dissertation I focused on the role ofmultidrug resistance (MDR) transporters in oocytes. embryos and larvae in model (sea urchin, starfish and urechis) systems. In the first chapter of this section l described species-specific differences in transporter expression and consequent differences in susceptibility to naturally degraded hydrocarbons. In the second chapter I hypothesized that the reason for organic susceptibility in echinoderms is not due to the absence of a transporter. but rather due to the specificity of the predominant transporter family expressed in each species. Consistent with this hypothesis I showed that the predominant type of transporter expressed in echinoderm oocytes and embryos bear homology to the multidrug resistance associated protein (MRP) family rather than the multidrug resistance (P-gp/MDR) family. I examined the potential consequences of this difference for transporter substrate specificity and we measured the level of MRP expression during early development. I show that echinoderms possess a characteristic MRP transport phenotype that is activated at fertilization.

Gene regulation during embryogenesis is required for executing the program fordevelopment, as well as responding to the environment in a context-specific manner. Nuclear receptors (NRs) are transcription factors that modulate gene expression, often in response to xenobiotics. However, NRs role in early development remains less understood. To address this gap, a marine model organism like the sea urchin, Strongylocentrotus purpuratus, is ideal for studying NRs during embryogenesis. This study screened 10 NRs important for cellular defense and localized mRNA through colorimetric and fluorescence whole mount in-situ hybridization at key developmental stages. With this initial screen, we found two NRs with localized expression patterns. These include: hnf1a, expressed primarily in the developing hindgut and later in larval gut and ciliary band and ppara1, which localized to the larval foregut and midgut. With hybridization chain reaction in-situ, ppara1 is confirmed to be localized in the gut along with ppargc1, a coactivator for ppara1. Coactivators are recruited after a NR is ligand bound and facilitates transactivation. NRs were further characterized with scRNA-seq data in the 72 hpf embryo and AlphaFold protein modeling to predict structure and binding. With scRNA-seq, rxr, a NR that heterodimerizes with ppara1, was found expressed in the larval gut. AlphaFold models the ligand binding domain helices and residues responsible for ligand binding and predicts Sp-PPAR:RXR heterodimer functions in binding to the pollutant tributyltin chloride. This study lays the conceptual framework for researching PPAR:RXR function to understand gene regulation by both endogenous and xenobiotic ligands.

ATP-binding cassette (ABC) transporters are large membrane proteins found in all organisms. They use ATP to power transport of diverse substrates across membranes, and the ABC-B, -C, and -G families make up the multidrug resistance (MDR) transporters. Here, I survey the expression of MDR transporters during development of the purple sea urchin, Strongylocentrotus purpuratus, and identify and characterize one transporter, Sp-ABCC5a (C5a) that has a role in developmental signaling required for hindgut morphogenesis.

From the unfertilized egg through the prism stage (~58 hours post-fertilization, hpf), I detected transcripts of 20 MDR transporters with predicted roles in cell signaling, lysosomal and mitochondrial homeostasis, potassium channel regulation, pigmentation, and xenobiotic efflux. The protective transporter ABCB1a is ubiquitously expressed throughout embryogenesis in all cells, and the protein localizes to apical membranes at the interface between the embryo and external environment. In contrast, expression of C5a, a previously uncharacterized transporter, is first detected at hatching and peaks after gastrulation, and the protein localizes to basolateral membranes. I propose that profiling spatiotemporal expression and subcellular protein localization may identify transporters with roles in protection, homeostasis, and signaling.

As expected, efflux activity of C5a does not support a role in protection; C5a is not a broad-spectrum chemical effluxor, and it discriminates between structurally similar compounds. C5a expression is restricted to aboral non-skeletogenic mesoderm (NSM) cells (pigment cells) and is dependent on delta/notch signaling from the skeletogenic mesoderm (SM) as well as the aboral NSM regulatory gene, glial cells missing. During gastrulation, C5a protein traffics to the plasma membrane of pigment cells, while these cells migrate away from the archenteron to embed in the aboral ectoderm.

Knockdown of C5a leads to embryos with differentiated pigment cells but abnormal protrusion of the hindgut, which is seen in ~90% of embryos by the late prism stage (~eight hours after C5a protein expression peaks). The ABCC5 substrate cAMP rescues the prolapse in a dose-dependent manner, and in control embryos causes hyper-invagination. The cAMP-producing enzyme soluble adenylyl cyclase (sAC) is expressed in tandem with C5a, with peak expression in pigment cells just after gastrulation. Blocking sAC activity with the specific inhibitor KH7 impairs gastrulation. Together these data suggest that C5a-mediated efflux of sAC-derived cAMP from pigment cells controls late invagination of the hindgut. This study elucidates a novel role for an ABCC/MRP transporter in mesoderm-endoderm signaling during embryogenesis.

The sea urchin faces a variety of environmental factors in the ocean, including small molecules. These small molecules can enter the cell and affect development, homeostasis, immunity, and cellular functions. Transcription factors play many roles, including the regulation of how these molecules enter and exit cells by altering expression of membrane proteins. Nuclear receptors (NRs) that aid in responding to small molecules by binding to these ligands, causing a downstream effect of altering the gene expression of target genes. In this thesis, I study a particular group of NRs, the Hepatocyte Nuclear Factors (HNF), which have been shown to play a role in responding to stimuli and to maintain metabolism and regulate development. Sea urchins need to adapt to dynamic environmental conditions by responding to specific stimuli. HNFs have been well studied in mammalian models, but there is a lack of a full description of HNFs within echinoderms. This is particularly useful to further understand these transcription factors in small molecules transport in a genetically enabled species. Here, I have identified and localized the four major types of HNFs - HNF1, HNF3, HNF4, and HNF6 within the sea urchin, Lytechinus pictus during early development.

Animal primordial germ cells (PGCs) that become eggs and sperm pass their genomes onto the next generation. Little is known about how PGCs are protected from toxic chemicals. In this dissertation, I explore the function and activity of ATP binding cassette (ABC) transporters in PGCs of the sea urchin Strongylocentrotus purpuratus known as small micromeres (Smics). I found that Smics down-regulate the activity of two ABC transporter families that confer multidrug resistance to chemotherapeutic resistant cancers. Nascent Smic plasma membranes are formed in the 16-cell embryo, which have reduced ABCB activity. Other echinoid species, including Lytechinus pictus and Dendraster excentricus also produce Smics with reduced ABCB-mediated efflux activity, showing that this reduction in activity is conserved within euechinoids. ABC transporters may also play a developmental role in addition to a protective role. To understand why PGCs would remove protective transporters from their plasma membranes, I grew embryos in ABC transporter inhibitors. Smics in inhibitor-treated embryos display altered left/right coelomic pouch distributions in the pluteus larva. To confirm that ABC transporters efflux chemoattractants that serve to guide Smics along the left/right axis, I completed a study that is now the first characterization of PGC migration in the sea urchin embryo. In addition to passively riding on the elongating archenteron, the eight Smics are highly motile and move independently on the tip of the archenteron. Throughout gastrulation, the eight Smics produce cortical blebs and filopodia, hallmarks of migratory PGCs in other organisms. When the ABC transporter ABCB1a is overexpressed in the Smics, the production of blebs and filopodia is severely reduced and Smics do not migrate to the two coelomic pouches as fast as control cells or those cells expressing efflux null ABCB1a. Moreover, when ABCB1a is expressed in Smics, they become significantly more left fated. In summary, the reduction of ABC transporter activity, including ABCB1a activity in Smics, is necessary for their directed migration and morphogenesis, presumably through the detection of morphogenetic signals effluxed by ABC transporters from other cells.

Metamorphosis is a radical morphological and environmental transition from a larval phase to an adult. This process is utilized by ~80% of all animal species and is a particularly common life history strategy among marine invertebrates. It has been well established that across species developmentally competent marine larvae utilize bacterial cues to indicate a suitable habitat conducive to adult survival and reproduction. Despite the high incidence of bacterially induced metamorphosis, the underlying mechanisms of how animals directly sense these signals remain under-studied, especially in Echinoderms (e.g., sea urchins). This study aims to a investigate component of the innate immune system, antimicrobial peptides (AMPs), as a potential candidate for connecting bacterial presence to metamorphosis. To explore AMPs as a player in bacterially induced metamorphosis, I utilized the rapid and synchronously developing sea urchin Lytechnius pictus. I first identified a homolog of the Echinoderm specific AMP Strongylocin 2 in L. pictus. I found that LpStrongylocin 2 (LpS2l) is only transcribed in early larvae in the event of bacterial infection. In nascent late-stage larvae, LpS2l transcription is upregulated and accumulates within cells in developing adult structures, likely a subtype of blastocoelar cells. The spatiotemporal regulation of LpS2l transcription in L. pictus larvae suggests a role for AMPs in the development of new structures and recognizing metamorphosis-inducing bacterial cues. Future experiments will be aimed at assessing functional role of LpS2l in metamorphosis by disrupting transcription using CRISPR/Cas9 sgRNA guides designed in this study.