Developmental evidence for serial homology of the vertebrate jaw and gill arch skeleton

Abstract

Gegenbaur's classical hypothesis of jaw-gill arch serial homology is widely cited, but remains unsupported by either palaeontological evidence (for example, a series of fossils reflecting the stepwise transformation of a gill arch into a jaw) or developmental genetic data (for example, shared molecular mechanisms underlying segment identity in the mandibular, hyoid and gill arch endoskeletons). Here we show that nested expression of Dlx genes--the 'Dlx code' that specifies upper and lower jaw identity in mammals and teleosts--is a primitive feature of the mandibular, hyoid and gill arches of jawed vertebrates. Using fate-mapping techniques, we demonstrate that the principal dorsal and ventral endoskeletal segments of the jaw, hyoid and gill arches of the skate Leucoraja erinacea derive from molecularly equivalent mesenchymal domains of combinatorial Dlx gene expression. Our data suggest that vertebrate jaw, hyoid and gill arch cartilages are serially homologous, and were primitively patterned dorsoventrally by a common Dlx blueprint.

Figure 1. Bayesian phylogenetic analysis of the gnathostome Dlx family

The skate Leucoraja erinacea, the shark Scyliorhinus canicula and the paddlefish Polyodon spathula each possess six Dlx orthologues (in bold). Our analysis recovered six Dlx clades, each containing a single mouse (Mus), leopard shark (Triakis), skate (Leucoraja), dogfish (Scyliorhinus) and paddlefish (Polyodon) Dlx orthologue. All sequences (with the exception of L. erinacea, S. canicula and P. spathula sequences) were taken from the alignment of Stock. A modified sequence alignment (including translated L. erinacea, S. canicula and P. spathula sequence fragments) was generated using the ClustalW algorhithm in MacVector, and phylogenetic analysis was performed using MrBayes 3.2. The analysis ran for 2,000,000 MCMC generations with a sampling frequency of 100. Numbers at the bases of clades are Bayesian posterior probabilities.

Figure 2. Nested Dlx expression in the pharyngeal arches of elasmobranchs. (a-n)

Wholemount in situ hybridization reveals that in the mandibular, hyoid and gill arches of the skate L. erinacea, (a,b) Dlx1-2, (c,d) Dlx5-6, and (e,f) Dlx3-4 are expressed in progressively more ventrally-restricted domains at stage 27, resulting in (g) nested, combinatorial Dlx gene expression (i.e., a Dlx code) in all pharyngeal arches. Similarly nested expression of (h,i) Dlx1-2, (j,k) Dlx5-6 and (l,m) Dlx3-4 is observed in the mandibular, hyoid and gill arches of the shark S. canicula at stage 24, establishing (n) a Dlx code in all pharyngeal arches. (o-u) Vibratome sections after in situ hybridization reveal that pharyngeal arch expression of Dlx1-6 is restricted to the neural crest-derived mesenchyme and excluded from ectoderm, endoderm and the mesodermal core. 1-4, gill arches 1-4; e, eye; ect, ectoderm; end, endoderm; h, hyoid arch; m, mandibular arch; mes, mesoderm; nc, neural crest-derived mesenchyme; o, otic vesicle. Scale bars: (a-f) = 500μm; (h-m) = 500μm; (o-t) = 25μm.

Figure 3. Nested Dlx expression in the pharyngeal arches of paddlefish

Viewed laterally, wholemount in situ hybridization in stage 34 P. spathula embryos reveals expression of (a,b) Dlx1-2, (c,d) Dlx5-6, and (e,f) Dlx3-4 in progressively more ventrally-restricted domains in the hyoid and gill arches, resulting in a nested Dlx code in these arches, schematized in (g). Frontal views of the same embryos reveal similarly nested expression patterns of (h,i) Dlx1-2, (j,k) Dlx5-6 and (l,m) Dlx3-4 in the mandibular arch, schematized in (n). Sections reveal that paddlefish pharyngeal arch expression of (o-u) Dlx1-6 is restricted to the neural crest-derived mesenchyme, and excluded from the ectoderm, endoderm and mesodermal core. 1-3, gill arches 1-3; e, eye; ect, ectoderm; end, endoderm; h, hyoid arch; m, mandibular arch; mes, mesoderm; mth, mouth; olf, olfactory organ; nc, neural crest-derived mesenchyme; o, otic vesicle. Scale bars: (a-f) = 200μm; (h-m) = 200μm; (o-t) = 50μm.

Figure 4. Fate of Dlx-coded pharyngeal arch mesenchyme in skate

(a) Focal DiI-labelling of the dorsal (Dlx1-2-expressing) mesenchyme of the mandibular, hyoid and first gill arch resulted in (b) regions of DiI-positive chondrocytes (red dots) concentrated in the dorsal segments of the jaw, hyoid and gill arch skeleton, e.g. (c) in the palatoquadrate but (d) not in Meckel’s cartilage; (e) in the hyomandibula but (f) not in the pseudoceratohyal; and (g) in the epibranchial but (h) not in the ceratobranchial. Conversely, (i) focal DiI-labelling of ventral (Dlx1-6-expressing) mesenchyme of the mandibular, hyoid and first gill arch resulted in (j) regions of DiI-positive chondrocytes (red dots) concentrated in the ventral segments of the jaw, hyoid and gill arch skeleton, e.g. (k) not in the palatoquadrate, but (l) in Meckel’s cartilage; (m) not in the hyomandibula, but (n) in the pseudoceratohyal; and (o) not in the epibranchial, but (p) in the ceratobranchial. (q) Focal DiI-labelling of intermediate (Dlx1, 2, 5 and 6-expressing) mandibular, hyoid and gill arch mesenchyme resulted in (r) DiI-positive chondrocytes (red dots) concentrated at the point of articulation between the dorsal and ventral segments of the jaw, hyoid and gill arch skeleton, e.g. (s-u) at the articulation between the palatoquadrate and Meckel’s cartilage; (v,w) in the hyomandibula and the pseudoceratohyal; and (x-z) at the articulation between the epibranchial and ceratobranchial. Dorsal and ventral skeletal elements of the mandibular, hyoid and first gill arch, therefore, arise from equivalent domains of combinatorial Dlx expression. All dorsal-ventral image pairs are from the same individual. Cb, ceratobranchial; Ch, pseudoceratohyal; e, eye; Eb, epibranchial; ga, gill arch; ha, hyoid arch; Hm, hyomandibula; ma, mandibular arch; Mk, Meckel’s cartilage; o, otic vesicle; Pq, palatoquadrate. Scale bars: (a,i,q) = 200μm; (c-h),(k-p),(s-z) = 25μm.

Figure 5. Evolution of the Dlx code and serial homology of gnathostome pharyngeal endoskeletal elements

The Dlx code arose along the gnathostome stem, and was primitively deployed in all pharyngeal arches. The evolutionary relationship between the Dlx code of gnathostomes and the nested expression of DlxA-D in the pharyngeal arches of lamprey remains unclear. Dorsal (Dlx1-2-expressing) and ventral (Dlx1-6-expressing) domains of the Dlx code would have primitively given rise to dorsal “epimandibular”, “epihyal” and “epibranchial” elements and ventral “ceratomandibular”, “ceratohyal” and “ceratobranchial” elements (in the mandibular, hyoid and gill arches, respectively), while intermediate (Dlx1, 2, 5 and 6-expressing) domains would have given rise to the region of articulation between these elements. The primitive role for the Dlx code in patterning the mandibular, hyoid and gill arch endoskeletal segments has been conserved in elasmobranchs, and presumably in non-teleost actinopterygians (e.g. paddlefish), while post-hyoid arch expression of the Dlx code has been modified or obscured in amniotes (e.g. mouse), and possibly in teleosts. at, ala temporalis; cb, ceratobranchials; “ch”, hypothetical ceratohyal; ch, ceratohyal; “cm”, hypothetical ceratomandibula; eb, epibranchials; “eh”, hypothetical epihyal; “em”, hypothetical epimandibula; hm, hyomandibula; in, incus; mk, Meckel’s cartilage; pq, palatoquadrate; sp, styloid process; st, stapes.