During development, the hypothalamus emerges from the ventral diencephalon and is regionalised into several distinct functional domains. Each domain is characterised by a different combination of transcription factors, including Nkx2.1, Nkx2.2, Pax6, and Rx, which are expressed in the presumptive hypothalamus and its surrounding regions, and play critical roles in defining each area. Here, we recapitulated the molecular networks formed by the gradient of Sonic Hedgehog (Shh) and the aforementioned transcription factors. Using combinatorial experimental systems of directed neural differentiation of mouse embryonic stem (ES) cells, as well as a reporter mouse line and gene overexpression in chick embryos, we deciphered the regulation of transcription factors by different Shh signal intensities. We then used CRISPR/Cas9 mutagenesis to demonstrate the mutual repression between Nkx2.1 and Nkx2.2 in a cell-autonomous manner; however, they induce each other in a non-cell-autonomous manner. Moreover, Rx resides upstream of all these transcription factors and determines the location of the hypothalamic region. Our findings suggest that Shh signalling and its downstream transcription network are required for hypothalamic regionalisation and establishment.

Understanding the molecular mechanisms leading to retinal development is of great interest for both basic scientific and clinical applications. Several signaling molecules and transcription factors involved in retinal development have been isolated and analyzed; however, determining the direct impact of the loss of a specific molecule is problematic, due to difficulties in identifying the corresponding cellular lineages in different individuals. Here, we conducted genome‐wide expression analysis with embryonic stem (ES) cells devoid of the Rx gene, which encodes one of several homeobox transcription factors essential for retinal development. We performed three‐dimensional differentiation of wild‐type and mutant cells and compared their gene‐expression profiles. The mutant tissue failed to differentiate into the retinal lineage and exhibited precocious expression of genes characteristic of neuronal cells. Together, these results suggest that Rx expression is an important biomarker of the retinal lineage and that it helps regulates appropriate differentiation stages.

During development, the hypothalamus emerges from the ventral diencephalon of the neural tube and is regionalised into several distinct functional domains. Each domain is characterised by different combinations of transcription factors, the expression of which is regulated by signalling molecules and downstream transcriptional networks. Transcription factors, including Nkx2.1, Nkx2.2, Pax6 and Rx, are expressed in the presumptive hypothalamus and its surrounding regions from an early developmental stage and play critical roles in the development of these areas. However, the regulation of transcription factor expression and the details of the transcriptional network among them have not been fully elucidated.As early hypothalamus development takes place in the ventral region of the forebrain where Sonic Hedgehog (Shh) is expressed, we focused on the relationship between Shh and its downstream transcription factors and investigated the transcriptional regulation along the dorsal-ventral axis. Using a reporter mouse line, in vitro neural differentiation of mouse embryonic stem cells and gene overexpression in chick embryos, we found that Pax6, Nkx2.1 and Nkx2.2 are regulated epistatically by different Shh signal intensities. Nkx2.1 and Nkx2.2 mutually repress each other; however, they induce each other in a non-cell-autonomous manner. Moreover, Rx resides upstream of all these transcription factors and determines the location of the hypothalamic region along the dorsal-ventral and anterior-posterior regulations. Finally, we found that the Shh signal demarcates the diencephalic region from the retinal area. Our findings suggest that Shh signalling, and its downstream transcription network, are required for hypothalamic regionalisation and establishment of diencephalic cell fate.