Signal-dependent regulation of the sea urchin skeletogenic gene regulatory network

The endoskeleton of the sea urchin embryo is produced by primary mesenchyme cells (PMCs). Maternal inputs activate a complex gene regulatory network (GRN) in the PMC lineage in a cell-autonomous fashion during early development, initially creating a uniform population of prospective skeletonforming cells. Previous studies showed that at post-blastula stages of development, several effector genes in the network exhibit non-uniform patterns of expression, suggesting that their regulation becomes subject to local, extrinsic cues. Other studies have identified the VEGF and MAPK pathways as regulators of PMC migration, gene expression, and biomineralization. In this study, we used whole mount in situ hybridization (WMISH) to examine the spatial expression patterns of 39 PMC-specific/enriched mRNAs in Strongylocentrotus purpuratus embryos at the late gastrula, early prism and pluteus stages. We found that all 39 mRNAs (including several regulatory genes) showed non-uniform patterns of expression within the PMC syncytium, revealing a global shift in the regulation of the skeletogenic GRN from a cellautonomous to a signal-dependent mode. In general, localized regions of elevated gene expression corresponded to sites of rapid biomineral deposition. We used a VEGFR inhibitor (axitinib) and a MEK inhibitor (U0126) to show that VEGF signaling and the MAPK pathway are essential for maintaining high levels of gene expression in PMCs at the tips of rods that extend from the ventral region of the embryo. These inhibitors affected gene expression in the PMCs in similar ways, suggesting that VEGF acts via the MAPK pathway. In contrast, axitinib and U0126 did not affect the localized expression of genes in PMCs at the tips of the body rods, which form on the dorsal side of the embryo. Our results therefore indicate that multiple signaling pathways regulate the skeletogenic GRN during late stages of embryogenesisVEGF/MAPK signaling on the ventral side and a separate, unidentified pathway on the dorsal side. These two signaling pathways appear to be activated sequentially (ventral followed by dorsal) and many effector genes are subject to regulation by both pathways. (C) 2014 Elsevier B.V. All rights reserved.