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

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.

Gremlin 2 regulates distinct roles of BMP and Endothelin 1 signaling in dorsoventral patterning of the facial skeleton.

Patterning of the upper versus lower face involves generating distinct pre-skeletal identities along the dorsoventral (DV) axes of the pharyngeal arches. Whereas previous studies have shown roles for BMPs, Endothelin 1 (Edn1) and Jagged1b-Notch2 in DV patterning of the facial skeleton, how these pathways are integrated to generate different skeletal fates has remained unclear. Here, we show that BMP and Edn1 signaling have distinct roles in development of the ventral and intermediate skeletons, respectively, of the zebrafish face. Using transgenic gain-of-function approaches and cell-autonomy experiments, we find that BMPs strongly promote hand2 and msxe expression in ventral skeletal precursors, while Edn1 promotes the expression of nkx3.2 and three Dlx genes (dlx3b, dlx5a and dlx6a) in intermediate precursors. Furthermore, Edn1 and Jagged1b pattern the intermediate and dorsal facial skeletons in part by inducing the BMP antagonist Gremlin 2 (Grem2), which restricts BMP activity to the ventral-most face. We therefore propose a model in which later cross-inhibitory interactions between BMP and Edn1 signaling, in part mediated by Grem2, separate an initially homogenous ventral region into distinct ventral and intermediate skeletal precursor domains.

Combinatorial roles for BMPs and Endothelin 1 in patterning the dorsal-ventral axis of the craniofacial skeleton.

Bone morphogenetic proteins (BMPs) play crucial roles in craniofacial development but little is known about their interactions with other signals, such as Endothelin 1 (Edn1) and Jagged/Notch, which pattern the dorsal-ventral (DV) axis of the pharyngeal arches. Here, we use transgenic zebrafish to monitor and perturb BMP signaling during arch formation. With a BMP-responsive transgene, Tg(Bre:GFP), we show active BMP signaling in neural crest (NC)-derived skeletal precursors of the ventral arches, and in surrounding epithelia. Loss-of-function studies using a heat shock-inducible, dominant-negative BMP receptor 1a [Tg(hs70I:dnBmpr1a-GFP)] to bypass early roles show that BMP signaling is required for ventral arch development just after NC migration, the same stages at which we detect Tg(Bre:GFP). Inhibition of BMP signaling at these stages reduces expression of the ventral signal Edn1, as well as ventral-specific genes such as hand2 and dlx6a in the arches, and expands expression of the dorsal signal jag1b. This results in a loss or reduction of ventral and intermediate skeletal elements and a mis-shapen dorsal arch skeleton. Conversely, ectopic BMP causes dorsal expansion of ventral-specific gene expression and corresponding reductions/transformations of dorsal cartilages. Soon after NC migration, BMP is required to induce Edn1 and overexpression of either signal partially rescues ventral skeletal defects in embryos deficient for the other. However, once arch primordia are established the effects of BMPs become restricted to more ventral and anterior (palate) domains, which do not depend on Edn1. This suggests that BMPs act upstream and in parallel to Edn1 to promote ventral fates in the arches during early DV patterning, but later acquire distinct roles that further subdivide the identities of NC cells to pattern the craniofacial skeleton.

Jagged-Notch signaling ensures dorsal skeletal identity in the vertebrate face.

The development of the vertebrate face relies on the regionalization of neural crest-derived skeletal precursors along the dorsoventral (DV) axis. Here we show that Jagged-Notch signaling ensures dorsal identity within the hyoid and mandibular components of the facial skeleton by repressing ventral fates. In a genetic screen in zebrafish, we identified a loss-of-function mutation in jagged 1b (jag1b) that results in dorsal expansion of ventral gene expression and partial transformation of the dorsal hyoid skeleton to a ventral morphology. Conversely, misexpression of human jagged 1 (JAG1) represses ventral gene expression and dorsalizes the ventral hyoid and mandibular skeletons. We further show that jag1b is expressed specifically in dorsal skeletal precursors, where it acts through the Notch2 receptor to activate hey1 expression. Whereas Jagged-Notch positive feedback propagates jag1b expression throughout the dorsal domain, Endothelin 1 (Edn1) inhibits jag1b and hey1 expression in the ventral domain. Strikingly, reduction of Jag1b or Notch2 function partially rescues the ventral defects of edn1 mutants, indicating that Edn1 promotes facial skeleton development in part by inhibiting Jagged-Notch signaling in ventral skeletal precursors. Together, these results indicate a novel function of Jagged-Notch signaling in ensuring dorsal identity within broad fields of facial skeletal precursors.

Pharmacotherapy for cognitive impairment in a mouse model of Down syndrome.

Ts65Dn mice, a model for Down syndrome, have excessive inhibition in the dentate gyrus, a condition that could compromise synaptic plasticity and mnemonic processing. We show that chronic systemic treatment of these mice with GABAA antagonists at non-epileptic doses causes a persistent post-drug recovery of cognition and long-term potentiation. These results suggest that over-inhibition contributes to intellectual disabilities associated with Down syndrome and that GABAA antagonists may be useful therapeutic agents for this disorder.

Health insurance and education: major contributors to oral health inequalities in Colombia.

BACKGROUND: Health inequalities, including inequalities in oral health, are problems of social injustice worldwide. Evidence on this issue from low-income and middle-income countries is still needed. We aimed to examine the relationship between oral health and different dimensions of socioeconomic position (SEP) in Colombia, a very unequal society emerging from a long-lasting internal armed conflict. METHODS: Using data from the last Colombian Oral Health Survey (2014), we analysed inequalities in severe untreated caries (≥3 teeth), edentulousness (total tooth loss) and number of missing teeth. Inequalities by education, income, area-level SEP and health insurance scheme were estimated by the relative index of inequality and slope index of inequality (RII and SII, respectively). RESULTS: A general pattern of social gradients was observed and significant inequalities for all outcomes and SEP indicators were identified with RII and SII. Relative inequalities were larger for decay by health insurance scheme, with worse decay levels among the uninsured (RII: 2.57; 95% CI 2.11 to 3.13), and in edentulousness (RII: 3.23; 95% CI 1.88 to 5.55) and number of missing teeth (RII: 2.08; 95% CI 1.86 to 2.33) by education, with worse levels of these outcomes among the lower educated groups. Absolute inequalities followed the same pattern. Inequalities were larger in urban areas. CONCLUSION: Health insurance and education appear to be the main contributors to oral health inequalities in Colombia, posing challenges for designing public health strategies and social policies. Tackling health inequalities is crucial for a fairer society in a Colombian post-conflict era and our findings highlight the importance of investing in education policies and universal health care coverage.