DEV02228 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Active Shh-signaling is required for BrdU incorporation at later periods of neural tube development in dorsal and ventral areas of the neural tube. (A-C) HH14 stage chick embryos were electroporated in ovo with Ptc1^Dloop2, pulse labelled with bromodeoxyuridine (BrdU) (4 hours prior to fixation) and analysed 48 hours PE for GFP and BrdU expression. (A) The electroporated side of the neural tube is smaller than the control side. (B,C) Double immunostaining with anti-GFP (green) and anti-BrdU (red) revealed the presence of only a few double stained cells (yellow) in the dorsal (B) and the ventral (C) neural tube. (D) Quantitative analysis of BrdU/GFP double labelled cells in: ventral and dorsal neural tube (n=2) (***P<0.0001). Error bars indicate s.d. (E-G) HH18 stage chick embryos were analysed 48 hours PE for GFP and BrdU expression. (E) The electroporated side of the neural tube is smaller than the control side. (F,G) Double immunostaining with anti-GFP (green) and anti-BrdU (red) revealed the presence of only a few double stained cells (yellow) in the dorsal (F) and the ventral (G) neural tube. (H) Quantitative analysis of BrdU/GFP double labelled cells in ventral and dorsal neural tube. n=2-5; ***P<0.0001; error bars indicate s.d. (I,J) HH18 stage chick embryos electroporated with Ptc1^Dloop2 were analysed 48 hours PE for the TUNEL reaction. (I) GFP expression on the electroporated side of the spinal cord. (I,J) Increased TUNEL staining was observed in the electroporated side as compared with the control side. j is an enlargement of J.

• Supplemental Figure 2 -

  Fig. S2. Activation of the Shh-pathway by electroporation of a dominant-negative form of PKA induces cell fate changes and overproliferation. HH11/12 stage embryos were electroporated with dnPKA/GFP and assayed 12, 24 and 48 hours PE for the indicated markers. (A-C) Electroporation of dnPKA causes cell autonomous cell fate changes including repression of the dorsal marker Pax7 at 24 hours PE (B) but not at 12 hours PE (A), and activation of the motor neuron marker MNR2 (C). (D-G) Electroporation of dnPKA causes overgrowth of the electroporated side that requires at least 48 hours PE. BrdU immunostaining 12 hours PE (D) and 24 hours PE. At 48 hours PE, there is increased proliferation in the dorsal neural tube, revealed by anti-BrdU (F) and by anti-pH3 (G). (H) Quantitative analysis of total BrdU labelled cells in electroporated versus non-electroporated ventral and dorsal neural tubes after a 1 hour BrdU pulse (n=3). (I) Quantitative analysis of BrdU/GFP double labelled cells 24 hours PE (n=4). *P<0.05, **P<0.005, P<0.0001; error bars indicate s.d.