DEV039180 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

• Supplemental Table S2 -

• Supplemental Table S3 -

• Supplemental Figure S1 -

  Fig. S1. Comparison of Nepro among different species. (A) The alignment of mouse (Mus musculus NP_666084), rat (Rattus norvegicus NP_001009678), human (Homo sapiens NP_056227) and chimpanzee (Pan troglodytes XP_001157643) Nepro protein sequences. (B) The alignment of mouse, frog (Xenopus laevis NP_001087674) and zebrafish (Danio rerio XP_001331100) Nepro protein sequences. The Nepro sequence of each species was analyzed using Protein Blast (http://blast.ncbi.nlm.nih.gov/). Graded alignments were made with the ClustalX program. Identical residues are shown on a black background, and similar residues are shown on a gray background. Conserved regions are underlined: QVEQC motif (orange), hydrophobic region (blue) and DDIDDIF motif (green). The positions of the NLS are conserved among mammals but not in frogs or fish (boxed with red). Three types of NLS Nepro mutants were generated by PCR-based mutagenesis using the QuikChange MultiSite-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). RQRR and RKPQRK (amino acid residues 442-445 and 455-460) are to be replaced by AQAA and AAPQAA, respectively. A separate mutant has both of the mutations.

• Supplemental Figure S2 -

  Fig. S2. Nepro-transfected cells in the VZ are not differentiated neurons. The neocortex 3 days after transfection of Eyfp alone as a control (left) and Eyfp with Nepro (right) at E13.5. IHC using the anti-βIII-tubulin antibody (magenta). Cells transfected with Eyfp alone migrated into the CP and were labeled with βIII-tubulin. Nepro-transfected cells in the VZ were not positive for βIII-tubulin. Scale bar: 50 µm.

• Supplemental Figure S3 -

  Fig. S3. Nepro siRNA or ΔNepro-misexpressing cells are not properly maintained in the VZ. IHC using the anti-Nestin antibody (magenta). The neocortex 2 days after transfection of Eyfp as a control and Eyfp with Nepro siRNA or ΔNepro at E11.5. Nepro siRNA or ΔNepro-misexpressing cells in the CP were not labeled with the anti-Nestin antibody. Scale bar: 100 µm.

• Supplemental Figure S4 -

  Fig. S4. Nepro represses expression of a proneural gene, Mash1. Expression of Mash1 in the neocortex was analyzed by ISH. The neocortex 12 hours after transfection of Eyfp as a control and Eyfp with Nepro, Hes5 or caNotch at E13.0. Scale bar: 200 µm.

• Supplemental Figure S5 -

  Fig. S5. Nepro is regulated by the canonical Notch signaling pathway. The expression of Nepro and Hes1 was analyzed by ISH. (A) The neocortex 16 hours after transfection of Eyfp with control siRNA or Rbpjκ siRNA at E11.5. (B) The neocortex 16 hours after transfection of Eyfp as a control and Eyfp with DN-Maml1 at E11.5. Scale bar: 200 µm.

• Supplemental Figure S6 -

  Fig. S6. There is no cross-regulation between Nepro and Hes1. The expression of Nepro and Hes1 was analyzed by ISH. (A) The neocortex 12 hours after transfection of Eyfp as a control and Eyfp with Hes1 or Nepro at E13.0. (B) The neocortex 16 hours after transfection of Eyfp with control or Nepro siRNA at E11.5. Scale bars: 200 µm.

• Supplemental Figure S7 -

  Fig. S7. Nepro siRNA and ΔNepro block the function of caNotch. The neocortex 2 days after transfection of Eyfp and caNotch with or without Nepro siRNA and ΔNepro at E11.5. IHC using anti-GFP (green) and anti-Tbr1 (magenta) antibodies. Scale bar: 100 µm.

• Supplemental Figure S8 -

  Fig. S8. The combination of Nepro and Hes maintains NPCs in the presence of L-685,458. The neocortex 2 days after transfection of Eyfp as a control and Eyfp with Hes1, Hey1 and/or Nepro at E11.5. IHC using anti-GFP (green) and anti-Ki67 (magenta) antibodies. Scale bar: 50 µm.

• Supplemental Figure S9 -

  Fig. S9. The combination of Nepro and Hes maintains NPCs in the presence of DN-Maml1. The neocortex 2 days after transfection of Eyfp as a control and Eyfp with Hes and/or Nepro at E11.5. Precocious differentiation was caused by blocking the Notch pathway using DN-Maml1. In the presence of DN-Maml1, co-misexpression of Hes1 and Nepro was able to maintain NPCs, whereas either misexpression of Hes1 or Nepro was not sufficient. Scale bar: 50 µm.