A Novel Mouse Fgfr2 Mutant, Hobbyhorse (hob), Exhibits Complete XY Gonadal Sex Reversal

Abstract

The secreted molecule fibroblast growth factor 9 (FGF9) plays a critical role in testis determination in the mouse. In embryonic gonadal somatic cells it is required for maintenance of SOX9 expression, a key determinant of Sertoli cell fate. Conditional gene targeting studies have identified FGFR2 as the main gonadal receptor for FGF9 during sex determination. However, such studies can be complicated by inefficient and variable deletion of floxed alleles, depending on the choice of Cre deleter strain. Here, we report a novel, constitutive allele of Fgfr2, hobbyhorse (hob), which was identified in an ENU-based forward genetic screen for novel testis-determining loci. Fgr2hob is caused by a C to T mutation in the invariant exon 7, resulting in a polypeptide with a mis-sense mutation at position 263 (Pro263Ser) in the third extracellular immunoglobulin-like domain of FGFR2. Mutant homozygous embryos show severe limb and lung defects and, when on the sensitised C57BL/6J (B6) genetic background, undergo complete XY gonadal sex reversal associated with failure to maintain expression of Sox9. Genetic crosses employing a null mutant of Fgfr2 suggest that Fgr2hob is a hypomorphic allele, affecting both the FGFR2b and FGFR2c splice isoforms of the receptor. We exploited the consistent phenotype of this constitutive mutant by analysing MAPK signalling at the sex-determining stage of gonad development, but no significant abnormalities in mutant embryos were detected.

Cell fate commitment during mammalian sex determination

“…Fgf signaling functions to antagonize Wnt signaling [45]. Elimination of the expression of Fgf9 or its receptor Fgfr2 (fibroblast growth factor receptor 2), leads to upregulation of the Wnt pathway and male-to-female sex reversal [44,52–54]. On the other hand, elimination of Wnt4/Rspo1/β-catenin leads to female-to-male sex reversal near birth [27,31–33], suggesting that the Fgf9 and Wnt4 signaling pathways have antagonistic roles in sex determination.…”

Protein Detection by Simple Western™ Analysis

“…The instrument of choice can (a) perform protein separation (size or charge), (b) immobilize the separated protein onto the capillary wall, (c) immuno-probe the target protein using a primary and secondary antibody, (d) perform washes between antibody addition, and (e) develop, identify, and quantitate the signal ( see Fig. 1 ) Pam Sigger et al [ 4 ] used the Peggy Sue system to detect mitogen-activated protein kinase (MAPK) signaling proteins, the methods and materials of which are described below.…”

Signaling Pathways Involved in Mammalian Sex Determination and Gonad Development

“…Knockout of Fgf9 or its receptor Fgfr2 resulted in the upregulation of Wnt4 expression and testis-to-ovary sex reversal [Colvin et al, 2001;Kim et al, 2006Kim et al, , 2007BagheriFam et al, 2008;Siggers et al, 2014]. Similarly, partial ovary-to-testis sex reversal was observed when Wnt4 [Vainio et al, 1999;Jeays-Ward et al, 2003], Rspo1 [Chassot et al, 2008;Tomizuka et al, 2008], or β-catenin [Manuylov et al, 2008;Liu et al, 2009] was ablated.…”

“…1 C). Mice homozygous for a null mutation in Fgf9 or Fgfr2 exhibit male-to-female sex reversal, with all testis-specific cellular events being disrupted, including cell proliferation, mesonephric cell migration, Sertoli cell differentiation, and testis cord formation [Colvin et al, 2001;Kim et al, 2007;Bagheri-Fam et al, 2008;Siggers et al, 2014]. Accordingly, mutations in FGFR2 in human have been identified that result in 46,XY DSD [Bagheri-Fam et al, 2015].…”