BMPs and chordin regulate patterning of the directive axis in a sea anemone

Abstract

The TGF-beta molecules Dpp/BMP2/4/7 and their antagonist Sog/Chd play a conserved role in establishing the dorso-ventral (DV) axis in bilaterians. Homologues of BMPs and the antagonist, Chordin, have been isolated from Cnidaria and show a striking asymmetric expression pattern with respect to the primary oral-aboral (OA) axis. We used Morpholino knockdowns of Nematostella dpp (bmp2/4), bmp5-8, chordin, and tolloid to investigate their function during early development of the sea anemone Nematostella vectensis. Molecular analysis of the BMP Morpholino phenotypes revealed an upregulated and radialized expression of bmps and chordin in ectoderm and endoderm indicating a negative feedback loop. Our data further suggest that BMP signaling is required for symmetry breaking of bmp and chordin expression during gastrulation. While bmps and chordin marker genes of the ectodermal OA axis extended aborally, other ectodermal markers of the OA axis were not significantly affected. By contrast, expression of other endodermal marker genes marking both the OA and the directive axis were abolished. Our data suggest that the logic of BMP2/4 signaling and the BMP antagonist, Chordin, differs significantly between Cnidaria and Bilateria, yet the double negative feedback loop detected in Nematostella bears systemic similarities with part of the regulatory network of the DV axis patterning system in amphibians.

Fig. 1.

Dpp signaling regulates symmetry break of dpp and chordin expression during gastrulation. In gastrula stage embryos (24 h) dpp knockdown leads to a radialized expression of dpp (B) and chd (D), both of which are asymmetrically expressed on the same side in controls (A and C), whereas treatment with recombinant human BMP2 protein virtually abolishes chordin expression showing that chordin depends on BMP signaling (E and F). Asterisk marks the oral end. (Scale bar, 100 μm.)

Fig. 2.

Knockdown of dpp, bmp5–8, and chd affects all asymmetrically expressed endodermal genes. Controls show the typical asymmetric expression of dpp (A), bmp5–8 (F), chd (K) along one side of the planula and gdf5-like (P) and hoxE (U) on the opposite side. (Z) Gbx is expressed laterally between dpp and gdf5-like in two broad domains. Note that the specimen in Z is oriented 90° rotated to show the two “lateral” expression domains of gbx. Dpp and bmp5–8 knockdowns, as well as the double knockdown, result in a radialized expression of dpp, bmp5–8 and chd. Furthermore; a shift of the ectodermal dpp and bmp5–8 expression toward the aboral pole is visible in these knockdowns (B–D, G–I, L–N). In contrast, expression of gdf5-like, hoxE, and gbx is abolished (Q–S, V–X, A′–C′). Chd knockdowns show an abolishment of gene expression of all genes tested (E, J, T, Y, D′) except for chd itself (O), where the expression is radialized but weaker than normal. All specimens shown are 52-h post fertilization planula larva. (Scale bar, 100 μM.)

Fig. 3.

BMP pathway inhibition results in differential phenotypes of the OA axis in ectoderm and endoderm. (A–I′) Expression of marker genes of the OA axis in controls, dpp MO, bmp5–8 MO, double dpp/BMP5–8 MO, and chd MO. (A–E) Outer pharynx expression of fgf8A is unaltered in morphants. (F–J) SoxB1 expression in the pharynx but not in the aboral pole is abolished in Morpholino-injected animals. (K–O) Expression of nk2.1 in the inner pharynx is abolished in all Morpholino-injected animals. (P–T) Wnt2 expression and (U–Y) fgf1A expression are unaltered. (Z–D′) Twist and (E′–I′) endodermal otxC expression along the OA axis are abolished in Morpholino-injected embryos. Note that otxC is expressed in two domains, oral ectodermal, and aboral endodermal (E′). Its endodermal expression is abolished after bmp5-8, dpp-bmp5-8-double, and chd knockdown (G′–I′) but remains expressed weakly in the oral region and the expression shifts to the aboral ectoderm after dpp knockdown (F′). All specimens shown are 52 h post fertilization, except in Z–D′ which are 76 hpf. (Scale bar 100 μM.)

Fig. 4.

Ectopic overexpression of chd leads to misexpression of dpp and bmp5–8. Animals injected with a construct carrying the chd gene under the control of a 1 kb actin upstream promoter show transient ectopic expression of chd at 52 hpf (B). Controls expressing mCherry under the same promoter show no altered expression of chd (A). The expression of dpp and bmp5–8 is altered only in animals injected with the chd plasmid (D and F), whereas the control plasmid did not lead to an alteration in expression (C and E). All specimens shown are 52 h post fertilization.

Fig. 5.

Model for the functional relationships of BMP5–8, Dpp and Chd. (A) Molecular interactions of BMPs and BMP modulators in the Xenopus embryo. Note that on both, the ventral and the dorsal side, pairs of BMPs and BMP antagonists are expressed. The green square highlights the double negative feedback loop of ADMP and Chordin. Scheme modified from ref. . (B) Molecular interactions of Chordin and the BMP2/4 homolog DPP (as well as BMP5–8) in the Nematostella embryo. Note that the double negative feedback loop is reminiscent of the molecular interactions in the Spemann organizer (green square).