Manipulation of Signal Gradient and Transcription Factors Recapitulates: Multiple Hypothalamic Identities

Abstract

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.

Acquisition of neural fate by combination of BMP blockade and chromatin modification

“…Herein, we differentiated ES cells into forebrain-type neural progenitor cells by using a three-dimensional differentiation system. The differentiation medium, namely, growth factor-free chemically defined differentiation medium (gfCDM), 49 , 50 , 51 which contains no unidentified growth factors to which the effects of exogenously treated growth factors can be easily evaluated. With this differentiation protocol, Nanog expression was drastically reduced ( Figures 1 A and 1B) compared to that in ES cells four days after the start of differentiation (hereafter denoted as day 4), suggesting that the cells entered the somatic cell state.…”

“…Furthermore, according to the extension of the differentiation until day 7 with ChIR99021 at day 4 onwards, Pax6 ( Figures 2 L and 2M) and Rax ( Figures 2 N and 2O) were still not expressed in the Phc1 -KO cells, with abundant Nanog expression ( Figures 2 P and 2Q). Treatment with SAG on day 3 was carried out to attempt ventral diencephalic differentiation, 49 , 51 but the Phc1 -KO cells did not express any early hypothalamus cells positive for Nkx2.1 at day 7 ( Figures S2 A and S2B). Together, these findings suggest that Phc1 -KO cells have essentially lost the competence to initiate neural differentiation and essentially abandoned neural differentiation.…”

“…In this protocol, the diencephalic area, including the prethalamic and thalamic areas, was generated unless the differentiation was modified by the addition of exogenous signals. 51 At the same concentration of 0.5 μM, ChIR99021 (Sigma) was treated from day 4 onward to form the retinal structure 55 and SAG (Selleck) at day 3 for the hypothalamus, 51 respectively. BMP4 ( Figure 1 ; R&D Systems) and LDN193189 ( Figures 5 H–5M; Selleck) were used when necessary at 10 ng/ml and 50 nM, respectively, at the start of differentiation, and the cells were collected for analysis at day 4.…”