DEV01230 Supplemental Figures

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Abnormal morphology and proliferation of spinal neuroepithelium in Gli2^–/–;Gli3^xt/xt mutant embryos at E10.5. (A,B) BrdU staining in thoracic regions of wild-type and Gli2^–/–;Gli3^xt/xt double mutant embryo with morphological defect (arrowhead). (D,E) Cyclin D1 and Ngn1 expression. Cyclin D1 marks cells in G1 phase, while Ngn1 is expressed in cells after undergoing their terminal mitosis and is co-expressed with CycD1 in the lateral VZ in wild-type embryos (yellow cells). In Gli2^–/–;Gli3^xt/xt mutants, Ngn1 and cyclin D1 expression is similar to wild type along the mediolateral axis, although expression extends ventrally abnormally across the midline. (G,H) Very few cells expressing phosphorylated caspase 3, which indicates apoptotic cells, are detected in either wild type or Gli2^–/–;Gli3^xt/xt mutants. (C,F) Quantification of cells expressing BrdU in wild type and Gli2^–/–;Gli3^xt/xt mutants (excluding regions with expanded neuroepithelium) in thoracic (C) and lumbar (F). Counts were made by comparing BrdU+ cells in dorsal (defined by region expressing Pax7) versus ventral (non-Pax7 expressing) VZ. Numbers of proliferating cells were similar in both wild type (open bars) and Gli2^–/–;Gli3^xt/xt mutants (hatched) embryos (n=4 embryos, four sections counted in each). All sections from E10.5 embryos. BrdU was given 1 hour before sacrifice. (I,J) Cyclin D1 expression is normally confined to cells dorsal to MNs in wild-type embryos (I). In Gli2^–/–;Gli3^xt/xt mutants, cyclin D1 expands into the ventral midline but is not expressed in Olig2+ pMN cells (J).

• Supplemental Figure 2 -

  Fig. S2. (A-F) Co-localization of GFP (Gli2DN-term or Gli3DN-term constructs) with induced Nkx2.2 in the dorsal spinal cord showing cell-autonomous induction of Nkx2.2 expression. (G-L) Co-transfection of N-terminally truncated Gli2 and ptcDloop2 in the chick neural tube. (G,G¢) Transfection of ptcDloop2 alone blocks Nkx2.2 in the p3 domain. (J) Transfection of ptcDloop2 alone activates Pax7 expression ventral to its normal domain (yellow cells). (H,I,K,L) Co-transfection of Gli2DN-term and ptcDloop2 induces Nkx2.2 expression and suppresses Pax7 expression in transfected cells (arrowheads). Similar results were obtained with Gli3DN-term co-transfections.