Competition between Jagged-Notch and Endothelin1 Signaling Selectively Restricts Cartilage Formation in the Zebrafish Upper Face

Abstract

The intricate shaping of the facial skeleton is essential for function of the vertebrate jaw and middle ear. While much has been learned about the signaling pathways and transcription factors that control facial patterning, the downstream cellular mechanisms dictating skeletal shapes have remained unclear. Here we present genetic evidence in zebrafish that three major signaling pathways − Jagged-Notch, Endothelin1 (Edn1), and Bmp − regulate the pattern of facial cartilage and bone formation by controlling the timing of cartilage differentiation along the dorsoventral axis of the pharyngeal arches. A genomic analysis of purified facial skeletal precursors in mutant and overexpression embryos revealed a core set of differentiation genes that were commonly repressed by Jagged-Notch and induced by Edn1. Further analysis of the pre-cartilage condensation gene barx1, as well as in vivo imaging of cartilage differentiation, revealed that cartilage forms first in regions of high Edn1 and low Jagged-Notch activity. Consistent with a role of Jagged-Notch signaling in restricting cartilage differentiation, loss of Notch pathway components resulted in expanded barx1 expression in the dorsal arches, with mutation of barx1 rescuing some aspects of dorsal skeletal patterning in jag1b mutants. We also identified prrx1a and prrx1b as negative Edn1 and positive Bmp targets that function in parallel to Jagged-Notch signaling to restrict the formation of dorsal barx1+ pre-cartilage condensations. Simultaneous loss of jag1b and prrx1a/b better rescued lower facial defects of edn1 mutants than loss of either pathway alone, showing that combined overactivation of Jagged-Notch and Bmp/Prrx1 pathways contribute to the absence of cartilage differentiation in the edn1 mutant lower face. These findings support a model in which Notch-mediated restriction of cartilage differentiation, particularly in the second pharyngeal arch, helps to establish a distinct skeletal pattern in the upper face.

Essential Role of Nr2f Nuclear Receptors in Patterning the Vertebrate Upper Jaw

“…Hierarchical clustering of transcriptome data grouped the two independent nr2f2; nr2f5 mutant samples with a previously published Edn1-misexpressing sample, whereas the Nr2f5-misexpressing sample clustered with two edn1 mutants (Fig. 4B) (Barske et al, 2016, Askary et al, 2017). Thus, Nr2fs and Edn1 act oppositely to regulate both jaw molecular identity and skeletal composition/morphology.…”

“…Less is known about upper jaw regulation. Although Jagged-Notch signaling restricts mandibular gene expression in parts of the upper first and second arches, maxillary-derived portions of the upper jaw develop normally in zebrafish and mice with reduced Jagged-Notch signaling (Zuniga et al, 2010, Humphreys et al, 2012, Barske et al, 2016, Teng et al, 2017). Six1 mutant mice show a partial transformation of the posterior end of the maxilla into a mandible-like structure, but this phenotype is attributable to an upstream role for Six1 in limiting Edn1 expression within the dorsal pharyngeal endoderm (Tavares et al, 2017).…”

Systems biology of facial development: contributions of ectoderm and mesenchyme

“…Its functional network (Fig 9B) shows that 7 of its 9 genes are involved in gene expression, including four transcription factors involved in OFCs, Barx1, Dlx1, Lhx8 and Sim2, as well as Lhx6 and the histone deacetylase co-repressor complex members Mab21l1 and Mab21l2. Four of these 9 genes are involved in establishing the dental lamina (Denaxa et al, 2009; Grigoriou et al, 1998; Mitsiadis and Drouin, 2008), while Ndnf, Mab21l2, Lhx6, Lhx8b, Barx1 and probably also Dlx1a are regulated by the interaction of the Edn1 and Notch signaling pathways that restricts cartilage primordia in the upper face in zebrafish (Barske et al, 2016). Because there is no obvious role of the dental lamina in earlier facial morphogenesis, we interpret this module as a jaw patterning program, possibly specific to the maxilla.…”

Requirement for Jagged1-Notch2 signaling in patterning the bones of the mouse and human middle ear

“…In a genetic screen in zebrafish, we previously identified a loss-of-function mutation in the JAG1 homolog, jag1b , which resulted in specific malformations of the palatoquadrate and hyomandibular cartilages 3 . Subsequently, we found that Jag1b works through the Notch2 and Notch3 receptors to regulate bone and cartilage differentiation in the dorsal portions of the mandibular and hyoid arches, regions from which the incus and stapes bones arise in mammals 4 . We therefore asked in this study whether loss of Jagged-Notch signaling might similarly disrupt development of the stapes and incus bones.

Figure 1Craniofacial and middle ear defects in mice deficient for Jag1 or Notch2 .

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