Gender-Difference in Hair Length as Revealed by Crispr-Based Production of Long-Haired Mice with Dysfunctional FGF5 Mutations

Abstract

Fibroblast growth factor 5 (FGF5) is an important molecule required for the transition from anagen to catagen phase of the mammalian hair cycle. We previously reported that Syrian hamsters harboring a 1-bp deletion in the Fgf5 gene exhibit excessive hair growth in males. Herein, we generated Fgf5 mutant mice using genome editing via oviductal nucleic acid delivery (GONAD)/improved GONAD (i-GONAD), an in vivo genome editing system used to target early embryos present in the oviductal lumen, to study gender differences in hair length in mutant mice. The two lines (Fgf5go-malc), one with a 2-bp deletion (c.552_553del) and the other with a 1-bp insertion (c.552_553insA) in exon 3 of Fgf5, were successfully established. Each mutation was predicted to disrupt a part of the FGF domain through frameshift mutation (p.Glu184ValfsX128 or p.Glu184ArgfsX128). Fgf5go-malc1 mice had heterogeneously distributed longer hairs than wild-type mice (C57BL/6J). Notably, this change was more evident in males than in females (p < 0.0001). Immunohistochemical analysis revealed the presence of FGF5 protein in the dermal papilla and outer root sheath of the hair follicles from C57BL/6J and Fgf5go-malc1 mice. Histological analysis revealed that the prolonged anagen phase might be the cause of accelerated hair growth in Fgf5go-malc1 mice.

Keywords: CRISPR/Cas9; GONAD; fibroblast growth factor 5; gender difference; genome editing; hair follicle cycle; indels; long-haired mice.

Figure 1

Characterization of mutations in Fgf5^go-malc mice. (A) Sequence of the synthesized guide RNA (gRNA). The arrow indicates the gRNA target site within exon 3 of the Fgf5 gene. The underlined sequence depicts the protospacer adjacent motif (PAM). (B) Electropherograms showing mutations in the two Fgf5^go-malc mouse lines (Fgf5^go-malc1 and Fgf5^go-malc2). The red arrowhead indicates indels in the Fgf5 gene. A “GA” deletion and “A” insertion (red squares) were found in Fgf5^go-malc1 and Fgf5^go-malc2 mice, respectively. B6, C57BL/6J. (C) Alignment of the partial Fgf5 cDNA sequences in the B6, Fgf5^go-malc1, and Fgf5^go-malc2 mice. The underlined region indicates the positions of the 2-bp deletion and 1-bp insertion mutations in Fgf5^go-malc1 and Fgf5^go-malc2 mice, respectively. (D) Alignment of part of the predicted FGF5 amino acid sequence. The arrowhead indicates the start position of frameshift mutations that resulted in the production of abnormal or defective protein products. (E) Schematic representation of the wild-type (WT) and mutant FGF domains (shown by black boxes) in Fgf5^go-malc mice and Fgf5^malc hamsters. FGF5S is shown at the bottom. The gray line indicates the amino acid sequence of abnormal FGF5 expressed in Fgf5^go-malc1 mice. The mutant FGF5 protein expressed in Fgf5^go-malc1 mice was longer than the WT FGF5 by 48 amino acids.

Figure 2

Long-haired phenotype in Fgf5^go-malc1 mice. (A) Gross appearance of the hair in B6 and Fgf5^go-malc1 mice. White and black arrows and arrowheads indicate representative positions of longer hairs in the neck, buttock, and whiskers, respectively. B6, C57BL/6J. (B) Measurement of hair length in B6 (C57BL/6J) and Fgf5^go-malc1 mice. Black and gray indicate hair length in male (M) and female (F) B6. Blue and red circles indicate hair length in male and female Fgf5^go-malc1 mice. The mice used were all littermates from B6 or Fgf5^go-malc1 mice. More than 30 hairs harvested from three different mice (n = 3) were examined and shown as hair length (mm). ****: p < 0.0001; n.s.: no significant differences. (C) Hair length distribution in the littermates [male (blue bars) and female (red bars)] of Fgf5^go-malc1 mice.

Figure 3

Analysis of FGF5 and FGF5S expression in Fgf5^go-malc1 mice. (A) Semi-quantitative RT-PCR analysis of Fgf5 and Fgf5s transcripts in the skin. M: 100-bp DNA ladder. D.W.: distilled water. (B) Relative expression of Fgf5 (upper graphs) and Fgf5s (bottom graphs) transcripts in the skin (left graphs) and brain (right graphs) of C57BL/6J (B6) and Fgf5^go-malc1 mice. Black and gray bars indicate values of relative expression levels of Fgf5 and Fgf5s transcripts in B6 and Fgf5^cr mice, respectively (n = 3; geometric means ± standard deviation). n.s.: no significant differences. (C) Immunohistochemical localization of FGF5 protein (red) in the skin of B6 (upper panels) and Fgf5^go-malc1 mice (bottom panels). Nuclei were counterstained with DAPI (blue). Arrows and arrowheads indicate the dermal papilla (Dp) and outer root sheath (Ors) in the hair follicles, respectively. Scale bar = 100 µm.

Figure 4

Histological evaluation of the anagen phase in Fgf5^go-malc1 mice. Hematoxylin-eosin (H&E)-stained skin sections from C57BL/6J (B6) (left two lanes) and Fgf5^go-malc1 mice (right two lanes) at P13 (A) and P21 (B). Within these two lanes, images of male and female mice are shown on the left or right side, respectively. At P21, TUNEL staining was also performed (C). Black arrows and arrowheads show the dermal papilla (Dp) and outer root sheath (Ors), respectively, and white arrows and arrowheads indicate hair clubs (Hc) and apoptosis cells, respectively. Scale bar = 200 µm.