Further Development 13.8: Transcriptional Cross-Repression by the Downstream Shh and TGF-β Effector Proteins

Neural Tube Formation and Patterning

In vertebrates, the main downstream effectors of Shh signaling are the Gli family of transcription factors, which function as repressors or activators based, respectively, on the absence or presence of Shh (see Figure 4.22 in the textbook to review the Hedgehog pathway). Therefore, Shh from the notochord and floor plate is transduced into a ventral-to-dorsal gradient of Gli activators to Gli repressors. It is interesting that the pattern of cells with Gli activator function in the mouse neural tube was shown not only to be a gradient, but also to change over time (see Further Development 13.7 online; Balaskas et al. 2012; Cohen et al. 2015). An early expansion of Gli activator function coincided with the initial induction of the broad and overlapping expression of progenitor cell transcription factors, yet Gli activity was not maintained over the course of cell differentiation in the neural tube. Despite this reduction in Shh signaling over time, the domains of progenitor-specific transcription factors still became highly refined, with tight borders between each domain. This result suggests that this level and duration of Shh are sufficient for cell specification, which is the case only in the context of a robust gene regulator network that maintains the Shh-induced pattern of gene expression (reviewed in Briscoe and Small 2015).Gene regulatory networks of progenitor cells play a direct role in reinforcing, refining, and maintaining progenitor cell fates through the mechanism of transcriptional cross-repression, whereby transcription factors repress each other. Gain- and loss-of-function manipulations of transcription factors in progenitor cells have demonstrated that different transcription factors, such as Olig2 and Nkx2.2, which are expressed in adjacent domains, can mutually repress each other’s expression, thereby helping define the borders between adjacent domains (Figure 1; Balaskas et al. 2012). Transcriptional cross-repression integrated into a model that includes Shh provides a mechanism for a cell to “remember” the Shh signal and, consequently, its position in the neural tube (Figure 2; see also Figure 1F).

**Figure 1** Transcriptional cross-repression in neural progenitor cells. (A–D) Transverse sections of the mouse neural tube labeled for Nkx2.2, Olig2, and GFP-expressing cells. (B) Loss of *Olig2* leads to expansion of the Nkx2.2 domain (compare the brackets in A and B). (C) Gain of *Olig2* function by ectopically expressing the gene through electroporation represses Nkx2.2 expression (arrow, yellow outline). (D) Loss of the *Pax6* gene leads to a marked expansion of Nkx2.2 at early and later time points (compare the brackets in A with the brackets and arrows in C and D), yet Olig2 is lost only at the later time point (80 hph). Abbreviations: hph, hours post headfold stage; hpe, hours post electroporation. (E) Shorthand explanation of the experimental manipulation and its results (above line) and the conclusion that can be drawn from these results (below line). (F) The gene regulatory network combining the Gli (activated by the Hedgehog pathway), Olig2, Nkx2.2, and Pax6 transcription factors.

**Figure 2** Model for interpreting the Shh morphogen gradient. Signal-mediated patterning of the ventral portion of the neural tube. At the earliest time of induction (t₀– t₁), Shh from the notochord (green triangles) induces Gli (purple) in the floor plate cells. This action is not sufficient to activate Olig2 or Nkx2.2 or to repress Pax6. As development ensues, Gli is able to induce Olig2, which inhibits Nkx2.2 and Pax6. As the most ventral cells experience higher concentrations of Shh for longer periods, Nkx2.2 is activated and suppresses Olig2. This pattern can be retained even when Gli levels decrease.

Literature Cited

Balaskas, N. and 7 others. 2012. Gene regulatory logic for reading the Sonic Hedgehog signaling gradient in the vertebrate neural tube. Cell 148: 273–284.
PubMed Link

Briscoe, J. and S. Small. 2015. Morphogen rules: Design principles of gradient-mediated embryo patterning. Development 142: 3996–4009.
PubMed Link

Cohen, M., A. Kicheva, A. Ribeiro, R. Blassberg, K. M. Page, C. P. Barnes, and J. Briscoe. 2015. Ptch1 and Gli regulate Shh signalling dynamics via multiple mechanisms. Nat. Commun. 6: 6709.
PubMed Link

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