DEV032128 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Reduced cell cycle progression and cell cycle exit in Cux2 mutants. (A) Cyclin D1 immunohistochemistry in the dorsal spinal cord at E11.5. (B) Cux2 levels in a control Cux2^neo/+ spinal cord at E11.5. (C) Co-expression of cyclin D1 (red) and Cux2 (green) in the lateral-most spinal cord progenitors of the vz at E11.5. (D) Reduced cyclin D1 levels in the dorsal spinal cord of Cux2^neo/neo mutants at E11.5 (white line). (E) Quantification of the effect of Cux2 loss on cyclin D1-positive cells in the dorsal spinal cord at E11.5 (*, P<0.01). (F) Expression of p57^Kip2 protein in nascent neurons undergoing cell cycle exit in the iz at E11.5. (G) High Cux2 levels in the iz at E11.5. (H) Overlay of p57^Kip2 and Cux2 in the iz at E11.5, demonstrating Cux2 expression in nascent interneurons undergoing cell cycle withdrawal. (I) Reduction of p57^Kip2 levels in Cux2^neo/neo mutants at E11.5. (J) Quantification of the effect of Cux2 loss on p57^Kip2-positive nascent neurons (*, P<0.01). iz, intermediate zone; vz, ventricular zone.

• Supplemental Figure S2 -

  Fig. S2. Mash1 is not a direct Cux2 target gene. (A-C) Mash1 (A, red) and Cux2 (B, green) levels alone, and merged (C), in the dorsal spinal cord at E11.5. (C) Mash1 and Cux2 are co-expressed in dorsal interneuron progenitors at the lateral edge of the vz (arrows). (D) Quantification of the average number of Cux2/Mash1 double-positive cells in the dorsal neural tube at E11.4 (16.4%, n=6). (E) ChIP assay on E12.5 brain extracts demonstrating weak or non-significant Cux2 binding to the transcriptionally active Mash1 proximal region. (Top) A 251 bp fragment from the Mash1 proximal upstream region was amplified from E12.5 mouse embryonic brain extracts following incubation with rabbit IgGs (negative control), anti-RNA polymerase II antibody (RNA pol II; positive control), anti-histone H3 antibody (positive control), or a rabbit anti-Cux2 antibody. Additional controls for amplification include water, E12.5 brain extract DNA prior to incubation with antibodies (input) and genomic DNA. (Bottom) A 471 bp distal amplicon 1.3 kb downstream of the Mash1 transcription start site was chosen as a control region.

• Supplemental Figure S3 -

  Fig. S3. The Notch effector gene Rbpjκ is required for Cux2 mRNA expression. (A) Cux2 mRNA expression in the forebrain (fb) and neural tube (nt) of a wild-type E9.5 embryo. (B) Severe reduction of Cux2 transcripts in an Rbpjκ^−/− mutant at E9.5. (C) Transverse section across the neural tube (axial level identified by line in A) demonstrating normal Cux2 mRNA levels in a wild-type E9.5 embryo. (D) Transverse sections across the neural tube (axial level indicated by line in B) of an Rbpjκ^−/− mutant at E9.5 showing greatly attenuated Cux2 expression in the neuroepithelium.

• Supplemental Figure S4 -

  Fig. S4. Constitutive NICD expression induces neural progenitor maintenance and inhibits proneural expression. (A) Expression of the Notch target transcription factor Hes1 in neural progenitors in a non-GFP-positive control dorsal spinal cord at E11.5 (n=6). Hes1 is required for the maintenance of neural progenitors. (B-D) Induction of Hes1 (B, red) in the dorsal spinal cord following Wnt1-Cre-mediated activation of NICD-ires-EGFP (n=8; C,D). (C) GFP levels indicating activation of the NICD-ires-EGFP transgene. (D) Merge of GFP (green) and Hes1 (red) levels in an induced NICD-ires-EGFP R26R transgenic spinal cord showing ectopic co-expression (yellow). (E) Levels of the proneural transcription factor Mash1 in a non-GFP-positive control dorsal spinal cord at E11.5 (n=6). (F-H) NICD-ires-EGFP overexpression by the Wnt1-Cre driver repressed Mash1 levels in GFP-positive dorsal spinal cord progenitors at E11.5 (n=8). Mash1 (E, red) and GFP (G, green) levels following Wnt1-Cre-mediated activation of NICD-ires-EGFP. (H) Merge of GFP (green) and Mash1 (red) levels in an induced NICD-ires-EGFP R26R transgenic spinal cord showing repression of proneural expression.

• Supplemental Figure S5 -

  Fig. S5. Constitutive NICD expression inhibits cell cycle exit in the spinal cord. (A-C) P27^Kip1 levels in E11.5 dorsal spinal cords of a control non-GFP-positive embryo (n=6; A) and a Wnt1-Cre-driven NICD-ires-EGFP transgenic embryo (n=8; B,C). P27^Kip1-positive cells (B, red) are reduced in the dorsal spinal cord overexpressing NICD-ires-EGFP (C, green). (D-F) P57^Kip2 levels in E11.5 dorsal spinal cords of a control non-GFP-positive embryo (n=6; D) and Wnt1-Cre-driven NICD-ires-EGFP transgenic embryo (n=8; E,F). p57^Kip2-positive cells (D, red) are reduced in the dorsal spinal cord overexpressing NICD-ires-EGFP (F, green).

• Supplemental Figure S6 -

  Fig. S6. Reduced commissural interneuron formation in the dorsal spinal cord following constitutive NICD overexpression. (A-D) Expression of Brn3a in dorsal interneurons of E11.5 non-GFP-positive control (n=6; A) versus Wnt1-Cre-driven NICD-ires-EGFP transgenic embryos (n=8; B-D). (B) Reduced Brn3a-positive interneurons following NICD-ires-EGFP overexpression (C,D; green) in the dorsal spinal cord at E11.5. (E-H) Lhx2 expression in E11.5 commissural interneurons of non-GFP control (n=6; E) versus Wnt1-Cre-driven NICD-ires-EGFP transgenic (F-H) embryos (n=8). (F) Near complete loss of Lhx2-positive commissural interneurons (F, red) following NICD-ires-EGFP overexpression (G,H; green). (I-L) Math1 expression in E11.5 commissural interneurons of non-GFP control (n=6; I, red) versus Wnt1-Cre-driven NICD-ires-EGFP transgenic (J-L) embryos (n=8). (J) Reduction of Math1-positive populations (J, arrow) following constitutive overexpression of NICD-ires-EGFP in the dorsal neural tube (K,L; green, arrow in L).