Signal Dependent Regulation of Skeletogenesis in the Sea Urchin Embryo

Morphogenesis, the process by which the tissues and organs of the embryo are properly shaped, is a fundamental feature of development. In the sea urchin, the formation of the calcified enodoskeleton is a major morphogenetic event. Differentiation of the skeletogenic primary mesenchyme cells (PMCs) has been considered to occur in two phases: the autonomous specification of PMCs followed by signal dependent patterning of PMCs and the embryonic skeleton they produce. Autonomous specification creates a homogenous population of PMCs, but the later differentiation of these cells is influenced by extrinsic signals that provide essential positional information. Recent studies showed that ectodermal growth factors are critically involved in the guidance of PMC migration and skeletal differentiation. However, a better understanding of the various signaling pathways that regulate skeletogenesis and their role in PMC gene expression remains to be established. This study examines the regulation of morphogenesis by signaling pathways, using skeletogenesis in the sea urchin embryo as a model. The aim of this study was to identify and study the roles of extrinsic signals in regulating PMC gene expression, focusing on the later, signal dependent phase of PMC differentiation. By analyzing and classifying spatial expression patterns of 39 genes preferentially expressed in PMCs, I find that: 1) these genes are expressed non uniformly within the PMC syncytium, reflecting a widespread influence of locally activated signals; 2) regions with elevated gene expression correlate with sites of rapid biomineral deposition at each stage; 3) non uniform expression of genes within the PMC syncytium is controlled by multiple signal in a precise temporal sequence. I also provide evidence that ectoderm derived VEGF signaling regulates gene expression in PMCs via the MAPK pathway on the ventral side of the embryo. Additionally, my work has identified an essential role for TGF b signaling in skeletogenesis. Previous studies indicate that a complete repertoire of TGF b signaling components is present in the sea urchin genome and TgfbrII mRNA is preferentially expressed in PMCs at the early gastrula stage. In this study, I show that TgfbrII mRNA is specifically expressed in the PMC lineage from the hatched blastula to the mid gastrula stage. Perturbation experiments indicate that TgfbrII is activated by the single, sensu stricto TGF b ligand in sea urchins and is required for skeletogenesis in the sea urchin embryo. I also show that the late activity of Alk4/5/7, the putative Type I receptor, regulates skeletogenesis in a dose dependent manner. Isolation and in vitro culture of PMCs demonstrates that both Alk4/5/7 and TgfbrII function cell autonomously in these cells. I provide evidence that TGF b TgfbrII signaling is not involved in dorsal ventral axis patterning or PMC specification; instead, this pathway plays a selective role in later skeletal patterning. Taken as a whole, my research demonstrates that skeletogenesis is regulated by a much more diverse suite of signaling pathways than was previously appreciated. These findings significantly expand our understanding of the complex regulation of skeletal morphogenesis by extrinsic signals during embryonic development.