Gene and Protein Structure

FOXL2 is a 376 amino acid protein encoded by a single-exon gene of 2.7kb, the encoded protein contains a highly conserved 110 amino acid forkhead box DNA binding domain for which it was names.^[2] This forkhead domain consists of a helix-turn-helix motif made primarily by three alpha helices and two large loops, or wings as they are sometimes called.^[3] One of the interesting features of the forkhead domain is also a 14 amino acid polyalanine (poly-A) tract which, though its role has not fully been elucidated, is an incredibly important domain. The poly-A tract is of significance due to it being a mutational hotspot in the critical DNA binding domain of the protein – 93% of all in-frame mutations (making up 33% of all mutations in the protein) are expansions of the poly-A tract.^[2][6][7]

Protein Function

Ovary Formation and Maintenance

During embryonic ovary development a bipotential gonadal ridge forms from lateral mesoderm adjacent to the developing kidneys. At this stage the ridge is in an undifferentiated state and basally expresses Wnt1, Sox9, Wnt4, Lhx9, FGF9, GATA4 and SF1. In females the presence of a second X chromosome causes Wnt4 and R-spondin 1 to be upregulated, therefore promoting female ovary development. These factors induce the expression of FOXL2 which then suppresses Sox9 (the factor responsible for testicular cell development). With the expression of Wnt4 and R-spondin 1 bi-potential ridge cells begin to differentiate into granulosa and thecal cells which will form the basis of mature follicles in adult ovaries.

WNT4 and R-spondin 1 block the expression of Sox9 in female embryos.^[8] FoxL2 is expressed once the ovaries are determined.^[9] When germ cells enter meiosis 1 at embryonic day 13.5, the cells are now committed to ovarian production and being to develop oocytes. Then these cells are held at the dictyate phase at birth . On embryonic day 15, FIGα (an oocyte-specific transcription factor present in females only) is expressed in germ cells and it signals the production of zona pellucida protein.^[10] As this occurs in the germ cells, on embryonic day 11.5 somatic cells express Wnt4 which induces the production of follistatin. On embryonic day 12, FoxL2 is expressed which stimulates the differentiation of pregranulosa cells. A mutation or disruption at this stage can lead to the failure of transitioning of granulosa cells from squamous to cuboidal stage, this stops folliculogenesis and leads to early depletion of follicles resulting in premature ovary failure. This reveals that FOXL2 is important for proper differentiation of granulosa cells during folliculogenesis. Furthermore, on embryonic day 16, WNT4 and follistatin both work together to antagonize the formation of testis vasculature and maintain the survival of female germ cells.^[10]

After birth, zona pellucida proteins, whose production is induced by FIGα, couple the oocyte and granulosa cells. FIGα and FOXL2 work together to form primordial follicles, in which FIGα is responsible for recruiting granulosa cells and FOXL2 is responsible for further differentiation of pregranulosa cells. Throughout female adulthood, FoxL2 is known to be continuously expressed in the granulosa cells and it is responsible for granulosa cell differentiation and proliferation and plays a role in ovary maintenance and female phenotype.^[10][8]

Eyelid Formation

In mice, the eyelid begins develops between embryonic days 13.5 and 15.5. Experimental results reveal that during this time, FoxL2 is expressed in the margins and protruding ridges of eyelids forming outside the cornea and the mesenchyme around the optic cup. At embryonic day 15.5, the eyelid is developing and FoxL2 is expressed in the periocular mesenchyme. The eyelid develops from the surface of the ectoderm fold that rises from the frontonasal and maxillary process. The eyelid begins to lengthen and cover the cornea as the mesenchyme proliferates. As the eyelids lengthen from each side, they fuse and later separate but remain closed until after birth.^[14] In eyelid development, FOXL2 is found to be responsible for the development and differentiation of the superior levator eyelid muscle.^[15]

Pituitary Gland

During embryonic development, the anterior neural ridge develops the anterior pituitary and the neural ridge forms the hypothalamus.^[16] During the development of the pituitary gland in mice, FoxL2 is strictly expressed in the ventral part of the Rathke’s pouch on embryonic day 10.5.^[16][14] The restricted expression allows a gradient formation of transcription factors which determine the pituitary cell lineage determination.^[14] FoxL2 is also expressed in the pituitary primordium and oral ectoderm.^[16] Furthermore, FoxL2 is continuously expressed in the anterior pituitary throughout development and into adulthood, where it is important for promoting pituitary cell differentiation and the maintenance of the pituitary gland.^[21]

Roles in BPES

Blepharophimosis-Ptosis-Epicanthus inversus Syndrome BPES is an autosomal dominant disorder caused by mutations in the FOXL2 gene.^[2][9] The disorder is characterized by small palpebral fissures (blepharophimosis), drooping eyelids (ptosis) and a small skinfold running inward from the lower lid (epicanthus inversus). In females BPES I patients develop premature ovarian failure, defined as early onset menopause (before the age of 40), low levels of circulating estrogen and elevated gonadotropin concentrations, while in BPES II individuals have the same characteristics without the premature ovarian failure.^[24]

BPES I

Although there are many mutations leading to BPES I, the most common is caused by stop codons in FoxL2 that interrupts translation, producing truncated proteins missing the C-terminal sequence 3’ to the forkhead domain. This is believed to cause loss of some or all function of the protein, contributing to its autosomal dominant inheritance.^[24]

FoxL2 is a very important gene expressed in the developing eyes, ovaries and pituitary gland. During eye development, two ectodermal folds containing mesenchymal cores grow over the developing cornea and fuse. These folds eventually form the eyelids, which undergo a subsequent re-opening after birth. This process is mediated by the expression of FoxL2, which is found in highest concentration around the mouse cornea and all protruding ridges (which form the basis of eyelid development). Patients with BPES I however have local deficiencies of FOXL2, which causes underdevelopment of eyelid muscles and the supporting structures. It is common to see fibrosis and hypoactivity of the levator palpebrae superior muscles which results in an inability to properly lift the eyelid. This causes patients to overuse frontalis muscles in order to lift eyelids, resulting in an increased vertical brow width. Patients also attempt to oppose these physical ailments by tipping their head back in a characteristic manner to increase visual efficiency.^[24]

In ovary development FoxL2 is expressed in the follicular cells and oppose Sox9 expression, therefore maintaining their identity as thecal and granulosa cells through adulthood. In patients with BPES I there is a local deficiency of FOXL2 causing a multitude of issues ultimately resulting in premature ovarian failure (defined as early onset menopause). Because FOXL2 is involved in the differentiation of bi-potential somatic cells into granulosa and thecal cells, deficiencies can result in insufficient recruitment of follicle cells, therefore resulting in an overall decreased primordial follicle pool (total egg pool). FOXL2 is also involved in regulating actavin- BA and AMG, which are two proteins that inhibit follicles from transitioning between the immature and mature state. When FOXL2 is unable to regulate these proteins more eggs are recruited at the onset of ovulation and therefore the primordial follicle pool (egg pool) is depleted at a much faster rate. Since FOXL2 is also believed to affect GNRH receptors in the pituitary, less FSH and LH are produced, therefore once again affecting the health and maintenance of the primordial follicle pool.

In order to treat patients with BPES I eye surgeries may be performed to reconstruct eye morphology, and eggs may be frozen for later use in vitro fertilization. Although there is a lot of work out there being done on BPES patients, more research is needed to better understand the full mechanism and workings of the disorder.

BPES II

One cause of BPES II is from mutations in the FoxL2 gene where there may be a duplication of 10 more alanine residues in the poly-A domain causing elongation of the protein. This mutation results in eye malformation however, premature ovarian failure is not associated with this mutation.^[24]

Avenues for Future Research

For our big experiment we are hoping to resolve the issue of physical and psychological identity in trans-genders. Specifically we are attempting to provide a solution to those females who wish to transform into males. We will do this by blocking the action of FOXL2 so that SOX9 is expressed causing follicular cells to differentiate into male gonadal tissue. This should be accompanied by the secretion of testosterone at concentrations comparable to regular XY males. This offers an alternative to hormone injections and gives females an even greater sense of identity as they begin to acquire male genitalia tissue.

FOXL2 genes are expressed in granulosa and theca cells in the female ovaries. During development FOXL2 is necessary for the bi-potential gonadal cells to differentiate into follicular cells opposed to testicular tissue (sertoli and lydig cells). FOXL2 works by promoting female tissue development by suppressing SOX9; a transcription factor necessary for male tissue development. In our experiment we will attempt to inhibit FOXL2 protein expression by silencing the gene with siRNA. With FOXL2 repressed, SOX9 will be expressed causing granulosa and theca cells to differentiate into Lydig and Sertoli cells. A similar experiment was conducted in Mice, and found that when FOXL2 was deleted using the drug Tamoxifen, adult granulosa and theca cells did in fact develop into Sertoli like cells. Therefore emphasizing the fact that granulosa and thecal cells retain plasticity into adulthood.^[30]

We will conduct our experiment using mice as our test subjects. We will inject ovary specific siRNA directly into the ovaries of 2 month old female mice. We expect the siRNA to be taken up into granulosa and thecal cells. The siRNA being complementary to the FOXL2 coding region, will bind to FOXL2 mRNA, cleaving and destructing it so that the FOXL2 protein is not translated. The siRNA remains unharmed in this process, therefore able to go on and look for more mRNA molecules. Even if FOXL2 is still transcribed, siRNA present in the cell will continue to degrade mRNA resulting in a silenced FOXL2 gene.

In order to test the progress of our experiment we will take blood samples one month after siRNA injections to test for testosterone. We expect that XX mice will show higher than normal levels of testosterone as the hormone is produced in Sertoli cells. We will also use fluorescently tagged antibodies targeted for SOX9 and conduct fluorescent microscopy on tissue sections to see if the antibodies are localized to the differentiating tissue.

Some problems associated with our experiment are that FOXL2 expression will be affected in other organ systems such as the pituitary causing other issues associated with hormone production. Another potential issue is that we will create infertility in our test subjects, which could create problems when attempting to extend our experiment to humans.

If the experiment proves successful we will be able to help females around the world re-establish their physical identity. This could be another step forward in gender re-assignment processes allowing humans to ultimately choose their desired sex.

Another method of testing the effects of silencing FoxL2 expressiong would be to use the same transgenic mouse concept as described in Chow et al. “A doxycycline-inducible, tissue-specific aromatase-expressing transgenic mouse”, this would allow tissue specific silencing of FoxL2, and thus give better results. In this method we take an anti-sense cDNA of FoxL2, which when expressed would cause post-transcriptional gene silencing of FoxL2, and connect it to a tetracycline-responsive promoter – this promoter is inducible by the presence of the molecule doxycycline. The promoter has been further modified to also require a transactivator (in this case called rtTA) for proper activation of transcription. The transactivator is connected to a separate ovary-specific promoter, to ensure that it is only present in the ovaries. Neither the transactivator nor the presence of doxycycline are sufficient on their own for expression of the anti-sense FoxL2 gene, both are necessary for successful activation. ^[31]

Once these two constructs have been made they can be microinjected into the nucleus of separate mice eggs and allowed to develop into single-transgenic mice. These mice can then be backcrossed to achieve homozygosity before being crossed to each other to form the double transgenic mice.

These double-transgenic mice can be fed doxycyline, which will allow expression of the anti-sense cDNA FoxL2 gene anywhere there is rtTA present – which, due to its promoter being ovary specific will occur only in the ovaries. Because of this both factors necessary for proper transcription of the anti-sense FoxL2 are only present in the ovary there will be no expression anywhere else in the organism – removing an important possible source of confounding variables.

Using RT-qPCR expression levels of the anti-sense FoxL2, FoxL2, and other molecules such as testosterone and Sox9 can be tested to determine if the post-transcriptional gene silencing of FoxL2 is successful, and if so what effects it has on those important sex-specific genes. Following a period of time the ovaries could then also be biopsied to check for possible changes in cell type such as to sertoli cells.

Controls should also be run in the cases where there is no administration of doxycycline, no gene attached to the tetracycline-responsive promoter, or no presence of the transactivator. This would be to determine there are no unintended effects of any of these factors alone.

Should the experiment prove to be successful we would expect to see severely reduced levels of FOXL2, and increased levels of transcription of Sox9 and testosterone. In a biopsy we would also expect to see some sertoli and lydig cells in the ovaries.

Further Reading

References