Expression and Transcriptional Targets of TGFß-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-ß) signaling regulates both sea urchin and vertebrates' skeletogenesis. Here, we study the upstream regulation and identify transcriptional targets of TGF-ß in the Mediterranean Sea urchin species, Paracentrotus lividus. TGF-ßRII is transiently active in the skeletogenic cells downstream of vascular endothelial growth factor (VEGF) signaling, in P. lividus. Continuous perturbation of TGF-ßRII activity significantly impairs skeletal elongation and the expression of key skeletogenic genes. Perturbation of TGF-ßRII after skeletal initiation leads to a delay in skeletal elongation and minor changes in gene expression. TGF-ß targets are distinct from its transcriptional targets during vertebrates' bone formation, suggesting that the role of TGF-ß in biomineralization in these two phyla results from convergent evolution.

ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis.

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates' biomineralizing cells, yet, little is known on ROCK's role in invertebrates' biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

The tolerance to hypoxia is defined by a time-sensitive response of the gene regulatory network in sea urchin embryos.

Deoxygenation, the reduction of oxygen level in the oceans induced by global warming and anthropogenic disturbances, is a major threat to marine life. This change in oxygen level could be especially harmful to marine embryos that use endogenous hypoxia and redox gradients as morphogens during normal development. Here, we show that the tolerance to hypoxic conditions changes between different developmental stages of the sea urchin embryo, possibly due to the structure of the gene regulatory networks (GRNs). We demonstrate that during normal development, the bone morphogenetic protein (BMP) pathway restricts the activity of the vascular endothelial growth factor (VEGF) pathway to two lateral domains and this restriction controls proper skeletal patterning. Hypoxia applied during early development strongly perturbs the activity of Nodal and BMP pathways that affect the VEGF pathway, dorsal-ventral (DV) and skeletogenic patterning. These pathways are largely unaffected by hypoxia applied after DV-axis formation. We propose that the use of redox and hypoxia as morphogens makes the sea urchin embryo highly sensitive to environmental hypoxia during early development, but the GRN structure provides higher tolerance to hypoxia at later stages.

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.

Possible cooption of a VEGF-driven tubulogenesis program for biomineralization in echinoderms.

Biomineralization is the process by which living organisms use minerals to form hard structures that protect and support them. Biomineralization is believed to have evolved rapidly and independently in different phyla utilizing preexisting components. The mechanistic understanding of the regulatory networks that drive biomineralization and their evolution is far from clear. Sea urchin skeletogenesis is an excellent model system for studying both gene regulation and mineral uptake and deposition. The sea urchin calcite spicules are formed within a tubular cavity generated by the skeletogenic cells controlled by vascular endothelial growth factor (VEGF) signaling. The VEGF pathway is essential for biomineralization in echinoderms, while in many other phyla, across metazoans, it controls tubulogenesis and vascularization. Despite the critical role of VEGF signaling in sea urchin spiculogenesis, the downstream program it activates was largely unknown. Here we study the cellular and molecular machinery activated by the VEGF pathway during sea urchin spiculogenesis and reveal multiple parallels to the regulation of vertebrate vascularization. Human VEGF rescues sea urchin VEGF knockdown, vesicle deposition into an internal cavity plays a significant role in both systems, and sea urchin VEGF signaling activates hundreds of genes, including biomineralization and interestingly, vascularization genes. Moreover, five upstream transcription factors and three signaling genes that drive spiculogenesis are homologous to vertebrate factors that control vascularization. Overall, our findings suggest that sea urchin spiculogenesis and vertebrate vascularization diverged from a common ancestral tubulogenesis program, broadly adapted for vascularization and specifically coopted for biomineralization in the echinoderm phylum.