Figure 3

Nodal Signaling Is Upstream of PCP-Driven Convergence and Extension, which Drives Elongation

(A–C) The first signaling event that polarizes to a single point within the pescoid is that of Nodal signaling, demonstrated through (A–C) polarization of phospho-Smad 2/3 activity. This is shown in composite color images and as pSmad2/3 signal inverted images (A’–C’; 2 hpc n = 4/8; 3 hpc n = 5/8; 5 hpc n = 4/6; total with polarized signal/total imaged).

(D and E) We also observe polarized (D) ndr1 and (E) ndr2 expression in the elongation at 5hpc (ndr1 n = 4/6; ndr2 n = 4/6; expression observed/total imaged; total with polarized signal/total imaged).

(F–I) Treatment with the Nodal inhibitor SB-505124 between (G) 1 and 3 hpc inhibits elongation of the explants and (H) to a lesser extent when applied between 5 and 7 hpc when compared to controls at 7 hpc (F and I).

(J) The PCP components wnt11f2 and fzd2 are observed expressed in the elongating end of the pescoid with the spatial organization of these domains reflected in (J’) surface renderings of the HCR signal.

(K and K’) Inhibition of Nodal signaling between 1 and 3 hpc results in loss of wnt11f2 and frz2 expression.

(L–O) Inhibition of convergence and extension movements using (M) blebbistatin or (N) injection at the one-cell stage of a dominant-negative dishevelled construct blocks elongation when compared to controls (L and O), further supporting that elongation is caused by convergence and extension movements.

Elongation is demonstrated not be to be caused by polarized cell division in Figure S2. Explant sizes and the ability to form mesoderm are shown in Figure S3. Polarization of Nodal signaling with the absence of polarized β-catenin or FGF (ppERK) is further shown in Figure S4. Further time-lapse data from signal reporters are available in Video S4. Scale bar represents 50 μm.