Distinct regulatory states control the elongation of individual skeletal rods in the sea urchin embryo.

Understanding how gene regulatory networks (GRNs) control developmental progression is a key to the mechanistic understanding of morphogenesis. The sea urchin larval skeletogenesis provides an excellent platform to tackle this question. In the early stages of sea urchin skeletogenesis, skeletogenic genes are uniformly expressed in the skeletogenic lineage. Yet, during skeletal elongation, skeletogenic genes are expressed in distinct spatial sub-domains. The regulation of differential gene expression during late skeletogenesis is not well understood. Here we reveal the dynamic expression of the skeletogenic regulatory genes that define a specific regulatory state for each pair of skeletal rods, in the sea urchin Paracentrotus lividus. The vascular endothelial growth factor (VEGF) signaling, essential for skeleton formation, specifically controls the migration of cells that form the postoral and distal anterolateral skeletogenic rods. VEGF signaling also controls the expression of regulatory genes in cells at the tips of the postoral rods, including the transcription factors Pitx1 and MyoD1. Pitx1 activity is required for normal skeletal elongation and for the expression of some of VEGF target genes. Our study illuminates the fine-tuning of the regulatory system during the transition from early to late skeletogenesis that gives rise to rod-specific regulatory states.

Tarsis K ，Gildor T ，Morgulis M ，Ben-Tabou de-Leon S ... -  《-》

The biological regulation of sea urchin larval skeletogenesis - From genes to biomineralized tissue.

Biomineralization is the process in which soft organic tissues use minerals to produce shells, skeletons and teeth for various functions such as protection and physical support. The ability of the cells to control the time and place of crystal nucleation as well as crystal orientation and stiffness is far beyond the state-of-the art of human technologies. Thus, understanding the biological control of biomineralization will promote our understanding of embryo development as well as provide novel approaches for material engineering. Sea urchin larval skeletogenesis offers an excellent platform for functional analyses of both the molecular control system and mineral uptake and deposition. Here we describe the current understanding of the genetic, molecular and cellular processes that underlie sea urchin larval skeletogenesis. We portray the regulatory genes that define the specification of the skeletogenic cells and drive the various morphogenetic processes that occur in the skeletogenic lineage, including: epithelial to mesenchymal transition, cell migration, spicule cavity formation and mineral deposition into the spicule cavity. We describe recent characterizations of the size, motion and mineral concentration of the calcium-bearing vesicles in the skeletogenic cells. We review the distinct specification states within the skeletogenic lineage that drive localized skeletal growth at the tips of the spicules. Finally, we discuss the surprising similarity between the regulatory network and cellular processes that drive sea urchin skeletogenesis and those that control vertebrate vascularization. Overall, we illustrate the novel insights on the biological regulation and evolution of biomineralization, gained from studies of the sea urchin larval skeletogenesis.

Gildor T ，Winter MR ，Layous M ，Hijaze E ，Ben-Tabou de-Leon S ... -  《-》

VEGF signaling activates the matrix metalloproteinases, MmpL7 and MmpL5 at the sites of active skeletal growth and MmpL7 regulates skeletal elongation.

Organisms can uptake minerals, shape them in different forms and generate teeth, skeletons or shells that support and protect them. Mineral uptake, trafficking and nucleation are tightly regulated by the biomineralizing cells through networks of specialized proteins. Specifically, matrix metalloproteases (MMPs) digest various extracellular substrates and allow for mineralization in the vertebrates' teeth and bones, but little is known about their role in invertebrates' systems. The sea urchin embryo provides an excellent invertebrate model for genetic and molecular studies of biomineralization. MMP inhibition prevents the growth of the calcite spicules of the sea urchin larval skeleton, however, the molecular mechanisms and genes that underlie this response are not well understood. Here we study the spatial expression and regulation of two membrane type MMPs that were found to be occluded in the sea urchin spicules, Pl-MmpL7 and Pl-MmpL5, and investigate the function of Pl-MmpL7 in skeletogenesis. The inhibition of MMPs does not change the volume of the calcium vesicles in the skeletogenic cells. The expression of Pl-MmpL7 and Pl-MmpL5 is regulated by the Vascular Endothelial Growth Factor (VEGF) signaling, from the time of skeleton initiation and on. The expression of these genes is localized to the subsets of skeletogenic cells where active spicule growth occurs throughout skeletogenesis. Downregulation of Pl-MmpL7 expression delays the growth of the skeletal rods and in some cases, strongly perturbs skeletal shape. The localized expression of Pl-MmpL7 and Pl-MmpL5 to the active growth zone and the effect of Pl-MmpL7 perturbations on skeletal growth, suggest that these genes are essential for normal spicule elongation in the sea urchin embryo.

Morgulis M ，Winter MR ，Shternhell L ，Gildor T ，Ben-Tabou de-Leon S ... -  《-》

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 skeleton-forming 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 cell-autonomous 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 embryogenesis-VEGF/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.

Sun Z ，Ettensohn CA 《-》

Expression and Transcriptional Targets of TGFβ-RII in Paracentrotus lividus Larval Skeletogenesis.

Organisms from the five kingdoms of life use minerals to harden their tissues and make teeth, shells and skeletons, in the process of biomineralization. The sea urchin larval skeleton is an excellent system to study the biological regulation of biomineralization and its evolution. The gene regulatory network (GRN) that controls sea urchin skeletogenesis is known in great details and shows similarity to the GRN that controls vertebrates' vascularization while it is quite distinct from the GRN that drives vertebrates' bone formation. Yet, transforming growth factor beta (TGF-β) signaling regulates both sea urchin and vertebrates' skeletogenesis. Here, we study the upstream regulation and identify transcriptional targets of TGF-β in the Mediterranean Sea urchin species, Paracentrotus lividus. TGF-βRII is transiently active in the skeletogenic cells downstream of vascular endothelial growth factor (VEGF) signaling, in P. lividus. Continuous perturbation of TGF-βRII activity significantly impairs skeletal elongation and the expression of key skeletogenic genes. Perturbation of TGF-βRII after skeletal initiation leads to a delay in skeletal elongation and minor changes in gene expression. TGF-β targets are distinct from its transcriptional targets during vertebrates' bone formation, suggesting that the role of TGF-β in biomineralization in these two phyla results from convergent evolution.

Goloe D ，Gildor T ，Ben-Tabou de-Leon S 《-》