Hoxa4 expression in developing mouse hair follicles and skin

Hoxa4 expression in developing mouse hair follicles and skin

Mechanisms of Development 99 (2000) 153±157 www.elsevier.com/locate/modo Gene expression pattern Hoxa4 expression in developing mouse hair follicle...

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Mechanisms of Development 99 (2000) 153±157

www.elsevier.com/locate/modo

Gene expression pattern

Hoxa4 expression in developing mouse hair follicles and skin Alan I. Packer a,1, Daniel Jane-wit a,1, Leon McLean a, Andrei A. Panteleyev b, Angela M. Christiano a,b, Debra J. Wolgemuth a,c,d,e,f,* a

Department of Genetics and Development, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA b Department of Dermatology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA c Department of Obstetrics and Gynecology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA d Center for Reproductive Sciences, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA e Institute of Human Nutrition, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA f Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA Received 3 August 2000; received in revised form 1 September 2000; accepted 6 September 2000

Abstract We have examined the expression of the Hoxa4 gene in embryonic vibrissae and developing and cycling postnatal pelage hair follicles by digoxigenin-based in situ hybridization. Hoxa4 expression is ®rst seen in E13.5 vibrissae throughout the follicle placode. From E15.5 to E18.5 its expression is restricted to Henle's layer of the inner root sheath. Postnatally, Hoxa4 expression is observed at all stages of developing pelage follicles, from P0 to P4. Sites of expression include both inner and outer root sheaths, matrix cells, and the interfollicular epidermis. Hoxa4 is not expressed in hair follicles after P4. Hoxb4, however, is expressed both in developing follicles at P2 and in catagen at P19, suggesting differential expression of these two paralogous genes in the hair follicle cycle. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hoxa4; Hoxb4; Hox genes; Hair follicle; Outer root sheath; Henle's layer; Inner root sheath; Matrix; Dermal papilla; Skin; In situ hybridization

1. Results and discussion The homeobox-containing Hox genes have been implicated in the development of a variety of organs and cell types in multicellular organisms, both in the embryo and in the adult (reviewed in Favier and DolleÂ, 1997). Although this evolutionarily conserved family of transcription factors has a clear role in axial and appendicular patterning, Hox gene expression has also been observed in structures or cells where their function is more likely to be in differentiation and/or morphogenesis. This includes expression in the skin, and particularly in the developing and cycling hair follicles of mice and humans. For example, expression of each of the group 4 Hox paralogues as well as eight of the nine genes from the Hox-B locus have been detected by ribonuclease protection assay in skin samples from mouse embryos at day 17 (E17) and day 18 (E18) of gestation (Detmer et al., 1993). In the human, HOXA4 was among the HOX genes whose expression was detected in fetal, neonatal, and adult skin by RT-PCR analysis (Stelnicki et al., 1998). HOXA5 * Corresponding author. Tel.: 11-212-305-7900; fax: 11-212-305-6084. E-mail address: [email protected] (D.J. Wolgemuth). 1 A.I.P. and D.J.-w. contributed equally to this work.

and HOXA7 transcripts were also identi®ed in a similar temporal pattern, while other HOX genes are expressed in the skin in a more restricted manner, including HOXA6, HOXA10, HOXA11, HOXB1, HOXB3, HOXB6, HOXB7, and HOXC4 (Stelnicki et al., 1998). HOXD1 has been detected in the germinative epithelial cells of the hair follicle matrix (Reynolds et al., 1994). Several of the mouse Hox genes whose expression patterns in the skin have been determined in situ, including Hoxc8 (Bierberich et al., 1991), Hoxd9, Hoxd11, and Hoxd13 in hair follicles (Kanzler et al., 1994), have been observed in a pattern consistent with the spatial colinearity of expression seen in other lineages (reviewed in Krumlauf, 1994). Perhaps the most intriguing report of Hox function in skin to date has been that of the targeted deletion of Hoxc13, which renders the null mice hairless (Godwin and Capecchi, 1998). Given our continuing interest in the expression and function of the mouse Hoxa4 gene (Wolgemuth et al., 1987, 1989; Behringer et al., 1993; Horan et al., 1994; Packer et al., 2000) and the growing interest in the potential function of Hox genes and other patterning genes in skin and hair follicle development (reviewed in Haake and Goldsmith, 1997; Millar, 1997), we decided to examine the expression of Hoxa4 in mouse skin in situ. In embryonic vibrissae,

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Fig. 1. Expression of Hoxa4 and Hoxb4 in mouse embryonic and neonatal vibrissae determined by digoxigenin-based in situ hybridization. (A±C) Hoxa4 expression is observed throughout the developing vibrissa follicles at E13.5. (D) A schematic cross-section of a developing vibrissa (from Bereiter-Hahn et al., 1984). (E±G) In cross-sections from E15.5 to E18.5, Hoxa4 expression is restricted to Henle's layer of the inner root sheath (arrows). No expression is seen in Huxley's layer of the inner root sheath or in the outer root sheath (ORS) or connective tissue sheath (CTS). Non-speci®c staining of the hair shaft itself is seen occasionally, as in panel (G). (H) No staining is seen with a control Hoxa4 sense probe. (I) No staining is seen in a section from the maxillary region of a Hoxa4 null embryo. (J) Periodic acid-Schiff (PAS) staining of a sagittal section of a vibrissa follicle at P0. Staining is observed in the outer root sheath (ORS), but not in the inner root sheath (IRS). (K) A sagittal section of a vibrissa follicle at E16.5 shows expression in the ORS (arrow), as in (J), with occasional non-speci®c staining of the hair shaft itself. (L,M) Hoxa4 expression in vibrissa follicles at P0 is restricted to the developing IRS.

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Fig. 2. Expression of Hoxa4 and Hoxb4 in mouse developing and cycling postnatal pelage hair follicles determined by digoxigenin-based in situ hybridization. (A) Hoxa4 expression in hair follicles from dorsal skin at P2. Expression is seen in follicles from several stages (2±4) and appears to be most abundant in the matrix cells. Expression is also observed in the interfollicular epidermis. (B) Robust expression of Hoxa4 in the matrix cells at P2. Note the absence of expression in the region of the dermal papilla (DP). (C) Hoxa4 expression in a stage 7±8 follicle at P2, as staged by the position of the hair shaft. Note again the expression in the interfollicular epidermis on either side of the hair shaft. (D) No expression is observed in hair follicles from Hoxa4 null mice. The brown staining that is seen corresponds to the melanin granules. (E,F) No Hoxa4 expression is observed in mid-anagen follicles from P5.5 mice. Again, the brown staining corresponds to melanin granules. (G) No Hoxa4 expression is observed in catagen follicles at P17. (H) No Hoxa4 expression is observed in telogen follicles at 8 weeks of age. The brown staining corresponds to the remainder of the hair shaft. Light staining in the surrounding sheath is equivalent to background levels. (I) Hoxb4 expression in the IRS of developing follicles at P2. (J) Hoxb4 expression in a follicle in the catagen stage of the hair follicle cycle at P19.

Hoxa4 expression was ®rst detected at E13.5 throughout the developing hair follicle placode (Fig. 1A). Expression at E13.5 was also observed in the lower portion of the vibrissa follicle peg adjacent to the dermal papilla (Fig. 1B) and in the innermost layer of the matrix (Fig. 1C). Thereafter (E15.5± E18.5), Hoxa4 expression in the embryonic vibrissae exhibited a consistent and precise pattern of expression, being restricted to Henle's layer of the inner root sheath (Fig. 1D±G). No staining was seen with a Hoxa4 sense probe (Fig. 1H) or in sections from Hoxa4 mutant mice (Fig. 1I).

In order to de®ne better the various layers in sagittal sections, we stained vibrissae follicles at P0 with periodic acid-Schiff (PAS) stain, which is known to be speci®c for the outer root sheath (ORS). This result (Fig. 1J) clearly de®nes the ORS and the adjacent, unstained inner root sheath (IRS). Hoxa4 expression was found in the same layer of the developing vibrissa follicles at E16.5 (Fig. 1K) and at P0 (Fig. 1L,M). Expression of a paralogous gene, Hoxb4, was also observed in Henle's layer in vibrissae (data not shown). We also examined Hoxa4 expression both in developing

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and cycling pelage follicles at various times postnatally. Pelage follicles form the overhair and underfur along the murine torso. Their development is initiated around parturition and continues until P4±5 when the hair shaft pierces the surface of the skin. Hair follicle development at this time is characterized by eight stages as de®ned by Hardy (reviewed in Hardy, 1992). Thereafter, follicular cycling is initiated, consisting of anagen, catagen, and telogen stages. In sections at P2, Hoxa4 expression is observed in hair follicles at all stages of their development, including the IRS precursor cells at stage 2±4 (Fig. 2A), the matrix cells at stage 4 (Fig. 2A,B), and the IRS and ORS stage at 7±8 (Fig. 2C). Hoxa4 expression was also found in the interfollicular epidermis (Fig. 2A,C). No expression was seen in hair follicles from Hoxa4 mutant mice (Fig. 2D; negative control). We then examined sections containing mid-anagen cycling follicles at P5.5 (Fig. 2E,F), late anagen cycling follicles at P9 (data not shown), catagen cycling follicles at P17 (Fig. 2G) and telogen cycling follicles at 8 weeks of age (Fig. 2H). Hoxa4 expression above background levels was not observed in any cycling follicles (Fig. 2E±H). We also observed no Hoxa4 expression in sections of skin from mice at 8 weeks of age, either 3 or 5 days after depilation (data not shown). As a positive control, sections from a wild-type P2 mouse were processed in parallel and probed with a keratin 1 probe, revealing the expected expression in the interfollicular epidermis (data not shown). Hoxb4 expression was observed both in developing follicles at P2 (Fig. 2I) and in cycling follicles in catagen at P19 (Fig. 2J), suggesting a difference in the expression and potential roles of these paralogous genes in hair follicle morphogenesis. 2. Materials and methods 2.1. Tissues and mice Skin samples were dissected and processed as described previously (Behringer et al., 1993; Panteleyev et al., 1997). For timed matings, the day of the detection of the vaginal plug was considered embryonic day 0.5 and the developmental stage was determined according to Theiler (1972). Genotyping of Hoxa4 mutant mice was carried out as described previously (Horan et al., 1994). 2.2. In situ hybridization and PAS staining Non-radioactive (digoxigenin) in situ hybridization was carried out on 6 mM sections according to Komminoth (1992). Preparation of the digoxigenin-labeled Hoxa4speci®c riboprobe was carried out as described previously (Packer et al., 2000). The region of Hoxb4 used for riboprobe preparation was as described in Watrin and Wolgemuth (1993). The mouse keratin 1-speci®c riboprobe was derived from a 268 bp template originally described in Panteleyev et al. (1997). PAS staining was performed using a periodic

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