Normal sex differentiation depends largely on the healthy development of the bipotential gonad,
which is identical in both sexes during early stages of embryonic development. Sex differentiation
towards the female phenotype is initiated by the expression of pro-ovarian genes, among which
Forkhead Box L2 (FOXL2) is an important regulator. Moreover, FOXL2 was found to be one of the
genes most widely implicated in female disorders of sex development (DSD). However, there is a
lack of understanding regarding its precise role during ovarian differentiation and development.
In order to study the gene during early gonadal development, human embryonic stem cells (hESCs)
were used as a model. An inducible FOXL2 activation line was generated in vitro, by applying the
CRISPR/Cas9 technique in combination with the tetON and destabilized DHFR systems. The cells
were also subjected to gonadal differentiation, based on a previously established protocol.
The results showed that the establishment of the activation line was successful, and expression of
FOXL2 could only be observed in cells that were treated with trimethoprim and doxycycline.
Similar findings were observed in the differentiated activator cells, as again only the induced cells
expressed FOXL2. On the other hand, both induced and non-induced differentiated cells showed
expression of bipotential gonadal marker genes LHX9, EMX2, GATA4 and WT1. However, in the
induced cells a lower relative expression of these markers could be observed. Therefore it seems
that relative expression of bipotential gonadal markers was affected by FOXL2 activation. The
expression of female gonadal marker genes RSPO1, FSHR, WNT4, AMH and FST was not
influenced by FOXL2 activation during gonadal differentiation, as most of the markers showed
similar levels of expression in both induced and non-induced cells. Therefore further research needs
to be conducted to determine optimal time point of FOXL2 activation during differentiation.
Nevertheless, an in vitro model could be generated, which could help in the future to further study
the role of FOXL2 in gonadal differentiation, and to better understand pathological mechanisms
underlying female DSDs.
