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.

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12Biomineralization is the process in which living organisms use minerals to form hard 13 structures that protect and support them. Biomineralization is believed to have evolved 14 rapidly and independently in different phyla utilizing existing components used for other 15 purposes. The mechanistic understanding of the regulatory networks that drive 16 biomineralization and their evolution is far from clear. The sea urchin skeletogenesis is an 17 excellent model system for studying both gene regulation and mineral uptake and deposition. 18The sea urchin calcite spicules are formed within a tubular cavity generated by the 19 skeletogenic cells under the control the vascular endothelial growth factor (VEGF) signaling. 20The VEGF pathway controls tubulogenesis and vascularization across metazoans while its 21 regulation of biomineralization was only observed in echinoderms. Despite the critical role of 22 for vascularization and specifically co-opted for biomineralization in the echinoderm phylum. 33 Significance statement 36The sea urchin calcite spicules and vertebrate blood vessels are quite distinct in their function, 37 yet both have a tubular structure and are controlled by the vascular endothelial growth factor 38 (VEGF) pathway. Here we study the downstream program by which VEGF pathway drives 39 sea urchin spiculogenesis and find remarkable similarities to the control of vertebrate 40 vascularization. The similarities are observed both in the upstream gene regulatory network, 41 in the downstream effector genes and the cellular processes that VEGF signaling controls at 42 the site of the calcite spicule formation. We speculate that sea urchin spiculogenesis and 43 vertebrate vascularization diverged from a common ancestral tubulogenesis program that was 44 co-opted for biomineralization in the echinoderm phylum. 45 46 3 47

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Deoxygenation, the reduction of oxygen level in the oceans induced by global warming and anthropogenic disturbances, is a major threat to marine life. Acute diurnal changes in oxygen levels could be especially harmful to vertebrate and sea urchin embryos that utilize endogenous hypoxia gradients to drive morphogenetic events during normal development. Here we show that the tolerance to hypoxic conditions changes between different developmental stages of the sea urchin embryo, due to the structure of the gene regulatory network (GRN). Specifically, Nodal signaling, bone morphogenetic protein (BMP) and the vascular endothelial growth factor (VEGF) pathways, are strongly disturbed by hypoxia during early embryogenesis, but are largely unaffected by hypoxia applied after dorsal-ventral axis formation. These pathways regulate hypoxia-induced vascularization in vertebrates which could suggest that they are a part of an evolutionary conserved program that uses hypoxia to drive morphogenesis. We propose that the structure of the GRN, that includes positive and negative feedback and feedforward loops, increases its resilience to changes of the initial hypoxia gradients and could help the embryos tolerate transient hypoxic conditions.