Figure 2. RA is responsible for the loss of sensitivity of dorsal NT progenitors to BMP during the transition from NC to RP.

(A-D’) Immunostaining for pSmad1/5/9. Embryos at E2.5 (27-8ss) were co-electroporated with RARα403, Cyp26A1 or with a control PCAGG vector, along with GFP, and analyzed at 35ss (control only, NC stage) and at E4. Note the presence of BMP activity in premigratory NC (A) and its absence in the RP of control embryos (N = 8, B-B’). Whereas only ventrally located dorsal interneurons are positive in control embryos, the RP domain itself is positive in Cyp26A1 (N = 8) and RARα403-electroporated embryos (N = 7, C-C',D-D'). (E) Quantification of pSmad staining intensity (measured area includes dorsal interneurons). Imaging and quantification were performed at somite levels 24–26. Four-to-21 sections per embryo were analyzed. *p < 0.05, **p < 0.005, one-way ANOVA with post-hoc Tukey’s tests. (F–O) In situ hybridization of embryos electroporated with Cyp26a1 or RARα403 at E2.5 (27 ss) and analyzed at E4. Asterisks mark the RP domain. (F–I) ISH for BAMBI, showing downregulation in the RP of Cyp26A1- and RARα403-treated embryos. (I) For quantification of the intensity of BAMBI, 6-to-21 sections per embryo were analyzed at somite levels 24–26. N = 7,7 and 6 embryos for control, Cyp26A1 and RARα403, respectively. *p < 0.05, ****p < 0.0001, one-way ANOVA with post-hoc Tukey’s tests. (J–L) ISH for hairy1, showing downregulation in the RP of RARα403-treated embryos. (L) For quantification of the intensity of hairy1, 7–18 sections per embryo were analyzed at somite levels 24–26. N = 7 embryos for each group. **p < 0.005, Student’s unpaired t-test. (M–O) ISH for Grem1, showing downregulation in the RP of RARα403-treated embryos. For quantification of the intensity of Grem1, 11–27 sections per embryo were analyzed at somite levels 24–26. N = 8 embryos for each group. **p < 0.005 via Student’s unpaired t-test. Abbreviations, dI1, dorsal interneurons 1, RP, roof plate. Scale bar, 50 μm.

Figure 2—figure supplement 1. Electroporation of Cyp26A1 or RARα403 into the NT effectively downregulates RA activity.

(A–D) Measurement of RA activity using RARE-RFP (magenta) in embryos electroporated with Cyp26a1, RARα403 or control GFP (green). NTs were co-electroporated bilaterally at E2 (23-24ss) and analyzed 30 hr later. GFP was delivered along with the reporter construct and was used to assess the relative intensity of RFP fluorescence on whole-embryo images (D). N = 7, 7 and 6 embryos for control, Cyp26A1 and RARα403 groups, respectively. ****p < 0.0001, one-way ANOVA with post-hoc Tukey’s tests. (E–F) The same experimental conditions as in (A–D) were applied to unilaterally deliver RARE-AP (electroporated side facing the top). Note the robust AP staining in the control embryo, compared to the weak staining in the RARα403-treated embryo. N = 4/4 for both groups. Scale bar, 100 μm.

Figure 2—figure supplement 2. RA promotes the downregulation of Wnt signaling in the nascent RP.

(A–C) A destabilized Wnt reporter, 12XTopFlash-d2EGFP, was electroporated bilaterally into the NT of E2.5 embryos (27 ss), and analyzed at E3 (35 ss, NC stage, control only) and at E4 (RP stage). RFP was co-electroporated to mark the electroporated region. Note the presence of Wnt activity in the premigratory NC (A-A’, arrowheads) compared to its absence in the control RP (B-B’). Wnt activity is upregulated in RARα403-electroporated embryos compared to RFP only controls. Note the GFP⁺ cells in the RARα403-treated embryo (C-C’, arrowheads). (D) Measurement of d2EGFP fluorescence signal intensity. N = 9 embryos for control NC and RP groups, and N = 8 for the RARα403-treated group, 12 sections were analyzed per embryo at somite levels 24–26. ****p < 0.0001, *p < 0.05, one-way ANOVA with post-hoc Tukey’s test. Abbreviations, NT, neural tube. Scale bar, 50 μm.