Nick Warr

Bacterial artificial chromosomes (BACs) offer a means of manipulating gene expression and tagging gene products in the mammalian genome without the need to alter endogenous gene structure and risk deleterious phenotypic consequences. However, for a BAC clone to be useful for such purposes it must be shown to contain all the regulatory elements required for normal gene expression and allow phenotypic rescue in the absence of an endogenous gene. Here, we report identification of a functional BAC containing Gadd45g, a gene implicated in DNA repair, DNA demethylation and testis determination in mice and exhibiting a broad pattern of embryonic expression. Mouse fetuses lacking the endogenous Gadd45g gene undergo normal testis development in the presence of the Gadd45g BAC transgene. Moreover, a survey of embryonic Gadd45g expression from the BAC reveals that all reported sites of expression are maintained. This functional BAC can now be used for subsequent manipulation of the Gadd45g gene with the confidence that regulatory elements required for embryonic expression, including testis determination, are present. We describe the generation and characterisation of a Gadd45g-mCherry fluorescent reporter exhibiting strong expression in developing gonads and neural tissue, recapitulating endogenous gene expression, as evidence of this.

Significance: Sex determination involves antagonistic interactions between the testis-determining (SRY-SOX9-FGF9) and ovary-promoting (RSPO1-WNT/β-catenin-FOXL2) pathways, but the underlying molecular mechanisms remain unclear. We show that ZNRF3, an E3 ubiquitin ligase that inhibits WNT signaling and is a direct target of RSPO1-mediated membrane clearance, is testis-determining in mice. Testis determination defects in the absence of ZNRF3 arise due to ectopic canonical WNT signaling in XY gonads at the sex-determining stage. We identify human ZNRF3 sequence variants in cases of 46,XY disorders of sex development with XY female presentation. In vitro functional assays show that these variants disrupt ZNRF3 function. Our data reveal a sex-determining role for ZNRF3 and indicate that interactions between ZNRF3 and RSPO1 regulate mammalian sex determination.

Testis determination in mammals is initiated by expression of SRY in somatic cells of the embryonic gonad. Genetic analyses in the mouse have reveale

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. Fgr2^hob 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 Fgr2^hob 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.

Disorders of sex development in the human population range in severity from mild genital defects to gonadal sex reversal. XY female development has been associated with heterozygous mutations in several genes, including SOX9, WT1 and MAP3K1. In contrast, XY sex reversal in mice usually requires complete absence of testis-determining gene products. One exception to this involves T-associated sex reversal (Tas), a phenomenon characterized by the formation of ovotestes or ovaries in XY mice hemizygous for the hairpin-tail (T^hp) or T-Orleans (T^Orl) deletions on proximal mouse chromosome 17. We recently reported that mice heterozygous for a null allele of Map3k4, which resides in the T^hp deletion, exhibit XY ovotestis development and occasional gonadal sex reversal on the sensitized C57BL/6J-Y^AKR (B6-Y^AKR) genetic background, reminiscent of the Tas phenotype. However, these experiments did not exclude the possibility that loss of other loci in the T^hp deletion, or other effects of the deletion itself, might contribute to Tas. Here, we show that disruption to Sry expression underlies XY gonadal defects in B6-Y^AKR embryos harbouring the T^hp deletion and that a functional Map3k4 bacterial artificial chromosome rescues these abnormalities by re-establishing a normal Sry expression profile. These data demonstrate that Map3k4 haploinsufficiency is the cause of T-associated sex reversal and that levels of this signalling molecule are a major determinant of the expression profile of Sry.

Otitis media with effusion (OME) is the most common cause of hearing loss in children and tympanostomy to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of OM are known to have a very significant genetic component, however, until recently little was known of the underlying genes involved. The identification of mouse models of chronic OM has indicated a role of transforming growth factor beta (TGFβ) signalling and its impact on responses to hypoxia in the inflamed middle ear. We have, therefore, investigated the role of TGFβ signalling and identified and characterized a new model of chronic OM carrying a mutation in the gene for transforming growth interacting factor 1 (Tgif1). Tgif1 homozygous mutant mice have significantly raised auditory thresholds due to a conductive deafness arising from a chronic effusion starting at around 3 weeks of age. The OM is accompanied by a significant thickening of the middle ear mucosa lining, expansion of mucin-secreting goblet cell populations and raised levels of vascular endothelial growth factor, TNF-α and IL-1β in ear fluids. We also identified downstream effects on TGFβ signalling in middle ear epithelia at the time of development of chronic OM. Both phosphorylated SMAD2 and p21 levels were lowered in the homozygous mutant, demonstrating a suppression of the TGFβ pathway. The identification and characterization of the Tgif mutant supports the role of TGFβ signalling in the development of chronic OM and provides an important candidate gene for genetic studies in the human population.

Summary: The ZIC transcription factors are key mediators of embryonic development and ZIC3 is the gene most commonly associated with situs defects (heterotaxy) in humans. Half of patient ZIC3 mutations introduce a premature termination codon (PTC). In vivo, PTC-containing transcripts might be targeted for nonsense-mediated decay (NMD). NMD efficiency is known to vary greatly between transcripts, tissues and individuals and it is possible that differences in survival of PTC-containing transcripts partially explain the striking phenotypic variability that characterizes ZIC3-associated congenital defects. For example, the PTC-containing transcripts might encode a C-terminally truncated protein that retains partial function or that dominantly interferes with other ZIC family members. Here we describe the katun (Ka) mouse mutant, which harbours a mutation in the Zic3 gene that results in a PTC. At the time of axis formation there is no discernible decrease in this PTC-containing transcript in vivo, indicating that the mammalian Zic3 transcript is relatively insensitive to NMD, prompting the need to re-examine the molecular function of the truncated proteins predicted from human studies and to determine whether the N-terminal portion of ZIC3 possesses dominant-negative capabilities. A combination of in vitro studies and analysis of the Ka phenotype indicate it is a null allele of Zic3 and that the N-terminal portion of ZIC3 does not encode a dominant-negative molecule. Heterotaxy in patients with PTC-containing ZIC3 transcripts probably arises due to loss of ZIC3 function alone.

The mammalian gonad is adapted for the production of germ cells and is an endocrine gland that controls sexual maturation and fertility. Gonadal sex reversal, namely, the development of ovaries in an XY individual or testes in an XX, has fascinated biologists for decades. The phenomenon suggests the existence of genetic suppressors of the male and female developmental pathways and molecular genetic studies, particularly in the mouse, have revealed controlled antagonism at the core of mammalian sex determination. Both testis and ovary determination represent design solutions to a number of problems: how to generate cells with the right properties to populate the organ primordium; how to produce distinct organs from an initially bipotential primordium; how to pattern an organ when the expression of key cell fate determinants is initiated only in a discrete region of the primordium and extends to other regions asynchronously; how to coordinate the interaction between distinct cell types in time and space and stabilize the resulting morphology; and how to maintain the differentiated state of the organ throughout the adult period. Some of these, and related problems, are common to organogenesis in general; some are distinctive to gonad development. In this review, we discuss recent studies of the molecular and cellular events underlying testis and ovary development, with an emphasis on the phenomenon of gonadal sex reversal and its causes in mice and humans. Finally, we discuss sex‐determining loci and disorders of sex development in humans and the future of research in this important area. WIREs Dev Biol 2012, 1:559–577. doi: 10.1002/wdev.42This article is categorized under: Gene Expression and Transcriptional Hierarchies > Sex Determination Signaling Pathways > Cell Fate Signaling Birth Defects > Organ Anomalies