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First published online 16 January 2008
doi: 10.1242/dev.012708

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

¹ Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
² Tsukuba Safety Assessment Laboratories, Banyu Pharmaceutical Company Limited, 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan.
³ Department of Developmental Medical Technology (Sankyo), Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
⁴ Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120, Heidelberg, Germany.

^* Author for correspondence (e-mail: kuri-tky{at}umin.ac.jp)

Accepted 15 November 2007

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/G₁₁-deficient mice, indicate that the dorsoventral axis patterning of pharyngeal arches is regulated by the Ednra-selective, G_q/G₁₁-dependent signaling, while the formation of the distal pharyngeal region is under the control of a G_q/G₁₁-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.

Key words: Endothelin, G protein-coupled receptor, Pharyngeal arch, Neural Crest, Mouse

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