New perspectives on pharyngeal dorsoventral patterning in development and evolution of the vertebrate jaw

Abstract

Patterning of the vertebrate facial skeleton involves the progressive partitioning of neural-crest-derived skeletal precursors into distinct subpopulations along the anteroposterior (AP) and dorsoventral (DV) axes. Recent evidence suggests that complex interactions between multiple signaling pathways, in particular Endothelin-1 (Edn1), Bone Morphogenetic Protein (BMP), and Jagged-Notch, are needed to pattern skeletal precursors along the DV axis. Rather than directly determining the morphology of individual skeletal elements, these signals appear to act through several families of transcription factors, including Dlx, Msx, and Hand, to establish dynamic zones of skeletal differentiation. Provocatively, this patterning mechanism is largely conserved from mouse and zebrafish to the jawless vertebrate, lamprey. This implies that the diversification of the vertebrate facial skeleton, including the evolution of the jaw, was driven largely by modifications downstream of a conversed pharyngeal DV patterning program.

Figure 1. Regional patterning of the facial skeletons of zebrafish and mouse

A and B, Lateral views of a 6 dpf larval zebrafish skull (A) and a P0 mouse skull (B, courtesy of Michael Depew). Alcian Blue labels cartilage and Alizarin Red labels mineralized bone and teeth. C and D, Schematics of the AP origins of select skeletal elements. Hox-negative maxillary and mandibular arch-derived elements are not shaded, with progressive shades of gray showing increasing numbers of Hox genes being expressed in more posterior hyoid and branchial arches. E and F, Schematics show a meristic series of skeletal elements along the DV axis (proximal-distal in mouse). Elements are classically divided into five repeating units in each arch, which are designated by the following prefixes from dorsal to ventral: pharyngo-, epi-, cerato-, hypo-, and basi-. The suffixes –mandibular, -hyal, and –branchial (1–5) refer to the first, second, and more posterior arches in the AP series, respectively. Many of the elements are named by combining these descriptors, e.g. the ceratohyal element of the more ventral second arch. Pharyngobranchial and epibranchial elements (dotted lines) are not apparent in 6 dpf zebrafish larvae but develop later. Although not present in wild-type zebrafish, a basi-mandibular element (dotted line, not shaded) can form in certain zebrafish mutants and other species such as dogfish (Balczerski et al., 2012). Putative maxillary-derived elements are shown in gray, and the neurocranium/skull to which the facial skeleton articulates is not shaded. Zebrafish abbreviations: Bh, basihyal. Br, branchiostegal ray bone. Ch, ceratohyal. Hm, hyomandibular. Ih, interhyal. Mc, Meckel’s cartilage. Op, opercular bone. Pq, palatoquadrate. Ptp, pterygoid process. Sy, symplectic. Mouse abbreviations: Dnt, dentary. Gh, greater horn of the hyoid bone. Hy, hyoid bone. In, incus. Jg, jugal. Lh, lesser horn of the hyoid bone. lIn, lower incisor. Ma, malleus. Mc, Meckel’s cartilage. Mx, maxilla. Pmx, premaxilla. Pt, palatine. Rtp, retrotympanic process. Sq, squamosal. Sp, styloid process. St, stapes. Tc, tracheal cartilage. uIn, upper incisor.

Figure 2. Signaling networks in arch DV patterning

A and B, Sketches of arch-stage zebrafish (A) and mouse (B) embryos show the initial locations of the maxillary prominences (gray), dorsal mandibular and hyoid arches (green), and ventral mandibular and hyoid arches (red). C and D, Schematics of later DV gene expression domains in 36 hpf zebrafish (C) and E10.5 mouse (D) arches. E, In the early arches (24 hpf in zebrafish and E9.0 in mouse), Edn1 and Bmp4 from the epithelia function largely redundantly to initiate mesenchymal gene expression (colored boxes) in a common ventral domain. F, As arch development proceeds (shown here for 36 hpf in zebrafish and E10.5 in mouse), a network of signaling interactions refines DV gene expression. Interactions are a composite of data from multiple vertebrates, primarily fish and mouse. Black lines are transcriptional interactions and red lines protein-protein interactions.

Figure 3. Dynamic DV zones during facial skeletal development

A–C, At 36 hpf (A), dlx5a:GFP expression (green) marks ventral cells of each arch within the NCC-derived sox10:dsRed positive mesenchyme (red) of zebrafish. At 6 dpf (B and C), dlx5a:GFP-expressing cells contribute to portions of every cartilage, with the exception of the ventral-most tip of the Ch cartilage (outlined), the dorsal tip of the Pq cartilage, and the majority of the Hm cartilage. By 6 dpf, early sox10:dsRed NCC expression has weakened and strong expression is now seen in chondrocytes. D–F, hand2:GFP expression is restricted to the ventral-most arch NCCs at 36 hpf (D), as well as a subset of sox10:dsRed-negative epithelial cells. By 6 dpf (E and F), hand2:GFP-expressing cells are found within and around the distal-most tip of the Ch cartilage, with progressively weaker expression towards more dorsal regions. G, Schematic of dynamic DV gene expression in the developing Ch cartilage. At early stages (24 hpf), Dlx3–6 genes, msxe, and hand2 are largely co-expressed in ventral NCC-derived precursors of the Ch cartilage. By 36 hpf, ventral expression has resolved into three domains: 1) a dorsal intermediate Dlx3–6 domain, 2) a ventral-intermediate domain expressing both msxe and Dlx3–6 genes, and 3) a ventral domain expressing hand2. Note all regions also express Dlx1/2. By around 52 hpf, chondrogenesis begins in intermediate NCCs expressing Dlx3–6 (blue outlines depict chondrocytes) while hand2- and msxe-positive NCCs remain as an undifferentiated growth zone that lengthens the future cartilage. At later stages (e.g. 6 dpf), the chondrogenic domain expands ventrally as hand2- and msxe-positive precursors become depleted.

Figure 4. The larval lamprey pharyngeal skeleton and vertebrate phylogeny

A, Larval lamprey pharyngeal skeleton at 35 dpf visualized by Alcian Blue staining. B, The AP origins of lamprey pharyngeal skeleton elements. Hox negative pre mandibular and first arch-derived elements are unshaded, with progressively darker shades of gray indicating increasing numbers of Hox genes in the second, third, and posterior branchial arches. C, The components of the larval lamprey pharyngeal skeleton. The skeleton of the upper lip (Ul), lower lip (Ll), first arch (Pa1), second arch (Pa2), and ventral pharynx (Vm) consists of mucocartilage. A fused meshwork of cellular cartilage rods supports the remaining posterior arches. This “branchial basket” includes the horizontal subchordal (Sc) and hypobranchial (Hb) bars and vertical branchial bars (Bb). Projecting anteriorly from the branchial bars are the epitrematic (Ep) and hypotrematic (Hp) processes. D, Phylogeny illustrating the relationships between jawed (gnathostome) and jawless (agnathan) vertebrates, the cephalochordates, urochordates, and hemichordates. Lampreys are members of the most basal vertebrate group, the cyclostomes, which also includes hagfish. The last common ancestor of lamprey and hagfish diverged from the lineage leading to the jawless fossil ostracoderms and the gnathostomes near the time of vertebrate origins.

Figure 5. DV polarity in the developing lamprey pharyngeal skeleton

A, DV-restricted gene expression in the nascent lamprey pharyngeal skeleton at Tahara st. 26.5 (about 13 dpf) (Tahara, 1988). Only the pre-mandibular (maxillary) region and pharyngeal arches 1–3 are shown. B, Presumed derivatives of these domains in a 35 dpf larva. Though all skeletal tissue in the pre-mandibular domain and the first two arches is mucocartilage, gene expression suggests it is divided into molecularly distinct subpopulations. Dynamic expression of Dlx paralogs, Msx, and Hand in NCCs of the 3^rd (and other posterior arches) corresponds to different DV components of the branchial basket. C, Differentiated branchial cartilage at 17, 18, and 20 dpf as visualized by Alcian Blue-induced green fluorescence. Adapted with permission from (Martin et al., 2009). As in gnathostomes, differentiation of lamprey pharyngeal cartilage begins in the intermediate domain where all Dlx genes are expressed simultaneously in the absence of Msx or Hand. At later stages, the zone of differentiated cells expands dorsally and ventrally. D, Diagram showing the distribution of cellular cartilage subtypes and mucocartilage along the DV axis in the posterior portion of the lamprey pharyngeal skeleton.