Jaw morphogenesis is a complex event mediated by inductive signals that establish and maintain the distinct developmental domains required for formation of hinged jaws, the defining feature of gnathostomes. The mandibular portion of pharyngeal arch 1 is patterned dorsally by Jagged-Notch signaling and ventrally by endothelin receptor A (EDNRA) signaling. Loss of EDNRA signaling disrupts normal ventral gene expression, the result of which is homeotic transformation of the mandible into a maxilla-like structure. However, loss of Jagged-Notch signaling does not result in significant changes in maxillary development. Here we show in mouse that the transcription factor SIX1 regulates dorsal arch development not only by inducing dorsal Jag1 expression but also by inhibiting endothelin 1 (Edn1) expression in the pharyngeal endoderm of the dorsal arch, thus preventing dorsal EDNRA signaling. In the absence of SIX1, but not JAG1, aberrant EDNRA signaling in the dorsal domain results in partial duplication of the mandible. Together, our results illustrate that SIX1 is the central mediator of dorsal mandibular arch identity, thus ensuring separation of bone development between the upper and lower jaws.

Development of the vertebrate face requires the coordinated regulation of patterning cues throughout the pharyngeal arches of the developing embryo. Populated by cranial neural crest cells (NCCs) originating in the dorsal neural tube (Le Douarin, 1982; Noden, 1983), NCCs receive patterning signals from the surrounding arch ectoderm and endoderm that establish their positional identity (Clouthier et al., 2010, 2013; Medeiros and Crump, 2012), dividing the arches into dorsal (proximal), intermediate and ventral (distal) domains (Clouthier et al., 2010; Clouthier and Schilling, 2004; Medeiros and Crump, 2012).

Patterning in the intermediate and ventral domains of the mandibular portion of arch 1 is established in large part by endothelin receptor A (EDNRA) signaling within NCCs, arising when arch ectoderm-derived endothelin 1 (EDN1) binds to the EDNRAs on NCCs (Clouthier et al., 1998; Nair et al., 2007; Tavares et al., 2012). This signaling initiates a gene expression cascade that establishes the identity of NCCs in the intermediate/ventral mandibular arch and results in the formation of lower jaw and middle ear structures. Loss of EDNRA signaling leads to homeotic transformation of the mandibular bone into a maxilla-like structure along with other dorsal-ventral duplications (Kimmel et al., 2003; Ozeki et al., 2004; Ruest et al., 2004), events that are preceded by disrupted expression of EDNRA signaling network genes and a ventral expansion of a dorsal domain gene expression profile (Alexander et al., 2011; Clouthier et al., 1998, 2000; Miller et al., 2003; Nair et al., 2007; Ozeki et al., 2004; Ruest et al., 2004; Sato et al., 2008b). This expansion is achieved in part through upregulated Jagged-Notch signaling, the mechanism reported to be responsible for establishing dorsal NCC identity in zebrafish (Zuniga et al., 2010). In this model, Ednra signaling normally represses jag1b expression (Zuniga et al., 2011, 2010), while Jagged-Notch signaling prevents expansion of Ednra-dependent gene expression into the dorsal arch (Barske et al., 2016; Zuniga et al., 2010).

Although Edn1 expression is not observed rostral to the mandibular arch (Clouthier et al., 1998), aberrant EDNRA signaling in maxillary NCCs leads to homeotic transformation of the maxilla into a mandible-like structure (Sato et al., 2008b; Tavares and Clouthier, 2015). These changes are accompanied by an upregulation of a ventral/intermediate gene expression profile in the dorsal mandibular arch domain and maxillary prominence (Sato et al., 2008b; Tavares and Clouthier, 2015; Zuniga et al., 2011). By contrast, overexpression of jag1b in zebrafish embryos results in downregulation of ventral arch gene expression (Zuniga et al., 2010). However, unlike changes observed in more caudal arches, loss of jag1b expression in zebrafish does not lead to homeotic transformation of dorsal structures in arch 1 (Barske et al., 2016; Zuniga et al., 2010). Similarly, conditional inactivation of Jag1 in mouse NCCs leads to the development of a shortened maxilla but not to homeotic changes (Humphreys et al., 2012).

The transcription factor SIX1, a member of the SIX family of transcription factors (Kawakami et al., 2000; Kumar, 2009), is involved in numerous developmental and disease-related events, with loss of SIX1 leading to defects in eye, ear, heart, rib, kidney and lower jaw development (Laclef et al., 2003; Ozaki et al., 2004; Xu et al., 2003; Zou et al., 2006, 2004; Guo et al., 2011). In humans, both SIX1 and its co-factor EYA1 have been implicated in branchiootic syndrome [BOS1, Online Mendelian Inheritance in Man (OMIM) 602588; and BOS3, OMIM 608389] and branchiootorenal syndrome (BOR1, OMIM 113650) (Lee et al., 2007; Orten et al., 2008; Ruf et al., 2003, 2004). These syndromes are characterized by hearing loss, defects in pharyngeal arch derivatives and renal anomalies. SIX1 is also involved in the metastatic progression of breast cancer cells and does so through the activation of several signaling networks, including Jagged-Notch signaling (Smith et al., 2012). SIX1 regulates otic vesicle and olfactory epithelium development in a similar manner (Bosman et al., 2009; Ikeda et al., 2010). This raises the intriguing possibility that SIX1 establishes or maintains Jagged-Notch signaling during pharyngeal arch development.

To investigate this hypothesis, we have examined the function of SIX1 and its relationship to both EDNRA and Jagged-Notch signaling during jaw morphogenesis in mouse. We find that the loss of Six1 leads to expansion of the maxilla into a rod-shaped bone, with the posterior end of the bone resembling a mandibular process. The formation of this bone occurs in part due to EDNRA signaling in the dorsal mandibular arch that arises from aberrant expression of endodermal pouch-expressed Edn1. Therefore, a primary function of SIX1 appears to be maintaining a dorsal mandibular arch domain that is free of EDNRA signaling to ensure that the region between the maxillary prominence and mandibular arch develops without intrusion of bone structures from the maxillary prominence.

Loss of Six1 leads to the formation of a novel bone in the zygomatic arch

To analyze SIX1 function in facial morphogenesis, we first examined embryos from one of two Six1 mutant strains that have been created, in which both exons of Six1 were targeted (Ozaki et al., 2004). Control and Six1−/− embryos were first collected at embryonic day (E) 18.5 and skull structures analyzed by both Alizarin Red/Alcian Blue staining (Fig. 1A-D′) and micro-computed tomography (micro-CT) (Fig. 1E,F). Six1−/− embryos presented with retrognathia (Fig. 1B,D) and previously described defects in the nasal bones, otic capsule, tympanic ring bone and middle ear ossicles (Fig. 1B) (Guo et al., 2011; Ozaki et al., 2004). However, the most striking change in Six1−/− embryos was the formation of a novel bone extending posteriorly from the maxilla. In mice, the zygomatic arch is normally composed of the zygomatic process of the maxilla, the jugal bone and the zygomatic process of the squamosal bone (Fig. 1A,C,C′ and pseudo-colored red, blue and green, respectively, in 1E). In Six1−/− embryos, the anterior portion of the maxilla appeared similar to that of control embryos (Fig. 1B,D,D′,F). However, the zygomatic process of the maxilla became a thicker and longer rod-shaped bone (arrowhead in Fig. 1D,D′ and pseudo-colored red in 1F; arrowheads in 1F) and was capped in cartilage (arrows in Fig. 1B,D,D′), similar to mandibular processes (Fig. 1B,D). The jugal bone was present as a separate element or fused to the new maxillary bone (pseudo-colored blue in Fig. 1F).

Fig. 1.

Analysis of craniofacial defects in E18.5 Six1−/− mouse embryos. (A-D′) Alizarin Red (bone) and Alcian Blue (cartilage) staining of control (left column) and Six1−/− (right column) embryos. Representative embryos are shown in lateral (A,B) and ventral (C,C′,D,D′) views. In Six1−/− embryos, the maxilla (mx) extends posteriorly as a rod-shaped bone (arrowhead) that is capped in cartilage (arrow) (B,D,D′). Control, n=10; Six1−/−, n=8. (E,F) Micro-CT scans of control (E) and Six1−/− (F) embryos. The maxilla (red), jugal (blue) and zygomatic process of the squamosal (green) bones are pseudo-colored. Arrowheads denote elongated bone. (G,H) Hematoxylin and Eosin-stained frontal sections through the TMJ. In Six1−/− embryos, the end of the elongated maxillary bone (asterisk) resides within the mandibular fossa (f) and is covered in cartilage. In addition, the articular disk (d) bifurcates to also contact the elongate bone (arrow in H). Control, n=4; Six1−/−, n=4. Scale bars: 100 μm. cp, condylar process; h, hyoid; j, jugal bone; lp, lateral pterygoid muscle; md, mandible; n, nasal bone; oc, otic capsule; sq, squamosal bone; ty, tympanic ring.

Fig. 1.

Analysis of craniofacial defects in E18.5 Six1−/− mouse embryos. (A-D′) Alizarin Red (bone) and Alcian Blue (cartilage) staining of control (left column) and Six1−/− (right column) embryos. Representative embryos are shown in lateral (A,B) and ventral (C,C′,D,D′) views. In Six1−/− embryos, the maxilla (mx) extends posteriorly as a rod-shaped bone (arrowhead) that is capped in cartilage (arrow) (B,D,D′). Control, n=10; Six1−/−, n=8. (E,F) Micro-CT scans of control (E) and Six1−/− (F) embryos. The maxilla (red), jugal (blue) and zygomatic process of the squamosal (green) bones are pseudo-colored. Arrowheads denote elongated bone. (G,H) Hematoxylin and Eosin-stained frontal sections through the TMJ. In Six1−/− embryos, the end of the elongated maxillary bone (asterisk) resides within the mandibular fossa (f) and is covered in cartilage. In addition, the articular disk (d) bifurcates to also contact the elongate bone (arrow in H). Control, n=4; Six1−/−, n=4. Scale bars: 100 μm. cp, condylar process; h, hyoid; j, jugal bone; lp, lateral pterygoid muscle; md, mandible; n, nasal bone; oc, otic capsule; sq, squamosal bone; ty, tympanic ring.

The condylar process of the mandible rests in the mandibular fossa of the squamosal bone, with these two structures comprising the temporomandibular joint (TMJ). The articular disk rests between these two bones (Hanken and Hall, 1993). In Six1−/− embryos, the anterior end of the elongated bone ended at the mandibular fossa. In addition, whereas the articular disk overlaid the condylar process of the mandible in both control (Fig. 1G) and Six1−/− (Fig. 1H) embryos, the disk in mutant embryos bifurcated to also extend over the posterior end of the elongated bone (Fig. 1H, arrow). These findings suggest that loss of SIX1 leads to a partial transformation of the proximal maxilla into a structure, the posterior end of which resembles the posterior mandible. Although defects in the maxilla were not reported in the other Six1 mutant strain (Laclef et al., 2003), different targeting strategies and mouse genetic background could influence phenotypic penetrance.

Loss of Six1 disrupts expression of maxillary patterning genes

Misexpression of Edn1 in NCCs within the maxillary prominence results in complete homeotic transformation of the maxilla into a mandible (Sato et al., 2008a; Tavares and Clouthier, 2015). One method we have previously used to achieve this misexpression was through conditional activation of Edn1 expression in NCCs (Tavares and Clouthier, 2015) (Fig. 2C,C′) (see Materials and Methods). Interestingly, the bifurcation of the posterior end of the novel maxillary bone in Six1−/− embryos (Fig. 2B-B″) resembled the posterior end of the duplicated mandible in CBA-Edn1;Wnt1-Cre embryos (Fig. 2C-C″). For reference, the jugal bone was not bifurcated in control embryos (Fig. 2A-A″). To determine whether the maxillary changes reflected earlier changes in NCC patterning (Clouthier et al., 2010, 2013; Medeiros and Crump, 2012), we examined gene expression in E10.5 control and Six1−/− embryos. As a positive control for expanded EDNRA signaling, we also examined gene expression in CBA-Edn1;Wnt1-Cre embryos. Expression of Dlx3 (Fig. 2D-F), an intermediate domain marker (Tavares et al., 2012; Walker et al., 2006), and Dlx5 (Fig. 2G-I), a dorsal/intermediate marker (Talbot et al., 2010; Tavares et al., 2012), expanded into both the rostral mandibular arch and maxillary prominence in both Six1−/− (Fig. 2E,H) and CBA-Edn1;Wnt1-Cre (Fig. 2F,I) (Tavares and Clouthier, 2015) embryos. By contrast, aberrant Hand2 expression was not observed in the dorsal mandibular arch or maxillary prominence (Fig. 2K), although expression was decreased in the ventral mandibular arch (Fig. 2K and data not shown). In CBA-Edn1;Wnt1-Cre embryos, expanded Hand2 expression was observed in the maxillary prominence (Fig. 2L).

Fig. 2.

Similarities in facial structures and earlier gene expression patterns between Six1−/− and CBA-Edn1;Wnt1-Cre embryos. (A-C′′) Ventral view of representative E18.5 embryo skulls (A-C) and dissected views of the control maxilla (A′,A″), elongated bone in Six1−/− embryos (arrowhead in B′;B″) and duplicated mandible in CBA-Edn1;Wnt1-Cre embryos (C′,C″). Rotation of the dissected bones reveals two processes capped in cartilage (arrows) on the proximal end of the elongated bone (B″) and duplicated mandible (C″). Control, n=10; Six1−/−, n=8; CBA-Edn1;Wnt1-Cre, n=7. (D-R) Whole-mount ISH analysis of gene expression in E10.5 control (D,G,J,M,P), Six1−/− (E,H,K,N,Q) and CBA-Edn1;Wnt1-Cre (F,I,L,O,R) embryos. Arrows indicate regions of expanded gene expression; arrowheads indicate regions of reduced gene expression. (S) Quantification of gene expression in the proximal mandibular arch of control and Six1−/− embryos. n=3; error bars represent s.e.m.; two-tailed t-test, **P<0.01, ***P<0.001. 1, first pharyngeal arch; 2, second pharyngeal arch; j, jugal; lo, lamina obturans; md*, duplicated mandible; mx, maxilla; sq, squamosal bone. The images shown are representative of three embryos of each genotype.

Fig. 2.

Similarities in facial structures and earlier gene expression patterns between Six1−/− and CBA-Edn1;Wnt1-Cre embryos. (A-C′′) Ventral view of representative E18.5 embryo skulls (A-C) and dissected views of the control maxilla (A′,A″), elongated bone in Six1−/− embryos (arrowhead in B′;B″) and duplicated mandible in CBA-Edn1;Wnt1-Cre embryos (C′,C″). Rotation of the dissected bones reveals two processes capped in cartilage (arrows) on the proximal end of the elongated bone (B″) and duplicated mandible (C″). Control, n=10; Six1−/−, n=8; CBA-Edn1;Wnt1-Cre, n=7. (D-R) Whole-mount ISH analysis of gene expression in E10.5 control (D,G,J,M,P), Six1−/− (E,H,K,N,Q) and CBA-Edn1;Wnt1-Cre (F,I,L,O,R) embryos. Arrows indicate regions of expanded gene expression; arrowheads indicate regions of reduced gene expression. (S) Quantification of gene expression in the proximal mandibular arch of control and Six1−/− embryos. n=3; error bars represent s.e.m.; two-tailed t-test, **P<0.01, ***P<0.001. 1, first pharyngeal arch; 2, second pharyngeal arch; j, jugal; lo, lamina obturans; md*, duplicated mandible; mx, maxilla; sq, squamosal bone. The images shown are representative of three embryos of each genotype.

Loss of EDNRA signaling also results in ventral expansion of Dlx2 and Twist1 (Ruest et al., 2004). In both Six1−/− and CBA-Edn1;Wnt1-Cre embryos, the expression of Dlx2 (Fig. 2M-O) and Twist1 (Fig. 2P-R) was reduced in the maxillary prominence of Six1−/− embryos (Fig. 2N,Q), although the reduction was less severe than that observed in CBA-Edn1;Wnt1-Cre embryos (Fig. 2O,R). To quantify these changes, we used quantitative real-time PCR (qRT-PCR) analysis to assay the levels of gene expression in the dorsal mandibular arch of E10.5 embryos. Compared with control embryos, the expression of all five genes was significantly upregulated in Six1−/− embryos (Fig. 2S), indicating an upregulation of ventral gene expression in this region. It is important to note that the graph only depicts the relative expression of each gene between genotypes and does not reflect the level of expression between genes. Although Hand2 expression was elevated, as indicated by qRT-PCR, the relative expression was 50% lower than that of the other genes (data not shown). Together, these results illustrate that the Six1−/− phenotype is likely to result from similar changes in early NCC patterning as observed after Edn1 overexpression.

EDNRA and SIX1 genetically interact during NCC patterning

Owing to similarities in both phenotypic and gene expression changes in Six1−/− and CBA-Edn1;Wnt1-Cre embryos, we analyzed the expression of Six1 and its co-factor Eya1 in E10.5 CBA-Edn1;Wnt1-Cre and Ednra−/− embryos. In control embryos, Six1 (Fig. 3A) and Eya1 (Fig. 3E) were both expressed in the maxillary prominence, with Six1 expression also prominent in the dorsal mandibular arch adjacent to the first pharyngeal pouch (arrowhead in Fig. 3A). Expanded EDNRA signaling in CBA-Edn1;Wnt1-Cre embryos disrupted the expression of both Six1 and Eya1 in the maxillary prominence (Fig. 3B,F), with expression in the dorsal mandibular arch also decreased (arrowhead in Fig. 3B). By contrast, expression of both Six1 and Eya1 appeared to be expanded in the ventral mandibular arch in Ednra−/− embryos (Fig. 3C,G), with dorsal arch expression of Six1 appearing similar to that in controls (arrowhead in Fig. 1C). We also examined Six1/Eya1 expression in E10.5 Jag1fl/fl;Wnt1-Cre embryos, in which Jag1 expression was conditionally deleted in NCCs. For both genes, expression did not appear dramatically changed (Fig. 3D,H); this included the Six1 expression domain in the dorsal mandibular arch (arrowhead in Fig. 3D). These results suggest that Six1/Eya1 expression in the dorsal mandibular arch is not repressed by Jagged-Notch signaling during early arch patterning. We therefore examined Edn1 expression in E9.5 control and Six1−/− embryos, comparing it with Six1 expression. At this age, Six1 expression in control embryos was observed in the pharyngeal pouch endoderm and in the arch mesenchyme (Fig. 3I,I′). This inversely corresponded to areas of highest Edn1 expression in control embryos, observed in the arch endoderm (Fig. 3J,J′); expression in pouch endoderm and arch ectoderm was weaker (Fig. 3J′). In E9.5 Six1−/− embryos, ectodermal and endodermal Edn1 expression was increased in arches 1 and 2 (Fig. 3K,K′). To better quantify this change, qRT-PCR was used to assay the level of Edn1 expression in the dorsal mandibular arch of E10.5 embryos. Compared with control embryos, Edn1 expression was elevated ∼2-fold in Six1−/− embryos (Fig. 3L). Interestingly, the expression of Ednra was ∼50% lower, indicating that receptor levels might compensate for changes in ligand levels.

Fig. 3.

Genetic interaction between Six1 and Ednra. (A-H) Whole-mount ISH analysis of Six1 and Eya1 expression in E10.5 control (A,E), CBA-Edn1;Wnt1-Cre (B,F), Ednra−/− (C,G) and Jag1fl/fl;Wnt1-Cre (D,H) embryos. The Six1 expression domain in the dorsal mandibular arch is marked by yellow arrowheads. (I-K′) Analysis of E9.5 embryos following whole-mount (I,J,K) or sectional (I′,J′,K′) ISH. The plane of section for I′,J′,K′ is depicted by the dashed yellow lines on I,J,K, respectively. Six1 expression in control embryos is strongest in the pharyngeal pouch endoderm (en) of arches 1 and 2 (demarcated by arrows) (I,I′). Edn1 is expressed along the pharyngeal endoderm of control embryos, but is weaker in pharyngeal pouch endoderm (demarcated by arrows) (J,J′). Edn1 expression is enhanced in the endoderm and ectoderm (ec) of Six1−/−embryos (K,K′). The images shown are representative of three embryos of each genotype. (L) Quantification of Edn1 and Ednra expression in the proximal mandibular arch of E10.5 control and Six1−/− embryos. n=3; error bars represent s.e.m.; two-tailed t-test, ***P<0.001. (M-O′) Ventral view of E18.5 skulls from a Six1;Ednra allelic series shown in ventral (M,N,O) and dissected ventral (M′,N′,O′) views. The elongated bone observed in Six1−/−;Ednra+/+ embryos is denoted by an arrowhead. Six1+/+;Ednra+/+, n=10; Six1−/−;Ednra+/+, n=8; Six1−/−;Ednra+/−, n=7. md1, mandibular arch 1; mx1, maxillary prominence of arch 1; 1, arch 1; 2, arch 2; 3, arch 3; j, jugal; md, mandible; mx, maxilla; pl, palatine bone; ppe1, pharyngeal pouch endoderm; sq, squamosal bone.

Fig. 3.

Genetic interaction between Six1 and Ednra. (A-H) Whole-mount ISH analysis of Six1 and Eya1 expression in E10.5 control (A,E), CBA-Edn1;Wnt1-Cre (B,F), Ednra−/− (C,G) and Jag1fl/fl;Wnt1-Cre (D,H) embryos. The Six1 expression domain in the dorsal mandibular arch is marked by yellow arrowheads. (I-K′) Analysis of E9.5 embryos following whole-mount (I,J,K) or sectional (I′,J′,K′) ISH. The plane of section for I′,J′,K′ is depicted by the dashed yellow lines on I,J,K, respectively. Six1 expression in control embryos is strongest in the pharyngeal pouch endoderm (en) of arches 1 and 2 (demarcated by arrows) (I,I′). Edn1 is expressed along the pharyngeal endoderm of control embryos, but is weaker in pharyngeal pouch endoderm (demarcated by arrows) (J,J′). Edn1 expression is enhanced in the endoderm and ectoderm (ec) of Six1−/−embryos (K,K′). The images shown are representative of three embryos of each genotype. (L) Quantification of Edn1 and Ednra expression in the proximal mandibular arch of E10.5 control and Six1−/− embryos. n=3; error bars represent s.e.m.; two-tailed t-test, ***P<0.001. (M-O′) Ventral view of E18.5 skulls from a Six1;Ednra allelic series shown in ventral (M,N,O) and dissected ventral (M′,N′,O′) views. The elongated bone observed in Six1−/−;Ednra+/+ embryos is denoted by an arrowhead. Six1+/+;Ednra+/+, n=10; Six1−/−;Ednra+/+, n=8; Six1−/−;Ednra+/−, n=7. md1, mandibular arch 1; mx1, maxillary prominence of arch 1; 1, arch 1; 2, arch 2; 3, arch 3; j, jugal; md, mandible; mx, maxilla; pl, palatine bone; ppe1, pharyngeal pouch endoderm; sq, squamosal bone.

These reciprocal changes in gene expression suggest a genetic interaction between SIX1 and EDNRA. To test whether such an interaction exists, we examined whether reducing Ednra gene dosage would rescue the Six1−/− maxillary phenotype (Fig. 3N,N′). Removing one Ednra allele on the Six1−/− background completely rescued maxillary development in 42% (3/7) of Six1−/−;Ednra+/− embryos (Fig. 3O,O′), with the maxilla resembling the maxilla of control embryos (Fig. 3M,M′). In the remaining Six1−/−;Ednra+/− embryos, no change in the Six1−/− phenotype was observed, arguing the existence of modifier or background effects (data not shown). The maxillary defect was observed in 11/12 Six1−/− embryos examined (1/12 embryos had the defect on only one side), arguing that variable penetrance was not a major factor in the degree of phenotypic rescue. Removing an additional Ednra allele resulted in a rescue percentage of 100% (2/2) in Six1−/−;Ednra−/− embryos, although the significant defects in lower jaw development complicated analyses of these embryos (data not shown).

SIX1 is required for proper Jagged-Notch signaling

Jagged-Notch signaling patterns dorsal NCCs in the zebrafish arches, in part by repressing Ednra signaling (Zuniga et al., 2010). Because Six1 expression appeared normal in Jag1fl/fl;Wnt1-Cre mouse embryos, we examined whether a relationship exists between SIX1 and Jagged-Notch signaling that influences EDNRA signaling by analyzing the expression patterns of Jag1, Notch1, Notch2 and Hey1 in E9.5 and E10.5 control and Six1−/− embryos. Jag1 expression in control embryos was first detected at E9.5 in the endoderm of pharyngeal pouches 1 and 2 (Fig. 4A,A′); expression was similar in E10.5 control embryos (Fig. 4C), although expression extended into the arch mesenchyme adjacent to the pharyngeal endoderm of arch 1 (Fig. 4C′). In Six1−/− embryos, expression of Jag1 was decreased in pouch endoderm at E9.5 (Fig. 4B,B′) and in arch 1 mesenchyme at E10.5 (Fig. 4D,D′). Notch1 was weakly expressed in the first arch at both time points in control (Fig. 4E,G) and Six1−/− (Fig. 4F,H) embryos. By contrast, Notch2 expression in E9.5 control (Fig. 4I) and Six1−/− (Fig. 4J) embryos was detected in the first arch, with expression increased by E10.5 in the mesenchyme of the mandibular and maxillary regions of the first arch in both genotypes (Fig. 4K,L). Finally, expression of the Jagged-Notch mediator Hey1 was observed in a small area of the dorsal (hinge) mandibular arch mesenchyme that partially overlapped with the Jag1 expression domain at E9.5 (Fig. 4M,M′), and this Hey1 expression domain expanded at E10.5 (Fig. 4O,O′). Expression was decreased in Six1−/− embryos at E9.5 (Fig. 4N,N′) and E10.5 (Fig. 4P,P′). Quantitation of the Hey1 expression area in E10.5 Six1−/− embryos revealed a ∼70% decrease compared with the expression area in control embryos (Fig. 4Q).

Fig. 4.

SIX1 is required for proper Jag1 expression in the hinge region. (A-P′) Whole-mount ISH analysis of Jag1 (A-D), Notch1 (E-H), Notch2 (I-L) and Hey1 (M-P) in E9.5 (A,B,E,F,I,J,M,N) and E10.5 (C,D,G,H,K,L,O,P) embryos. (A′-D′) Section ISH of Jag1 expression; the plane of section is depicted by the dashed yellow lines in A-D. 1, arch 1; 2, arch 2. (M′-P′) Magnification of the boxed area shown in M-P. Blue lines demarcate the pharyngeal endoderm; dashed yellow lines demarcate the extent of Hey1 expression in the arch mesenchyme. The images shown are representative of four embryos of each genotype subjected to ISH. (Q) Quantification of the Hey1 expression domain in control and Six1−/− embryos shown in O-P′. n=4. (R,S) Quantification of mRNA levels in the dorsal mandibular arch of E9.5 (R) and E10.5 (S) control and Six1−/− embryos. n=3. Error bars represent s.e.m.; two-tailed t-test, **P<0.01, ***P<0.001, n.s., not significant.

Fig. 4.

SIX1 is required for proper Jag1 expression in the hinge region. (A-P′) Whole-mount ISH analysis of Jag1 (A-D), Notch1 (E-H), Notch2 (I-L) and Hey1 (M-P) in E9.5 (A,B,E,F,I,J,M,N) and E10.5 (C,D,G,H,K,L,O,P) embryos. (A′-D′) Section ISH of Jag1 expression; the plane of section is depicted by the dashed yellow lines in A-D. 1, arch 1; 2, arch 2. (M′-P′) Magnification of the boxed area shown in M-P. Blue lines demarcate the pharyngeal endoderm; dashed yellow lines demarcate the extent of Hey1 expression in the arch mesenchyme. The images shown are representative of four embryos of each genotype subjected to ISH. (Q) Quantification of the Hey1 expression domain in control and Six1−/− embryos shown in O-P′. n=4. (R,S) Quantification of mRNA levels in the dorsal mandibular arch of E9.5 (R) and E10.5 (S) control and Six1−/− embryos. n=3. Error bars represent s.e.m.; two-tailed t-test, **P<0.01, ***P<0.001, n.s., not significant.

We also examined gene expression using qRT-PCR with RNA isolated from the dorsal half of the mandibular arch of E9.5 (Fig. 4R) and E10.5 (Fig. 4S) control and Six1−/− embryos. At E9.5, expression of both Jag1 and Hey1 had decreased 30-40%, whereas expression of Notch1 and Notch2 did not significantly differ between genotypes. These findings mirrored those observed in in situ hybridization (ISH) analysis. At E10.5, Jag1 expression had decreased by ∼25%, whereas expression of Notch1, Notch2 and Hey1 had decreased 40-50% (Fig. 4S). Although the Jag1 and Hey1 results match those observed in ISH, the decrease in Notch1 and Notch2 suggests that expression changes are present in the dorsal mandibular arch but are below the sensitivity of ISH. Overall, these findings indicate that loss of SIX1 adversely affects Jagged-Notch signaling in the dorsal mandibular arch.

To further investigate the relationship between SIX1 and Jagged-Notch signaling, a Six1 expression construct was transfected into the mouse NCC line O9-1, with expression levels of Jagged-Notch and EDNRA signaling components subsequently assessed by qRT-PCR (Fig. 5A). Overexpression of Six1 resulted in a significant increase in Jag1 and Notch2 mRNA levels, whereas changes in Notch1 expression were not statistically significant. These findings support the idea that SIX1 regulates Jagged-Notch signaling. Similarly, expression of Six1 led to significant downregulation of Dlx3 and Dlx5 expression. In addition, Edn1 expression was also downregulated whereas expression of Ednra was upregulated (Fig. 5A). This pattern is opposite to that observed in Six1−/− embryos (Fig. 2E,H and Fig. 3K,L) and again indicates that SIX1 functions in part through regulating EDNRA signaling, most likely by regulating Edn1 expression.

Fig. 5.

SIX1 regulates Edn1 and Jag1 expression independently. (A) qRT-PCR analysis of gene expression in O9-1 cells following transfection of either a control vector or a Six1 expression vector. (B) qRT-PCR analysis of gene expression changes in O9-1 cells following transfection with a Six1 expression vector, treatment with EDN1, or both transfection with a Six1 expression vector and treatment with EDN1. n=3; error bars represent s.e.m.; two-tailed t-test *P<0.05, **P<0.01, ***P<0.001.

Fig. 5.

SIX1 regulates Edn1 and Jag1 expression independently. (A) qRT-PCR analysis of gene expression in O9-1 cells following transfection of either a control vector or a Six1 expression vector. (B) qRT-PCR analysis of gene expression changes in O9-1 cells following transfection with a Six1 expression vector, treatment with EDN1, or both transfection with a Six1 expression vector and treatment with EDN1. n=3; error bars represent s.e.m.; two-tailed t-test *P<0.05, **P<0.01, ***P<0.001.

While loss of SIX1 resulted in downregulation of Jag1 expression and upregulation of Edn1 expression, it is possible that decreased Jag1 expression was primarily caused by elevated EDNRA signaling as observed during normal intermediate/ventral mandibular arch patterning (Zuniga et al., 2010). To test this possibility, we examined whether SIX1 could induce Jag1 expression in the presence of EDN1 (Fig. 5B). We found that the addition of EDN1 to Six1-transfected cells prevented upregulation of Jag1 expression (Fig. 5B), suggesting that a key component of SIX1 induction of Jag1 expression is the repression of Edn1 expression.

Loss of SIX1 affects gene expression in the hinge region

Because loss of Six1 disrupts normal development of the posterior maxilla, we examined the expression of genes previously identified as playing a role in the development of this region. Zygomatic arch development requires PRRX1 and PRRX2 (Lu et al., 1999; ten Berge et al., 1998). Similarly, Prrx1a/b has recently been shown to work in parallel with Jagged-Notch signaling to pattern the dorsal region of zebrafish pharyngeal arches (Barske et al., 2016). This function involves Barx1, a protein that is required for zebrafish jaw joint formation (Nichols et al., 2013). In E10.5 control mouse embryos, Prrx1, Prrx2 and Barx1 were expressed in a similar fashion in the maxillary prominence and in the ventral and intermediate domains of the mandibular arch (Fig. 6A,D,G). Expression of each was excluded from the dorsal mandibular arch domain. In Six1−/− embryos, the expression of all three genes expanded into the dorsal region (Fig. 6B,E,H). In CBA-Edn1;Wnt1-Cre embryos, expression of Prrx1 (Fig. 6C) and Barx1 (Fig. 6I) had also expanded into the dorsal domain, even though there was a partial downregulation in the maxillary prominence. By contrast, Prrx2 (Fig. 6F) was downregulated in the maxillary prominence, suggesting that its upregulation in Six1−/− embryos is likely to be EDNRA independent. We also analyzed Pou3f3, the expression of which normally spans the maxillary prominence and dorsal mandibular arch (Fig. 6J). Expression of this gene was completely downregulated in CBA-Edn1;Wnt1-Cre embryos (Fig. 6L) and partially downregulated in Six1−/− embryos (Fig. 6K). To quantify these changes, we performed qRT-PCR using dorsal mandibular arch RNA from control and Six1−/− embryos. Supporting the ISH results, expression of Prrx1 and Prrx2 in Six1−/− embryos was elevated 3-fold, with Barx1 expression elevated 5-fold (Fig. 6M). Pou3f3 expression, appearing downregulated in ISH, was 50% lower in qRT-PCR (Fig. 6M). Taken together, these results show that loss of Six1 causes changes in the patterning of NCCs in the hinge/dorsal mandibular arch domain that are similar to, but less severe than, changes observed in CBA-Edn1;Wnt1-Cre animals.

Fig. 6.

Expression of early pharyngeal arch patterning genes. (A-L) Whole-mount ISH analysis of Prrx1, Prrx2, Barx1 and Pou3f3 in E10.5 control (A,D,G,J), Six1−/− (B,E,H,K) and CBA-Edn1;Wnt1-Cre (C,F,I,L) embryos. Arrows denote regions of expanded gene expression; arrowheads denote regions of reduced gene expression. (M) Quantification of Prrx1, Prrx2, Barx1 and Pou3f3 expression in the dorsal mandibular arch of E10.5 control and Six1−/− embryos. n=3; error bars represent s.e.m.; two-tailed t-test, ***P<0.001. md1, mandibular portion of arch 1; mx1, maxillary prominence of arch 1; 2, second pharyngeal arch.

Fig. 6.

Expression of early pharyngeal arch patterning genes. (A-L) Whole-mount ISH analysis of Prrx1, Prrx2, Barx1 and Pou3f3 in E10.5 control (A,D,G,J), Six1−/− (B,E,H,K) and CBA-Edn1;Wnt1-Cre (C,F,I,L) embryos. Arrows denote regions of expanded gene expression; arrowheads denote regions of reduced gene expression. (M) Quantification of Prrx1, Prrx2, Barx1 and Pou3f3 expression in the dorsal mandibular arch of E10.5 control and Six1−/− embryos. n=3; error bars represent s.e.m.; two-tailed t-test, ***P<0.001. md1, mandibular portion of arch 1; mx1, maxillary prominence of arch 1; 2, second pharyngeal arch.

As mentioned above, Prrx1a/b and Barx1 in zebrafish work in parallel with Jagged-Notch and Ednra signaling to establish the timing of chondrogenesis in the first and second arches, thus establishing the hinge region (Barske et al., 2016). Because expression of both Prrx1 and Prrx2 expands into the dorsal domain in the absence of SIX1, we examined whether this expansion correlated with precocious ossification in this domain. In E12.5 control embryos, Osterix (Osx), a transcription factor associated with early osteogenesis (Baek et al., 2013; Nakashima et al., 2002), was weakly expressed in the posterior maxillary region (Fig. 7A). By contrast, loss of SIX1 resulted in an apparent expansion of Osx expression in this region (Fig. 7B). Since this domain is likely to give rise to the novel maxillary bone, these findings are consistent with an association between the expansion of these genes and precocious ossification.

Fig. 7.

Upregulation of Osterix expression in Six1−/− embryos. Whole-mount ISH analysis of Osterix (Osx) expression in E12.5 control (A) and Six1−/− (B) embryos. Osx expression is more pronounced in the maxilla of Six1−/− embryos (B) than in control embryos (A) (arrows), whereas expression in the mandible appears similar between the two genotypes (arrowheads). The images shown are representative of three embryos of each genotype.

Fig. 7.

Upregulation of Osterix expression in Six1−/− embryos. Whole-mount ISH analysis of Osterix (Osx) expression in E12.5 control (A) and Six1−/− (B) embryos. Osx expression is more pronounced in the maxilla of Six1−/− embryos (B) than in control embryos (A) (arrows), whereas expression in the mandible appears similar between the two genotypes (arrowheads). The images shown are representative of three embryos of each genotype.

We have shown here that SIX1 normally represses Edn1 expression in the endoderm lining of both mandibular arch 1 and the first pharyngeal pouch, thus preventing EDNRA signaling in the adjacent NCC-derived mesenchyme of the dorsal mandibular arch, a region referred to as the hinge in the ‘hinge and caps’ model of pharyngeal arch development (Depew and Simpson, 2006; Depew et al., 2005; Tavares et al., 2012). At the same time, SIX1 induces Jagged-Notch signaling in the dorsal arch mesenchyme. This places SIX1 temporally upstream of both EDN1 and Jagged-Notch and explains why dorsal ventral patterning is maintained in arch 1 in the absence of Jagged-Notch signaling. Based on the Six1−/− phenotype, SIX1 action is essential to limit bone formation in the posterior maxilla during jaw morphogenesis.

Loss of Six1 causes a partial homeotic transformation of the maxilla

Aberrant EDNRA signaling throughout the first arch following misexpression of Edn1 in cranial NCCs results in homeotic transformation of the maxilla into a mandible-like structure (Sato et al., 2008a; Tavares and Clouthier, 2015). Although a complete homeotic transformation is not observed in Six1−/− embryos, early upregulation of some EDNRA signaling mediators in the dorsal mandibular arch is accompanied by later aberrant expansion of the maxilla posteriorly as a large rod-shaped bone, with its posterior end covered in cartilage. Such a structure is suggestive of a duplication of the proximal portion of the mandible, a hypothesis strengthened by the finding that removing one allele of Ednra in a Six1−/− background rescues the craniofacial phenotype in Six1−/− embryos. The absence of a complete duplication could be due to the fact that the Hand2 upregulation observed by qRT-PCR in these embryos is weak compared with that observed in CBA-Edn1;Wnt1-Cre embryos. Overexpression of Hand2 in NCCs, either through expression of Hand2 from the Ednra locus (EdnraHand2) (Sato et al., 2008b) or by overexpressing Hand2 in NCCs (Hand2NC) (Funato et al., 2016), produces a mandible duplication phenotype very similar to that observed following Edn1 expression in cranial NCCs (Sato et al., 2008a; Tavares and Clouthier, 2015). These findings indicate that ectopic expression of Hand2 in the maxillary prominence is required and sufficient for complete transformation of the maxilla into a mandible. A similar situation is observed in jag1b mutant zebrafish, in which loss of Jagged-Notch signaling does not result in expanded hand2 expression in the first arch, and dorsal-to-ventral transformation of cartilage elements is not observed (Zuniga et al., 2010). Further studies to understand the mechanism behind repression of Hand2 in the dorsal arch are required to clarify this point.

SIX1 regulates Edn1 expression in the dorsal mandibular arch

Jagged-Notch signaling during zebrafish embryogenesis is believed to establish a dorsal arch domain in at least the first and second pharyngeal arches, in part through restricting Ednra signaling to the intermediate domain through mechanisms that function downstream of actual receptor signaling (Alexander et al., 2011; Barske et al., 2016; Zuniga et al., 2011, 2010). However, as discussed above, dorsal-to-ventral transformation of dorsal first arch cartilage derivatives are not observed in jag1b mutants, which has led to the hypothesis that Jagged-Notch signaling has a more extensive role in establishing the identity of second arch NCCs (Barske et al., 2016; Zuniga et al., 2010). Defects in arch 1 structures are also not widely observed in mouse embryos in which Jagged-Notch signaling is disrupted. Although conditional loss of Jag1 in mouse NCCs (Jag1fl/fl;Wnt1-Cre embryos) resulted in shortening of the maxilla, a new posterior extension suggestive of a transformation was not observed (Humphreys et al., 2012). Similarly, the maxilla in embryos with an NCC-specific deletion of Rbpj (a molecule responsible for activation of the canonical Notch pathway) appears normal (Mead and Yutzey, 2012). In addition, no gross facial abnormalities were reported in mice containing a neural crest-specific deletion of Pofut1, the gene encoding protein O-fucosyltransferase 1 (POFUT1), whose modification of Notch is required for Notch signaling (Okamura and Saga, 2008), although detailed skeletal analysis was not presented.

Our current results provide an explanation for these findings: in Jagged-Notch pathway mutants, Six1 expression in the pharyngeal pouch endoderm is unaffected. Therefore, dorsal Edn1 expression is repressed, preventing both EDNRA signaling in the dorsal arch and subsequent arch repatterning (Fig. 8). Negative regulation of Edn1 expression by SIX1 is crucial to maintain dorsal NCC identity; although EDNRA is found in all cranial NCCs (Clouthier et al., 1998), EDN1 is normally derived from the intermediate and ventral ectoderm of the mandibular arch (Clouthier et al., 1998; Yanagisawa et al., 1998; Miller et al., 2000) and has a short half-life and limited diffusion potential (Yanagisawa, 1994). This means that any change in EDNRA signaling in the dorsal mandibular arch would likely require an EDN1 source very close to the dorsal domain. In addition, our in vitro data show that EDNRA signaling can block SIX1-induced Jag1 expression. Thus, these results indicate that while SIX1 signaling is crucial for NCC identity in the dorsal mandibular arch, Jagged-Notch signaling by itself is not, at least in mouse. Rather, SIX1-Jagged-Notch signaling ensures that the dorsal mandibular arch domain develops in an EDNRA-independent manner, thus demarcating the mammalian hinge region. This is clearly not the only function of Jagged-Notch signaling, as recent findings illustrate that Jagged-Notch signaling in zebrafish is crucial for later establishment of the sites and timing of arch chondrogenesis (Barske et al., 2016). The role of SIX1 in repressing Edn1 expression also explains why inactivating one copy of Ednra in Six1−/− embryos rescued the phenotype in almost 50% of Six1−/−;Ednra+/− embryos. Allelic reduction of Edn1 gene dosage (Edn1+/−) results in a 40% decrease in the expression of both Dlx5 and Dlx6 (Vieux-Rochas et al., 2010), indicating that the expression of these two genes is exquisitely sensitive to the level of EDNRA signaling and that decreasing Ednra dosage in Six1−/−;Ednra+/− embryos is likely to be sufficient to reduce their aberrant expression in the dorsal domain and thus reestablish normal developmental control in this region.

Fig. 8.

Model of dorsal (hinge) domain patterning by SIX1. Normal dorsal arch gene expression (represented by Jag1) is shown in blue; normal ventral gene expression induced by EDNRA signaling is shown in red. Loss of SIX1 leads to upregulation of EDN1 in the pharyngeal endoderm, which induces EDNRA signaling in the hinge region (shown in green). Subsequent repression of Jag1 expression by EDNRA signaling compounds loss of SIX1-induced Jag1 expression. Loss of Jag1 expression also leads to aberrant expression of genes involved in osteogenesis. While normal EDNRA signaling in the ventral arch and aberrant dorsal arch EDNRA signaling induce similar genes, the source of EDN1 is different for each region.

Fig. 8.

Model of dorsal (hinge) domain patterning by SIX1. Normal dorsal arch gene expression (represented by Jag1) is shown in blue; normal ventral gene expression induced by EDNRA signaling is shown in red. Loss of SIX1 leads to upregulation of EDN1 in the pharyngeal endoderm, which induces EDNRA signaling in the hinge region (shown in green). Subsequent repression of Jag1 expression by EDNRA signaling compounds loss of SIX1-induced Jag1 expression. Loss of Jag1 expression also leads to aberrant expression of genes involved in osteogenesis. While normal EDNRA signaling in the ventral arch and aberrant dorsal arch EDNRA signaling induce similar genes, the source of EDN1 is different for each region.

Potential functions of the SIX1-Jagged-Notch signaling axis in the hinge region

The ‘hinge and caps’ model has been proposed to explain how signals from the ventral arches influence development of more dorsal regions (Britanova et al., 2006; Depew and Compagnucci, 2008; Depew and Simpson, 2006; Depew et al., 2005; Tavares et al., 2012). In mammals, the caps of the first arch encompass the ventral aspects of the mandibular arch and the region surrounding the lambdoidal junction in the maxillary prominence, while the hinge region includes the dorsal portions of both arches and some of the intermediate domain of the mandibular arch (Depew et al., 2005). It is from this region that the TMJ arises (Kontges and Lumsden, 1996). Here we have shown that while the condylar process of the mandible and the mandibular fossa are normal in Six1−/− embryos, the new elongated maxillary bone extends into the mandibular fossa, with the articular disk bifurcating to encompass both the condylar process and the proximal end of the elongated bone. Although bifid mandibular condyle in humans has been reported (Khojastepour et al., 2015; Cho and Jung, 2013) and is observed in Foxc1−/− mouse embryos (Inman et al., 2013), to our knowledge this is the first example of a maxillary bone inserting into the TMJ and associating with the articular disk.

In our current model, SIX1 establishes a dorsal mandibular arch domain by inhibiting endodermal Edn1 expression, which prevents expansion of Dlx gene expression (Fig. 8). Jagged-Notch signaling induced by SIX1 contributes to this domain by repressing the expression of genes associated with ossification. Such a temporal buffer would thus allow controlled ossification in the maxilla without intrusion into the forming TMJ. This function of Jagged-Notch signaling appears to be conserved in the zebrafish arch, as dorsal expression of prrx1a/b and barx1 is repressed by Jagged-Notch signaling (Barske et al., 2016). However, there are also differences between species, as Prrx1 and Barx1 have overlapping expression domains in the mouse mandibular arch, whereas zebrafish barx1 expression is repressed by prrx1a/b (Barske et al., 2016). These differences might relate to different temporal functions, as many of the effects examined in our study occur during early NCC patterning, whereas the studies in zebrafish embryogenesis focused on later chondrogenesis (Barske et al., 2016). However, in mouse models lacking Prrx1 or both Prrx1 and Prrx2, intramembranous elements of the maxilla and TMJ do not form (Lu et al., 1999; ten Berge et al., 1998), suggesting that PRRX1/2 have roles in pattering the mammalian jaw apparatus.

While we have focused on SIX1 regulation of EDN1 and Jagged-Notch signaling during normal patterning of the dorsal mandibular arch, other molecules function in this region during facial morphogenesis, including FGFs, SHH, TBXs, TGFs and BMPs (reviewed by Chai and Maxson, 2006; Gou et al., 2015; Medeiros and Crump, 2012), with at least some interacting with SIX1 in this process. One example is FGF8, the expression of which in the dorsal mandibular arch is regulated by SIX1/EYA1 action (Guo et al., 2011), while SIX1 and EYA1 regulate GLI activators during development (Eisner et al., 2015). As Wnt signaling acting through R-spondin 2 (RSPO2) has been reported to work in an FGF8/EDN1 pathway during ventral mandibular arch patterning (Jin et al., 2011), it will be interesting to see how these pathways relate to the establishment or maintenance of the dorsal domain and/or TMJ development.

Mice

Generation and genotyping of Wnt1-Cre (Danielian et al., 1998), Six1+/− (Ozaki et al., 2004), CBA-Edn1 (Tavares and Clouthier, 2015), Jag1flox (Brooker et al., 2006) and Ednra+/− (Clouthier et al., 1998) mice have been previously described. The Six1+/− strain has been backcrossed onto the 129S6 background five generations. The Ednra+/− strain has been backcrossed onto the 129S6 background >12 generations. Briefly, CBA-Edn1 animals carry an Edn1 expression cassette that is separated from the CBA promoter by a strong stop cassette flanked by loxP sites. Breeding these mice with the Wnt1-Cre strain results in the removal of the stop cassette and thus expression of Edn1 in NCCs (Tavares and Clouthier, 2015). The sex of embryos was not determined. All experiments using mice were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Anschutz Medical Campus. Care of these mice followed local and national animal welfare law and guidelines. The University of Colorado Anschutz Medical Campus is certified by the Association for Assessment and Accreditation of Laboratory Animal Care.

Skeletal staining

Skeletal staining, analysis and photography of E18.5 embryos with Alizarin Red (bone) and Alcian Blue (cartilage) was performed as previously described (Ruest et al., 2004; Tavares et al., 2012). Four embryos of each genotype were examined for phenotype, with no variation observed between embryos of the same genotype.

Micro-CT and image processing

Embryos were imaged at the Small Animal Tomographic Analysis Facility at Seattle Children's Research Institute using a Skyscan model 1076 micro-computed tomograph (Bruker). Scans were collected at an isotropic resolution of 35.26 μm using the following parameters: no filter, 45 kV, 180 μA, 100 ms exposure, three frame averaging, 0.6° rotation step. All raw data were reconstructed using Nrecon V1.6.9.4 (Bruker) with consistent grayscale thresholding and the smoothing parameters set to 1. Reconstructed data were then rendered and assessed in 3D using Drishti V2.6 Volume Exploration software (Ajay Limaye, 2006; http://sf.anu.edu.au/Vizlab/drishti), again using consistent transfer function parameters to allow for comparison between specimens.

Whole-mount and section ISH

Whole-mount ISH analysis was performed as previously described (Clouthier et al., 1998). To detect bound probe, embryos were incubated in 4-nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) (Roche) except when detecting Six1 and Osx expression, where embryos were incubated with BM-Purple (Roche) (Tavares and Clouthier, 2015). Section ISH was performed as previously described (Vincentz et al., 2016). All ISH experiments were performed on a minimum of three mutant embryos. Three to four embryos of each genotype were examined for expression of each marker.

Quantification of the Hey1 expression domain

Determination of the Hey1 expression domain in control and Six1−/− embryos was performed in a randomized manner. E10.5 embryos were collected, with genotyping performed by a second individual. Four control and four Six1−/− embryos were provided for Hey1 ISH. Following completion of ISH, the area of Hey1 expression in the dorsal domain of the mandibular arch of all eight embryos was measured using ImageJ (NIH). Genotypes were then revealed and data compiled. Statistical analysis was conducted using Excel (Microsoft), with significance calculated using an unpaired two-tailed t-test.

Histology

The collection, staining and analysis of E18.5 embryos were performed as previously described (Barron et al., 2011). Four embryos of each genotype were examined for phenotype, with no variation observed between embryos of the same genotype.

Cell culture

O9-1 cells (Ishii et al., 2012) (EMD Millipore) were cultured on dishes coated with Matrigel in Complete ES Cell Medium (EMD Millipore) supplemented with 25 ng/ml FGF2 (EMD Millipore) at 37°C and 5% CO2. Cells were only used between passages 2 and 8. Antibiotics and antifungal agents were not used during culture and no signs of contamination were observed.

Six1 overexpression and in vitro EDN1 treatment

The Six1 overexpression plasmid pfSix1 has been previously described (Ford et al., 1998). An empty pFLAG-CMV-2 vector was used as a control for transfection experiments. O9-1 cells were seeded into 12-well plates and transfected 24 h later. Then, 0.8 µg of either control plasmid or pfSix1 were transfected into O9-1 cells using X-tremeGENE 9 DNA Transfection Reagent (Sigma-Aldrich). Transfection complexes were made according to the manufacturer's recommendations, added to culture media and incubated for 48 h. In some experiments, EDN1 (Sigma-Aldrich) was added to a final concentration of 10 nM in the culture medium with the transfection complexes. Experiments were performed in duplicate (technical replicate), with each transfection experiment performed three to four times (biological replicate).

RNA collection and qRT-PCR

RNA collection from dissected E9.5 and E10.5 mouse dorsal mandibular arches and from O9-1 cells was performed as previously described (Barron et al., 2011). qRT-PCR was performed using 5 ng cDNA with the Quantitect SYBR Green PCR Kit (Qiagen) and Quantitect assay primers (Qiagen). qRT-PCR of each biological replicate was performed in triplicate. PCR and data analysis were performed using a CFX Connect thermocycler (Bio-Rad). Statistical analysis was conducted using Excel, with significance calculated using an unpaired two-tailed t-test.

We thank Holly Buttermore and Camilla Teng for technical assistance and Peter Dempsey and the Clouthier/Ford labs for helpful discussions.

Author contributions

Conceptualization: A.L.P.T., D.E.C.; Methodology: A.L.P.T., D.E.C.; Software: T.C.C.; Validation: R.M.M.; Investigation: A.L.P.T.; Resources: T.C.C., R.M.M., H.L.F.; Data curation: A.L.P.T., T.C.C., R.M.M., D.E.C.; Writing - original draft: A.L.P.T., H.L.F., D.E.C.; Writing - review & editing: A.L.P.T., H.L.F., D.E.C.; Visualization: T.C.C., D.E.C.; Supervision: D.E.C.; Project administration: D.E.C.; Funding acquisition: D.E.C.

Funding

This work was supported by the National Institutes of Health (DE018899 and DE023050 to D.E.C. and CA095277 to H.L.F.) and by an endowment for Pediatric Craniofacial Research from the Laurel Foundation (T.C.C.). Deposited in PMC for release after 12 months.

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Competing interests

The authors declare no competing or financial interests.