The use of nudibranchs as experimental species for neuroethology has been pivotal to elucidating the basis of behavior. These shell-less molluscs are particularly well-suited, given their swimming and crawling behaviors and large, identifiable neurons. However, little is known about early life stages: how the neural system controls behavior during embryonic, larval, and juvenile stages remains unclear, mostly due to the intractability of embryos in species currently used for behavioral studies. Understanding central nervous system development and the formation of identifiable neurons are crucial for neuroethological studies. However, the embryonic origins of the central nervous system and the appearance of identified neurons after metamorphosis are unknown. Therefore, the aeolid nudibranch Berghia stephanieae has been established as an experimental system; this species' embryos are accessible, enabling the study of brain development. This study reports on methodology for labeling the central nervous system with various antibodies that recognize neurotransmitters, creates the most detailed larval and juvenile staging system in a nudibranch to date, and describes the expression patterns of neurotransmitters in the brain during these stages. The post-metamorphic juvenile brain is organized similarly to the adult brains of nudibranch neuroethological models. Gangliogensis of the larval and juvenile central nervous systems is described and quantified. Additionally, the central and peripheral nervous systems are described in detail throughout distinct larval and juvenile stages. Finally, landmark neurons were identified in post-metamorphic stages. These results provide a detailed atlas of neural development and quantification of those growth through ganglion cell counts and immunoreactive cell counts throughout time.

The central question underlying all four chapters of this thesis is to develop a deeper understanding of the developmental and evolutionary basis for biomineralization in Mollusca. To address this, a model organism is essential; specifically, a molluscan species that has: (i) identified candidate genes involved in shell development, (ii) expression atlases of shell gene patterns during development, and (iii) established functional gene perturbation techniques for studying shell genes during early development. I use the slipper snail, Crepidula, to meet these requirements. In Chapter 1, I conduct proteomic and transcriptomic analyses on shells and mantle tissue to identify shell matrix proteins within the shell's extracellular matrix. In Chapter 2, I assess the evolutionary age of Crepidula shell matrix proteins and determine the localization of shell matrix genes in shell gland cells during larval shell development. In Chapter 3, I develop techniques for CRISPR/Cas9 gene perturbation in Crepidula, demonstrating the first shell matrix protein gene knockout in a molluscan species. Finally, in Chapter 4, I examine a species outside of gastropods, specifically within Polyplacophora, and develop animal husbandry and larval biology techniques for studying shell plate formation in a local chiton species. Together, the work presented here helps lay the foundation for Crepidula as a capable model for conducting mechanistic studies on shell matrix proteins and their regulators.

The development of the sea urchin larval body plan is well-understood from extensive studies of embryonic patterning. However, fewer studies have investigated the late larval stages during which the unique pentaradial adult body plan develops. Ancestral and evolved traits exist both in the larval and juvenile echinoderm body plans, so studying juvenile development is necessary to understand echinoderm evolution. In chapter 1, we apply hybridization chain reaction (HCR) in situs to the fast-developing and transparent sea urchin Lytechinus pictus, with a focus on skeletogenic cells. First, we use HCR to provide a detailed staging scheme for the localization of skeletogenic cells around the rudiment and show that many skeletogenic cells unassociated with larval rods appear outside of the rudiment prior to appearing inside. Second, we find co-expression of blastocoelar and skeletogenic cell markers around juvenile skeleton located outside of the rudiment, which is consistent with data showing that cells from the non-skeletogenic mesoderm embryonic lineage reprogram into juvenile skeletogenic cells. Third, we show that downstream biomineralization genes have similar expression patterns during larval and juvenile skeletogenesis, suggesting some conservation of skeletogenic mechanisms in the larva and juvenile. In chapter 2, we use a fluorescent protein that is bright when injected into L pictus and identify cell-type specific reporters to provide tools to lineage trace and understand cell movement prior to and during metamorphosis. This work sets the foundation for subsequent studies of the late larva of L. pictus to better understand juvenile body plan development, patterning, and evolution.

The use of model organisms has been instrumental in furthering our understanding of biology as a whole; however, the use of additional organisms among different phylogenetic branches are needed to better understand the evolution of biological processes, traits, cell types, and genes. In recent years, due to its feasibility in culturing and established molecular techniques, the molluscan nudibranch Berghia stephanieae has emerged as a “non-model organism” to study a wide variety of biological processes. One barrier to Berghia research is the lack of a foundational understanding of Berghia biology, including the molecular mechanisms underlying novel tissue and cell type development. To lessen this barrier, this study combines three NGS datasets with in situ hybridization chain reaction to identify and characterize tissue and cell types in juvenile Berghia. Three novel tissues, the digestive gland, cnidosac, and rhinophore, were characterized through marker gene expression. A literature review of all single-cell RNA sequencing studies performed in molluscs was done, and a workflow for identifying cell types in our Berghia scRNA-seq data was established. From this data, four different cell types, including two hemocyte populations, neurons, ectomesoderm-derived secretory cells, were identified and characterized. Identification of these cell types will allow for future comparisons with additional scRNA-seq datasets to address questions about the evolution of these cell types. Overall, this work provides a foundational understanding of the molecular mechanisms behind tissues and cell types in juvenile development in the nudibranch Berghia stephanieae, enhancing its usefulness as a model organism.

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