Hairless Streaks in Cattle Implicate TSR2 in Early Hair Follicle Formation

Abstract

Four related cows showed hairless streaks on various parts of the body with no correlation to the pigmentation pattern. The stripes occurred in a consistent pattern resembling the lines of Blaschko. The non-syndromic hairlessness phenotype observed occurred across three generations of a single family and was compatible with an X-linked mode of inheritance. Linkage analysis and subsequent whole genome sequencing of one affected female identified two perfectly associated non-synonymous sequence variants in the critical interval on bovine chromosome X. Both variants occurred in complete linkage disequilibrium and were absent in more than 3900 controls. An ERCC6L missense mutation was predicted to cause an amino acid substitution of a non-conserved residue. Analysis in mice showed no specific Ercc6l expression pattern related to hair follicle development and therefore ERCC6L was not considered as causative gene. A point mutation at the 5'-splice junction of exon 5 of the TSR2, 20S rRNA accumulation, homolog (S. cerevisiae), gene led to the production of two mutant transcripts, both of which contain a frameshift and generate a premature stop codon predicted to truncate approximately 25% of the protein. Interestingly, in addition to the presence of both physiological TSR2 transcripts, the two mutant transcripts were predominantly detected in the hairless skin of the affected cows. Immunohistochemistry, using an antibody against the N-terminal part of the bovine protein demonstrated the specific expression of the TSR2 protein in the skin and the hair of the affected and the control cows as well as in bovine fetal skin and hair. The RNA hybridization in situ showed that Tsr2 was expressed in pre- and post-natal phases of hair follicle development in mice. Mammalian TSR2 proteins are highly conserved and are known to be broadly expressed, but their precise in vivo functions are poorly understood. Thus, by dissecting a naturally occurring mutation in a domestic animal species, we identified TSR2 as a regulator of hair follicle development.

Fig 1. Streaked hairlessness in Pezzata Rossa cattle.

(A) The sharply demarcated hairless areas are clearly distinguishable. (B) The V-shaped pattern on the back is illustrated. (C) Note that the hairless pattern is unrelated to coat color. The animal shown corresponds to case 1 in Fig 2A.

Fig 2. Histopathology of skin samples from a cow with streaked hairlessness.

(A) Haired skin of an affected cow (case 1 in Fig 2A), showing no abnormalities in hair follicle size and distribution. (B) Border between the haired and the hairless skin. Note the presence of normal and abnormal hair follicles. Hair follicles of normal size with bulbs reaching into the subcutis were adjacent to dysplastic follicles characterized by a distorted contour and a smaller diameter (arrow). Sebaceous glands are present. (C) Loss of normal hair follicles and sebaceous glands in the hairless skin whereas the sweat glands (asterisk) are all present. Haematoxylin and eosin staining, magnifications 20X.

Fig 3. X-linked inheritance of bovine streaked hairlessness.

(A) Family tree of four affected females (black symbols). The males are indicated by squares, the females by circles. Deduced X chromosome haplotypes are shown colored below the individuals. Y chromosomes are shown in grey. Note the red haplotype spanning the entire chromosome X is shared by all the affected animals. In one of the two non-affected male offspring of case 1, a recombinant haplotype was detected which helped to exclude the proximal part of the X chromosome. The position of both genes (TSR2 and ERCC6L) containing disease-associated variants are shown. (B) Genome regions showing positive LOD scores in linkage analysis are shown in orange.

Fig 4. A TSR2 splice site mutation leads to aberrant splicing in hairless skin.

(A) An RT-PCR analysis of TSR2 using primers located in exons 3 and 5 on the cDNA of the bovine skin of affected and unaffected animals. The lower band corresponds to the wild type transcript and was present in all the tissue samples examined. A second larger PCR product was present predominantly in the hairless skin of the affected cows. (B) Sequence analysis of the RT-PCR products revealed the presence of two wild type transcripts (wt1 and wt2) and two mutant transcripts (mt1 and mt2). The second wild type transcript includes 9 additional nucleotides of exon 4b. The 5'-splice junction mutation of exon 5 is indicated by the red arrow. Note that the splice acceptor site mutation results in two alterations: intron 4 retention and the alternative usage of a cryptic splice site in exon 5. (C) Bovine TSR2 protein. The conserved protein domain (WGG) is shown in light green and the predicted loss of the C-terminus is indicated in red. The terminal 19 amino acids are conserved in mammals, indicating a possible function role.

Fig 5. Expression of TSR2 protein in adult bovine skin.

Immunohistochemistry carried out on skin biopsies using an anti-bovine TSR2 antibody. (A, B) Normally haired control cow (longitudinal section). A predominantly nuclear signal in the epidermal zone is present. Note that the bulge shows a particularly stronger signal (arrow). (C, D) Haired skin of an affected cow (transverse section). (E, F) Hairless skin of the same affected cow. The TSR2 expression in the haired skin corresponds to the expression in the normal cow; it is fainter in dysplastic hair follicles and absent in the inner root sheath of the affected skin (asterisk).

Fig 6. Expression of TSR2 protein in fetal bovine skin.

Immunohistochemistry on wild-type fetal skin biopsies using an anti-bovine TSR2 antibody. The TSR2 protein is expressed in the epidermis and developing hair follicle. (A, B) Skin of a fetus at ~177 days of gestation (longitudinal section). (C, D) Skin of a fetus at ~230 days of gestation (longitudinal section). (E, F) Skin of a fetus at ~268 days of gestation (transverse section). Note the strong signal on the root sheath (day 230, arrow) and bulge (day 268, asterisk).

Fig 7. Tsr2 is expressed in murine hair follicles.

(A-D): Whole mount in situ hybridization of a mouse embryo at embryonic day (E) E14.5 with a digoxigenin-labeled Tsr2 antisense (AS) (A, C) and sense (S) (B, D) probe. C and D are close-ups of the inserts shown in A and B, respectively. (E-L): In situ hybridization with a ³⁵S-labeled Tsr2 antisense probe (E, G, I, K) during embryonic (E18.5), and postnatal (PN) morphogenesis (PN8), and at the onset of anagen (PN20). A sense probe (F, J, H, L) was used as a control. I and H are close-ups of the inserts shown in G and J, respectively. Arrowheads mark the expression of Tsr2 in the growing part of the hair follicle where proliferating cells reside. Scale bars are 200 (C, D) and 50 (E-L) μm.