Morphogenesis, or the development of tissues, structures, and organs, is at the heart of embryonic development. Morphogenesis is a complex, multi-tissue process that requires coordinated cellular communication, migration, and differentiation; due to this complexity, the mechanisms that underlie morphogenesis remain poorly understood. The sea urchin embryo is morphologically and genetically more simple than most other developmental model organisms, and is optically transparent, making it a highly tractable system in which to study morphogenetic processes. The sea urchin larval endoskeleton is a biomineral that is secreted by primary mesenchyme cells (PMCs). The PMCs ingress into the embryo and remain individual, mesenchymal cells that migrate into a stereotypic three-dimensional (3-D) pattern within the blastocoel, prefiguring the form of the ensuing skeleton, which they subsequently secrete. PMC positioning is directed by cues originating in the overlying ectoderm; however, the molecular identity of those cues has remained unknown. The work described in this dissertation combines systems-level approaches with in vivo 3-D spatial analysis to identify novel skeletal patterning genes and to define their functional roles in skeletal patterning. A transcriptomics-based screen identified numerous novel candidate skeletal patterning cues. Of those cues, two were further pursued for detailed functional studies. First, the sulfate transporter SLC26a2/7 (SLC) was found to promote ventral accumulation of sulfated proteoglycans that is both necessary and sufficient to attract PMCs to the ventral territory for ventral skeleton formation. Second, the enzyme 5-lipoxygenase (LOX) was found to be required for ventral and rotational skeletal patterning, and its product, 5(S)-HETE was found to be a chemoattractant for PMCs, thereby identifying a novel role for lipoxygenase enzymes in embryonic patterning and morphogenesis. Recent work from other groups has demonstrated that PMCs diversify their gene expression profiles during skeletal patterning, implying that PMC diversification is involved in skeletal patterning, likely as a response to locally distinct spatial cues. The studies herein identify Tbx2/3 and Pks2 as important PMC subset-specific genes whose spatial expression is modulated by SLC and LOX, respectively. Together, these results provide new mechanistic insights that define our molecular understanding of the regulation of sea urchin ventral skeletal patterning.

Continuous efforts have been directed towards controlled calcium carbonate biomineral synthesis in recent years. Compared to their inorganic counterparts, biominerals are more tensile in industrial applications, biocompatible with scientific designs, and sustainable for the environment. Most current approaches for synthetic biomineral production rely heavily on sophisticated engineering techniques to constrain the physical property of their crystals, which limits the adaptability of these products. Here, we proposed a novel approach to synthesize calcium carbonate biominerals by reproducing skeletogenesis of the sea urchin larva in vitro using common cellular and molecular methods. Skeleton formation in Lytechinus variegatus sea urchin embryos is a highly coordinated event, where ectodermal cells in different domains express distinct patterning cues that are received by adjacent primary mesenchyme cells (PMCs), which in turn secrete the skeleton. Our group and others have identified a range of skeletal patterning cues, and based on our current understanding of the mechanism, we envisioned a synthetic ectoderm culture using defined ectodermal lineages that, when combined with PMCs, will direct the synthetic production of skeletal structures. Here we have developed a detailed protocol for establishing such as ectoderm culture and have begun initial experiments towards this goal. Future deployment of this protocol will provide invaluable insights into the mechanism of skeletal patterning in sea urchins, as well as an unprecedented system for customized synthetic calcium carbonate biomineral production. Finally, improving our mechanistic understanding of skeletal patterning in echinoderms has the potential to shed light on analogous biomineralization processes in other species as well. / 2024-10-03T00:00:00Z