Insights from the amphioxus genome on the origin of vertebrate neural crest

Abstract

The emergence of the neural crest has been proposed to play a key role in early vertebrate evolution by remodeling the chordate head into a "new head" that enabled early vertebrates to shift from filter feeding to active predation. Here we show that the genome of the basal chordate, amphioxus, contains homologs of most vertebrate genes implicated in a putative neural crest gene regulatory network (NC-GRN) for neural crest development. Our survey of gene expression shows that early inducing signals, neural plate border patterning genes, and melanocyte differentiation genes appear conserved. Furthermore, exogenous BMP affects expression of amphioxus neural plate border genes as in vertebrates, suggesting that conserved signals specify the neural plate border throughout chordates. In contrast to this core conservation, many neural crest specifier genes are not expressed at the amphioxus neural plate/tube border, raising the intriguing possibility that this level of the network was co-opted during vertebrate evolution. Consistent with this, the regulatory region of AmphiFoxD, homologous to the vertebrate neural crest specifier FoxD3, drives tissue-specific reporter expression in chick mesoderm, but not neural crest. Thus, evolution of a new regulatory element may have allowed co-option of this gene to the NC-GRN.

Figure 1.

Expression of amphioxus genes of inductive signals during late gastrula stage. Anterior is perpendicular to the plane of the page in the blastopore view and at the top in the dorsal view. Dorsal is up in the blastopore view. Scale bar, 50 μm. Amphioxus BMP2/4 is expressed throughout the ectoderm and mesendoderm, except the dorsal axial mesoderm and ectoderm, and the BMP antagonist Chordin is expressed in complementary domain in the most dorsal-medial mesendorem and ectoderm. Fgf8/17/18 is expressed in the dorsal axial mesendoderm underlying the prospective neural plate, and Wnt8 is expressed in the paraxial mesoderm and ventral mesendoderm. Notch is expressed throughout the entire mesendoderm and in the prospective neural plate, and its ligand Delta is expressed in the paraxial dorsal mesoderm.

Figure 2.

Expression of amphioxus neural plate border specifier genes is effected by exogenous zebrafish Bmp4 (zBmp4) protein. Anterior is perpendicular to the plane of the page in the blastopore view and at the top in the dorsal view. The medial boundaries of gene expression in dorsal ectoderm are indicated by arrows (A) or arrowheads (B). Scale bar, 50 μm. (A) In normal late gastrula stage embryos, AP2, Dlx, and Msx are expressed throughout the epidermal ectoderm. Msx is also strongly expressed at the neural plate border. Zic and Pax3/7 expression in the dorsal ectoderm marks the neural plate border, and SoxB1-a expression is in the entire neural plate. During the early neurula stage, the edges of the epidermal ectoderm dissociate from the neuroectoderm and migrate toward the midline. This process is shown in the blastopore view of early neurula embryos. (B) In zBmp4 treated embryos, expression of epidermal markers AP2 and Dlx is greatly expanded, suggesting that the entire dorsal ectoderm is transformed to epidermal fate. The expression of neural plate border markers Msx and Zic is converged to the dorsal midline; expression of another neural plate border marker Pax3/7 and neural plate marker SoxB1-a is lost. In addition, no neurulation process can be observed in zBmp4 treated embryos.

Figure 3.

Expression of amphioxus homologues of neural crest specifier genes and reporter gene expression mediated by amphioxus FoxD (AmphiFoxD) regulatory region in chick embryos. (A) Anterior is perpendicular to the plane of the page in the blastopore view and at the top in the dorsal view. Dorsal is up in the blastopore view. Scale bar, 50 μm. Amphioxus Snail is expressed in the entire prospective neural plate and paraxial dorsal mesoderm in late gastrula stage. During the early neurula stage, Snail is down-regulated in the neural plate and transiently expressed at the neural plate border. SoxE is expressed in the dorsal mesendoderm at the border of future axial and paraxial mesoderm. FoxD is expressed in the dorsal axial mesoderm, paraxial mesoderm, and the anterior neural plate. Amphioxus Myc is expressed in the dorsal paraxial mesoderm and ventral mesendoderm. (B–E) Reporter gene expression in mesoderm derivatives and hindbrain directed by AmphiFoxD in chick embryos. Rostral is to the top in B, and dorsal to the top in C–E. EGFP+ cells have yellow or orange nuclei due to coexpression of RFP. (B) Whole-mount image of stage 11 chick embryo expressing RFP in all electroporated cells and EGFP under the control of the AmphiFoxD regulatory region; EGFP+ cells are seen in somites (white arrows, shown at higher power in inset), paraxial mesoderm, and hindbrain (black arrows). (C–E) Cross-sections showing expression of EGFP in the hindbrain (C, black arrows), somites (D, arrows), and notochord (E, arrow). No expression of EGFP is seen in premigratory or migratory neural crest cells (C, white arrows). (NT) neural tube; (S) somite; (No) notochord.

Figure 4.

Expression of amphioxus homologs of neural crest effector genes and summary for putative neural border gene network in amphioxus. (A) Expression of Mitf, tyrosinase, Tyrp-a, and Tyrp-b in late neurula stage embryos. Side views are shown here; anterior is at left and dorsal is up. Scale bar, 50 μm. All four of these genes are coexpressed in the neural tube at the level where the first primary pigment spot will form. (B) In amphioxus, gene expression and BMP signaling perturbation suggest that the processes of dorsal ectoderm patterning and neural border specification are conserved between vertebrates and amphioxus. In addition, the differentiation gene battery for pigment cell development appears conserved in both vertebrates and amphioxus. However, at intermediate levels of their respective neural border networks, the two groups diverge. Amphioxus lacks neural border expression of most neural crest specifiers, as well as the effector subcircuit controlling neural crest delamination and migration, consistent with a lack of bona fide neural crest cells in this lineage.