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Foxl2 function in ovarian development

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Abstract

Foxl2 is a forkhead transcription factor essential for proper reproductive function in females. Human patients carrying mutations in the FOXL2 gene display blepharophimosis/ptosis/epicanthus inversus syndrome (BPES), an autosomal dominant disease associated with eyelid defects and premature ovarian failure in females. Recently, animal models for BPES have been developed that in combination with a catalogue of human FOXL2 mutations provide further insight into its molecular function. Mice homozygous mutant for Foxl2 display craniofacial malformations and female infertility. The analysis of the murine phenotype has revealed that Foxl2 is required for granulosa cell function. These ovarian somatic cells surround and nourish the oocyte and play an important role in follicle formation and activation. Mutations upstream of FOXL2 in humans, not affecting the coding sequence itself, have also been shown to cause BPES, which points to the existence of a distant regulatory element necessary for proper gene expression. The same regulatory sequences may be deleted in the goat polled intersex syndrome (PIS), in which FoxL2 expression is severely reduced. Sequence comparison of FoxL2 from several vertebrate species has shown that it is a highly conserved gene involved in ovary development. Thus, the detailed understanding of Foxl2 function and regulation and the identification of its transcriptional targets may open new avenues for the treatment of female infertility in the future.

Introduction

Foxl2 was first identified in 1998 as a novel member of the winged helix or forkhead family of transcription factors in a screen for genes involved in mouse pituitary gland development. P-Frk, as it was initially named (for pituitary forkhead factor), showed differential expression in the most ventral part of Rathke’s pouch, coincident with signaling gradient boundaries specifying the gonadotroph lineage [1].

The FOX (Forkhead box) family of transcription factors shares a common DNA binding domain of up to 110 amino acids whose helix–turn–helix structure resembles a butterfly, hence the alternative name “winged helix.” Outside of the conserved forkhead domain, there is little similarity between family members which include transcriptional activators as well as repressors [2].

In humans (and in mice), the forkhead family consists of 39 members, which influence a diverse range of biological processes. For instance, forkhead genes are necessary for the establishment of the body axis, for the development of tissues from all three germ layers, for metabolic processes as well as cell cycle control [2]. Currently, eight different human developmental disorders have been associated with mutations in forkhead genes, with a striking prevalence of eye abnormalities. Namely, mutations in FOXC1, FOXC2, FOXE3, and FOXL2 lead to ocular phenotypes in mice and humans [3].

The amino acid sequences of the human FOXL2 and mouse Foxl2 genes are more than 95% identical [4]. The murine Foxl2 protein consists of 375 amino acids and so far has no predicted functional domains apart from the DNA binding domain and a 14 amino acids polyalanine tract [5], [6]. Polyalanine tracts (poly-A) are often found in association with transcriptional repression but there is no evidence so far that alanine tracts have repressive function per se [7]. The human genome comprises an estimate of ∼300 genes bearing polyalanine stretches with a surprising bias for the occurrence of poly-A within transcription factors, suggesting a role for alanine-rich regions in transcriptional control [8]. Current speculations include mechanisms in which polyalanine tracts serve to properly orient different domains of the protein with respect to each other, thus being required for proper protein–protein or protein–DNA interactions [7] (Fig. 1).

Section snippets

Blepharophimosis/ptosis/epicanthus inversus syndrome

In 2001, Crisponi et al. [5] reported that loss of a functional FOXL2 allele is underlying the human blepharophimosis/ptosis/epicanthus inversus syndrome (BPES). This autosomal dominant disorder manifests itself in small palpebral fissures (blepharophimosis), drooping eyelids (ptosis), a small skinfold running inward from the lower lid (epicanthus inversus), and a broad nasal bridge (telecanthus). In BPES type I, these craniofacial abnormalities are associated with premature ovarian failure

Regulation of FOXL2 expression

Apart from the minimal or core promoter which lies directly 5′ of the transcriptional start site of the gene and which is necessary for the assembly of the RNA synthesizing protein complex, many genes require additional cis-acting regulatory elements that serve as transcription factor binding sites for their correct spatiotemporal expression. However, in some cases these elements can be quite far from the transcriptional start site and lie upstream, within introns, or downstream of the gene.

The goat PIS syndrome

Interestingly, FoxL2 gene function is also compromised in the goat polled/intersex syndrome (PIS). The PIS mutation leads to the absence of horns in male and female goats (polledness) and is inherited in a dominant fashion. Intriguingly, it also causes XX female-to-male sex reversal in a recessive manner [26]. The autosomal locus underlying the goat syndrome was mapped to a region homologous to the human chromosome band 3q23 which harbours the FOXL2 gene. An 11.7 kb DNA element containing mainly

Foxl2 function in mouse development

Consistent with human BPES in which ovary and eyelid development are affected, the mouse Foxl2 gene was shown to be expressed in embryonic eyelids and ovarian follicles [5]. We have further characterized the murine expression pattern of Foxl2 during ovary organogenesis and folliculogenesis using a LacZ knock-in strategy. Foxl2 expression is sexually dimorphic, starting at 12.5 dpc in female gonads but is not detectable in developing male gonads. This explains why male BPES patients do not

Molecular mechanism of Foxl2 function

In contrast to the wealth of phenotypic characterizations and reports on Foxl2 function in vivo, not much is known about the molecular mechanism of Foxl2 action. One speculation is that Foxl2 regulates TGFβ-related signaling pathways involved in both eyelid development and ovarian function, such as activin/inhibin. The mouse mutant for Inhbb also shows eyelid defects, is born with open eyes, and has a reproductive phenotype [9], [40]. Interaction between forkhead transcription factors and

Evolutionary conservation of FoxL2

The study of evolutionarily conserved mechanisms of sex determination and gonadogenesis has yielded important insights for reproductive biology [51]. Numerous publications have therefore addressed the question whether FoxL2 acts as a conserved factor for ovary development in different organisms. The FoxL2 sequence is very similar among all mammals studied, with nearly identical proteins found in human, mouse, rat, rabbit, goat, cow, and pig [4], [52]. Remarkably, all of those orthologues have

Outlook

Mouse genetics will continue to be useful to elucidate the molecular mechanisms of Foxl2 function. The generation of a floxed Foxl2 allele in combination with tissue-specific Cre lines should allow a more detailed dissection of the roles Foxl2 plays in ovarian, craniofacial, and pituitary gland development. Similarly, mice genetically modified to harbour Foxl2 alleles with an expansion or deletion of the polyalanine tract (in combination with in vitro studies) could shed light on the purpose of

Acknowledgment

This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG, TR-341) (M.T.).

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