A deuterostome origin of the Spemann organiser suggested by Nodal and ADMPs functions in Echinoderms

Abstract

During development of chordates, establishment of the body plan relies on the activity of an organizing centre located on the dorsal side of the embryo that patterns the embryo and induces neural tissue. Intriguingly, the evolutionary origin of this crucial signalling centre remains unclear and whether analogous organizers regulate D/V patterning in other deuterostome or protostome phyla is not known. Here we provide evidence that the ventral ectoderm of the sea urchin embryo is a long-range organizing centre that shares several fundamental properties with the Spemann organizer: the ability to induce duplicated embryonic axes when ectopically induced, the ability to induce neural fate in neighbouring tissues and the ability to finely regulate the level of BMP signalling by using an autoregulatory expansion-repression mechanism. These findings suggest that the evolutionary origin of the Spemann organizer is more ancient than previously thought and that it may possibly be traced back to the common ancestor of deuterostomes.

Figure 1. Ectopic Nodal generates Siamese pluteus larvae with two skeletons, two oral lobes, two mouths, two dorsal sides and two ciliary bands.

(A) Eggs injected with a nodal morpholino and RLDX were re-injected at the four-cell stage, into one randomly chosen or into two opposite blastomeres with either mRNA encoding the diffusible ligand Nodal or the Alk4/5/7QD activated Nodal receptor together with FLDX as a lineage tracer. (B) Phenotypes induced by a single or double source of Nodal signalling in a nodal morphant background. (a,b,e,f) Typical morphology of 72 h rescued embryos derived from a single injection of nodal (a,b) or alk4/5/7QD (e,f) mRNA. Note that the progeny of the injected cell contributes exclusively to the ventral ectoderm, mesoderm and gut. (c,d,g–j) Lateral views of 72 h embryos doubly injected with either mRNA encoding Nodal or the activated Nodal receptor together with FLDX. (c,d) In the case of nodal, the progeny of the injected blastomeres (green) are facing in opposite directions each forming two short oral arms and an oral lobe. The oral lobes derived from each injection clone are fused back to back in the animal-most region formed of ciliary band-like ectoderm. Two pairs of spicules are present in these embryos. (g–j) Two embryos (g,h,i,j) doubly injected with the alk4/5/7QD mRNA. The larvae are viewed as in C. The two ventral sides are respectively on the left and right sides of the picture. Note the two well visible ciliary bands in the embryo in j (white arrows). (k–p) Siamese larvae derived from a double injection of alk4/5/7QD mRNA into two opposite blastomeres at the four-cell stage (without prior injection of the Nodal morpholino). The close up in m–p show the two stomodeal invaginations before (m) or after (n–p) fusion with the tip of the gut. (C,D) Schemes depicting lateral views (C) or animal pole views (D) of nodal morphants rescued by a single (left) or double (right) injection of nodal or alk4/5/7QD mRNA. The progeny of injection clones is highlighted in green. Note that instead of converging towards a unique dorsal apex, the two spicules that originate from a given ventral side diverge to join two different dorsal apex. Scale bar, 20 μm in a–l; and 5 μm in m–p. FLDX, Fluoresceinated Dextran; RLDX, Rhodamine Dextran.

Figure 2. Ectopic Nodal respecifies the ectoderm, induces ectopic neural tissues and reorganizes patterning of the skeleton and secondary mesoderm.

(A) Double injection of alk4/5/7QD mRNA duplicated the territories expressing nodal, chordin, foxA and induced ectopic territories expressing the PMC cluster marker sm30 and the lateral ectoderm markers pax2/5/8, and fgfA. It also duplicated the dorsal territory expressing hox7 and the ventral and dorsal secondary mesodermal lineages expressing gata1/2/3 and gcm. Finally, ectopic alk4/5/7QD triggered formation of two sharply delimited ciliary bands that expressed foxG and onecut. (B) Cell lineage analysis of embryos doubly injected with alk4/5/7QD mRNA. In situ hybridization (dark blue) of chordin, onecut and hox7. The red color identifies the clone of alk4/5/7QD expressing cells. Note that while chordin is induced within the clone of injection, hox7 instead is induced non-autonomously, at 90 °C of the clone of injected cells. Note that the belts of cells expressing onecut partially overlap with the clone of injected cells in most of the ciliary band. Also note the almost perfect congruence of the alk4/5/7QD expressing territory and onecut expressing territory in the vegetal pole region. (C) Confocal observation of embryos following fluorescent in situ hybridizations with the ciliary band marker onecut in control and double axis gastrulae. (a) Control embryo seen from the animal pole express onecut in a ring of thick cells around the ventral ectoderm. onecut is shown in red, outline of the embryo observed using transmission is in blue. onecut expression is absent in the ventral ectoderm (black arrows) and the dorsal ectoderm (white arrows). (b–d) Three-dimensional reconstruction of a nodal morphant gastrula obtained by double injection of alk4/5/7QD. red, onecut expression. green, progeny of the alk4/5/7QD injected clones. g, gut. (e,f) onecut (red), as seen on the focal plane A (see b), is expressed in the whole ectoderm, but is absent in two regions (black arrows) corresponding to the progeny of injected blastomere (green) (e). (g,h) onecut, as seen on the vegetal focal plane B (see b), is expressed through most of the ectoderm, but is absent from two regions (white arrows) found in between the progeny of the injected clones (green) that correspond to the presumptive dorsal ectoderm (g). Scale bar, 20 μm in A–C.

Figure 3. The pattern of Phospho-Smad1/5/8 in embryos with diametrically opposing sources of BMP2/4 provides evidence for shuttling of BMP2/4.

(A) Phospho-Smad1/5/8 immunostaining at the mesenchyme blastula stage of control embryos (a) or nodal morphants (b). Strong nuclear staining is observed in a D/V gradient in one half of the control embryo, both in the ectoderm and in skeletogenic mesenchyme cells (a). Most of the signal disappears in the nodal morphants (b). (B) Phospho-Smad1/5/8 immunostaining of nodal morphants rescued by single injection of alk4/5/7QD mRNA at the 4-cell stage (c–g) and nodal morphants rescued by double injection of alk4/5/7QD mRNA (h-l). (c,h). The nodal morpholino was injected together with RLDX and the alk4/5/7QD mRNA with FLDX. Both tracers can be seen in the fully rescued embryo in (c). Single injection of alk4/5/7QD mRNA rescues psmad1/5/8 staining in nodal morphants on the opposite side of the injection clone (c–g) while double injection of alk4/5/7 mRNA results in nuclear pSmad1/5/8 staining in two groups of cells located in lateral regions (h–l). Note that, unlike rescued embryos displaying high pSmad 1/5/8 signalling opposite to the ventral side (b,c), the highest intensity of pSmad1/5/8 staining is found in the midline separating the two organizers. Embryos in c, d, h and i are confocal stacks acquired with the exact same parameters. Embryos in e–g and j–l are maximum projections of confocal stacks acquired with the exact same parameters. Embryos in g and l are the same as in f and I but using the Fiji ‘Thermal' lookup table. (C) Accumulation of pSmad1/5/8 in the lateral regions of the doubly injected embryos requires Chordin function. Chordin morphants doubly injected with alk4/5/7QD do not show the strong bilateral pSmad staining but show instead a uniform and low staining. (D) Scheme summarizing the pattern of pSmad1/5/8 of wild type embryos (a) or of embryos doubly injected with Alk4/5/7QD (b). Both local repression of BMP signalling ventrally by Chordin and active shuttling of BMP2/4 from the ventral side to the dorsal side shape the steep gradient of pSmad1/5/8. Note that this configuration is partially similar to that of a Drosophila embryo in which Chordin produced in two ventrolateral territories shuttles Dpp to the dorsal midline. Scale bar, 10 μm.

Figure 4. Phylogenetic analysis of sea urchin and various metazoan TGFβ ligands reveals ADMP gene duplication in Ambulacrarians.

Bayesian analysis of ADMP ligands. Numbers above nodes correspond to posterior probabilities above 50%. Each branch of the tree is coloured according to the major phyla to which it belongs. Scale bar unit for branch length is the number of substitutions per site. Abbreviations for species name are the following. Ambulacrarian species: Parli: Paracentrotus lividus; Ptyfl: Ptychodera flava; Sacko: Saccoglossus kowalevskii; Strpu: Strongylocentrotus purpuratus. Annelid species: Capte: Capitella teleta; Helro: Helobdella robusta. Arachnid species: Ixosc: Ixodes scapularis; Metoc: Metaseiulus occidentalis; Stemi: Stegodyphus mimosarum. Cephalochordate specie: Brafl: Branchiostoma floridae. Crustacean specie: Dappu: Daphnia pulex. Insect species: Apido: Apis dorsata; Apifl: Apis florea; Apime: Apis mellifera; Nasvi: Nasonia vitripennis; Zoone: Zootermopsis nevadensis. Mollusc species: Aplca: Aplysia californica; Cragi: Crassostrea gigas; Lotgi: Lottia gigantea. Placozoan specie: Triad: Trichoplax adhaerens. Platyhelminth species: Echgr: Echinococcus granulosus; Hymmi: Hymenolepis microstoma; Macli: Macrostomum lignano; Schme: Schmidtea mediterranea. Tunicate specie: Cioin: Ciona intestinalis. Vertebrate species: Danre: Danio rerio; Galga: Gallus gallus; Xenla: Xenopus laevis. Acoel specie: Hofmi: Hofstenia miamia. This subtree was extracted from a phylogenetic tree including 192 TGFβ ligands provided in Supplementary Fig. 2. The complete list of accession numbers is provided in the supplementary methods.

Figure 5. admp1 is expressed in the ventral organizer while admp2 is expressed in the dorsal-vegetal ectoderm.

(a) admp1 expression starts at mesenchyme blastula, first in a few ventral ectodermal cells near the animal pole, then it spreads vegetally to occupy a territory on the midline of the ventral ectoderm. At prism stage, novel territories expressing admp1 appear in the animal pole region and in the ventral-most endoderm. In embryos with duplicated D/V axes, admp1 is expressed at the centre of each oral lobe. (b) admp2 expression is initiated at the late blastula stage in the dorsal-vegetal plate. During gastrulation admp2 expression persists in a thin layer of dorsal-vegetal ectodermal cells and at prism stage in cells that will give rise to the dorsal apex of the pluteus larva. In embryos with duplicated D/V axes, admp2 is expressed in two opposing territories corresponding to the duplicated dorsal-vegetal ectoderm. Scale bar, 10 μm.

Figure 6. Opposite regulation of admp1 and admp2 by BMP signalling.

(a) admp1 expression is abolished in nodal morphants and following inhibition of Nodal signalling by SB431542. admp1 expression is also suppressed by overexpression of nodal but it is strongly upregulated in nickel treated embryos. (b) Expression of admp1 at gastrula stage in control embryos and following knockdown of various components of the BMP signalling pathway as indicated. Blocking BMP signalling using either a morpholino targeting bmp2/4, or the BMP type I receptor alk3/6, or the BMP type I receptor alk1/2 causes a partial extension of admp1 while blocking translation of both alk3/6 and alk1/2 transcripts with a combination of morpholinos causes a massive extension of admp1 expression to most of the ectoderm. (c) A massive derepression of admp1 is also observed after combining inhibition of bmp2/4 mRNA translation and overexpression of nodal. (d) Expression of admp2 is expanded ventrally following treatment with recombinant BMP4 and suppressed by overexpression of nodal or treatment with nickel. Expression of admp2 persists in a thin layer of dorsal-vegetal cells following inhibition of Nodal signalling by treatment with SB431542. LV, lateral view; VV, vegetal view; FV, frontal view, AV, animal view. Scale bar, 10 μm.

Figure 7. admp1 and admp2 act as prototypical BMP ligands but display divergent activities in specific contexts.

(a) Morphological phenotypes resulting from overexpression of admp1 and admp2. Overexpression of admp1 or admp2 at 1,000 μg ml⁻¹ completely radialized the embryos at gastrula stage. Note the presence of multiple spicule rudiments and the axial position of the gut in admp1 and admp2 overexpressing gastrulae. However, at pluteus stage, most embryos overexpressing admp1 or admp2 recovered a largely normal D/V axes and developed into normal pluteus. A fraction of these larvae displayed minor skeletal defects such as ectopic spicules elements. (b) Overexpression of admp2 suppressed formation of the ciliary band of SB431542 treated embryos and promoted growth and elongation of the skeleton resulting in a phenotype similar to that caused by overexpression of bmp2/4 or of bmp5/8. Overexpression of admp1 but not of admp2 in a chordin morpholino background caused 100% of the embryos to develop with a Nodal loss-of-function phenotype. (c) chordin expression is reduced in embryos overexpressing admp1 or in chordin morphants and eliminated in chordin morphants overexpressing admp1. (d) Both ADMP1 and ADMP2 promote pSmad1/5/8 signalling. pSmad1/5/8 antibody staining in control embryos and in embryos injected with admp1 or admp2 mRNAs. While the pSmad1/5/8 signal is restricted to the dorsal side of control embryos at mesenchyme blastula stage, overexpression of admp1 or of admp2 induces massive and strong phosphorylation of Smad1/5/8 in the whole ectoderm. In addition, overexpression of admp2, but not of admp1, induces strong pSmad1/5/8 in the primary mesenchymal cells. (e) Molecular analysis of admp1 or admp2 overexpressing embryos. At mesenchyme blastula stage, overexpression of admp1 or admp2 suppresses the expression of nodal and expands the expression of the dorsal marker genes smad6 and tbx2/3.admp1 overexpression also expands admp2 while overexpression of admp2 expands wnt5. Scale bar, 10 μm.

Figure 8. ADMP1 and ADMP2 are required for high level of BMP signalling and cooperate with BMP2/4 to build the dorsal apex.

(a) Morphological phenotypes caused by knocking-down ADMP1 or ADMP2 with morpholino oligonucleotides. Injection of morpholinos targeting the translation start site (Mo1) or the 5' UTR (Mo2) of admp1 or admp2 disrupts D/V patterning resulting in embryos that do not elongate along the ventral or dorsal sides. (b) Knocking-down admp1 with admp1-Mo2 increases the size of the chordin expressing territory consistent with the idea that ADMP1 antagonizes Nodal. The dorsal marker gene msx is also downregulated in admp1 morphants. (c) Knocking-down admp2 with admp2-Mo1 specifically eliminated the expression of marker genes expressed in the dorsal-vegetal and lateral ectoderm including fgfA, pax2/5/8, irxA and msx and suppressed expression of sm30 in the PMC clusters. (d)Morpholinos targeting either admp1 or admp2 strongly reduced pSmad1/5/8 staining in the presumptive dorsal-vegetal ectoderm and mesoderm but knocking-down admp2 with admp2-Mo1 more strongly diminished pSmad1/5/8 staining in the PMCs compared with knocking-down admp1 with admp1-Mo2. Scale bar, 10 μm.

Figure 9. ADMP1 and ADMP2 act in synergy with BMP2/4 to repress formation of the ciliary band on the dorsal side.

(a) Blocking translation of BMP2/4 causes a dramatic expansion of the ciliary band on the dorsal side while partially inhibiting BMP2/4 causes a truncation of the dorsal apex. Co-injection of low doses of the bmp2/4 together with low, sub-optimal, doses of the admp1 or admp2 morpholinos produces a strong BMP loss-of-function phenotype indicating that these two ADMP ligands cooperate with BMP2/4 during D/V axis formation. Note the similarity of the phenotypes of embryos injected with high doses of the BMP2/4 morpholino or co-injected with the admp1+bmp2/4 or admp2+bmp2/4 morpholinos at sub-optimal doses. (b) Expression of chordin, onecut and hox7 in embryos co-injected with low doses of the bmp2/4 and admp1 or bmp2/4 and admp2 morpholinos. Note the dramatic dorsal expansion of the ciliary band marker onecut, the loss of hox7 and the partial expansion of chordin following co-injection of either combination of the two morpholinos at low doses. Scale bar, 10 μm.

Figure 10. Homologous gene regulatory networks drive formation of the D/V organizer in chordates and in the sea urchin.

(a) Topology of the BMP-ADMP-Chordin network in chordates and echinoderms. In chordates, BMP2/4/7 ligands are expressed in a ventral signalling centre while admp is expressed on the opposite side. BMP signalling on the ventral side promotes expression of BMP ligands and represses admp expression, (repression). Nodal signalling in the Spemann organizer promotes expression of ADMP and Chordin. Chordin then shuttles BMP and ADMP ligands towards the ventral side where they activate BMP signalling (expansion). Chordin inhibits ADMP signalling dorsally. In the sea urchin, Nodal also positively regulates the expression of chordin and of an organizer specific BMP ligand, admp1, but Nodal also controls the expression of bmp2/4. Chordin inhibits ADMP and BMP2/4 signalling ventrally and promotes translocation of these ligands to the opposite dorsal side where they activate BMP signalling (expansion). BMP signalling on the dorsal side does not activate bmp2/4 expression but induces admp2, which can in turn autoregulate. BMP signalling on the side opposite to the organizer represses expression of admp1 in the organizer by an unknown mechanism (repression). A historical continuity of the D/V organizer of chordates and echinoderms is suggested by the striking conservation at the level of the whole gene regulatory network (GRN) which includes key transcription factors and signalling molecules such as nodal, lefty, bmp2/4, chordin, admp, not, lim1 and HN3β/foxA. Both GRNs require Wnt and Univin/Vg1 signalling to start and the activity of both GRNs can endow cells with organizer-like properties and coherently induce a whole set of tissues along the D/V axis. Finally, both organizers are involved in induction of neural tissue mainly through BMP inhibition and also possibly through direct induction by Nodal in the case of the sea urchin embryo. (b) Phylogeny of deuterostomes and phyla where a D/V organizer was characterized functionally. The red dot indicates the presence of a D/V organizer. A D/V organizer is present in all chordates, with perhaps the exception of tunicates. A D/V organizer is also present in echinoderms, suggesting that the evolutionary origin of the organizer may be traced back, at least, to the common ancestor of deuterostomes.