Opposing Nodal/Vg1 and BMP signals mediate axial patterning in embryos of the basal chordate amphioxus

Abstract

The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution.

Fig. 1

Expression of Vg1, Nodal, Lefty, Cerberus and Blimp1 in normal amphioxus embryos. Animal pole or anterior to left except as noted. Arrowheads = anterior limits of expression. (A-H) Vg1. (A) 2-cell stage. (B) 4-cell stage; dorsal view. (C) mid-blastula. (D) mid-gastrula; side view, blastopore at right. (E) Dorsal view of mid-gastrula in D. (F) Mid-neurula (15 hrs); side view. (G) Dorsal view of embryo in F. Expression in 4 left anterior somites; blastopore at right. (H) Late neurula (20 hrs); dorsal view; expression in left somites. (I-Q). Nodal. (I) Fertilized egg. Arrow shows second polar body. (J) 2-cell stage. (K) 8-cell stage. (L) Mid-blastula. (M) Surface view of blastula in K. (N) Late blastula; optical cross section. (O) Mid-gastrula. Side view; blastopore at right. (P). Blastopore view of the gastrula in O. (Q) Dorsal view of the gastrula in O. (R-Z) Lefty. (R) Very early blastula. (S) early-mid blastula. (T) Mid-blastula. (U) surface view of the embryo in T. (V) Vegetal pole view of the embryo in V. (W) Very early gastrula. (X) Mid-gastrula. (Y) Blastoporal view of late gastrula. (Z) Dorsal view of late gastrula. (A’-D’). Cerberus. (A’) Mid-gastrula; optical cross-section; animal pole at top. (B’) Mid-gastrula; blastopore at right. (C’) Early neurula; side view and (D’) dorsal view. Cerberus expression in anterior right paraxial mesoderm. (E’-H’) Blimp1. (E’) Mid-blastula. (F’). Mid-gastrula. Blastopore at right. (G’) Late gastrula. (H’) Early neurula.

Fig. 2

Human Activin protein dorsal/anteriorizes amphioxus embryos. 10 ng/ml human activin applied at the early blastula eliminates expression of the ventral marker Evx and expands those of the anterior marker FoxQ2 and dorsal markers Chordin and Gsc. The entire ectoderm is specified as neural; the forebrain expressing Otx is expanded. (A-C, E-G) Mid-gastrulae; optical cross-sections. (D, H) Mid-gastrulae; side views, blastopore at right. (I-P) Mid-neurulae (15 hr); side view. Anterior at left. (R-U) Cross-sections through levels indicated in I, J, M, N. nc = nerve cord, nt = notochord, s = somites, e = endoderm. Gene markers as indicated.

Fig. 3

Inhibition of Nodal/Vg1 signaling ventral/posteriorizes amphioxus embryos. 50 μM SB505124, which inhibits signaling by Nodal, Vg1 and Activin, was added at the early blastula unless otherwise indicated. Side views. Anterior to left. (A-J) Mid-gastrula stage. Expression of dorsal and anterior markers except for FoxQ2 is eliminated. (K-H’) Mid-late neurulae. The earlier SB505124 is added, the more severe the ventralization and the greater the foreshortening.

Fig. 4

Activin protein rescues the ventral/posteriorizing defects in embryos overexpressing BMP2/4. Early mid-neurulae (14 hrs). Gene markers as indicated (A-H) Injection of amphioxus BMP2/4 mRNA eliminates dorsal and anterior structures. Embryos are severely foreshortened. (I-K). Human activin protein (10 ng/ml) added to blastulae dorsal/anteriorizes embryos, causing an expanded anterior end (J). (K) Embryos injected with amphioxus BMP2/4 mRNA and treated with human Activin protein at the blastula stage are either partially rescued with restoration of a narrower anterior end and a decreased domain of posterior Brachyury expression, but no notochord (14/21 embryos) or nearly completely rescued with restoration of the notochord as well (7/21).

Fig. 5

Knockdown of Chordin truncates the head. Side views, anterior to left, except dorsal views in S,V and cross sections in A’, D’. (A, D, G, J, M, P) Mid gastrulae. (B, E, H, K, N, Q, T, W, A’, B’,D’, E’) Mid-neurulae (15 hrs). (U, X) Late neurulae (22 hrs). (C, F, I, L, O, R, U, X, C’ F’) Early larvae (36 hrs). (A,D) Chordin expression is unaffected. (B,C,E,F) Otx expression in the CNS is reduced (arrow). (G,J) Cerberus expression is eliminated. (H,K,I,L) FoxQ2 expression eliminated. (M, P) Sox1/2/3 expression in CNS unaffected. (N, O, Q, R) Wnt3 expression unaffected, but the forebrain/midbrain, which normally does not express Wnt3, is reduced. (S,V) Late gastrula; dorsal views. BMP2/4 partially down regulated in neural plate. (T,W,U,X) Hex expression in anterior endoderm reduced. Arrowhead in U,X indicates presumptive endostyle (A’, F’) Brachyury expression in notochord largely unaffected (A’-F’). In some neurulae (D’, E’), the domain extends anteriorly. A’-A’ and D’, D’ indicate levels of cross sections in A’ and D’.

Fig. 6

Overexpression of Cerberus mRNA or knockdown of Blimp1 ventralizes and posteriorizes amphioxus embryos. Side views. Anterior at left. Expression of gene markers as noted. (A-H) Overexpression of Cerberus eliminates dorsal, anterior structures. Early mid-neurulae (15 hrs). (A-D) Injection of control mRNA. (E-H) Injected with Cerberus mRNA. (I-X) MO knockdown of Blimp eliminates the anterior part of the head. (I,M,K,O,Q,U,S,W) Early mid-neurulae (15 hrs). (J,N,L,P,R,V,T,X) Early larva (36 hrs). (I-L,Q-T) Embryos injected with control MO. (M-P,U-X) Injected with Blimp1 MO.

Fig. 7

Experiments in Xenopus show conserved protein functions. (A-F) Injections into marginal zones of 4-cell Xenopus embryos except for B. (A) Uninjected control. (B) 10pg/cell amphioxus Cerberus mRNA in D4 blastomere at 32 cell stage induces secondary axis. (C) 100pg/cell amphioxus Blimp1 mRNA expands ventral structures. (D) 500 pg/cell amphioxus Blimp1 mRNA reduces head structures and expands ventral ones. (E) 100pg/cell amphioxus BMP2/4 mRNA ventralizes. (F) 10pg/cell amphioxus Chordin mRNA enlarges the cement gland. (G-J) Assays of amphioxus proteins in Xenopus animal caps. (G) BMP responsive reporter assay. Amphioxus Chordin and Cerberus suppress signaling by Xenopus BMP4, as monitored by transcription from an Xvent2 reporter. (H) Amphioxus Blimp1 induces transcription from a Nodal-responsive (Xmix2) reporter construct. (I) Nodal-responsive assay. Amphioxus Cerberus suppresses amphioxus Nodal. (J) Activin response assay. Amphioxus Cerberus suppresses Xenopus Activin signaling. Experiments done twice with comparable results; error bars ± 1 s.d.

Fig. 8

Diagram of axial patterning genes in early amphioxus development of amphioxus. In the late blastula, Nodal/Vg1 signaling, presumably opposed by Lefty marks the future dorsal side (D) of the embryo, while FoxQ2 marks the anterior ectoderm. At the onset of gastrulation, dorsal Nodal/Vg1 signaling becomes opposed by BMP signaling in the mesendoderm. BMP signaling is modulated by several dorsally and anteriorly expressed genes. Wnt8 is expressed throughout the future mesendoderm, most strongly in the rim of the forming blastopore, which will be the posterior pole of the embryo. By the early gastrula, additional modulators of Nodal/Vg1 and BMP signaling, Evx, a marker of ventral ectoderm, and the neuroectoderm marker Sox1/2/3 are first expressed. A= anterior pole, An = animal pole, P= posterior pole, V= ventral.