The amphioxus SoxB family: implications for the evolution of vertebrate placodes

Abstract

Cranial placodes are regions of thickened ectoderm that give rise to sense organs and ganglia in the vertebrate head. Homologous structures are proposed to exist in urochordates, but have not been found in cephalochordates, suggesting the first chordates lacked placodes. SoxB genes are expressed in discrete subsets of vertebrate placodes. To investigate how placodes arose and diversified in the vertebrate lineage we isolated the complete set of SoxB genes from amphioxus and analyzed their expression in embryos and larvae. We find that while amphioxus possesses a single SoxB2 gene, it has three SoxB1 paralogs. Like vertebrate SoxB1 genes, one of these paralogs is expressed in non-neural ectoderm destined to give rise to sensory cells. When considered in the context of other amphioxus placode marker orthologs, amphioxus SoxB1 expression suggests a diversity of sensory cell types utilizing distinct placode-type gene programs was present in the first chordates. Our data supports a model for placode evolution and diversification whereby the full complement of vertebrate placodes evolved by serial recruitment of distinct sensory cell specification programs to anterior pre-placodal ectoderm.

Keywords: Evolution; amphioxus; chordates; development; placodes; vertebrates.

Figure 1

Phylogenetic tree and alignment of deuterostome SoxB proteins. (A) Phylogenetic tree created using the Neighbor-Joining method with chicken Sox8 as the outgroup. Black numbers at branch bases are confidence values derived from 1000 bootstrap resamplings of the alignment data. Sequence distance is indicated at the bottom left as substitutions per base. Numbers in green are quartet-puzzling reliability scores from Maximum Likelihood analysis of the same alignment. Amphioxus SoxB1c and amphioxus SoxB2 group with their respective vertebrate homologs. Amphioxus SoxB1a and SoxB1b group together outside a clade including SoxB1c and vertebrate and sea urchin SoxB1 genes. In both analyses, Ciona SoxB1 failed to group with the other SoxB1 genes. (B) Alignment of amphioxus, urchin, Ciona and chick SoxB1 proteins. While all proteins are highly conserved in N-terminal HMG domain, the C-terminal transactivation domain is only conserved in chick Sox2, urchin SoxB1 and amphioxus SoxB1c. Amphioxus SoxB1a, SoxB1b and Ciona SoxB1 all have divergent transactivation domains, suggesting a similar loss of functionality.

Figure 2

Embryonic expression of amphioxus SoxB1b. Anterior is to the left (A) Side view of 9-hour early neurula. SoxB1b expression is seen throughout the neural plate (arrow). (B) Optical cross section through 9-hour early neurula at approximately the level of b in A (arrow). (C) Side view of 12-hour neurula. SoxB1b is expressed in the neural plate as it rolls-up to form the neural tube (arrow). (D) Optical cross section through 12-hour neurula at the level of c in D. SoxB1b transcripts are detected in the neural plate (arrow) as it is overgrown by epidermal ectoderm.

Figure 3

Embryonic expression of amphioxus SoxB1c. Anterior is to the left. (A) Side view of 9-hour early neurula. SoxB1c expression is seen in a patch of neurectoderm near the blastopore (arrow) and in anterior endoderm (arrowhead). (B) Dorsal view of 12-hour neurula, expression in restricted areas of the rostral (arrow) and caudal (arrowhead) neural plate. (C) Side view of 12-hour neurula. SoxB1c transcripts persist in the anterior gut (arrow). (D) Side view of 18-hour late neurula, focused in the plane of the epidermis. Scattered SoxB1c labelled cells are seen in the epidermal ectoderm (arrow). Out of focus, SoxB1c expression has expanded throughout the entire neural tube (double arrowheads) and marks the foregut (arrowhead). (E) Optical cross section through a bisected 18-hour neurula at the level of e in D. Strong expression is seen in the neural tube (arrowhead) and foregut (arrow). (F) Optical cross section through a bisected 18-hour neurula at the approximate the level of f in D. Arrowheads point to SoxB1c-positive cells in epidermis. (G) Side view of 24-hour late neurula. Expression in the neural tube (double arrowheads) and foregut (arrowhead) persists, and a band of expression also appears in the hindgut (arrow). (H) Side view of 36-hour late larva. Neural tube (double arrowheads) and hindgut expression have ceased while high levels of transcripts remain in the foregut (arrowhead).

Figure 4

Embryonic expression of amphioxus SoxB2. Anterior is to the left. (A) Side view of 9-hour neurula. SoxB2 transcripts are seen in the caudal-most neurectoderm (arrow) and weakly throughout the ventral mesendoderm (arrowhead). (B) Dorsal view of 9-hour early neurula. SoxB2 transcripts are seen in a small region of neurectoderm bordering the blastopore (arrow). (C) Dorsal view of 12-hour neurula. SoxB2 expression has begun to expand into the anterior neural plate (arrow). (D) Side view of 15-hour neurula. High levels of SoxB2 transcripts are detected throughout the neural tube (arrow). Lower levels are seen in the gut (arrowhead). (E) Optical cross section through a bissected 15-hour neurula at the level of e in D. Strong SoxB2 signal is observed in the neural tube (arrow), while lower levels persist in the gut (arrowhead). By 24 hours, detectable SoxB2 expression has ceased (not shown).

Figure 5

SoxB1c expression in oral ectoderm at early larval stages. Anterior is to the left (A) The head of a 2-day old larva focused in the plane of the epidermis. Expression is apparent in ectoderm surrounding the newly formed mouth (arrow). Out of focus is staining in the underlying pharyngeal endoderm (arrowhead).(B) In 3-day larvae, SoxB1c continues to mark ectoderm around the mouth corresponding to the future location of oral spine cells and neurons. (C) Schematic of oral spine cells, sc, and oral nerve plexus, onp, in 12-14 day larvae modified from Lacalli.

Figure 6

Expression of amphioxus placode marker homologs in subsets of epidermal sensory cells. SoxB1c, Pitx, Msx, Pax6, Msx, Six1/2, Six3/6, Eya, Coe, and Brn3 homologs mark partially overlapping epidermal domains which give rise to putative sensory cells. Interestingly, the rostro-caudal extent of their expression corresponds roughly to that of their vertebrate cognates in cranial placodes. Though morphologically similar, epidermal sensory cells in the trunk (red) appear deploy different sets of genes. Taken together, these data suggest ancient functions for placode genes in sensory cell specification and a high level of sensory cell diversification in the first chordates.

Figure 7

A model for the evolution of placodal diversity. Gene expression in amphioxus suggests placode marker homologs functioned primitively to specify an array of epidermal sensory cell fates in the first chordates (A). In the vertebrates lineage these genes were serially recruited to the anterior ectoderm, driving diversification of a pre-placodal primordium into the full complement of vertebrate placodes . This may have occurred by a loss of responsiveness to retinoic acid patternins mechanisms and a gain of responsiveness to inductive signals in the head. (B) Urochordates may have diverged at an intermediate phase in this process and thus possess some composite placodes putatively homologous to vertebrate cranial placodes, as well as epidermal sensory cells (C). Alternately, urochordate and vertebrate placodes may represent parallel structures derived separately from similar sets of epidermal sensory cells.