Fig. 2. Effects of ectopic Shh signaling on regional patterning in the telencephalon. Embryos were injected with ActSmo-expressing virus at E9.5 and analyzed at E14.5. Histochemical staining for PLAP reveals the location of virally infected cells co-expressing ActSmo (A,D,G,I,K,N). Adjacent sections were assayed for regionally expressed homeodomain genes (A-J) or for the expression of downstream targets of Shh signaling pathway (K-O). (A-C) Confocal immunohistochemistry for PLAP (A) and Pax6 (C) proteins within the lateral neocortex shows a repression of Pax6 expression in clusters of cells infected by ActSmo (asterisks). (G-J) Coronal sections of ActSmo-infected brains stained for ventral genes expression. Ectopic Nkx2.1 expression is restricted to the LGE and lateral neocortex (H, arrows), whereas Gsh2 expression can be induced throughout the neocortex (J, arrows). Dotted bracket illustrates weaker levels of induction within the medial neocortex. Sagittal sections (D-F) reveal a similar restriction along the rostrocaudal extent of the telencephalon. Asterisks in D-J represent the sites of endogenous expression. (K-O) Ectopic expression of ActSmo induces the upregulation of Gli1 and Ptch expression all along the dorsoventral (L,M, arrows and bracket) or anteroposterior (O) axes, showing that the telencephalon has the competency to respond to Shh signaling at this age. The mismatch between areas of viral infection and Ptch or Gli1 induction reflects the fact that these are visualized in adjacent sections. This is unavoidable because of the incompatibility of PLAP histochemistry and in situ hybridization. (P,Q) Schematic representations of a sagittal (P) and coronal (Q) section showing Nkx2.1 and Gsh2 endogenous domains of expression as well as the regions where their expression can be induced by Shh signaling.

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