Cary GA et al. (2020), Systematic comparison of sea urchin and sea sta...

ECB-ART-49450

Nat Commun 2020 Dec 04;111:6235. doi: 10.1038/s41467-020-20023-4.

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Systematic comparison of sea urchin and sea star developmental gene regulatory networks explains how novelty is incorporated in early development.

Cary GA , McCauley BS , Zueva O , Pattinato J , Longabaugh W , Hinman VF .

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The extensive array of morphological diversity among animal taxa represents the product of millions of years of evolution. Morphology is the output of development, therefore phenotypic evolution arises from changes to the topology of the gene regulatory networks (GRNs) that control the highly coordinated process of embryogenesis. A particular challenge in understanding the origins of animal diversity lies in determining how GRNs incorporate novelty while preserving the overall stability of the network, and hence, embryonic viability. Here we assemble a comprehensive GRN for endomesoderm specification in the sea star from zygote through gastrulation that corresponds to the GRN for sea urchin development of equivalent territories and stages. Comparison of the GRNs identifies how novelty is incorporated in early development. We show how the GRN is resilient to the introduction of a transcription factor, pmar1, the inclusion of which leads to a switch between two stable modes of Delta-Notch signaling. Signaling pathways can function in multiple modes and we propose that GRN changes that lead to switches between modes may be a common evolutionary mechanism for changes in embryogenesis. Our data additionally proposes a model in which evolutionarily conserved network motifs, or kernels, may function throughout development to stabilize these signaling transitions.

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	Fig. 1. Sea star hesC is positively regulated downstream of the mesoderm kernel and is co-expressed with delta.Sea star hesC and delta transcripts are co-expressed in the vegetal mesoderm (a) until mid-gastrula stage (b), or 36 hours post fertilization (hpf). Sea star hesC and blimp1 are expressed in partially non-overlapping domains during blastula stage (c) and morpholino knockdown (KD) of blimp1 results in an expansion of the expression domain of hesC (d). Morpholino knockdown of sea star Tgif produces a mesoderm-specific decrease in hesC expression (e). Schematic showing non-overlapping expression domains of sea urchin hesC and delta transcripts at blastula stage (f). Regulatory inputs to the hesC gene that are specific to sea urchin embryos, sea star embryos, and those that are common to both (g). Data shown are double fluorescent WMISH showing both hesC expression (green) and either expression of delta or blimp1 (magenta) (a–c) and colorimetric WMISH (d, e). Data are representative of two biologically independent experiments consisting of at least ten embryos each. Scale bar represents 50 μm; applicable to all images in the panel.
	Fig. 2. Testing lateral inhibition of delta and hesC by inhibition of Notch signaling (DAPT) and morpholino knockdown (KD) of HesC.Using DAPT, an inhibitor of the proteolytic gamma-secretase necessary for notch signal transduction, we observe both a down-regulation of hesC (c) and an upregulation of delta transcripts (g). Importantly the down-regulation of hesC is phenocopied by injection of a morpholino targeting the delta transcript into one of the first two blastomeres (b). Knockdown of HesC with an antisense morpholino yields an upregulation of both delta (e) and hesC transcripts. Lateral inhibition network showing relationships tested by previous experiments (h); red letters indicate figure panel above supporting connection. All images are colorimetric WMISH with the probes to the indicated genes. Images are representative of two biologically independent experiments consisting of at least ten embryos each. Scale bar represents 50 μm; applicable to all images in the panel. Numbers in the lower left corner of (c, e, and g) represent normalized log2 fold-change values of perturbed expression compared with control (i.e., a, d, and f) as measured by qPCR.
	Fig. 3. Segregation of mesodermal subtypes into interleaved cells by 36 hpf.Sea star ets1 and six3 transcripts are co-expressed in the vegetal mesoderm of blastula stage embryos (a, b) at 24 h post fertilization (hpf). The expression of the ets1 transcript was assessed every 2 h from the onset of gastrulation by colorimetric WMISH. At 32 hpf the expression of ets1 is uniform throughout the mesoderm (c). At 36 hpf there is a discontinuity in the expression of ets1 transcript (d, asterisks). Patches of ets1 expression become more distinct by 40–42 hpf (e, line), and ets1 expressing cells start to ingress beginning at 46 hpf (f). Cells expressing ets1 transcript (green) at the tip of the archenteron are adjacent to cells with no detectable ets1 expression (g), using fluorescent WMISH with a DAPI counterstain (blue), and are interleaved by cells expressing six3 transcript (magenta) (h). Cells expressing ets1 transcript also express the transcript encoding the Delta ligand (i). Data are representative of two biologically independent experiments consisting of at least ten embryos each. Scale bars represent 50 μm; scale bar in (a) applicable to (a–f) and (i), scale bar in (g) applicable in (g–h), which show a magnified region at the tip of the archenteron.
	Fig. 4. Testing the lateral inhibition model of mesodermal subtype segregation.The expression of ets1 transcript (magenta) appears in a salt-and-pepper distribution throughout the mesoderm at 36 h post fertilization (hpf) by fluorescent WMISH (a) with DAPI stained nuclei (white). Treatment with the Notch inhibitor DAPT beginning at the 2-cell stage results in a uniform expression of ets1 in this territory (b). By 48 hpf, the mesenchyme cells expressing ets1 and erg (c, e) ingress into the blastocoel while cells that do not ingress express pax6 and six3 (g, i). DAPT treatment results in an increase in cells expressing ets1 and erg (d, f) and a reduction in cells expressing pax6 and six3 (h, j). There is also a consistent morphological shift with an increase in the number of ingressing cells and a reduction in the epithelium when Notch signaling is blocked. Data shown in (c–j) are colorimetric WMISH. Data are representative of two biologically independent experiments consisting of at least ten embryos each. Scale bar represents 50 μm; applicable to all images in the panel.
	Fig. 5. Subcircuit including pax6, six3, eya, dach1, and six1/2 involved in sea star coelomogenesis.a Data shown are colorimetric WMISH using the probes indicated on the left in the conditions listed along the top. Scale bar represents 50 μm; applicable to all images in the panel. six3 expression is normally distributed throughout the mesodermal bulb of the archenteron at 48 hpf, while pax6, six1/2, dach1, and eya are normally expressed at the posterior aspect of the mesodermal bulb, having been cleared earlier from anterior regions of the mesoderm. Phenotypic effect of the perturbation of each gene is indicated, including no difference (nd), increase (↑), decrease (↓). The number of embryos assessed and percent of embryos expressing the phenotype are also reported. Some reported phenotypes are localized to the top of the archenteron and are highlighted (dashed line); e.g., the effect of Pax6 knockdown on six3 expression is reported specifically for the anterior region of archenteron (dashed line). Some Pax6 knockdown embryos exhibited a bifurcated archenteron (e.g., boxed panel, “split archenteron”). Data are representative of two biologically independent experiments consisting of at least ten embryos each and specific phenotype counts are detailed in Supplementary Table 3. These results enabled the construction of a sub-network for sea star coelomic epithelium (b) and we note a similar regulatory sub-network in both sea star and sea urchin coelomic mesoderm, which is strikingly similar to the retinal determination gene network (RDGN) in Drosophila.
	Fig. 6. Evolutionary constraint of network kernels permits alterations to the surrounding network.A synthesis of key aspects of the GRN from sea urchins and sea stars is shown (a). Genes (nodes) are shown in the territories (colored boxes) in which they are expressed. Edges show regulation by the originating upstream factor and are either positive (arrow) or repressive (bar). Signaling across cell types are indicated as double arrow heads, and Delta to Notch signals are boxed. Genes and links that are unique to sea urchin embryos are colored purple, those specific to sea stars are green, and those in common are black. Network kernels are highlighted (yellow) as are distinct sub-circuits (pink), including the sea urchin-specific double-negative gate (i.e., Pmar and HesC, purple outline) and sea star-specific positive regulation of HesC (i.e., Tgif and HesC, green outline). Grayed out backgrounds indicate entire network circuits that are absent in sea stars. Our model of GRN evolution is depicted (b) showing that network kernels are constrained regions whereas both up and down the hierarchy the network is capable of change.

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