DEV015115 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Levels of Prox1 protein expression in the developing dentate gyrus. (A,B) Sections through the hippocampus of a P0 wild-type brain showing Prox1 immunolabeled cells in grey mode (A) or two-colour code (B) to better visualise different levels of immunolabeling intensity (see Materials and methods). Using the Metamorph and ImageJ softwares, cells with values between 30 and 70 (in blue in B) were counted as Prox1^low and cells with values between 100 and 250 (in red in B) as Prox1^high. Scale bars: 50 µm.

• Supplemental Figure S2 -

  Fig. S2. Prox1^low-expressing cells are progenitors. Sections through the hippocampus of P0 wild-type mice immunolabelled for Ki67 (red), Prox1 (green) and DAPI (blue). Arrows indicate double-labelled Prox1^low/Ki67 cells, while arrowheads indicate Prox1^high-expressing cells only. Only Prox1^low cells express Ki67 and therefore are dividing progenitors. Scale bars: 20 µm.

• Supplemental Figure S3 -

  Fig. S3. Neurod1 protein is not expressed in progenitors. (A,B) Sections through the hippocampus of E18.5 wild-type embryos showing the expression of Neurod1 transcripts by in situ hybridization (A) and of Neurod1 protein by immunohistochemistry (B) and BrdU-labelled nuclei (brown in A, green in B). Arrows indicate Neurod1-expressing cells having incorporated BrdU. BrdU⁺ progenitors express Neurod1 transcripts but not Neurod1 protein. Scale bars: 20 µm.

• Supplemental Figure S4 -

  Fig. S4. Loss of Mash1 does not affect neurogenesis in the developing dentate gyrus. Sections at three different coronal levels through the dentate gyrus of E18.5 wild-type (A-A′′), Mash1 mutant (B-B′′), Ngn2 mutant (C-C′′) and Mash1; Ngn2 double mutant (D-D′′) embryos showing the expression of Prox1 protein. (A-D) The pictures in grey allow better visualization of the different levels of Prox1 protein. Insets show the same sections with DAPI staining. Scale bars: 50 µm.

• Supplemental Figure S5 -

  Fig. S5. Loss of Mash1 does not affect progenitor proliferation or glial development in the dentate gyrus. Sections through the hippocampus of P0 wild-type (A-C) and Mash1^Δ/Δ (A′-C′) brains showing that similar numbers of dividing cells labelled by Ki67 and phosphohistone H3⁺ (pH3⁺) cells (A,A′) and similar levels of expression of the astrocyte marker GFAP (B,B′) and the oligodendrocyte precursor marker PDGFRα (C-C′) are observed in wild-type (A-C) and Mash1-mutant dentate gyrus (A′-C′). Scale bars: 50 µm.

• Supplemental Figure S6 -

  Fig. S6. Oligodendocyte precursor numbers are unaffected by the loss of Ngn2 and/or Mash1 in the hippocampal region. Sections through the hippocampus of E18.5 wild-type (A), Mash1 mutant (A′), Ngn2 mutant (A′′) and Mash1; Ngn2 double mutant (A′′′) embryos showing the expression of Olig2 protein. (B) Histograms representing the number of cells expressing Olig2 in the different genotypes shown in A-A′′′. Scale bars: 50 µm.

• Supplemental Figure S7 -

  Fig. S7. The bHLH genes Math2 and Math3 are expressed in the developing dentate gyrus. Sections through the hippocampus of E18.5 Ngn2^KIGFP/+ (A,B,) or Ngn2^KIGFP/GFP (A′,B′) embryos showing the expression of Math2 (A,A′) and Math3 (B,B′) by RNA in situ hybridization. (A1-B1′) are higher magnifications of the insets in (A-B′). The expression of Math3 is maintained in the secondary and tertiary matrix cells that persist in Ngn2 mutants. Scale bars: 100 µm.

• Supplemental Figure S8 -

  Fig. S8. Abnormal distribution of progenitors in the postnatal Ngn2 mutant dentate gyrus. Sections through the hippocampus of P21 Ngn2^KIGFP/+ (A) or Ngn2^KIGFP/KIGFP (A′) brains showing the position of GFP⁺ cells with respect to NeuN⁺ neurons in the granular layer. Note the distribution of GFP⁺ cells along the subgranular layer in the Ngn2^KIGFP/+ DG compared with the abnormal position of GFP⁺ cells at the junction of the two blades in in the Ngn2^KIGFP/GFP DG. (A1-A′1) High magnifications of the areas outlined in A-A′. Scale bars: 50 µm.

• Supplemental Figure S9 -

  Fig. S9. Lack of overt change in neurotransmission phenotype in the developing Ngn2 mutant dentate gyrus. Sections through the hippocampus of E18.5 Ngn2^KIGFP/+ (A,B) or Ngn2^KIGFP/KIGFP (A′,B′) embryos showing the expression of Glur6 (A,A′) and Gad67 (B,B′) by RNA in situ hybridization. No obvious change in expression of these glutamatergic and GABAergic markers, respectively, is observed in Ngn2 mutant DG. Scale bars: 50 µm.