Figure 4

PCP-Dependent Elongation Is Required for Regulating Exposure to BMP and Wnt Activity

(A–F) Expression of bmp7 (A–C) and TCF::GFP (D–F) as a time course at 2 hpc, 5 hpc, and 7 hpc reveals both signaling domains are spread across the explant evenly at early time points and are restricted to either end of the explant by 7 hpc. TCF::GFP assayed by HCR against GFP mRNA for immediate reporter activity readout is shown (2 hpc bmp7 2/3, tcf.:gfp 4/6; 5 hpc bmp7 5/5, tcf.:gfp 10/10; 7 hpc bmp7 4/4, tcf.:gfp 4/5; expression observed/total imaged).

(G–M, O–R, and T–W) The time course reveals the isolation of the BMP and Wnt domains to either end of the explant allows expression of hindbrain marker (G–I) krox20 in a characteristic two-stripe pattern. (G) Initially no expression is observed (n = 0/6; expression/total imaged), followed by (H) expression of a single stripe (n = 7/8; expression/total imaged) at 7 hpc and then (I) two stripes by 10 hpc (n = 5/8 expression/total imaged). Inhibition of convergence and extension using dominant-negative dishevelled injected at the one-cell stage (O–R) and treatment with 2.5 μM blebbistatin (T–W) reveals that the Wnt/TCF and BMP domains do not separate as observed in the (J–M) control.

(N, S, and X) Description of these profile quantitatively through normalization of the long axis of the explant and normalization of signal intensity between 0 and 1 (n = minimum 7 per condition; line represents mean). The lack of low BMP moderate Wnt/TCF domain can be observed in the central region of the explants compared to control. (N) displays a control profile, (S) displays the profile of a DEP+ explant, and (X) displays a profile of an explant treated with Blebbistatin.

(N’, S’, and X’) The lack of low BMP moderate Wnt/TCF domains results in significantly altered patterns of krox20 with no double-stripe patterns observed other than in controls. Scale bar represents 50 μm. Time-lapse data from signal reporters are available in Video S4.