Induction of the hair growth phase in postnatal mice by localized transient expression of Sonic hedgehog (original) (raw)

Shh expression. Several lines of evidence demonstrated that administration of AdShh led to expression of the Shh coding sequences, resulting in production of a functional Shh protein. First, Northern analysis of A549 cells infected with the Ad vector encoding murine Shh demonstrated transcripts of approximately 3.0 kb, corresponding to the sum of the full-length Shh cDNA (2.6 kb) with associated vector-derived 5′- and 3′-untranslated regions (∼400 bp; Figure 1a). In contrast, Northern analysis of naive A549 cells or A549 cells infected with AdNull demonstrated no Shh transcripts. Second, immunoblot analysis demonstrated that the AdShh-infected A549 cells produced a 19 kDa processed form of the murine Shh protein (Figure 1b). Third, Shh expression directed by AdShh could also be detected in vivo by Northern analysis. In naive C57BL/6 mouse skin, upregulation of the endogenous 2.6-kb Shh gene expression was observed at experimental day 7 when mouse skin entered early anagen (Figure 1c). By day 14, endogenous Shh gene expression was diminished. C57BL/6 mouse skin injected with AdNull showed a pattern of Shh expression similar to that seen in naive skin. In contrast, AdShh-injected mouse skin showed strong Shh gene expression at day 3 after injection (the AdShh-encoded transcript is 3.0 kb, larger than the 2.6-kb endogenous Shh seen in the controls at day 7). At day 3 and more clearly at day 5, 2 distinct bands were observed in close proximity. Vector-encoded message appeared at 3.0 kb. The lower band at 2.6 kb corresponded to endogenous Shh transcript that had the same size as that seen in the nontreated mouse skin. At day 7, endogenous signal exceeded vector-encoded signal, and at day 14 the upper band derived from AdShh could no longer be detected. These results raise the possibility that exogenous Shh expression derived from AdShh induced the endogenous Shh expression as early as day 3 after injection (postnatal day 22), when naive skin showed no endogenous Shh mRNA expression.

Functional expression of Shh by administration of AdShh in vitro and in vivFigure 1

Functional expression of Shh by administration of AdShh in vitro and in vivo in postnatal day 19 C57BL/6 mice. (a) Shh mRNA after in vitro infection of A549 epithelial cells with AdShh. A549 cells (106) were infected (20 moi) with the AdNull control vector or AdShh. After 2 days, RNA was analyzed by Northern blot with a 32P-labeled Shh cDNA probe. Uninfected cells (naive) and AdNull-infected cells were negative, whereas AdShh-infected cells contained a transcript of approximately 3.0 kb corresponding to the vector-encoded Shh transgene. Equal RNA loading was confirmed by analysis of GAPDH mRNA. (b) Shh protein expressed in vitro. Protein from A549 cells infected with AdNull or AdShh as described in a was analyzed by immunoblot for Shh protein. AdShh-infected, but not naive or AdNull-infected, A549 cells showed a 19-kDa immunoreactive band with anti-Shh. Equal protein loading was confirmed by Coomassie blue staining of an identically loaded gel (not shown). (c) Time course demonstrating expression of Shh, Ptc, and Gli1 mRNA in vivo. Dorsal skin was collected from naive C57BL/6 mice or C57BL/6 mice at postnatal days 19, 22, 24, 26, and 33 after intradermal injection on day 19 with AdNull or AdShh (108 PFU for either vector). Skin was analyzed for Shh, Ptc, or Gli1 mRNA by Northern analysis on experimental days 0, 3, 5, 7, and 14. Expression of the 3.0-kb mRNA vector-encoded transcript (upper arrow) and the 2.6-kb endogenous Shh transcript (lower arrows) was detected in AdShh-injected mice, but the vector-derived transcript temporally preceded the endogenous transcript. Marked upregulation of Ptc and Gli1 gene expression was detected in AdShh-injected mice on experimental days 3 and 5, when low levels of expression of these genes were seen in naive and AdNull-treated mice. Equal RNA loading was confirmed by analysis of GAPDH mRNA.

Finally, in addition to Shh, the AdShh vector induced in vivo expression of mRNA transcripts of both Ptc and Gli1, two of the mediators that act as downstream effectors in the Shh signaling pathway (2, 13, 23). Naive and AdNull-injected skin showed upregulation of both Ptc and Gli1 transcripts on experimental day 7 and day 14, on which days endogenous Shh was also upregulated, suggesting that the Shh signaling pathway is normally activated during the hair cycle. In contrast, AdShh-injected skin showed remarkable upregulation of both Ptc and Gli1 gene expression on experimental days 3, 5, and 7, parallel to the Shh expression pattern. Administration of the control AdNull vector appeared to shift expression of endogenous Shh, Ptc, and Gli1 from predominantly day 7 to expression on both days 7 and 14.

In situ hybridization. To establish the localization of Shh expression on day 3 after administration of AdShh, paraffin-embedded sections were hybridized with either antisense or sense [33P]UTP riboprobes, and were evaluated using a photographic emulsion. As a positive control, anagen skin was evaluated for Shh transcripts. In anagen skin, Shh expression was limited to the matrix of the hair follicle bulb and was often observed on the anterior side of the hair follicle (Figure 2, a and b), in agreement with previous reports in postnatal skin (34). Naive telogen skin (postnatal day 22), as well as skin injected with AdNull, showed no detectable Shh transcript using the sense probe (Figure 2, c and d). In contrast, AdShh-injected skin showed intense Shh transcript expression with antisense, but not sense riboprobes, in diverse cell types including keratinocytes in hair follicles, mesenchymal cells in dermal papillae, fibroblasts in the dermis and the subdermal fascia, and muscle (Figure 2, e-i).

Localization of Shh expression in skin by in situ hybridization before andFigure 2

Localization of Shh expression in skin by in situ hybridization before and after administration of AdShh. Paraffin sections of mouse skin during its natural anagen period (postnatal day 33) compared with postnatal day 22 naive mouse skin or skin of mice injected on postnatal day 19 with PBS, AdNull, or AdShh. The sections were analyzed by in situ hybridization using [33P]UTP-labeled antisense and sense Shh riboprobes. After hybridization, sites of probe binding were identified using a photographic emulsion, and tissue was stained with hematoxylin and eosin. Arrows indicate positive staining for Shh mRNA. Closed arrowheads indicate melanosomes in the hair follicle. Open arrowheads indicate melanin in hair shafts. (a) Anagen skin from a naive 33-day-old mouse (positive control) hybridized with an antisense Shh complementary to Shh mRNA. (b) Adjacent tissue section of anagen skin hybridized with a sense Shh probe. (c) Naive postnatal day 22 skin hybridized with an antisense Shh probe. (d) Postnatal day 22 skin 3 days after injection with 108 PFU of AdNull hybridized with an antisense Shh probe. (e) Postnatal day 22 skin 3 days after injection of 108 PFU of AdShh hybridized with an antisense Shh probe. (f) Postnatal day 22 skin 3 days after injection with 108 PFU of AdShh hybridized with a sense Shh probe. (gi) High-magnification examples of cells in AdShh-injected mouse skin hybridized with antisense Shh probe. Scale bar: 50 μm.

Hair growth. Hallmarks of anagen include thickening of the dermal and epidermal layers of the skin, increased size of hair follicles (and thus increased area occupied by follicles in a histologic section), extension of follicles deep into the dermal adipose tissue, and initiation of melanin synthesis (1, 51). Histology of mouse dorsal skin was evaluated in telogen/early anagen (7 days after intradermal injection of AdShh into postnatal day 19 catagen/telogen mouse skin) at the site of injection, and in dorsal skin at a site distant from the site of injection. As controls, naive, PBS-injected, and AdNull-injected skin were analyzed. Compared with control skin, both the dermis and epidermis of AdShh-injected skin showed characteristics of anagen, including thickening, hair follicles that were large, and extension of the follicles deep into the adipose layer of the dermis (Figure 3a). No derangements of the hair follicles were noted in AdShh-injected skin. No abnormal proliferation of basal cells was observed, nor was there evidence of basal cell carcinoma. All hair follicles maintained native orientation along the cephalocaudal axis, indicating that follicle polarity was not altered. The border of the injection site at 7 days after administration of AdShh revealed a sharp contrast in the hair follicle cycle between late anagen in the injection site and early anagen in the adjacent skin (Figure 3b). Quantitative analysis of hair follicle area as a percentage of total skin area (dermal plus epidermal) showed that AdShh induced an increase in follicle area at the site of injection (Figure 3c). In agreement with the histologic data, the expression level for mRNA transcripts of the hair-specific keratin ghHb-1, a molecular marker for anagen hair growth (52), was higher in AdShh-injected animals 7 days after treatment than in naive animals or animals injected with PBS or AdNull (Figure 3d). This is consistent with keratinocyte growth and differentiation induced by administration of AdShh. Melanogenesis was evident in anagen skin as a brown/black area in the matrix of the hair follicle bulb. After injection of AdShh, but not AdNull, the melanin in hair follicle bulbs in unstained paraffin sections was visible, indicating active hair shaft synthesis (Figure 3, e and f). Melanin production requires activity of tyrosinase, an enzyme in the biosynthetic pathway of melanin (51). RNA from skin of AdShh-injected mice 7 days after treatment showed greater levels of tyrosinase mRNA transcripts than did comparable samples from naive, PBS-injected, or AdNull-injected control mice (Figure 3g).

Induction of hair follicle growth and melanogenesis after intradermal adminFigure 3

Induction of hair follicle growth and melanogenesis after intradermal administration of AdShh. AdShh, AdNull, or PBS was administered to the dorsal skin of postnatal day 19 C57BL/6 mice as in Figure 1c, and analyses were performed on postnatal day 26. (a) Histologic evaluation 7 days after vector administration. The AdShh-injected skin has increased depth of the dermal layer, increased follicle length, and increased follicle area compared with the naive, PBS, and AdNull controls. The changes associated with AdShh injection were limited to the area of injection (injection site vs. distant site [control]). (b) Histologic evaluation of the border of the injection site 7 days after vector administration. Hair follicles in the injection site are in late anagen; hair follicles in adjacent skin are in early anagen. A corresponding change in skin thickness was noted. (c) Quantitation of follicle area as a percentage of the total dermal/epidermal area using digital imaging and pixel area integration. Each data point represents an area measurement from a representative field. Data from 3 fields are shown per mouse; data from 2 mice are shown per treatment. (d) Northern analysis showing enhanced expression of the hair-specific keratin ghHb-1 gene induced by AdShh in vivo relative to the naive, PBS, and AdNull controls. Equal RNA loading shown by probing for GAPDH mRNA. (e and f) Melanin expression in hair follicle. Accumulation of melanin was evaluated by bright-field microscopy of unstained paraffin sections. Skin at the site of injection of AdShh (f), but not AdNull-injected skin (e), showed increased melanin in hair follicle bulbs. Skin of PBS-injected and naive control mice was similar to skin of AdNull-injected mice (not shown). (g) Northern analysis showing upregulation of melanogenesis-related tyrosinase gene expression after administration of AdShh relative to the naive, PBS, and AdNull controls. Equal RNA loading was confirmed by probing for GAPDH mRNA. Scale bars: 200 μm.

The histologic and molecular evidence indicated that skin was induced to enter anagen when exposed to elevated expression of Shh caused by intradermal administration of AdShh. A corollary to this finding would be that treatment to accelerate the hair follicle cycle’s entry to anagen would produce new hair shafts sooner than untreated skin would. To test this hypothesis, dorsal skin of postnatal day 19 C57BL/6 mice were injected with AdShh and compared with naive mice or mice that received PBS or AdNull injections. Five days after injection, the hair was dyed with blonde hair dye to mark existing hair. On postnatal day 26 (experimental day 7), mouse hair was carefully clipped and examined for induction of the melanogenesis associated with anagen skin. The dorsal skin of naive mice (not shown), PBS-injected mice (not shown), and AdNull-injected mice (Figure 4, a and b) was of a uniform color, indicating that the dorsal skin appeared similar with respect to the phase of the follicle growth cycle. In contrast, AdShh-injected mouse skin showed a distinct oval of darkened skin in the area of intradermal injection; uninjected skin from the same mice was not affected (Figure 4, a and b; Table 1). On postnatal day 33 (experimental day 14), the area of melanogenesis had produced new hair shafts (note the striking appearance of black hair), whereas the remainder of the dorsal skin had only begun to enter anagen (note darkening of the dorsal skin in Figure 4, c and d; Table 1). In contrast, AdNull-injected animals maintained a uniform hair growth cycle on their dorsal surface (Figure 4, c and d).

Hair growth in C57BL/6 mice after intradermal administration of AdShh durinFigure 4

Hair growth in C57BL/6 mice after intradermal administration of AdShh during first telogen. AdShh, AdNull, or PBS was administered to the dorsal skin of postnatal day 19 C57BL/6 mice. Five days after administration, dorsal hair was bleached with blonde hair dye to provide contrast for assessing new growth of the natural black hair of the mice. On day 7, the dorsal hair was clipped. (a and b) Mice at day 7 after administration of AdShh. Shown are 2 example pairs. The injection site in AdNull-treated animals was indistinct (left-side mice), whereas melanogenesis was evident at the site of AdShh injection in AdShh-treated mice (right-side mice). (c and d) Mice at day 14 after administration of AdShh. Shown are 2 example pairs. The injection site in AdNull-treated animals was indistinct (left-side mice). New hair growth is seen (note black color relative to preexisting dyed hair) at the site of AdShh injection (right-side mice). (e) Lateral aspect of day 14 AdShh-treated mouse showing length of hair growth at injection site. (f) High magnification of injection site showing new black hair and preexisting blonde-dyed hair. Scale bar: 2 mm. (g) Scanning electron microscopic analysis of normal C57BL/6 mouse hair shaft. (h) Scanning electron microscopic analysis of C57BL/6 mouse hair shaft induced by intradermal injection of AdShh (day 14 after vector administration). Scale bar: 10 μm. (i) Spatial distribution of transgene expression. Adβgal (108 PFU), an E1–E3– Ad vector expressing the Escherichia coli βgal marker gene, was injected intradermally on the dorsal surface of postnatal day 19 C57BL/6 mice. On day 0 and on day 2, a single strip of dorsal skin along the cephalocaudal axis was harvested, divided into 5 equal 2.5-mm segments (see diagram below graph; 0 = site of infection), and assayed for βgal activity (data from 3 animals are shown). The gray area corresponds to the size and position of the wheal (7–10 mm in diameter) formed at the site of injection. Data are expressed as βgal activity per mg protein. The anatomic distribution of the marker gene is similar to that of the melanogenesis and new hair growth observed in b and c.

Table 1

AdShh-mediated acceleration of hair follicle maturation

The newly produced hair shafts in AdShh-treated mice at the gross morphologic level, as well as at the dissecting microscopic and scanning electronic microscopic levels, demonstrated that the newly produced hair shafts had normal hair structures (Figure 4, e–h). The size of the affected area correlated with the size of the dermal wheal formed during intradermal administration of the vector. The hypothesis that transgene expression was limited to the area of the dermal wheal was independently confirmed using an Ad vector expressing the marker gene βgal (Adβgal, 108 PFU/20 μL), administered to dorsal skin of postnatal day 19 C57BL/6 mice. Two days later, a 12.5-mm length of skin (2.5 mm wide) was dissected, divided into 5 parts, and analyzed for βgal activity. Transgene expression was limited to an area similar in size to the original wheal, with a slight broadening of the expression, indicating that the vector may have spread from the site of injection as the wheal resolved (Figure 4i).

The data indicate that administration of AdShh induced the hair cycle in first skin telogen to enter anagen phase at the site of injection. To demonstrate that AdShh would function in a similar fashion in second telogen, mice were injected with AdShh at 8 weeks of age. Analysis of melanogenesis in mouse skin 10 days after injection showed that AdShh induced melanogenesis, whereas control mice remained in telogen (Figure 5a). Histologic analysis of second telogen skin showed characteristics of anagen at the site of injection of AdShh, but not in control mice (Figure 5, b–e).

Hair growth in C57BL/6 mice after intradermal administration of AdShh durinFigure 5

Hair growth in C57BL/6 mice after intradermal administration of AdShh during second telogen. AdShh, AdNull, or PBS was administered to the dorsal skin of 8-week-old C57BL/6 mice. Mice were dyed and clipped as described in Figure 3, and were evaluated on experimental day 10 after vector administration for melanogenesis. Dorsal skin was subsequently collected, embedded in paraffin, and sectioned along the cephalocaudal axis. (a) Melanogenesis induced by AdShh. AdShh-treated mice (right pair of mice) but not AdNull-treated mice (left pair of mice) showed melanogenesis at the site of injection 7 days after treatment. (be) Histologic evaluation of area of AdShh injection. (b) Naive mouse skin. (c) PBS-injected mouse skin. (d) AdNull-injected mouse skin. (e) AdShh-injected mouse skin. Note the increased thickness and increased area of hair follicles similar to that in Figure 3. Scale bar: 200 μm.

Assessment of the AdShh-injected animals (n = 5 for each treatment) 6 months after administration of the AdShh vector revealed no gross differences compared with AdNull-injected mice (Figure 6a) or other controls (PBS-injected or naive mice, not shown). Histologic examination of dorsal skin 6 months after AdShh administration revealed no abnormalities compared with AdNull-injected, PBS-injected, or naive mice (Figure 6, b–e).

Macroscopic and histologic assessment of AdShh-injected mice 6 months afterFigure 6

Macroscopic and histologic assessment of AdShh-injected mice 6 months after administration of AdShh. (a) Macroscopic assessment of PBS-, AdNull-, and AdShh-injected mice. For long-term studies, mice were not exposed to hair dye, but were clipped once at the time of vector administration. No differences were noted among groups. (be) Histologic assessment. Shown are representative sections from skin of a naive mouse and of PBS-, AdNull-, and AdShh-injected mice 6 months after administration. No abnormalities were observed at the injection site at this time. Three mice were examined for each treatment. Scale bar: 200 μm.