Ancestral regulatory circuits governing ectoderm patterning downstream of Nodal and BMP2/4 revealed by gene regulatory network analysis in an echinoderm

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.

Figure 1. Gene expression profiles of transcription factors and signaling molecules analyzed in this study.

(A) Spatial and temporal expression profiles. The expression of 21 genes encoding transcription factors (goosecoid, nk2.2, tbx2.3, nk1, foxA, brachyury, foxG, onecut/hnf6, irxA, hox7, dlx, smad6, msx, id, oasis, deadringer, otx, gfi, pax2/5/8, atbf1, unc4), 13 signaling molecules (nodal, lefty, bmp2/4, chordin, fgfA, fgfr1, glypican5, admp2, bmp1, wnt8, univin, wnt5, Delta), 2 RNA binding proteins (rkhd, ptb), 3 differentiation genes (cyIIIa, 29D, tubulinß3) and a mitochondrial gene (cytochrome oxidase) is depicted above a scheme of early development of the sea urchin embryo. The genes are classified into 5 groups according to the timing of their expression. (1) Maternal cytochrome oxidase transcripts show a graded distribution in cleaving embryos. (2,3) Starting at the early blastula stage, nodal and lefty are the first zygotic genes to be expressed in a restricted pattern along the D/V axis, followed by bmp2/4 and goosecoid before hatching (4,5). (6–9) After hatching, expression of fgfr1 and chordin is initiated ventrally while nk2.2 and tbx2/3 start to be expressed dorsally. (10–26) At mesenchyme blastula stage foxA, brachyury, foxG, Delta, nk1, onecut, fgfA, glypican5, irxA, hox7, dlx, smad6, msx, id, oasis, admp2, and CyIII start to be expressed in a restricted pattern. (27–36) A restricted expression is established slightly later at the early gastrula stage for ptb, bmp1, dri, otx, rkhd, gfi1, pax2/5/8, wnt8, univin, and for atbf1, unc4 and wnt5 (37–39). (40–43) tubulinß3 and 29D are expressed in the presumptive ciliary band or dorsal ectoderm starting at prism/early pluteus stages while foxG is expressed in a ventral subdomain of the ciliary band and Delta in individual cells within the ciliary band and facial ectoderm. (B) (1) Two color double in situ hybridization showing that foxG is expressed ventrally. (2,15,16) nk2.2 is expressed in two discrete regions along the D/V axis. At the late blastula stage, the territory with the strongest expression is located on the ventral side while at mesenchyme blastula stage, the highest level of transcripts is detected on the dorsal side. (3–6) Two color double in situ hybridization with chordin shows that dlx, msx, irxA and smad6 are expressed on the dorsal side. (7–13) One color double in situs hybridizations confirm that id and hox7 are expressed on the dorsal side. (C) At late gastrula stage, differences in expression of marker genes along the A/V and D/V axes identify differently specified territories within the ventral ectoderm. (1)The NK1 homeobox gene is expressed in a trapezoidal domain that abuts the stomodeal domain and the lateral ectodermal regions that express fgfA and pax2/5/8. (2) brachyury is expressed in a group of 30 cells located in the center of the ventral ectoderm and that likely constitute the stomodeal precursors. (3–5) This group of cells is surrounded by a large belt of goosecoid and foxG expressing cells that are themselves surrounded by a thinner belt of onecut/hnf6 expressing ciliary band precursors. (D) Nested expression domains are also apparent within the dorsal ectoderm and ciliary band. (1) irxA is expressed in a medial sub domain of the dorsal ectoderm that abuts the ciliary band. Note that irxA is also expressed in the stomodeal region at this stage. (2,3) Genes like admp2 and msx are expressed in nested patterns in the dorsal most region that corresponds to the presumptive apex of the larva. (4) nk2.2, like tbx2/3, is expressed in most cells of the dorsal ectoderm. (5,6) Onecut is expressed in the whole ciliary band, while pax2/5/8 is expressed in the vegetal portion of the presumptive ciliary band. (E) A set of ectodermal genes including onecut/hnf6, glypican5, fgfA, univin and wnt8 are expressed broadly in the ectoderm at blastula stages and subsequently restricted to either the dorsal ectoderm (glypican5) or the ciliary band (onecut/hnf6, fgfA, univin, wnt8). (F) Expression of several ciliary band genes including onecut, gfi1, foxG, egip, fgfA, pax2/5/8, univin, wnt8, dri, otx and of the dorsal marker glypican5 critically relies on the activity of the transcription factor SoxB1. V, ventral, D, dorsal, L, lateral. lv, lateral view, vv, vegetal pole view, fv, frontal view.

Figure 2. Overexpression of nodal represses the expression of ciliary band and dorsal marker genes and expands the expression of ventral markers genes.

(A) morphology of nodal overexpressing embryos at 72h. (B) treatment with recombinant Nodal protein induces ectopic expression of nodal in a large belt of ectodermal cells. (C) Injection of nodal mRNA (200–400 µg/ml) caused the expression of most ventral marker genes to become radial. In most cases, however, the animal pole domain appeared to be resistant to ectopic expression of nodal. Overexpression of nodal also radialized the expression of dri, bmp1 and univin, which are expressed in a broad domain encompassing the presumptive ventral ectoderm and ciliary band. In contrast, Nodal strongly antagonized the expression of ciliary band marker genes such as wnt8, fgfA, pax2/5/8, foxG, egip, onecut, gfi1, otx, tubulinß3 or Delta in the equatorial region. Nodal overexpression efficiently repressed the expression of all the genes expressed in the dorsal ectoderm but did not affect the expression of marker genes of the animal pole (tubulinß3, foxQ2, Delta). Note that, starting at gastrula stage, irxA is expressed in a patch of animal-ventral cells that likely corresponds to the upper part of the presumptive stomodeum. Therefore in nodal overexpressing embryos, this territory becomes radial and forms a belt of cells near the animal pole. Also note that while a ciliary band does form in the vegetal region of nodal overexpressing embryos, as shown by the expression of ciliary band genes around the blastopore, no ciliary band forms in the vegetal region of lefty morphants as indicated by the absence of tubulinß3 expression. In contrast, nodal overexpression does not affect the expression of animal pole marker genes such as foxQ2. lv, lateral view, vv, vegetal pole view, av, animal pole view, fv, frontal view.

Figure 3. Overactivation of BMP signaling eliminates the expression of ventral and ciliary band marker genes and expands the dorsal territory.

(A) Morphology of BMP2/4 overexpressing or BMP4 treated embryos at 72h. Embryos with overactivated BMP signaling are elongated and covered with a thin and squamous ectoderm and possess ectopic spicules. (B) Expression of ventral, dorsal and ciliary band marker genes in embryos misexpressing BMP2/4. The results presented were obtained using treatments with recombinant BMP4 protein except one experiment in which an activated BMP receptor (Alk3/6QD) was used. For all these genes, identical results were obtained by overexpression of BMP2/4 mRNA. Overactivation of BMP2/4 signaling eliminated the expression of ventral markers and of ciliary band genes such as bmp1, foxG, onecut/hnf6, otx, gfi1, tubulinß3, egip, deadringer, univin, fgfA, and pax2/5/8. Overactivation of BMP signaling dramatically expanded the expression of all the dorsal marker genes and eliminated the expression of markers of the animal pole domain such as foxQ2. lv, lateral view, vv, vegetal pole view, av, animal pole view, fv, frontal view.

Figure 4. nodal, lefty, bmp2/4, chordin, goosecoid, nk2.2, and fgfr1 are direct targets of Nodal while tbx2/3, nk2.2, and smad6 are direct targets of BMP2/4 signaling.

(A,B) Embryos at the late blastula stage were treated for two hours with recombinant Nodal or BMP4 protein in the presence or absence of puromycin. At the end of the treatment the embryos were fixed and the expression of the indicated genes was analyzed by in situ hybridization. In control experiments, DMSO or the translational inhibitor were added alone. (A) A short treatment with recombinant Nodal protein induced strong ectopic expression of early expressed genes such as nodal, lefty, bmp2/4, chordin, goosecoid, nk2.2 and fgfr1. This ectopic expression was still observed in the presence of protein synthesis inhibitor indicating that these genes are early targets of Nodal signaling. In contrast, this 2h treatment with Nodal did not induce ectopic expression of genes expressed later such as foxA and only induced a partial expansion of brachyury expression, an effect that disappeared in the presence of the protein synthesis inhibitor. These genes are therefore likely secondary targets of Nodal signaling. Note that in control embryos the width of the brachyury expression territory encompasses 8–9 cells (red bar) while in Nodal treated embryos the width of this stomodeal field increases to about 12–15 cells. In embryos treated with Nodal and the translation inhibitor, the width of the stomodeal field is similar to that in control embryos. (B) Similarly, BMP4 treatment induced massive ectopic expression of the early expressed genes tbx2/3, nk2.2 and smad6 even in the presence of a translational inhibitor but failed to induce ectopic expression of dorsal markers genes expressed later. tbx2/3, nk2.2 and smad6 are therefore likely direct targets of BMP2/4 signaling while the other dorsally expressed genes are likely indirect targets whose expression requires protein synthesis downstream of activation of the BMP receptors. Identical results were obtained by using emetine as translational inhibitor. lv, lateral view, vv, vegetal pole view fv, frontal view.

Figure 5. Blocking Nodal function prevents expression of ventral and dorsal marker genes in the presumptive ectoderm and causes massive ectopic expression of ciliary band genes.

(A) morphology of nodal morphants at 72h. Note that most of the ectoderm of these embryos differentiates into a thick ciliated ectoderm that resembles the ciliary band ectoderm. (B–D) Embryos were injected with a Nodal morpholino or treated with the Nodal receptor inhibitor SB431542 and the expression of ventral, dorsal or ciliary band genes was analyzed at the relevant stages. (B,C) All the ventral and all the dorsal marker genes tested required Nodal to be expressed in the presumptive ventral and presumptive dorsal ectoderm respectively. However, in the nodal morphants, a number of genes expressed dorsally continued to be expressed in the ectoderm derived from the vegetal region and/or in the mesendoderm including tbx2/3, id, irxA, nk2.2, atbf1, admp2 and 29D (the residual expression of tbx2/3 is not visible here since it is mostly visible at gastrula stages). This indicates that in the nodal morphants there is a residual D/V polarity with the vegetal most ectoderm adopting a dorsal identity. (D) Inhibition of Nodal signaling caused a massive ectopic expression of ciliary band genes throughout the ectoderm. Note that genes expressed throughout the ciliary band territory such as bmp1, otx, onecut, gfi1, dri, or tubulinß3 are ectopically expressed throughout the ectoderm of Nodal morphants. Genes that are expressed in sub domains of the ciliary band such as pax2/5/8, fgfA, univin or wnt8 are also ectopically expressed and display a radial expression but in accordance with their normal animal-vegetal boundaries. The neural marker Delta and the ciliary band antigen 295, which in control embryos labels the ciliated cuboidal cells of the ciliary band, are also expressed ectopically throughout the thick ciliated ectoderm typical of nodal morphants. lv, lateral view, vv, vegetal pole view, fv, frontal view. Scale bar: 100µm.

Figure 6. Blocking BMP2/4 or Alk3/6 signaling strongly downregulates the expression of dorsal genes and causes massive ectopic expression of ciliary band marker genes on the dorsal side.

(A) Morphology of alk3/6 and BMP2/4 morphants at 72h. Ventral structures such as the stomodeum do form in embryos injected with the alk3/6 or BMP2/4 morpholinos but dorsal structures such as the apex fail to differentiate. (B–D) Embryos were injected with BMP2/4 or Alk3/6 morpholinos and the expression of ventral, dorsal or ciliary band marker genes was analyzed at the relevant stages. (B) Inhibition of BMP2/4 signaling does not interfere with the expression of ventral genes. (C) The expression of all the dorsal marker genes is strongly reduced or abolished following inhibition of BMP2/4 or Alk3/6 function. A residual expression in the vegetal most region is still observed for certain genes such as irxA, wnt5, id. (D) Inhibition of Alk3/6 or BMP2/4 causes a striking ectopic expression of ciliary band genes throughout the dorsal ectoderm. lv, lateral view, vv, vegetal pole view, av, animal pole view, fv, frontal view. Scale bar: 100µm.

Figure 7. Epistasis analysis of ventral genes: goosecoid as a key regulator of brachyury and foxA expression and a repressor of ciliary band genes.

(A) Morphology of goosecoid morphants at 48h. Interfering with goosecoid function strongly delays gastrulation and produces partially radialized embryos as reported previously . bmp2/4 and chordin are expressed normally in these embryos but expression of brachyury and foxA is lost while wnt8, univin and foxG and are ectopically expressed within the ventral ectoderm. (B) Injection of goosecoid mRNA at 250 µg/ml produced radialized embryos as reported previously . Overexpression of goosecoid caused ectopic expression of brachyury, foxA and deadringer throughout the ectoderm. Overexpression of goosecoid also led to repression of dorsal markers genes such as hox7 and msx and of ciliary band marker gene expression as shown for wnt8, univin, foxG, egip, gfi1 and onecut/hnf6. Note the dose dependent repression of onecut/hnf6, with low doses (40–100 µg/ml) causing a dorsal shift of the expression domain of onecut/hnf6 and high doses (250 µg/ml) leading to complete repression. (C) Embryos injected with low doses (0.3 mM) of the foxA morpholino lacked a stomodeum as reported previously. At higher doses (0.6 mM), the foxA morpholino strongly interfered with gastrulation resulting in embryos with no gut or a small exogastrulated gut. foxA morphants had a normal expression of chordin but lacked expression of brachyury and foxA. (D) brachyury morphants, like foxA morphants, lacked a stomodeum and did not express foxA in the presumptive stomodeal region. lv, lateral view, vv, vegetal pole view, fv, frontal view.

Figure 8. Epistasis analysis of dorsal genes: irxA as a repressor of ciliary band gene expression downstream of tbx2/3.

(A) tbx2/3 morphants are partially radialized and do not elongate along the D/V axis. The expression of ventral genes such as chordin, foxA, brachyury, onecut/hnf6 and of ciliary band genes such as pax2/5/8 and fgfA is largely normal in these embryos. In contrast, expression of dorsal marker genes such as msx, dlx, irxA and atbf1 is lost at gastrula stages. (B) irxA morphants are partially radialized and occasionally show an ectopic ciliary band like thickened region within the dorsal region. Expression of onecut/hnf6 is dramatically expanded towards the dorsal side of irxA morphants. (C) Onecut/hnf6 morphants are slightly radialized but do form a morphologically recognizable ciliary band. In these embryos, the expression of ciliary band genes gfi1 is absent while the expression of pax2/5/8, foxG or deadringer is strongly reduced. lv, lateral view, vv, vegetal pole view, av, animal pole view, dv, dorsal view, fv, frontal view.

Figure 9. Regulation of D/V patterning by extracellular and intracellular modulators of Nodal and BMP signaling.

(A) Wnt8 regulates maintenance of nodal expression. At mesenchyme blastula stage, wnt8 starts to be expressed in a large belt of ectodermal cells within the vegetal hemisphere. At gastrula stage wnt8 expression is detected in the animal and vegetal hemispheres in two broad lateral stripes that flank the ventral ectoderm. In wnt8 morphants, expression of nodal is lost at mesenchyme blastula stage. (B) Chordin is essential for normal patterning along the D/V axis and plays a key role in restricting the expression of ciliary band genes. In chordin morphants, most of the ectoderm derived from the animal hemisphere differentiates into a ciliary band-like ectoderm. Expression of ventral (nodal, chordin, bmp2/4, goosecoid) and dorsal (msx, wnt5) marker genes is strongly downregulated in these embryos. A transient ectopic expression of tbx2/3 is detected at early mesenchyme blastula stage in these embryos followed by downregulation of the gene. Restriction of the expression of the ciliary band gene onecut/hnf6 is disrupted in chordin morphants and ectopic expression of this gene is detected in the ectoderm. (C) At high concentration (>220 µg/ml) smad6 mRNA suppressed the ectodermal expression of Nodal targets genes such as chordin and foxA and caused a dramatic ectopic expression of onecut/hnf6 throughout the ectoderm. At lower doses (<180 µg/ml), smad6 mRNA did not interfere with the expression of chordin but specifically antagonized with the expression of BMP target genes as msx and caused the ectopic expression of onecut/hnf6 in the dorsal ectoderm. lv, lateral view, vv, vegetal pole view, fv, frontal view.

Figure 10. Representation of the gene regulatory network regulating regionalization of the ectoderm of the sea urchin embryo.

(A) Biotapestry , diagram of the provisional gene regulatory network describing the regulatory interactions that have been identified in this study. Arrows indicate positive transcriptional activation. Flat arrows indicate repression. The colored boxes represent the spatial domains as indicated. The linkages between Nodal and its immediate direct target genes as well as the linkages between BMP2/4 and its direct target genes are shown as bold arrows. The gene regulatory linkages sustained by solid evidence are presented as solid lines. The gene regulatory linkages that are hypothetical or suspected to be indirect are represented as dotted lines. Except for the bold arrows, no assumption on whether these interactions are direct or indirect is made. The linkage between Nodal and Delta, expressed in single cells of the ciliary band, is not represented here. (B) Gene regulatory interactions within the ventral ectoderm highlighting the repressive action of Goosecoid on ciliary band genes. The genes that are inactive are represented in light grey. (C) Gene regulatory interactions within the dorsal ectoderm highlighting the repressive action of irxA on onecut/hnf6 and more generally of Nodal signaling on the expression of ciliary band genes.

Figure 11. Changes in identity of ectodermal territories following perturbations of Nodal or BMP signaling and novel model of ectoderm patterning.

Schemes describing the morphology of control embryos and perturbed embryos. (A) control embryo. The thick ciliated epithelium of the ciliary band is restricted to a belt of cells at the interface between the ventral and dorsal ectoderm. (B) Nodal morphant. Most of the ectoderm differentiates into an expanded large ciliary band. An animal pole domain is nevertheless present in these embryos as shown by the presence of the apical tuft and at the molecular level by the expression of apical domain marker genes. In these embryos, the ectoderm surrounding the blastopore differentiates into dorsal ectoderm. (C) embryo overexpressing Nodal. Most of the ectoderm differentiates into ventral ectoderm. A ciliary band-like ectoderm forms at the animal pole and in the ectoderm surrounding the blastopore. (D) BMP2/4 morphants. An ectopic ciliary band forms in the dorsal ectoderm in addition to the normal ciliary band. (E) bmp2/4 overexpressing embryo. All the ectoderm has a dorsal identity. The animal pole domain is largely absent. The triradiated stars represent the spicule rudiments. (F) Proposed model for regionalization of the ectoderm of the sea urchin embryo through restriction of the ciliary band fate by Nodal and BMP signaling. Maternal factors such as SoxB1 promote the early expression of ciliary band genes within the ectoderm. Nodal signaling on the ventral side promotes differentiation of the ventral ectoderm and stomodeum and represses the ciliary band fate probably through the activity of Goosecoid as well as of additional repressors. Nodal induces its antagonist Lefty, which diffuses away from the ventral ectoderm up to the presumptive ciliary band territory. Within the ventral ectoderm, Nodal induces expression of bmp2/4 and of its antagonist chordin. Chordin prevents BMP signaling within the ventral ectoderm and probably within the presumptive ciliary band region. At blastula stages, protein complexes containing BMP2/4 and Chordin can diffuse towards the dorsal side to specify dorsal fates. In the dorsal ectoderm, BMP signaling strongly repress the ciliary band fate partly by inducing the expression of the irxA repressor. A high level of MAP kinase activity resulting from FGFA signaling in the lateral ectoderm likely contributes to maintain a low level of Nodal and BMP signaling within the presumptive ciliary band region by phosphorylating Smad1/5/8 and Smad2/3 in the linker region, which inhibits their activity. The presence of Chordin and Lefty in the prospective ciliary band allows expression of ciliary band genes to be maintained in this region. The ectoderm surrounding the blastopore differentiates into dorsal ectoderm likely because it receives Wnt signals that antagonize GSK3 and promote BMP signaling. (G) In the absence of Nodal signaling, both the ventral and the dorsal inducing signals are not produced, ciliary band genes are not repressed and unrestricted MAP kinase signaling promotes differentiation of the ventral and dorsal ectoderm into neural ectoderm and ciliary band. The genes or proteins that are inactive are represented in light grey.