Mutations in gasdermin 3 cause aberrant differentiation of the hair follicle and sebaceous gland

Defolliculated (Dfl) is a spontaneous mouse mutant with a hair-loss phenotype that includes altered sebaceous gland differentiation, short hair shafts, aberrant catagen stage of the hair cycle, and eventual loss of the hair follicle. Recently a similar mutant, finnegan (Fgn), with an identical phenotype was discovered during a phenotypic screen for mutations induced by chemical mutagenesis. The gene underlying the phenotype of both finnegan and defolliculated has been mapped to chromosome 11 and here we show that both mice harbor mutations in gasdermin 3 (Gsdm3), a gene of unknown function. Gsdm3Dfl is a B2 insertion near the 3' splice site of exon 7 and Gsdm3Fgn is a point mutation T278P. To investigate the role of the gasdermin gene family an antiserum was raised to a peptide highly homologous to all three mouse gasdermins and human gasdermin. Immunohistochemical analysis revealed that gasdermins are expressed specifically in cells at advanced stages of differentiation in the upper epidermis, the differentiating inner root sheath and hair shaft and in the most mature sebocytes of the sebaceous gland and preputial, meibomium, ceruminous gland, and anal glands. This expression pattern suggests a role for gasdermins in differentiation of the epidermis and its appendages

Keratin K6irs is specific to the inner root sheath of hair follicles in mice and humans

Background Keratins are a multigene family of intermediate filament proteins that are differentially expressed in specific epithelial tissues. To date, no type II keratins specific for the inner root sheath of the human hair follicle have been identified. Objectives To characterize a novel type II keratin in mice and humans. Methods Gene sequences were aligned and compared by BLAST analysis. Genomic DNA and mRNA sequences were amplified by polymerase chain reaction (PCR) and confirmed by direct sequencing. Gene expression was analysed by reverse transcription (RT)–PCR in mouse and human tissues. A rabbit polyclonal antiserum was raised against a C-terminal peptide derived from the mouse K6irs protein. Protein expression in murine tissues was examined by immunoblotting and immunofluorescence. Results Analysis of human expressed sequence tag (EST) data generated by the Human Genome Project revealed a fragment of a novel cytokeratin mRNA with characteristic amino acid substitutions in the 2B domain. No further human ESTs were found in the database; however, the complete human gene was identified in the draft genome sequence and several mouse ESTs were identified, allowing assembly of the murine mRNA. Both species' mRNA sequences and the human gene were confirmed experimentally by PCR and direct sequencing. The human gene spans more than 16 kb of genomic DNA and is located in the type II keratin cluster on chromosome 12q. A comprehensive immunohistochemical survey of expression in the adult mouse by immunofluorescence revealed that this novel keratin is expressed only in the inner root sheath of the hair follicle. Immunoblotting of murine epidermal keratin extracts revealed that this protein is specific to the anagen phase of the hair cycle, as one would expect of an inner root sheath marker. In humans, expression of this keratin was confirmed by RT–PCR using mRNA derived from plucked anagen hairs and epidermal biopsy material. By this means, strong expression was detected in human hair follicles from scalp and eyebrow. Expression was also readily detected in human palmoplantar epidermis; however, no expression was detected in face skin despite the presence of fine hairs histologically. Conclusions This new keratin, designated K6irs, is a valuable histological marker for the inner root sheath of hair follicles in mice and humans. In addition, this keratin represents a new candidate gene for inherited structural hair defects such as loose anagen syndrome

Keratin 9 Is Required for the Structural Integrity and Terminal Differentiation of the Palmoplantar Epidermis

Keratin 9 (K9) is a type I intermediate filament protein whose expression is confined to the suprabasal layers of the palmoplantar epidermis. Although mutations in the K9 gene are known to cause epidermolytic palmoplantar keratoderma, a rare dominant-negative skin disorder, its functional significance is poorly understood. To gain insight into the physical requirement and importance of K9, we generated K9-deficient (Krt9−/−) mice. Here, we report that adult Krt9−/−mice develop calluses marked by hyperpigmentation that are exclusively localized to the stress-bearing footpads. Histological, immunohistochemical, and immunoblot analyses of these regions revealed hyperproliferation, impaired terminal differentiation, and abnormal expression of keratins K5, K14, and K2. Furthermore, the absence of K9 induces the stress-activated keratins K6 and K16. Importantly, mice heterozygous for the K9-null allele (Krt9+/−) show neither an overt nor histological phenotype, demonstrating that one Krt9 allele is sufficient for the developing normal palmoplantar epidermis. Together, our data demonstrate that complete ablation of K9 is not tolerable in vivo and that K9 is required for terminal differentiation and maintaining the mechanical integrity of palmoplantar epidermis

Keratin-Alginate Sponges Support Healing of Partial-Thickness Burns

Deep partial-thickness burns damage most of the dermis and can cause severe pain, scarring, and mortality if left untreated. This study serves to evaluate the effectiveness of crosslinked keratin–alginate composite sponges as dermal substitutes for deep partial-thickness burns. Crosslinked keratin–alginate sponges were tested for the ability to support human dermal fibroblasts in vitro and to support the closure and healing of partial-thickness burn wounds in Sus scrofa pigs. Keratin–alginate composite sponges supported the enhanced proliferation of human dermal fibroblasts compared to alginate-only sponges and exhibited decreased contraction in vitro when compared to keratin only sponges. As dermal substitutes in vivo, the sponges supported the expression of keratin 14, alpha-smooth muscle actin, and collagen IV within wound sites, comparable to collagen sponges. Keratin–alginate composite sponges supported the regeneration of basement membranes in the wounds more than in collagen-treated wounds and non-grafted controls, suggesting the subsequent development of pathological scar tissues may be minimized. Results from this study indicate that crosslinked keratin–alginate sponges are suitable alternative dermal substitutes for clinical applications in wound healing and skin regeneration

Defolliculated (Dfl): a dominant mouse mutation leading to poor sebaceous gland differentiation and total elimination of pelage follicles

Defolliculated is a novel spontaneous mouse mutation that maps to chromosome 11 close to the type I keratin locus. Histology shows abnormal differentiation of the sebaceous gland, with the sebocytes producing little or no sebum and undergoing abnormal cornification. The hair follicles fail to regress during catagen leading to abnormally long follicles. In contrast the hair shafts are shorter than normal, suggesting altered differentiation or proliferation of matrix cells during anagen. The shafts emerge from the follicle with cornified material still attached. The dermis contains increased numbers of immune cells, including T cells (CD4-positive), macrophages, and mast cells, at all time points examined. Complete elimination of all pelage and tail follicles occurs after two to three hair cycles, apparently by necrosis. Defolliculated may be a useful model for determining further functions of the sebaceous gland, and for understanding the regulation of catagen and hair follicle immunology

Generation and characterisation of keratin 7 (K7) knockout mice

Keratin 7 (K7) is a Type II member of the keratin superfamily and despite its widespread expression in different types of simple and transitional epithelia, its functional role in vivo remains elusive, in part due to the lack of any appropriate mouse models or any human diseases that are associated with KRT7 gene mutations. Using conventional gene targeting in mouse embryonic stem cells, we report here the generation and characterisation of the first K7 knockout mouse. Loss of K7 led to increased proliferation of the bladder urothelium although this was not associated with hyperplasia. K18, a presumptive type I assembly partner for K7, showed reduced expression in the bladder whereas K20, a marker of the terminally differentiated superficial urothelial cells was transcriptionally up-regulated. No other epithelia were seen to be adversely affected by the loss of K7 and western blot and immunofluorescence microscopy analysis revealed that the expression of K8, K18, K19 and K20 were not altered in the absence of K7, with the exception of the kidney where there was reduced K18 expression

Loss of K7 is associated with hyperproliferation but not hyperplasia of the bladder urothelium.

<p>Immunohistochemistry of bladder sections from wildtype (A, D), heterozygous (B, E) and homozygous K7 knockout mice (C, F) stained with a rabbit polyclonal antibody to K7 (A, B, C) and mouse monoclonal antibody MM1 to the cell proliferation marker Ki-67 (D, E, F). Arrowheads and insets in panels D and E indicate Ki-67 positive nuclei in wildtype (D) and heterozygous K7 knockout (E) bladder. More Ki-67 positive cell nuclei can be seen in the bladder of homozygous K7 knockout mice (arrowheads in F). Scale bars = 50 µm. G. Graph showing the percentage of Ki-67 positive urothelial cells in wildtype, heterozygous and homozygous K7 knockout mice (5 bladders per genotype). For each bladder, 10 random images were collected and an average of 1480 (SD +/−300) urothelial cell nuclei were counted. Standard errors (SE) are indicated by the capped lines. * indicates a <i>p</i> value of less than 0.05 (WT <i>p</i> = 0.01; HET <i>p</i> = 0.007). H. H&E stained sections of the bladder urothelium of wildtype, heterozygous and homozygous K7 knockout mice. Scale bar = 25 µm.</p

<i>Krt7</i> gene targeting strategy.

<p>A. Schematic diagram of the mouse <i>Krt7</i> gene and upstream sequences, only the proximal part of the gene encompassing exons 1 to 3 is shown. Filled black boxes denote exons 1, 2, 3. The long and short homology arms of the targeting vector are indicated by filled black rectangles. Restriction enzyme sites are as indicated and the open black boxes denote the locations of the DNA probes that were used for southern blotting at the 5′ and 3′ ends of recombination in targeted ES cells. B. Genotyping of K7 knockout mice, the wildtype allele is 743 bp, the targeted allele is 593 bp. C. RT-PCR analysis of cDNA from the bladder (lanes 1–3), lung (lanes 4–6), colon (lanes 7–9) and kidney (lanes 10–12) amplified with primers to full-length K7 (∼1.5 kb) and GAPDH (509 bp). Lanes 1, 4, 7 and 10 are from wildtype mice; lanes 2, 4, 6 and 8 are from heterozygote K7 knockout mice; lanes 3, 6, 9 and 12 are from homozygous K7 knockout mice. M = DNA size standards. D. Northern blot of bladder RNA from wildtype (+/+), heterozygous (+/−) and homozygous (–) mice detected with RNA probes to K7 and GAPDH. The position of the 18S ribosomal subunit is indicated by a black bar. E. Coomassie blue SDS-PAGE gel and western blot of cytoskeletal extracts prepared from the bladder (lanes 1, 2, 3), lung (lanes 4, 5, 6) and colon (lanes 7, 8, 9) of wildtype (lanes 1, 4, 7), heterozygous (lanes 2, 4, 6) and homozygous (lanes 3, 6, 9) K7 knockout mice probed with a C-terminal K7 antibody. M = molecular weight standards, sizes in kDa are as indicated.</p