Recombinase-mediated cassette exchange reveals the selective use of Gq/G11-dependent and -independent endothelin 1/endothelin type A receptor signaling in pharyngeal arch development

Abstract

The endothelin (Edn) system comprises three ligands (Edn1, Edn2 and Edn3) and their G-protein-coupled type A (Ednra) and type B (Ednrb) receptors. During embryogenesis, the Edn1/Ednra signaling is thought to regulate the dorsoventral axis patterning of pharyngeal arches via Dlx5/Dlx6 upregulation. To further clarify the underlying mechanism, we have established mice in which gene cassettes can be efficiently knocked-in into the Ednra locus using recombinase-mediated cassette exchange (RMCE) based on the Cre-lox system. The first homologous recombination introducing mutant lox-flanked Neo resulted in homeotic transformation of the lower jaw to an upper jaw, as expected. Subsequent RMCE-mediated knock-in of lacZ targeted its expression to the cranial/cardiac neural crest derivatives as well as in mesoderm-derived head mesenchyme. Knock-in of Ednra cDNA resulted in a complete rescue of craniofacial defects of Ednra-null mutants. By contrast, Ednrb cDNA could not rescue them except for the most distal pharyngeal structures. At early stages, the expression of Dlx5, Dlx6 and their downstream genes was downregulated and apoptotic cells distributed distally in the mandible of Ednrb-knock-in embryos. These results, together with similarity in craniofacial defects between Ednrb-knock-in mice and neural-crest-specific G␣ q /G␣ 11 -deficient mice, indicate that the dorsoventral axis patterning of pharyngeal arches is regulated by the Ednra-selective, G q /G 11 -dependent signaling, while the formation of the distal pharyngeal region is under the control of a G q /G 11 -independent signaling, which can be substituted by Ednrb. This RMCE-mediated knock-in system can serve as a useful tool for studies on gene functions in craniofacial development.

Preotic neural crest cells contribute to coronary artery smooth muscle involving endothelin signalling

“…S10). Ednra expression, mimicked by knocked-in EGFP expression 18,24 , was detected broadly in cardiomyocytes and aortic SMCs, and also in Wnt1-Cre-labelled cells in the condensed mesenchyme and close to the endocardium at E12.5 ( Fig. 5d-f).…”

“…The similarity of the Edn1-/Ednra-null phenotype in mouse to the NC ablation in chick suggests that the preotic NC is the target of Edn signalling in coronary remodelling. We have previously reported that Edn1 activates the G-protein-coupled receptor Ednra on preotic NCCs migrating into the first PA, and specifies mandibular identity through G q /G 11 -dependent signalling 18 . This and the present findings indicate that Edn1 activates different signalling and genetic programmes in preotic NCCs in different contexts.…”

“…Endothelin-1 (Edn1), a 21-amino acid peptide known as the most potent vasoconstrictor 16 , is a key regulator of craniofacial and cardiovascular development, acting mainly on NCCs expressing Edn receptor type-A (Ednra), a G-protein-coupled receptor [17][18][19] . Inactivation of the Edn1-Ednra signalling leads to mandibular-to-maxillary homeotic-like transformation and cardiovascular anomalies, involving the outflow tract and aortic arch arteries [20][21][22] .…”

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

“…Second, we cannot appreciate the phenotypic consequences of reduced gene expression; third we may fail to recognize the dynamics of cell-cell signaling and interactions, as these often require a nearly normal context. Such is the case of Edn1?Dlx signaling at the basis of the homeotic lower jaw transformation, to investigate which many studies have been carried out based on either loss-of-function (Acampora et al, 1999;Beverdam et al, 2002;Clouthier et al, 1998;Depew et al, 1999Depew et al, , 2002Kurihara et al, 1994;Ozeki et al, 2004;Sato et al, 2008a;Thomas et al, 1998;Yanagisawa et al, 2003) or gain-of-function mutants (Sato et al, 2008b). Here we provide quantitative data on the effects of allelic reduction of Edn1 and Dlx5;Dlx6 at different developmental stages.…”

The “Cardiac Neural Crest” Concept Revisited

“…The craniofacial and cardiovascular anomalies are attributed to the disordered development of cranial (preotic) and cardiac (postotic) NCCs, respectively. In craniofacial development, the Edn1-Ednra signaling activates Gα q -/Gα 11 -dependent pathway, resulting in the induction of Dlx5/ Dlx6, homeobox genes critical to ventral (mandibular) identity of the pharyngeal arches [10][11][12]. In cardiovascular development, the Edn1-/Ednra-null phenotype of aortic arch anomalies is independent of Dlx5/Dlx6 [13], indicating that the Edn1-Ednra signaling pathway appears differently involved in craniofacial and cardiac development.…”