The nude mutant gene Foxn1 is a HOXC13 regulatory target during hair follicle and nail differentiation - PubMed (original) (raw)
The nude mutant gene Foxn1 is a HOXC13 regulatory target during hair follicle and nail differentiation
Christopher S Potter et al. J Invest Dermatol. 2011 Apr.
Abstract
Among the Hox genes, homeobox C13 (Hoxc13) has been shown to be essential for proper hair shaft differentiation, as Hoxc13 gene-targeted (Hoxc13(tm1Mrc)) mice completely lack external hair. Because of the remarkable overt phenotypic parallels to the Foxn1(nu) (nude) mutant mice, we sought to determine whether Hoxc13 and forkhead box N1 (Foxn1) might act in a common pathway of hair follicle (HF) differentiation. We show that the alopecia exhibited by both the Hoxc13(tm1Mrc) and Foxn1(nu) mice is because of strikingly similar defects in hair shaft differentiation and that both mutants suffer from a severe nail dystrophy. These phenotypic similarities are consistent with the extensive overlap between Hoxc13 and Foxn1 expression patterns in the HF and the nail matrix. Furthermore, DNA microarray analysis of skin from Hoxc13(tm1Mrc) mice identified Foxn1 as significantly downregulated along with numerous hair keratin genes. This Foxn1 downregulation apparently reflects the loss of direct transcriptional control by HOXC13 as indicated by our results obtained through co-transfection and chromatin immunoprecipitation (ChIP) assays. As presented in the discussion, these data support a regulatory model of keratinocyte differentiation in which HOXC13-dependent activation of Foxn1 is part of a regulatory cascade controlling the expression of terminal differentiation markers.
Figures
Figure 1. Hoxc13 null and nude mice exhibit similar hair defects
Histological comparison of HFs in H&E –stained sections (10 μm) of dorsal skin from 5d p.n. mice of the following strains: (a–c) C57/BL6L-Tyrc-2J (controls); (d–g) Hoxc13 null (homozygous B6.129-Hoxc13tm1Mrc/Hoxc13tm1Mrc); (h–k) nude (Hsd-Foxn1nu/Foxn1nu). In all strains, HFs were uniformly in late anagen phase of the hair cycle. Note that in both Hoxc13 null and nude mice the hair shaft became twisted and distorted at the level of the sebaceous gland (sb) and failed to penetrate the epidermis (arrows in panels g and k). For further explanations, see text; m: medulla; mtx: matrix;
ost: osteum
; pctx: precortex; scale bars: 500 μm in panels a, d, h, and 10 μm in the remaining panels.
Figure 2. Nails of Hoxc13 null and nude mutant mice exhibit similar structural defects
Histological comparison of nails in H&E –stained sections (10 μm) of rear foot digits derived from the following mouse strains at 5d p.n.: (a–d) C57/BL6J-Tyrc-2J mice (controls); (e–h) Hoxc13 null (homozygous B6.Cg-Tyrc-2JHoxc13tm1Mrc); nude (Hsd-Foxn1nu/Foxn1nu). Compared to control mice Hoxc13 null and nude mice had similar defects including reduced nail matrix (mtx), a defective nail plate (np), and an extended stratum granulosum (SG) with its distal limits (marked by arrows) reaching into the matrix as explained in the text; cu: cuticle; nb: nail bed; pnf: proximal nail fold; SG: stratum granulosum scale bars: 200 μm, panels a, e, i, and 5 μm in the remaining panels; distal points to the right.
Figure 3. Disruption of Foxn1 expression pattern in HF and nail of Hoxc13 null mice
ISH analysis of Hoxc13 and Foxn1 expression patterns in hair and nail from C57BL/6J-Tyrc-2J (control), Hoxc13 null (homozygous B6.Cg-Tyrc-2JHoxc13tm1Mrc), and nude mice. (a, b) Hoxc13 and Foxn1 expression (bluish stain) overlapped in cortical/precortical regions of control HFs (yellow arrowheads). (c) Reduced Hoxc13 expression in HF matrix of Hoxc13 null mice. HFs of nude mice showed characteristic aspects of the Hoxc13 pattern (d), whereas Foxn1 expression was undetectable in HFs of Hoxc13 null mice (e); note twisted hair shafts in Hoxc13 null and nude mice (arrowheads in c, d). Hoxc13 expression in nail matrix (mtx) of control mice (f) mirrored the Foxn1 pattern (g). Hoxc13 expression was undetectable in nail matrix of Hoxc13 null mice (h) but weakly present (orange arrow) in nails of nude mice (i), and Foxn1 expression was undetectable in Hoxc13 null nail matrix (j). (k) Bar graph showing the relative 2.4-fold change in Foxn1 expression in Hoxc13 null versus control (wild type) mice as measured by RT-PCR. Scale bars: 50 μm, a–e, and 100 μm in f–j.
Figure 4. HOXC13 directly regulates Foxn1 expression
(a) HOXC13 consensus binding sequences (TTA/TATNPuPu, black bars) upstream of Foxn1 transcription start (angled arrow); putative binding sites (BS) 1 and 2 examined by ChIP and locations of PCR primers used for isolating the 5 kb region included in Foxn1-luc are indicated; boxed region: transcribed sequences; (b) Normalized luciferase activities resulting from co-transfection of C2C12 cells with Foxn1-luc and HOXC13 expression vectors as indicated; co-transfection with HOXC13Δhd-F, in which the homeodomain was deleted, resulted in expression levels similar to baseline levels. (c) Western blot analysis (top) using anti-FLAG antibodies confirmed expression of HOXC13-F and HOXC13Δhd-F in transfected C2C12 cells at approximately equal levels (n=3) as determined by densitometric quantification of chemiluminescent signals (see bar diagram below). (d) ChIP assays of C2C12 cells transfected with HOXC13-FLAG; PCR analysis of immunoprecipitated chromatin (F) indicated amplification of HOXC13-bound sequences specific for BS2 but not BS1 after 42 cycles; unprecipitated input DNA was used as positive control (I), while ChIP DNA from untransfected cells (N) and distilled water (W) were used as negative controls; additionally, control reactions using primers specific for Prx1 sequences containing no HOXC13 consensus binding sequences failed to yield PCR products with the same batch of immunoprecipitated DNA.
Figure 5. Models of HOXC13-controlled network motifs involved in regulating HF differentiation
Left:
schematics of three HOXC13-controlled feed-forward loop (FFL)-type network motifs (1), (2), and (3) as explained in the text; a HOXC13 negative feedback loop (Tkatchenko et al., 2001) is included in all three circuits but requires validation.
Right:
Schematic of bisected lower portion of anagen HF indicating the medulla (M), cortex (CTX) and cuticle (CU) of the hair shaft, and the internal root sheath (IRS) and outer root sheath (ORS) that transitions into the germinal layer (GL) surrounding the dermal papilla (DP); the germinal compartment in the lower matrix (GMC) contains proliferating, transit-amplifying cells (Langbein and Schweizer, 2005); the topography of regulatory network motifs shown on the left is indicated by the numbers.
References
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