DEV033480 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Gad67-GFP-positive cells in the CN. (A-C) Double immunolabeling with indicated antibodies in the adult DCN of Gad67-GFP mice. Arrowheads indicate cartwheel (A), Golgi (B) and ML-stellate (C) cells. Scale bars: 100 µm.

• Supplemental Figure S2 -

  Fig. S2. Ptf1a neuroepithelial domain and distribution of Gad67-GFP-positive cells in the E11.5 hindbrain. (A-C) Gad-67-positive cells (A) and X-gal-stained cells (B) were detected in the same section of the E11.5 hindbrain of the indicated genotype. (D) High magnification of the boxed region in C. Brackets with an asterisk indicate the Ptf1a neuroepithelial domain, which corresponds to the dorsally-located origin of the Gad67-GFP-positive cells. Scale bars: 200 µm.

• Supplemental Figure S3 -

  Fig. S3. BrdU incorporation experiment in Ptf1a mutants at E11.5. (A,B) Immunostaining with BrdU of E11.5 middle hindbrains of indicated genotypes, which received BrdU injections 1 hour before fixation. (C,D) X-gal staining to the same sections in A and B was performed after the immunostaining, in order to visualize the Ptf1a neuroepithelial domain. (E,F) Merged pictures of A, C (E) and B, D (F). Dotted lines represent the Ptf1a neuroepithelial domains. BrdU incorporation rates in the Ptf1a domain of the heterozygotes and homozygotes were 18.94±2.0 and 22.82±2.33 cells/10000 µm², respectively (mean ±s.e.m., Student’s t-test P>0.05, n=12). Scale bars: 100 µm.

• Supplemental Figure S4 -

  Fig. S4. Generation of Tg-Atoh1-Cre and Atoh1^CreERn mouse lines. (A) Structure of the transgene of Tg-Atoh1-Cre (see Materials and methods). (B) Diagram showing Atoh1 wild-type allele, targeting construct and Atoh1 mutant allele (Atoh1^CreERn). Black and white boxes in wild-type Atoh1 allele represent non-coding and coding Atoh1 exon sequences. Black arrowheads represent FRT sequences. Cre-ERT2, Cre recombinase cDNA with mutant estrogen ligand-binding domain; Neo, neomycin-resistance gene under control of the pgk promoter; RI, EcoRI; S, SacI. (C) Atoh1 expression in Atoh1^CreERn mutants. RT-PCR analysis on mRNA extracted from brain stems of indicated genotypes. G3PDH was used as a positive control. RT− is a negative control in which reverse transcriptase was not added in the process of cDNA synthesis.

• Supplemental Figure S5 -

  Fig. S5. Fate analyses of Atoh1 lineage cells in the Atoh1 mutants. (A,B) X-gal-stained transverse sections of the CN of indicated genotypes at E18.5. Pregnant mice were given a tamoxifen injection at E10.5. (A′,B′) High magnification of dotted boxes in A and B. Black dotted lines indicate outlines of the CN. Note that many cells in the choroid plexus are β-gal-positive in the Atoh1 mutants. (C-F) Transverse sections of the CN immunostained with anti-GABA (C,D), parvalbumin (E,F) and β-gal antibodies (C-F). Genotypes are indicated. (C′-F′) High magnification views of white dotted boxes in C-F. Arrowheads indicate cells that express β-gal but not GABA or parvalubumin; arrows highlight cells that express both β-gal and parvalbumin. Scale bars: A,B, 100 µm; C-F, 200 µm.