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. 2022 Apr 8;11:e72723. doi: 10.7554/eLife.72723

Figure 6. Overlapping expression in RP of both NC and RP markers upon inhibition of RA signalling.

(A-B’’’) Embryos co-electroporated at E2.5 (27ss) with either control PCAGG or RARα403 along with a GFP plasmid. Embryos were fixed at E4, and adjacent sections were in-situ hybridized for foxd3 or Rspo1 and then superimposed to reveal overlapping domains of gene expression. Dotted lines delineate the Rspo1 expression domain. In control embryos, no foxd3 expression was detected in the RP (N = 2, and see also Figure 3). In RARα403-treated embryos, overlapping expression was detected in all four embryos examined. Imaging and analysis were performed somite levels 24–26. (C-D”) Immunostaining for SNAI2 and fluorescent ISH for Rspo1 were combined on the same sections of embryos electroporated at E2.5 (27ss) with either control PCAGG or RARα403 and analyzed at E4. Dashed lines mark the Rspo1 expression domain. Note the presence of SNAI2+Rspo1+ cells in the RP in D”. (E-G”) Immunostaining for Sox9 and fluorescent ISH for Rspo1 were performed as above. Note the presence of Sox9+Rspo1+ cells in the RP in F” and G’’. Additionally, delaminating Sox9+Rspo1+ cells (arrowheads in G-G”) were apparent under experimental conditions. (H,I) Quantification of SNAI2+Rspo1+ and Sox9+Rspo1+ cells in the RP. Imaging and analysis were performed at somite levels 24–26. N = 8 and 8 for SNAI2 in controls and RARα403. N = 9 and 10 embryos for control and RARα403 groups stained with Sox9, respectively. ****p < 0.0001, Welch’s t-test. Abbreviations, NT, neural tube. Scale bar, 20 μm.

Figure 6—source data 1. Overlapping expression in RP of both NC and RP markers upon inhibition of RA signalling.

Figure 6.

Figure 6—figure supplement 1. Inhibition of RA signaling only partially affects expression of definitive RP markers.

Figure 6—figure supplement 1.

ISH to monitor expression of RP-specific genes. Embryos were electroporated at E2.5 (27 ss) with either control PCAGG or RARα403, together with GFP, and analyzed at E4. Asterisks depict the RP domain. Imaging and analysis were performed at somite levels 25–27. (A–C) ISH for Slit1 reveals downregulation in the RP of RARα403-treated embryos. (C) Data quantification, N = 8 embryos for either control or RARα403 groups, 8–19 sections per embryo were analyzed. **p < 0.005. (D–F) ISH for Rspo1. (F) Data quantification, N = 8 embryos for either control or RARα403 groups, 12–21 sections were analyzed per embryo. (G–I) ISH for NDP (Norrin). (I) Data quantification, N = 6 embryos for either control or RARα403 conditions, 6 sections were analyzed per embryo. (J–L) ISH for draxin. (L) Data quantification, N = 8 control embryos and N = 7 embryos that received RARα403; 10–17 sections were analyzed per embryo. Intensity comparisons of ISH staining presented in C, F, I, and L were carried out using Welch’s t-test. Scale bar, 50 μm.
Figure 6—figure supplement 1—source data 1. Inhibition of RA signaling only partially affects expression of definitive RP markers.
Figure 6—figure supplement 2. Gain of RA signaling does not cause a premature upregulation of RP-specific genes.

Figure 6—figure supplement 2.

(A–J) ISH for the RP-specific genes BAMBI and Rspo1, to assess their onset of expression in control and VP16-RARα−treated NTs.(A–E) Embryos were electroporated unilaterally at E2 (22-24ss) with either control PCAGG or VP16-RARα, together with GFP and analyzed at 32-33ss for expression of BAMBI. (A-D’) Images taken from axial levels positioned just rostral (B,D) or caudal (A,C) to the first appearance of BAMBI mRNA signal (somite level 25). Positive signal marked by arrowheads in B,D; no signal detected yet at levels of sections in A,C, highlighting similar expression dynamics of BAMBI in the dorsal NT in both treatments. (E) Quantification of serial sections expressing BAMBI. Analysis in the rostro (R)-caudal (C) direction revealed rostral sections with positive signal (blue) followed by caudal sections lacking BAMBI (light blue). Data are expressed as fraction of embryo length (# sections) from the caudal end of the wing bud to the tail. N = 6 and 5 embryos for control and VP16-RARα, respectively, Mann-Whitney test. (F–J) Embryos were electroporated bilaterally at E2.5 (27-28ss) with either control PCAGG or VP16-RARα, together with GFP and analyzed at 35-36ss for expression of Rspo1. (F-I’) Images taken from axial levels localized just rostral or caudal to the onset of Rspo1 expression (somite level 28). Note that Rspo1 begins to be faintly expressed in the rostral region in both treatments (G-G’,I-I’, arrowheads) but is absent from the caudal regions (F-F’,H-H’). (J) Serial quantification of the first section with positive Rspo1 signal out of the total number of sections. Data were monitored from the tail level to the rostral end of the wing bud. N = 8 and 7 embryos for control and VP16-RARα, respectively, Mann-Whitney test. Abbreviations, NT, neural tube. Scale bar, 50 μm.
Figure 6—figure supplement 2—source data 1. Figure 6—figure supplement 2 – Gain of RA signaling does not cause a premature upregulation of RP-specific genes.