Cre recombinase-regulated Endothelin1 transgenic mouse lines: Novel tools for analysis of embryonic and adult disorders

Abstract

Endothelin-1 (EDN1) influences both craniofacial and cardiovascular development and a number of adult physiological conditions by binding to one or both of the known endothelin receptors, thus initiating multiple signaling cascades. Animal models containing both conventional and conditional loss of the Edn1 gene have been used to dissect EDN1 function in both embryos and adults. However, while transgenic Edn1 over-expression or targeted genomic insertion of Edn1 has been performed to understand how elevated levels of Edn1 result in or exacerbate disease states, an animal model in which Edn1 over-expression can be achieved in a spatiotemporal-specific manner has not been reported. Here we describe the creation of Edn1 conditional over-expression transgenic mouse lines in which the chicken β-actin promoter and an Edn1 cDNA are separated by a strong stop sequence flanked by loxP sites. In the presence of Cre, the stop cassette is removed, leading to Edn1 expression. Using the Wnt1-Cre strain, in which Cre expression is targeted to the Wnt1-expressing domain of the central nervous system (CNS) from which neural crest cells (NCCs) arise, we show that stable CBA-Edn1 transgenic lines with varying EDN1 protein levels develop defects in NCC-derived tissues of the face, though the severity differs between lines. We also show that Edn1 expression can be achieved in other embryonic tissues utilizing other Cre strains, with this expression also resulting in developmental defects. CBA-Edn1 transgenic mice will be useful in investigating diverse aspects of EDN1-mediated-development and disease, including understanding how NCCs achieve and maintain a positional and functional identity and how aberrant EDN1 levels can lead to multiple physiological changes and diseases.

Negative regulation of Endothelin signaling by SIX1 is required for proper maxillary development

“…2E,H) and CBA-Edn1;Wnt1-Cre (Fig. 2F,I) (Tavares and Clouthier, 2015) embryos. By contrast, aberrant Hand2 expression was not observed in the dorsal mandibular arch or maxillary prominence (Fig.…”

“…Misexpression of Edn1 in NCCs within the maxillary prominence results in complete homeotic transformation of the maxilla into a mandible (Sato et al, 2008a;Tavares and Clouthier, 2015). One method we have previously used to achieve this misexpression was through conditional activation of Edn1 expression in NCCs (Tavares and Clouthier, 2015) (Fig. 2C,C′) (see Materials and Methods).…”

“…Although Edn1 expression is not observed rostral to the mandibular arch (Clouthier et al, 1998), aberrant EDNRA signaling in maxillary NCCs leads to homeotic transformation of the maxilla into a mandible-like structure (Sato et al, 2008b;Tavares and Clouthier, 2015). These changes are accompanied by an upregulation of a ventral/intermediate gene expression profile in the dorsal mandibular arch domain and maxillary prominence (Sato et al, 2008b;Tavares and Clouthier, 2015;Zuniga et al, 2011). By contrast, overexpression of jag1b in zebrafish embryos results in downregulation of ventral arch gene expression (Zuniga et al, 2010).…”

Developmental mechanisms of the tympanic membrane in mammals and non‐mammalian amniotes

“…It is also possible that the pharyngeal endoderm is involved in the development of the tympanic region. Overexpression of Edn1 signaling in the dorsal region of the PAs results in transformation of the upper jaw into a lower jaw-like structure (Sato et al 2008;Tavares and Clouthier 2015). Although Gsc expression is detected in the dorsal region of the PA1 in the mouse, only portions of the tympanic ring and gonial bone are duplicated, but no EAM appears in the upper jaw region (Sato et al 2008).…”

Transcriptomics reveals complex kinetics of dorsal–ventral patterning gene expression in the mandibular arch

“…Homeotic transformations along the D–V axis are also observed in Dlx5/6 and Hand2 mutant embryos (Barron et al, ; Beverdam et al, ; Depew, Lufkin, & Rubenstein, ; Miller, Schilling, Lee, Parker, & Kimmel, ; Talbot, Johnson, & Kimmel, ; Walker, Miller, Coffin Talbot, Stock, & Kimmel, ; Yanagisawa, Clouthier, Richardson, Charité, & Olson, ). In contrast, overexpression of Edn1 in the dorsal arch (zebrafish) or maxillary prominence (mouse) or loss of Nr2f leads to the upregulation of ventral gene expression and subsequent homeotic transformation of maxillary into more mandibular‐like structures (Alexander et al, ; Barske et al, ; Tavares & Clouthier, ; Zuniga, Rippen, Alexander, Schilling, & Crump, ). Bmps also act as morphogens during mandibular arch development, inducing Hand2 expression, at least in zebrafish (Alexander et al, ; Zuniga et al, ).…”