Structural features and sites of expression of a new murine 65 kD and 48 kD hair-related keratin pair, associated with a special type of parakeratotic epithelial differentiation

In the course of studies on local keratin phenotypes in the epidermis of the adult mouse, we have identified a new 65 kD and 48 kD keratin pair. In mouse skin, this keratin pair is only expressed in suprabasal cells of adult mouse tail scale epidermis which is characterized by the complete absence of a granular layer and the formation of a remarkably compact stratum corneum. A second site in which the 65 kD and 48 kD keratin pair is suprabasally expressed and whose morphology corresponds to that of tail scale epidermis is found in the posterior unit of the complex filiform papillae of mouse tongue. The causal relationship of the expression of the 65 kD and 48 kD keratins with this particular type of a non-pathological epithelial parakeratosis is emphasized by the suppression of the mRNA synthesis of the two keratins during retinoic acid mediated orthokeratotic conversion of tail scale epidermis. Apart from tail scale epidermis and the posterior unit of the filiform papillae, the 65 kD and 48 kD keratin pair is, however, also coexpressed with "hard" alpha keratins in suprabulbar cells of hair follicles and in suprabasal cells of the central core unit of the lingual filiform papillae. The non alpha-helical domains of the two new keratins are rich in cysteine and proline residues and lack the typical subdomains into which epithelial keratins of both types can be divided. This structural resemblance of the 65 kD and 48 kD keratins to "hard" alpha keratins is supported by comparative flexibility predictions for their non alpha-helical domains. Phylogenetic investigations then show that the 65 kD and 48 kD keratin pair has evolved together with hair keratins, but has diverged from these during evolution to constitute an independent branch of a pair of hair-related keratins. In view of this exceptional position of the 65 kD and 48 kD keratins within the keratin multigene family, their expression has apparently been adopted by rare anatomical sites in which an orthokeratinized stratum corneum would be too soft and a hard keratinized structure would be too rigid to meet the functional requirement of the respective epithelia.     

Expression of a dominant negative mutant of epidermal growth factor receptor in the epidermis of transgenic mice elicits striking alterations in hair follicle development and skin structure.

Epidermal growth factor receptor (EGFR) is a key regulator of keratinocyte biology. However, the physiological role of EGFR in vivo has not been well established. To analyze the role of EGFR in skin, we have generated transgenic mice expressing an EGFR dominant negative mutant in the basal layer of epidermis and outer root sheath of hair follicles. Mice expressing the mutant receptor display short and waved pelage hair and curly whiskers during the first weeks of age, but subsequently pelage and vibrissa hairs become progressively sparser and atrophic. Eventually, most mice present severe alopecia. Histological examination of the skin of transgenic mice shows striking alterations in the development of hair follicles, which fail to enter into catagen stage. These alterations eventually lead to necrosis and disappearance of the follicles, accompanied by strong infiltration of the skin with inflammatory elements. The interfollicular epidermis of these mice shows marked hyperplasia, expression of hyperproliferation-associated keratin K6 and increased 5-bromo-2-deoxyuridine incorporation. EGFR function was inhibited in transgenic skin keratinocytes, since in vivo and in vitro autophosphorylation of EGFR was almost completely abolished on EGF stimulation. These results implicate EGFR in the control of hair cycle progression, and provide new information about its role in epidermal growth and differentiation.     

Roles for PDGF-A and sonic hedgehog in development of mesenchymal components of the hair follicle.

Skin appendages, such as hair, develop as a result of complex reciprocal signaling between epithelial and mesenchymal cells. These interactions are not well understood at the molecular level. Platelet-derived growth factor-A (PDGF-A) is expressed in the developing epidermis and hair follicle epithelium, and its receptor PDGF-Ralpha is expressed in associated mesenchymal structures. Here we have characterized the skin and hair phenotypes of mice carrying a null mutation in the PDGF-A gene. Postnatal PDGF-A-/- mice developed thinner dermis, misshapen hair follicles, smaller dermal papillae, abnormal dermal sheaths and thinner hair, compared with wild-type siblings. BrdU labeling showed reduced cell proliferation in the dermis and in the dermal sheaths of PDGF-A-/- skin. PDGF-A-/- skin transplantation to nude mice led to abnormal hair formation, reproducing some of the features of the skin phenotype of PDGF-A-/- mice. Taken together, expression patterns and mutant phenotypes suggest that epidermal PDGF-A has a role in stimulating the proliferation of dermal mesenchymal cells that may contribute to the formation of dermal papillae, mesenchymal sheaths and dermal fibroblasts. Finally, we show that sonic hedgehog (shh)-/- mouse embryos have disrupted formation of dermal papillae. Such embryos fail to form pre-papilla aggregates of postmitotic PDGF-Ralpha-positive cells, suggesting that shh has a critical role in the assembly of the dermal papilla.     

Extensive Hair Shaft Growth after Mouse Whisker Follicle Isolation,Cryopreservation and Transplantation in Nude Mice

We previously demonstrated that whole hair follicles could be cryopreserved to maintain their stem-cells differentation potential. In the present study, we demonstrated that cryopreserved mouse whisker hair follicles maintain their hair growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. Cryopreserved hair follicles also maintained the hair follicle-associated-pluripotent (HAP) stem cells, evidenced by P75^NTR expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair shaft growth of cryopreserved hair follicles.     

Differential localization of distinct keratin mRNA-species in mouse tongue epithelium by in situ hybridization with specific cDNA probes

The tongue of the adult mouse is covered by a multilayered squamous epithelium which is continuous on the ventral surface, however interrupted on the dorsal surface by many filiform and few fungiform papillae. The filiform papillae themselves are subdivided into an anterior and posterior unit exhibiting different forms of keratinization. Thus, the entire epithelium shows a pronounced morphological diversity of well recognizable tissue units. We have used a highly sensitive in situ hybridization technique to investigate the differential expression of keratin mRNAs in the tongue epithelium. The hybridization probes used were cDNA restriction fragments complementary to the most specific 3'-regions of any given keratin mRNA. We could show that independent of the morphologically different tongue regions, all basal cells uniformly express the mRNA of a type I 52-kD keratin, typical also for basal cells of the epidermis. Immediately above the homogenous basal layer a vertically oriented specialization of the keratin expression occurs within the morphological tissue units. Thus the dorsal interpapillary and ventral epithelium express the mRNAs of a type II 57-kD and a type I 47-kD keratin pair. In contrast, in the anterior unit of the filiform papillae, only the 47-kD mRNA is present, indicating that this keratin may be coexpressed in tongue epithelium with different type II partners. In suprabasal cells of both, the fungiform papillae and the posterior unit of the filiform papillae, a mRNA of a type I 59-kD keratin could be detected; however, its type II 67-kD epidermal counterpart seems not to be present in these cells. Most surprisingly, in distinct cells of both types of papillae, a type I 50-kD keratin mRNA could be localized which usually is associated with epidermal hyperproliferation. In conclusion, the in situ hybridization technique applied has been proved to be a powerful method for detailed studies of differentiation processes, especially in morphologically complex epithelia.     

The catalog of human hair keratins. II. Expression of the six type II members in the hair follicle and the combined catalog of human type I and II keratins.

The human type II hair keratin subfamily consists of six individual members and can be divided into two groups. The group A members hHb1, hHb3, and hHb6 are structurally related, whereas group C members hHb2, hHb4, and hHb5 are rather distinct. Specific antisera against the individual hair keratins were used to establish the two-dimensional catalog of human type II hair keratins. In this catalog, hHb5 showed up as a series of isoelectric variants, well separated from a lower, more acidic, and complex protein streak containing isoelectric variants of hair keratins hHb1, hHb2, hHb3, and hHb6. Both in situ hybridization and immunohistochemistry on anagen hair follicles showed that hHb5 and hHb2 defined early stages of hair differentiation in the matrix (hHb5) and cuticle (hHb5 and hHb2), respectively. Although cuticular differentiation proceeded without the expression of further type II hair keratins, cortex cells simultaneously expressed hHb1, hHb3, and hHb6 at an advanced stage of differentiation. In contrast, hHb4, which is undetectable in hair follicle extracts and sections, could be identified as the largest and most alkaline member of this subfamily in cytoskeletal extracts of dorsal tongue. This hair keratin was localized in the posterior compartment of the tongue filiform papillae. Comparative analysis of type II with the previously published type I hair keratin expression profiles suggested specific, but more likely, random keratin-pairing principles during trichocyte differentiation. Finally, by combining the previously published type I hair keratin catalog with the type II hair keratin catalog and integrating both into the existing catalog of human epithelial keratins, we present a two-dimensional compilation of the presently known human keratins.     

A crucial role for Fgfr2-IIIb signalling in epidermal development and hair follicle patterning

To understand the role Fgf signalling in skin and hair follicle development, we analysed the phenotype of mice deficient for Fgfr2-IIIb and its main ligand Fgf10. These studies showed that the severe epidermal hypoplasia found in mice null for Fgfr2-IIIb is caused by a lack of the basal cell proliferation that normally results in a stratified epidermis. Although at term the epidermis of Fgfr2-IIIb null mice is only two to three cells thick, it expresses the classical markers of epidermal differentiation and establishes a functional barrier. Mice deficient for Fgf10 display a similar but less severe epidermal hypoplasia. By contrast, Fgfr2-IIIb-/-, but not Fgf10-/-, mice produced significantly fewer hair follicles, and their follicles were developmentally retarded. Following transplantation onto nude mice, grafts of Fgfr2-IIIb-/- skin showed impaired hair formation, with a decrease in hair density and the production of abnormal pelage hairs. Expression of Lef1, Shh and Bmp4 in the developing hair follicles of Fgfr2-IIIb-/- mice was similar to wild type. These results suggest that Fgf signalling positively regulates the number of keratinocytes needed to form a normal stratified epidermis and to initiate hair placode formation. In addition, Fgf signals are required for the growth and patterning of pelage hairs.     

Long-Term Extensive Ectopic Hair Growth on the Spinal Cord of Mice from Transplanted Whisker Follicles

We have previously demonstrated that hair follicles contain nestin-expressing pluripotent stem cells that can effect nerve and spinal cord repair upon transplantation. In the present study, isolated whisker follicles from nestin-driven green fluorescent protein (ND-GFP) mice were histocultured on Gelfoam for 3 weeks for the purpose of transplantation to the spinal cord to heal an induced injury. The hair shaft was cut off from Gelfoam-histocultured whisker follicles, and the remaining part of the whisker follicles containing GFP-nestin expressing pluripotent stem cells were transplanted into the injured spinal cord of nude mice, along with the Gelfoam. After 90 days, the mice were sacrificed and the spinal cord lesion was observed to have healed. ND-GFP expression was intense at the healed area of the spinal cord, as observed by fluorescence microscopy, demonstrating that the hair follicle stem cells were involved in healing the spinal cord. Unexpectedly, the transplanted whisker follicles sprouted out remarkably long hair shafts in the spinal cord during the 90 days after transplantation of Gelfoam whisker histocultures to the injured spine. The pigmented hair fibers, grown from the transplanted whisker histocultures, curved and enclosed the spinal cord. The unanticipated results demonstrate the great potential of hair growth after transplantation of Gelfoam hair follicle histocultures, even at an ectopic site.     

Wnt-10b secreted from lymphocytes promotes differentiation of skin epithelial cells

Wnt-10b was originally isolated from lymphoid tissue and is known to be involved in a wide range of biological actions, while recently it was found to be expressed early in the development of hair follicles. However, few studies have been conducted concerning the role of Wnt-10b with the differentiation of skin epithelial cells. To evaluate its role in epithelial differentiation, we purified Wnt-10b from the supernatant of a concanavalin A-stimulated lymphocyte culture using an affinity column and investigated its effects on the differentiation of adult mouse-derived primary skin epithelial cells (MPSEC). MPSEC cultured with Wnt-10b showed morphological changes from cuboidal to spindle-shaped with inhibited proliferation, and also obtained characteristics of the hair shaft and inner root sheath of the hair follicle, represented by red-colored Ayoub Shklar staining, and reactions to AE-13 and AE-15 as seen with immunocytology. Further, RT-PCR analysis demonstrated the expression of mRNA for keratin 1, keratin 2, loricrin, mHa5, and mHb5, in association with a decreased expression of the basal cell marker keratin 5, in Wnt-10b-treated MPSEC. In addition, involvement of the canonical Wnt signal pathway was demonstrated by a TCF reporter (pTOPFLASH) assay. These results suggest that Wnt-10b promotes the differentiation of MPSEC and may play an important role in hair follicle development by promoting differentiation of epithelial cells.     

Wnt-10b promotes differentiation of skin epithelial cells in vitro

To evaluate the role of Wnt-10b in epithelial differentiation, we investigated the effects of Wnt-10b on adult mouse-derived primary skin epithelial cells (MPSEC). Recombinant Wnt-10b protein (rWnt-10b) was prepared using a gene engineering technique and MPSEC were cultured in its presence, which resulted in morphological changes from cuboidal to spindle-shaped and inhibited their proliferation. Further, involvement of the canonical Wnt signal pathway was also observed. MPSEC treated with rWnt-10b showed characteristics of the hair shaft and inner root sheath of the hair follicle, in results of Ayoub Shklar staining and immunocytochemistry. Further, the cells expressed mRNA for differentiated epithelial cells, including keratin 1, keratin 2, loricrin, mHa5, and mHb5, in association with a decreased expression of the basal cell marker keratin 5. These results suggest that Wnt-10b promotes the differentiation of MPSEC.     

Structure and site of expression of a murine type II hair keratin

We present here a 1770 bp-long cDNA which encodes a murine type II keratin. Sequence comparisons of the keratin with those of various type II keratins expressed in mouse epidermis and internal stratified epithelia reveal that the new keratin is unrelated to epithelial keratins. Rather the structural organization of its amino- and carboxyterminal domains and the high content of cysteine and proline residues in these regions suggest that the keratin represents a murine type II hair keratin. This assumption was confirmed by in situ hybridization which localized the mRNA of the keratin in upper cells of the hair cortex and in suprabasal cells of the central core unit of filiform papillae of the tongue. Hybrid selection analyses revealed that the keratin has a molecular weight of 58 kD. It remains to be seen whether the keratin corresponds to MHb 3 or MHb 4.     

A dermal niche for multipotent adult skin-derived precursor cells

A fundamental question in stem cell research is whether cultured multipotent adult stem cells represent endogenous multipotent precursor cells. Here we address this question, focusing on SKPs, a cultured adult stem cell from the dermis that generates both neural and mesodermal progeny. We show that SKPs derive from endogenous adult dermal precursors that exhibit properties similar to embryonic neural-crest stem cells. We demonstrate that these endogenous SKPs can first be isolated from skin during embryogenesis and that they persist into adulthood, with a niche in the papillae of hair and whisker follicles. Furthermore, lineage analysis indicates that both hair and whisker follicle dermal papillae contain neural-crest-derived cells, and that SKPs from the whisker pad are of neural-crest origin. We propose that SKPs represent an endogenous embryonic precursor cell that arises in peripheral tissues such as skin during development and maintains multipotency into adulthood.     

Hoxa4 expression in developing mouse hair follicles and skin

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 first 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.