The ciliary protein C2cd3 is required for mandibular musculoskeletal tissue patterning.

The mandible is composed of several musculoskeletal tissues including bone, cartilage, and tendon that require precise patterning to ensure structural and functional integrity. Interestingly, most of these tissues are derived from one multipotent cell population called cranial neural crest cells (CNCCs). How CNCCs are properly instructed to differentiate into various tissue types remains nebulous. To better understand the mechanisms necessary for the patterning of mandibular musculoskeletal tissues we utilized the avian mutant talpid2 (ta2) which presents with several malformations of the facial skeleton including dysplastic tendons, mispatterned musculature, and bilateral ectopic cartilaginous processes extending off Meckel's cartilage. We found an ectopic epithelial BMP signaling domain in the ta2 mandibular prominence (MNP) that correlated with the subsequent expansion of SOX9+ cartilage precursors. These findings were validated with conditional murine models suggesting an evolutionarily conserved mechanism for CNCC-derived musculoskeletal patterning. Collectively, these data support a model in which cilia are required to define epithelial signal centers essential for proper musculoskeletal patterning of CNCC-derived mesenchyme.

Brooks EC ，Han SJY ，Bonatto Paese CL ，Lewis AA ，Aarnio-Peterson M ，Brugmann SA ... -  《-》

Using the avian mutant talpid2 as a disease model for understanding the oral-facial phenotypes of oral-facial-digital syndrome.

Oral-facial-digital syndrome (OFD) is a ciliopathy that is characterized by oral-facial abnormalities, including cleft lip and/or palate, broad nasal root, dental anomalies, micrognathia and glossal defects. In addition, these individuals have several other characteristic abnormalities that are typical of a ciliopathy, including polysyndactyly, polycystic kidneys and hypoplasia of the cerebellum. Recently, a subset of OFD cases in humans has been linked to mutations in the centriolar protein C2 Ca(2+)-dependent domain-containing 3 (C2CD3). Our previous work identified mutations in C2CD3 as the causal genetic lesion for the avian talpid(2) mutant. Based on this common genetic etiology, we re-examined the talpid(2) mutant biochemically and phenotypically for characteristics of OFD. We found that, as in OFD-affected individuals, protein-protein interactions between C2CD3 and oral-facial-digital syndrome 1 protein (OFD1) are reduced in talpid(2) cells. Furthermore, we found that all common phenotypes were conserved between OFD-affected individuals and avian talpid(2) mutants. In light of these findings, we utilized the talpid(2) model to examine the cellular basis for the oral-facial phenotypes present in OFD. Specifically, we examined the development and differentiation of cranial neural crest cells (CNCCs) when C2CD3-dependent ciliogenesis was impaired. Our studies suggest that although disruptions of C2CD3-dependent ciliogenesis do not affect CNCC specification or proliferation, CNCC migration and differentiation are disrupted. Loss of C2CD3-dependent ciliogenesis affects the dispersion and directional persistence of migratory CNCCs. Furthermore, loss of C2CD3-dependent ciliogenesis results in dysmorphic and enlarged CNCC-derived facial cartilages. Thus, these findings suggest that aberrant CNCC migration and differentiation could contribute to the pathology of oral-facial defects in OFD.

Schock EN ，Chang CF ，Struve JN ，Chang YT ，Chang J ，Delany ME ，Brugmann SA ... -  《-》

Role of Dlx genes in craniofacial morphogenesis: Dlx2 influences skeletal patterning by inducing ectomesenchymal aggregation in ovo.

Dlx homeodomain transcription factors are expressed in neural crest-derived mesenchyme of the pharyngeal arches and are required for patterning of the craniofacial skeleton. However, the cellular and molecular mechanisms by which Dlx factors control skeletogenesis in the facial primordia are unclear. We have investigated the function of Dlx2 and Dlx5 by sustained misexpression in ovo. We find that RCAS-Dlx2- and RCAS-Dlx5-infected avian embryos exhibit very similar patterns of local, stereotypical changes in skeletal development in the upper jaw. The changes include ectopic dermal bone along the jugal arch, and ectopic cartilages that develop between the quadrate and the trabecula. The ectopic cartilage associated with the trabecula is reminiscent of a normally occurring element in this region in some bird taxa. Analysis of the distribution of RCAS-Dlx2-infected cells suggests that Dlx2 induces aggregation of undifferentiated mesenchyme, which subsequently develops into the ectopic skeletal elements. Comparison of infected embryos with restricted or widespread misexpression, and of embryos in which Dlx genes were delivered to migratory or postmigratory neural crest, indicate that there are limited regions of competence in which the ectopic elements can arise. The site-specific differentiation program that the aggregates undergo may be dependent on local environmental signals. Our results suggest that Dlx factors mediate localization of ectomesenchymal subpopulations within the pharyngeal arches and in doing so define where skeletogenic condensations will arise. Consequently, variation in Dlx expression or activity may have influenced the morphology of jaw elements during vertebrate evolution.

Gordon CT ，Brinas IM ，Rodda FA ，Bendall AJ ，Farlie PG ... -  《-》

Metalloprotease-Dependent Attenuation of BMP Signaling Restricts Cardiac Neural Crest Cell Fate.

In higher vertebrates, cephalic neural crest cells (NCCs) form craniofacial skeleton by differentiating into chondrocytes and osteoblasts. A subpopulation of cephalic NCCs, cardiac NCCs (CNCCs), migrates to the heart. However, CNCCs mostly do not yield skeletogenic derivatives, and the molecular mechanisms of this fate restriction remain elusive. We identify a disintegrin and metalloprotease 19 (Adam19) as a position-specific fate regulator of NCCs. Adam19-depleted mice abnormally form NCC-derived cartilage in their hearts through the upregulation of Sox9 levels in CNCCs. Moreover, NCC-lineage-specific Sox9-overexpressing mice recapitulate CNCC chondrogenesis. In vitro experiments show that Adam19 mediates the cleavage of bone morphogenic protein (BMP) type I receptor Alk2 (Acvr1), whereas pharmacogenetic approaches reveal that Adam19 inhibits CNCC chondrogenesis by suppressing the BMP-Sox9 cascade, presumably through processing Alk2. These findings suggest a metalloprotease-dependent mechanism attenuating cellular responsiveness to BMP ligands, which is essential for both the positional restriction of NCC skeletogenesis and normal heart development.

Arai HN ，Sato F ，Yamamoto T ，Woltjen K ，Kiyonari H ，Yoshimoto Y ，Shukunami C ，Akiyama H ，Kist R ，Sehara-Fujisawa A ... -  《Cell Reports》

Dual functions for WNT5A during cartilage development and in disease.

Mouse and human genetic data suggests that Wnt5a is required for jaw development but the specific role in facial skeletogenesis is unknown. We mapped expression of WNT5A in the developing chicken skull and found that the highest expression was in early Meckel's cartilage but by stage 35 expression was decreased to background. We focused on chondrogenesis by targeting a retrovirus expressing WNT5A to the mandibular prominence prior to cell differentiation. Unexpectedly, there were no phenotypes in the first 6days following injection; however later the mandibular bones and Meckel's cartilage were reduced or missing on the treated side. To examine the effects on cartilage differentiation we treated micromass cultures from mandibular mesenchyme with Wnt5a-conditioned media (CM). Similar to in vivo viral data, cartilage differentiates normally, but, after 6days of culture, nearly all Alcian blue staining is lost. Collagen II and aggrecan were also decreased in treated cultures. The matrix loss was correlated with upregulation of metalloproteinases, MMP1, MMP13, and ADAMTS5 (codes for Aggrecanase). Moreover, Marimastat, an MMP and Aggrecanase inhibitor rescued cartilage matrix in Wnt5a-CM treated cultures. The pathways mediating these cartilage and RNA changes were investigated using luciferase assays. Wnt5a-CM was a potent inhibitor of the canonical pathway and strongly activated JNK/PCP signaling. To determine whether the matrix loss is mediated by repression of canonical signaling or activation of the JNK pathway we treated mandibular cultures with either DKK1, an antagonist of the canonical pathway, or a small molecule that antagonizes JNK signaling (TCS JNK 6o). DKK1 slightly increased cartilage formation and therefore suggested that the endogenous canonical signaling represses chondrogenesis. To test this further we added an excess of Wnt3a-CM and found that far fewer cartilage nodules differentiated. Since DKK1 did not mimic the effects of Wnt5a we excluded the canonical pathway from mediating the matrix loss phenotype. The JNK antagonist partially rescued the Wnt5a phenotype supporting this non-canonical pathway as the main mediator of the cartilage matrix degradation. Our study reveals two new roles for WNT5A in development and disease: 1) to repress canonical Wnt signaling in cartilage blastema in order to promote normal differentiation and 2) in conditions of excess to stimulate degradation of mature cartilage matrix via non-canonical pathways.

Hosseini-Farahabadi S ，Geetha-Loganathan P ，Fu K ，Nimmagadda S ，Yang HJ ，Richman JM ... -  《-》