Conditional ablation of GFR1 in postmigratory enteric neurons triggers unconventional neuronal death in the colon and causes a Hirschsprung's disease phenotype

DEV001388 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. GFRα1 expression during ENS development. GFRα1 expression was examined using GFRα1^GFP/+ animals. (A) Confocal microscope images of a cross-section of E13.5 midgut. GFRα1 was expressed in almost all neurons (revealed by TuJ1 expression) and in smooth muscle layers. (B) Wholemount preparations of P5 enteric plexus of the colon. GFRα1 expression was detected only weakly in approximately 35% of PGP9.5⁺ neurons (upper panels: arrowheads), whereas high levels of GFRα1 expression were detected in almost all glial cells revealed by S100β immunoreactivity (lower panels). Scale bars: 10 μm.

• Supplemental Figure S2 -

  Fig. S2. Generation of conditional GFRα1 allele. (A) Schematic representation of conditional gene targeting strategy. Exons are denoted by boxes. The targeting construct was designed to introduce a gene cassette consisting of floxed GFRα1 cDNA+poly(A) and followed by GFP+poly(A) and a neomycin-resistance (Neo) expression cassette (flanked by FRT sites) into the GFRα1 locus. The Neo cassette was excised by crossing to ACTB-Flpe transgenic mice (Rodriguez et al., 2000). Activation of Cre recombinase resulted in the removal of floxed GFRα1, simultaneously generating a knock-in reporter allele expressing GFP under the GFRα1 promoter (hereafter referred to as GFRα1 GFP-knock-in allele). (B) Southern blot analysis of tail DNAs. Wild-type, GFRα1^+/flox and GFRα1^flox/flox newborn mice DNAs were digested with BamHI and hybridized with probe A (shown in A). Wild-type and floxed alleles appeared as 3.8 and 5.7 kb bands, respectively. (C) Floxed GFRα1 and GFRα1 GFP-knock-in alleles identified by PCR using primers (P1, P2, P3) located at the positions shown on the diagram of the wild-type and floxed alleles (shown in A). (D) Validation of GFRα1 GFP-knock-in allele. Heterozygous GFRα1 GFP-knock-in mice (GFRα1^GFP/+) were obtained by crossing GFRα1^flox/+ mice with β-actin-Cre mice to examine expression patterns of GFRα1. Note the complete overlap between cells expressing GFRα1 protein (red) and those expressing GFP (green), validating GFP expression as a marker to reliably detect GFRα1-expressing cell populations in mice carrying the GFRα1 GFP-knock-in allele. Scale bar: 10 μm. B, BamHI; H, HindIII; RV, EcoRV.

• Supplemental Figure S3 -

  Fig. S3. GFRα1^GFP/GFP mouse embryos display the absence of kidneys and the ENS. (A) Whereas GFP-fluorescent kidneys are easily identifiable in GFRα1^GFP/+ embryos, no functional kidneys develop in GFRα1^GFP/GFP embryos (E15.5). (B) Acetylcholinesterase-staining of newborn mouse colon revealing the complete absence of the ENS in GFRα1^GFP/GFP animals.

• Supplemental Figure S4 -

  Fig. S4. Numbers of glial cells in the gut of cis-only mice (GFRα1^−/−, Ret^GFRα1/+). Glial cells were stained with anti-S100b antibody and cell numbers were counted in the myenteric layer of the small intestine and the colon. The mean numbers were obtained by averaging the cell numbers in 10 scanned fields (three mice at the age of two months).