Murine pluripotent stem cells with a homozygous knockout of Foxg1 show reduced differentiation towards cortical progenitors in vitro.

Foxg1 is a transcription factor critical for the development of the mammalian telencephalon. Foxg1 controls the proliferation of dorsal telencephalon progenitors and the specification of the ventral telencephalon. Homozygous knockout of Foxg1 in mice leads to severe microcephaly, attributed to premature differentiation of telencephalic progenitors, mainly of cortical progenitors. Here, we analyzed the influence of a Foxg1 knockout on differentiation of murine pluripotent stem cells (mPSCs) in an in vitro model of neuronal development. Murine PSCs were prone to neuronal differentiation in embryoid body like culture with minimal medium conditions, based on the intrinsic default of PSCs to develop into cortical progenitors. Differences between Foxg1 wildtype (Foxg1WT) and knockout (Foxg1KO) mPSCs were analyzed. Several mPSC lines with homozygous mutations in Foxg1 were produced using the CRISPR/Cas9 system leading to loss of functional domains. Analysis of mRNA expression using quantitative Real-Time (q) PCR revealed that Foxg1KO mPSCs expressed significantly less mRNA of Foxg1, Emx1, and VGlut1 compared to Foxg1WT controls, indicating reduced differentiation towards dorsal telencephalic progenitors. However, the size of the derived EB-like structures did not differ between Foxg1WT and Foxg1KO mPSCs. These results show that loss of dorsal telencephalic progenitors can be detected using a simple and rapid differentiation protocol. This study is a first hint that this differentiation method can be used to analyze even extreme phenotypes that are lethal in vivo.