Functional conservation of Gsdma cluster genes specifically duplicated in the mouse genome

Abstract

Mouse Gasdermin A3 (Gsdma3) is the causative gene for dominant skin mutations exhibiting alopecia. Mouse has two other Gsdma3-related genes, Gsdma and Gsdma2, whereas human and rat have only one related gene. To date, no skin mutation has been reported for human GSDMA and rat Gsdma as well as mouse Gsdma and Gsdma2. Therefore, it is possible that only Gsdma3 has gain-of-function type mutations to cause dominant skin phenotype. To elucidate functional divergence among the Gsdma-related genes in mice, and to infer the function of the human and rat orthologs, we examined in vivo function of mouse Gsdma by generating Gsdma knockout mice and transgenic mice that overexpress wild-type Gsdma or Gsdma harboring a point mutation (Alanine339Threonine). The Gsdma knockout mice shows no visible phenotype, indicating that Gsdma is not essential for differentiation of epidermal cells and maintenance of the hair cycle, and that Gsdma is expressed specifically both in the inner root sheath of hair follicles and in suprabasal cell layers, whereas Gsdma3 is expressed only in suprabasal layers. By contrast, both types of the transgenic mice exhibited epidermal hyperplasia resembling the Gsdma3 mutations, although the phenotype depended on the genetic background. These results indicate that the mouse Gsdma and Gsdma3 genes share common function to regulate epithelial maintenance and/or homeostasis, and suggest that the function of human GSDMA and rat Gsdma, which are orthologs of mouse Gsdma, is conserved as well.

Keywords: Gasdermin A; alopecia; duplication; knockout; transgenic.

Figure 1

Gsdma orthologous genes in human, rat and mouse. (A) The genome structure of the Gsdma orthologous gene in human, rat and mouse was constructed based on Genome Reference Consortium Human Build 37, RGSC_v3.4, and Genome Reference Consortium Mouse Build 38, respectively. Transcriptional orientation of each gene is represented by an arrow. Expression domains of each Gsdma orthologous gene are shown in boxes. (B) Unrooted phylogenetic tree of Gsdma orthologous genes in human, rat, and mouse. The tree is constructed using a neighbor-joining method based on the multiple alignment generated by the ClustalW program. The numbers indicate the bootstrap values based on 1000 runs. DFNA5 orthologs are used as outgroup sequences. The scale bar indicates the number of amino acid substitutions per site.

Figure 2

Generation and validation of the Gsdma KO mouse. (A) Schematic diagrams of wild-allele, LacZ knock-in targeting vector, and knock-in allele of the Gsdma gene. Exons are indicated as black boxes. loxP and frt sites are indicated as yellow and green triangles, respectively. The LacZ-pA fragment was inserted into the start codon in exon 2. EcoRI fragments and the probe for Southern blot analysis are indicated by horizontal bars with double arrows and horizontal bars. Location of primers used for genotyping are indicated by allows and labeled P1 and P2. (B) Southern blot analysis of EcoRI digested genomic DNA from wild-type (+/+), heterozygous (L/+), and homozygous (L/L) mice for the LacZ knock-in allele. The probe located in exon 9−10 detects 9.5- and 8.5-kb fragments of the wild and knock-in allele, respectively. The DNA fragments containing Gsdma2 and Gsdma3 are intact. M indicates a marker. (C) PCR analysis of genomic DNA from mice with each genotype. Genotype was determined by PCR using primers P1 and P2. This yielded fragments of 410 and 530 bp for the wild and knock-in allele, respectively. (D) Semiquantitative reverse-transcription PCR analysis of Gsdma, Gsdma2, Gsdma3, and LacZ genes using cDNAs prepared from skin, tongue, cardia, and gastric fundus of each genotype. Actin-beta (Actb) was amplified as a control. The number of PCR cycles is shown in parentheses. (E) Immunohistological detection of Gsdma protein in skin and stomach of wild-type and Gsdma^LacZ/LacZ mice at E18.5. K14 was used as a marker for the basal cell layer. An arrow and asterisks indicate specific and nonspecific signal, respectively. Scale bar indicates 50 μm.

Figure 3

Expression of LacZ controlled by Gsdma promoter during epidermal development. X-gal−stained sections were made from Gsdma^LacZ/+ skin collected during the developmental stage at E18.5 (A), anagen at P1 (B), and P5 (C); catagen at P13 (D) and P19 (E); and telogen at P19 (F). The same sections were made for footpad at P11 (G), meibomian gland at P5 (H), tongue epithelium at P9 (I) and cardia of stomach at P1 (J) in Gsdma^LacZ/+ mice. An inset in (C) shows a cross section of a hair follicle at the same stage of development. An arrow (G) indicates sweat glands.

Figure 4

Distribution of Gsdma and Gsdma3 proteins. Immunohistological detection of Gsdma and Gsdma3 proteins in skin of wild-type (A, C, E, and G) and Gsdma^LacZ/LacZ (B, D, F, and H) mice at P8. Magnified images are provided to show Gsdma protein (arrowheads) in the suprabasal cell layer. K14 and K71 were used as a marker for the basal cell layer and IRS, respectively. Scale bars are 25 μm.

Figure 5

Phenotype of Gsdma KO mouse. (A) Result of skin permeability assay in wild-type and Gsdma^LacZ/LacZ mice at the ages indicated. (B) HE stained sections of skin during the first hair cycle in wild-type and Gsdma^LacZ/LacZ mice. Scale bars are 100 μm.

Figure 6

Phenotype of K5-Gsdma TG mouse. (A) Schematic diagrams of Gsdma TG vector construct with human K5 promoter. Gsdma in the construct is either the wild-type or has the Rim3-type mutation, A339T. (B) Macroscopic phenotypes of mice with wild-type or A339T mutant Gsdma transgenes at 3 months of age. A white arrow indicates patchy rough coat. Hematoxylin and eosin−stained sections of skin (C) and cardia (D) from control mouse (Non-TG), wild-type and A339T TG mice at 3 months of age. Inflammatory cells are present in the dermal fat layer (Arrows in C). Scale bars are 100 μm.

Figure 7

Mice with K5-Gsdma transgene exhibit epidermal hyperplasia. Immunohistological staining of skin (A) and cardia (B) from control mouse (Non-TG), and mice with wild-type and A339T Gsdma transgene, at 3 months of age. The sections were stained with anti-GFP and anti-Gsdma antibodies or anti-Gsdma and anti-K14 antibodies. Nuclei were stained with ToPro3. Scale bars are 50 μm.