Elucidating timing and function of endothelin-A receptor signaling during craniofacial development using neural crest cell-specific gene deletion and receptor antagonism

Abstract

Cranial neural crest cells (NCCs) play an intimate role in craniofacial development. Multiple signaling cascades participate in patterning cranial NCCs, some of which are regulated by endothelin-A receptor (Ednra) signaling. Ednra−/− embryos die at birth from severe craniofacial defects resulting from disruption of neural crest cell patterning and differentiation. These defects include homeotic transformation of lower jaw structures into upper jaw-like structures, suggesting that some cephalic NCCs alter their “identity” in the absence of Ednra signaling. To elucidate the temporal necessity for Ednra signaling in vivo, we undertook two strategies. We first used a conditional knockout strategy in which mice containing a conditionally targeted Ednra allele (Ednrafl) were bred with mice from the Hand2-Cre and Wnt1-Cre transgenic mouse strains, two strains in which Cre expression occurs at different time periods within cranial NCCs. In our second approach, we used an Ednra-specific antagonist to treat wild type pregnant mice between embryonic days E8.0 and E10.0, a time frame encompassing the early migration and proliferation of cranial NCCs. The combined results suggest that Ednra function is crucial for NCC development between E8.25 and E9.0, a time period encompassing the arrival of NCCs in the arches and/or early post-migratory patterning. After this time period, Ednra signaling is dispensable. Interestingly, middle ear structures are enlarged and malformed in a majority of Ednrafl/fl;Wnt1-Cre embryos, instead resembling structures found in extinct predecessors of mammals. These observations suggest that the advent of Ednra signaling in cranial NCCs may have been a crucial event in the evolution of the mammalian middle ear ossicles.

Spatio‐temporal dynamics of gene expression of the Edn1‐Dlx5/6 pathway during development of the lower jaw

“…5c), Hand2 is activated through the established Edn1?Dlx6 pathway most probably involving the reported Dlx6-dependent Hand2 enhancer (Charite et al, 2001). Notably, inactivation of this enhancer results in defects in the medio-distal part of PA1 as suggested by our model (Yanagisawa et al, 2003) and by timed inhibition of Edn1 signaling using Ednra antagonists (Ruest and Clouthier, 2009). (4) Finally, in the proximal part of the E10.5 PA1 (grey in Fig.…”

Endothelin-2 signaling in the neural retina promotes the endothelial tip cell state and inhibits angiogenesis

“…For this purpose, we constructed a mouse line carrying a conditional null allele of Ednrb (Ednrb f ) (Fig. S1B), and we used a previously described Ednra f line (30). Because Pax6-α-Cre is active in the neural retina but not in the vasculature or astrocytes (Fig.…”

Downregulation of Dlx5 and Dlx6 expression by Hand2 is essential for initiation of tongue morphogenesis

“…, Cre and lacZ genotyping were performed as described previously (Ruest and Clouthier, 2009;Soriano, 1999;Hendershot et al, 2008).…”

“…Skeleton (Ruest and Clouthier, 2009) and cartilage staining was performed as described previously. Stained bone and cartilage preparations were analyzed and photographed using an Olympus SZX12 stereomicroscope fitted with a DP11 digital camera.…”