Nodal signaling is required for mesodermal and ventral but not for dorsal fates in the indirect developing hemichordate, Ptychodera flava

Abstract

Nodal signaling plays crucial roles in vertebrate developmental processes such as endoderm and mesoderm formation, and axial patterning events along the anteroposterior, dorsoventral and left-right axes. In echinoderms, Nodal plays an essential role in the establishment of the dorsoventral axis and left-right asymmetry, but not in endoderm or mesoderm induction. In protostomes, Nodal signaling appears to be involved only in establishing left-right asymmetry. Hence, it is hypothesized that Nodal signaling has been co-opted to pattern the dorsoventral axis of deuterostomes and for endoderm, mesoderm formation as well as anteroposterior patterning in chordates. Hemichordata, together with echinoderms, represent the sister taxon to chordates. In this study, we analyze the role of Nodal signaling in the indirect developing hemichordate Ptychodera flava. In particular, we show that during gastrulation nodal transcripts are detected in a ring of cells at the vegetal pole that gives rise to endomesoderm and in the ventral ectoderm at later stages of development. Inhibition of Nodal function disrupts dorsoventral fates and also blocks formation of the larval mesoderm. Interestingly, molecular analysis reveals that only mesodermal, apical and ventral gene expression is affected while the dorsal side appears to be patterned correctly. Taken together, this study suggests that the co-option of Nodal signaling in mesoderm formation and potentially in anteroposterior patterning has occurred prior to the emergence of chordates and that Nodal signaling on the ventral side is uncoupled from BMP signaling on the dorsal side, representing a major difference from the molecular mechanisms of dorsoventral patterning events in echinoderms.

Keywords: Ambulacraria; Dorsoventral axis; Evolution; Hemichordate; Mesoderm; Nodal pathway.

Fig. 1.

Spatial expression of novel tissue specific markers in Ptychodera flava. Spatial distribution of Pf-vent1, Pf-cripto, Pf-sfrp1/5, Pf-six3, Pf-hh, Pf-tsg and Pf-irxA transcripts during normal development analyzed by WISH. Expression of the inter apical-stomodeum domain expression of Pf-irxA is indicated by a single white arrowhead in (U). Ectodermal, mesodermal and endodermal expression domains of Pf-six3 are indicated by white arrows in (Y). All embryos in this and following figures are oriented animal to the top, vegetal to the bottom, ventral to the left and dorsal to the right if not stated otherwise.

Fig. 2.

Spatial expression of novel dorsal markers in Ptychodera flava. Spatial distribution of Pf-follistatin, Pf-admp2, Pf-id4, Pf-msx, Pf-mef, Pf-sprouty and Pf-xbp1 transcripts during normal development. Insert in P is vegetal view (vv) and in S and T are animal views (av).

Fig. 3.

Spatial expression of novel ventral markers in Ptychodera flava. Spatial distribution of Pf-oasis, Pf-vent2, Pf-lefty and Pf-nodal transcripts during normal development. Insert in H is a vegetal view (vv), inserts in K and L are animal views (av) and inserts in J, N, P are ventral views (vev).

Fig. 4.

pSmad1/2 and pERK1/2 activation pattern during normal P. flava development. (A-D) Spatial distribution of phospho-Smad1/5 (green) positive cells in relation to Hoechst (nuclei in red) counterstaining. Insert in C is an animal view. (E-H) Spatial distribution of phospho-Erk1/2 (white) positive cells in relation to Hoechst (nuclei in red) counterstaining. G and H are stacks of four Z-acquisitions to better show the activation profile of pERK (white arrowheads). Insert in G is an animal view. (I) Schematic representation of an early gastrula stage indicating the focal planes represented in J and K that show the spatial distribution of pSmad1/5 and pErk1/2 simultaneously (vv, vegetal view; av, animal view). White arrows indicate cells in which pSmad1/5 and pErk1/2 are detected in the same nuclei.

Fig. 5.

Phenotypes observed after SB431542, U0126, mNodal and zBmp4 treatments. Control embryos (A-E) or SB4315432 (F-J), U0126 (K-O) mNodal (P-T) and zBmp4 (U-Y) treated embryos. All images are lateral views, except E, O, T and Y that are dorsal views (dv) and J, which is an animal view (av). The asterisk indicates the mouth (ventral), the star the protocoel (mesoderm) and the square the hydropore (dorsal).

Fig. 6.

Analysis of pErk1/2 and pSmad1/5 activity after drug and recombinant protein treatments. Control embryos (A,A′,F,F′) and SB4315432 (B,B′,G,G′), U0126 (C,C′,H,H′) zBmp4 (D,D′,I,I′) or mNodal (E,E′,J,J′) treated embryos. (A–E) pErk1/2 staining alone or (A′–E′) counterstained with Hoechst to visualize DNA/nuclei. (F–J) pSmad1/5 staining alone or (F′–J′) counterstained with Hoechst to visualize DNA/nuclei. All images are pre-hatching larva oriented as described in Fig. 1. White arrowheads in (A,A′–E,E′) indicate the position of pErk1/2 positive cells. Dashed lines in (F′,F–H,H′) indicate the limit between the larva ventral and dorsal sides.

Fig. 7.

Effects of SB431542, U0126 and mNodal on specification of ectoderm, mesoderm and endoderm. WISH performed at the late gastrula/pre-hatching stage of control embryos (A–and SB431542 (I–P), U0126 (Q–X) or mNodal (Y–Zf) treated embryos. The effects of these treatments on the gene expression program of the ectoderm, mesoderm of endoderm were analyzed by WISH with the indicated probes.

Fig. 8.

Comparison of the role and molecular mechnaisms of Nodal signaling in ambulacrarians. (A) Identified roles of Nodal signaling in ambulacrarians. (B-E) diagrams representing the molecular mechanism underlying D/V patterning in ambulacrarians. Hemichordates: P. flava (indirect development) and S. kowalevskii (direct development) (this study; Lowe et al., 2006). Echinoderms: P. lividus (indirect development) and H. erythrogramma (direct development) (Duboc et al., 2004; Lapraz et al., 2009b; Saudemont et al., 2010; Smith et al., 2008; Su et al., 2009).