DEV029900 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Patterns of gene expression and neurotransmitter identity in the ventrolateral midbrain. (A-B′′) Colocalization of Gata2 with Nkx6-1 and Nkx2-2 was analyzed by IHC and confocal microscopy. Staining on single optical sections (a′,a′′,b′,b′′) and the respective merged images (A,B) are shown. (C-C′′) Colocalization of Nkx2-2 protein and Pax6 mRNA demonstrated by combined IHC (C′) and ISH (C′′). (D-D′′) Colocalization of Pax6 and Slc17a6 analyzed by combined ISH (D′) and IHC (D′′) and confocal microscopy. (E-G) In situ hybridization with Gad1, Slc17a6 and Pax6 probes on adjacent sections. The arrow and squared brackets indicate the region that coexpresses Slc17a6 and Pax6, but not Gad1.

• Supplemental Figure S2 -

  Fig. S2. Conditional inactivation of Gata2. (A) Mating scheme for the production of Gata2 mutant embryos and mice. (B) Recombination of the R26R reporter allele (Soriano, 1999) in the midbrain-r1 region by En1^Cre, as revealed by β-galactosidase staining of an E9.5 En1^Cre/+; R26R/+ embryo. (C,D) Whole-mount ISH with a Gata2 probe on E10.5 wild-type (WT) and Gata2^cko embryos demonstrates loss of Gata2 transcripts from the midbrain-r1 region. The arrows point to the remaining Gata2 expression in the diencephalon (di). (E,F) Gata2 IHC on coronal sections of E11.5 WT and Gata2^cko mutant embryos demonstrates loss of the Gata2 protein in the mutants. Scale bar: 100 μm.

  Reference

  Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.

• Supplemental Figure S3 -

  Fig. S3. Neural development in Gata2^cko mutants. (A-J) In situ hybridization with Gad2, Gad1, Gata3 and Isl1 probes on coronal sections of E11.5 and E13.5 wild-type (WT) and Gata2^cko midbrain. nIII, nucleus of the third cranial nerve. (K,L) IHC with anti-Olig2 antibody on coronal sections of E13.5 WT and Gata2^cko midbrain. Scale bars: 100 μm.

• Supplemental Figure S4 -

  Fig. S4. Cell proliferation and apoptosis in the Gata2^cko midbrain. (A,B,D,E) Anti-Sox2 and HuC/D IHC on coronal paraffin sections of E11.5 and E13.5 embryos demonstrates no obvious difference between wild-type (WT) and Gata2^cko midbrains. (C) The thickness of the Sox2-positive layer compared with that of the entire neuroepithelium was measured at the ventral border of the Helt expression domain n(embryos/genotype)=2, n(sections/embryo)=4. (F,G) Incorporation of BrdU in the E11.5 WT and Gata2^cko midbrain ventricular zone during a 2-hour labeling pulse detected by anti-BrdU IHC. (H) The ratio of BrdU-positive cells with respect to the total number of cells counted from the Helt expression domain n(embryos/genotype)=3, n(sections/embryo)=8; P=0.0012. (I,J) IHC staining for phospho-histone H3 and Helt in E11.5 WT and Gata2^cko midbrain. (K) The ratio of PH3-positive cells with respect to the total number of cells counted from the apical surface of the Helt expression domain n(embryos/genotype)=3, n(sections/embryo)=4. In summary, a very small, although statistically significant, reduction in the number of BrdU-positive nuclei was observed. At the same time, no change in the number of mitotic cells or relative numbers of progenitor/precursor cells was detected. Currently, we do not have an explanation for this discrepancy. However, even if the rate of cell proliferation were slightly different between WT and Gata2^cko mutants, it cannot explain the observed loss of GABAergic neurons in the mutants. (L,M) IHC analysis of p57 expression reveals no obvious difference between E11.5 WT and Gata2^cko midbrain. (N,O) IHC analysis of cleaved caspase 3 expression. Only very few caspase 3-positive cells were found in either genotype n(embryos/genotype)=2, n(sections/embryo)=4; white arrows. In addition, no obvious increase in the number of apoptotic nuclei was observed in DAPI-stained sections of Gata2^cko mutants. Scale bars: 100 μm.

• Supplemental Figure S5 -

  Fig. S5. GABAergic neurons in the midbrain-r1 region of perinatal Gata2^cko mutants. ISH with Gad1 (A,B, midsagittal; C,D, parasagittal), Gata3 (G,H) and Pitx2 (E,F) probes on sagittal sections of E17.5 wild-type and Gata2^cko embryos. The dashed lines represent the midbrain-r1 boundary, as defined by the posterior border of Otx2 expression determined on adjacent sections.