Hedgehog Activity Gradient in Combination with Transcription Network Confers Multiple Hypothalamic Identities

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.