Abstract
Echinoderms, which are phylogenetically related to vertebrates and produce large numbers of transparent embryos that can be experimentally manipulated, offer many advantages for the analysis of the gene regulatory networks (GRN) regulating germ layer formation. During development of the sea urchin embryo, the ectoderm is the source of signals that pattern all three germ layers along the dorsal-ventral axis. How this signaling center controls patterning and morphogenesis of the embryo is not understood. Here, we report a large-scale analysis of the GRN deployed in response to the activity of this signaling center in the embryos of the Mediterranean sea urchin Paracentrotus lividus, in which studies with high spatial resolution are possible. By using a combination of in situ hybridization screening, overexpression of mRNA, recombinant ligand treatments, and morpholino-based loss-of-function studies, we identified a cohort of transcription factors and signaling molecules expressed in the ventral ectoderm, dorsal ectoderm, and interposed neurogenic (“ciliary band”) region in response to the known key signaling molecules Nodal and BMP2/4 and defined the epistatic relationships between the most important genes. The resultant GRN showed a number of striking features. First, Nodal was found to be essential for the expression of all ventral and dorsal marker genes, and BMP2/4 for all dorsal genes. Second, goosecoid was identified as a central player in a regulatory sub-circuit controlling mouth formation, while tbx2/3 emerged as a critical factor for differentiation of the dorsal ectoderm. Finally, and unexpectedly, a neurogenic ectoderm regulatory circuit characterized by expression of “ciliary band” genes was triggered in the absence of TGF beta signaling. We propose a novel model for ectoderm regionalization, in which neural ectoderm is the default fate in the absence of TGF beta signaling, and suggest that the stomodeal and neural subcircuits that we uncovered may represent ancient regulatory pathways controlling embryonic patterning.
Highlights
It is becoming increasingly apparent that most developmental processes are controlled by dozens or hundreds of regulatory genes assembled into complex gene regulatory networks (GRNs), rather than by a small number of master genes
During early development of the common sea urchin Paracentrotus lividus, a signaling center located within the ventral ectoderm sends two key signals, Nodal and BMP2/4, that control patterning of the embryo along the whole dorsal-ventral axis
We identified direct targets of Nodal and BMP2/4 and identified several key regulators that mediate the effects of these factors and drive essential and probably ancient regulatory circuits that together constitute a transcriptional program controlling morphogenesis of the embryo
Summary
It is becoming increasingly apparent that most developmental processes are controlled by dozens or hundreds of regulatory genes assembled into complex gene regulatory networks (GRNs), rather than by a small number of master genes. Over the last few years, a number of GRNs have been elucidated, including regulatory networks that drive specification of germ layers or organs in various organisms [1,2,3,4,5,6,7]. Sea urchin embryos offer many advantages for GRN analysis [8]. Analysis of the first full sea urchin genome sequence from Strongylocentrotus purpuratus has revealed that echinoderms have a vast genetic repertoire but a low level of genetic redundancy, with almost all developmental regulatory genes being present as single copy [9]. Echinoderms occupy a basal position within the deuterostome lineage and are more related to chordates than most other invertebrate
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