Embryonic development evolves by balancing stringent morphological constraints with genetic and environmental variation. The design principle that allows developmental transcriptional programs to conserve embryonic morphology while adapting to environmental changes is still not fully understood. To address this fundamental challenge, we compare developmental transcriptomes of two sea urchin species, Paracentrotus lividus and Strongylocentrotus purpuratus, that shared a common ancestor about 40 million years ago and are geographically distant yet show similar morphology. We find that both developmental and housekeeping genes show highly dynamic and strongly conserved temporal expression patterns. The expression of other gene sets, including homeostasis and response genes, show divergent expression which could result from either evolutionary drift or adaptation to local environmental conditions. The interspecies correlations of developmental gene expressions are highest between morphologically similar developmental time points whereas the interspecies correlations of housekeeping gene expression are high between all the late zygotic time points. Relatedly, the position of the phylotypic stage varies between these two groups of genes: developmental gene expression shows highest conservation at mid-developmental stage, in agreement with the hourglass model while the conservation of housekeeping genes keeps increasing with developmental time. When all genes are combined, the relationship between conservation of gene expression and morphological similarity is partially masked by housekeeping genes and genes with diverged expression. Our study illustrates various transcriptional programs that coexist in the developing embryo and evolve under different constraints. Apparently, morphological constraints underlie the conservation of developmental gene expression while embryonic fitness requires the conservation of housekeeping gene expression and the species-specific adjustments of homeostasis gene expression. The distinct evolutionary forces acting on these transcriptional programs enable the conservation of similar body plans while allowing adaption.

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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.

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 utilize 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, bone morphogenetic protein (BMP) pathway restricts the activity of the vascular endothelial growth factor (VEGF) pathway to two lateral domains and by that controls proper skeletal patterning. Hypoxia applied during early development strongly perturbs the activity of Nodal and BMP pathways that affect 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.

Biomineralization, the ability of organisms to use minerals to harden their tissues, has attracted scientists from various disciplines to decipher the molecular mechanisms that control it. Many of these studies focus on the gene regulatory networks (GRNs) that control biomineralization and are apparently, phylum specific. Yet, downstream to the GRNs lays the cellular machinery that drives morphogenetic processes that are commonly used in biomineralization, such as, vesicular motion and secretion. The actomyosin network is a key regulator of these processes and participates in biomineralization across Eukaryotes, from unicellular organisms to vertebrates, yet, little is known about its regulation of biomineral growth. Here we reveal that the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK), controls the formation, growth and morphology of the calcite spicules in the sea urchin larva. We show that ROCK expression is elevated in the sea urchin skeletogenic cells and its inhibition impairs the organization of F-actin around the spicules and leads to skeletal loss. We discovered that ROCK inhibition after spicule formation, slows down mineral deposition, induces ectopic spicule branching and disrupts skeletogenic gene expression. We propose that ROCK and the actomyosin network are an essential part of the common biomineralization tool-kit in Eukaryotes.