'Cyclic alopecia' in Msx2 mutants: defects in hair cycling and hair shaft differentiation

Msx2-deficient mice exhibit progressive hair loss, starting at P14 and followed by successive cycles of wavelike regrowth and loss. During the hair cycle, Msx2 deficiency shortens anagen phase, but prolongs catagen and telogen. Msx2-deficient hair shafts are structurally abnormal. Molecular analyses suggest a Bmp4/Bmp2/Msx2/Foxn1 acidic hair keratin pathway is involved. These structurally abnormal hairs are easily dislodged in catagen implying a precocious exogen. Deficiency in Msx2 helps to reveal the distinctive skin domains on the same mouse. Each domain cycles asynchronously - although hairs within each skin domain cycle in synchronized waves. Thus, the combinatorial defects in hair cycling and differentiation, together with concealed skin domains, account for the cyclic alopecia phenotype.     

Development and evolution of the mammalian limb: adaptive diversification of nails, hooves, and claws

SUMMARY Paleontological evidence indicates that the evolutionary diversification of mammals early in the Cenozoic era was characterized by an adaptive radiation of distal limb structures. Likewise, neontological data show that morphological variation in distal limb integumentary appendages (e.g., nails, hooves, and claws) can be observed not only among distantly related mammalian taxa but also among closely related species within the same clade. Comparative analysis of nail, claw, and hoof morphogenesis reveals relatively subtle differences in mesenchymal and epithelial patterning underlying these adult differences in distal limb appendage morphology. Furthermore, studies of regulatory gene expression during vertebrate claw development demonstrate that many of the signaling molecules involved in patterning ectodermal derivatives such as teeth, hair, and feathers are also involved in organizing mammalian distal limb appendages. For example, Bmp4 signaling plays an important role during the recruitment of mesenchymal cells into the condensations forming the terminal phalanges, whereas Msx2 affects the length of nails and claws by suppressing proliferation of germinal epidermal cells. Evolutionary changes in the form of distal integumentary appendages may therefore result from changes in gene expression during formation of mesenchymal condensations ( Bmp4 , posterior Hox genes), induction of the claw fold and germinal matrix ( shh ), and/or proliferation of epidermal cells in the claw matrix ( Msx1 , Msx2 ). The prevalence of convergences and parallelisms in nail and claw structure among mammals underscores the existence of multiple morphogenetic pathways for evolutionary change in distal limb appendages.     

Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development

The homeobox-containing genes Msx1 and Msx2 are highly expressed in the limb field from the earliest stages of limb formation and, subsequently, in both the apical ectodermal ridge and underlying mesenchyme. However, mice homozygous for a null mutation in either Msx1 or Msx2 do not display abnormalities in limb development. By contrast, Msx1; Msx2 double mutants exhibit a severe limb phenotype. Our analysis indicates that these genes play a role in crucial processes during limb morphogenesis along all three axes. Double mutant limbs are shorter and lack anterior skeletal elements (radius/tibia, thumb/hallux). Gene expression analysis confirms that there is no formation of regions with anterior identity. This correlates with the absence of dorsoventral boundary specification in the anterior ectoderm, which precludes apical ectodermal ridge formation anteriorly. As a result, anterior mesenchyme is not maintained, leading to oligodactyly. Paradoxically, polydactyly is also frequent and appears to be associated with extended Fgf activity in the apical ectodermal ridge, which is maintained up to 14.5 dpc. This results in a major outgrowth of the mesenchyme anteriorly, which nevertheless maintains a posterior identity, and leads to formation of extra digits. These defects are interpreted in the context of an impairment of Bmp signalling.     

BMP4 rescues a non-cell-autonomous function of Msx1 in tooth development

The development of many organs depends on sequential epithelial-mesenchymal interactions, and the developing tooth germ provides a powerful model for elucidating the nature of these inductive tissue interactions. In Msx1-deficient mice, tooth development arrests at the bud stage when Msx1 is required for the expression of Bmp4 and Fgf3 in the dental mesenchyme (Bei, M. and Maas, R. (1998) Development 125, 4325-4333). To define the tissue requirements for Msx1 function, we performed tissue recombinations between wild-type and Msx1 mutant dental epithelium and mesenchyme. We show that through the E14.5 cap stage of tooth development, Msx1 is required in the dental mesenchyme for tooth formation. After the cap stage, however, tooth development becomes Msx1 independent, although our experiments identify a further late function of Msx1 in odontoblast and dental pulp survival. These results suggest that prior to the cap stage, the dental epithelium receives an Msx1-dependent signal from the dental mesenchyme that is necessary for tooth formation. To further test this hypothesis, Msx1 mutant tooth germs were first cultured with either BMP4 or with various FGFs for two days in vitro and then grown under the kidney capsule of syngeneic mice to permit completion of organogenesis and terminal differentiation. Previously, using an in vitro culture system, we showed that BMP4 stimulated the growth of Msx1 mutant dental epithelium (Chen, Y., Bei, M. Woo, I., Satokata, I. and Maas, R. (1996). Development 122, 3035-3044). Using the more powerful kidney capsule grafting procedure, we now show that when added to explanted Msx1-deficient tooth germs prior to grafting, BMP4 rescues Msx1 mutant tooth germs all the way to definitive stages of enamel and dentin formation. Collectively, these results establish a transient functional requirement for Msx1 in the dental mesenchyme that is almost fully supplied by BMP4 alone, and not by FGFs. In addition, they formally prove the postulated downstream relationship of BMP4 with respect to Msx1, establish the non-cell-autonomous nature of Msx1 during odontogenesis, and disclose an additional late survival function for Msx1 in odontoblasts and dental pulp.     

A nonsense mutation in MSX1 causes Witkop syndrome

Witkop syndrome, also known as tooth and nail syndrome (TNS), is a rare autosomal dominant disorder. Affected individuals have nail dysplasia and several congenitally missing teeth. To identify the gene responsible for TNS, we used candidate-gene linkage analysis in a three-generation family affected by the disorder. We found linkage between TNS and polymorphic markers surrounding the MSX1 locus. Direct sequencing and restriction-enzyme analysis revealed that a heterozygous stop mutation in the homeodomain of MSX1 cosegregated with the phenotype. In addition, histological analysis of Msx1-knockout mice, combined with a finding of Msx1 expression in mesenchyme of developing nail beds, revealed that not only was tooth development disrupted in these mice, but nail development was affected as well. Nail plates in Msx1-null mice were defective and were thinner than those of their wild-type littermates. The resemblance between the tooth and nail phenotype in the human family and that of Msx1-knockout mice strongly supports the conclusions that a nonsense mutation in MSX1 causes TNS and that Msx1 is critical for both tooth and nail development.     

Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b

The murine genome is known to have two keratin 6 (K6) genes, mouse K6 (MK6)a and MK6b. These genes display a complex expression pattern with constitutive expression in the epithelia of oral mucosa, hair follicles, and nail beds. We generated mice deficient for both genes through embryonic stem cell technology. The majority of MK6a/b-/- mice die of starvation within the first two weeks of life. This is due to a localized disintegration of the dorsal tongue epithelium, which results in the build up of a plaque of cell debris that severely impairs feeding. However, approximately 25% of MK6a/b-/- mice survive to adulthood. Remarkably, the surviving MK6a/b-/- mice have normal hair and nails. To our surprise, we discovered MK6 staining both in the hair follicle and the nail bed of MK6a/b-/- mice, indicating the presence of a third MK6 gene. We cloned this previously unknown murine keratin gene and found it to be highly homologous to human K6hf, which is expressed in hair follicles. We therefore termed this gene MK6 hair follicle (MK6hf). The presence of MK6hf in the MK6a/b-/- follicles and nails offers an explanation for the absence of hair and nail defects in MK6a/b-/- animals.     

MSX2 expression in the apical ectoderm ridge is regulated by an MSX2 and Dlx5 binding site.

The apical ectodermal ridge (AER) is a specialized ectodermal region essential for limb outgrowth. Msx2 expression patterns in limb development strongly suggest an important role for Msx2 in the AER. Our previous studies identified a 348-bp fragment of the chicken Msx2 gene with AER enhancer activity. In this study, the functions of four potential homeodomain binding TAAT sites in this enhancer were studied using transgenic mice and in vitro protein-DNA interactions. Transgenic studies indicate that the four TAAT sites are not redundant and that only the B-TAAT site is critical for AER enhancer activity. The expression patterns of Msx2 and Dlx5 genes in the AER suggest that they might be involved in the regulation of Msx2. In support of this hypothesis, we found that Msx2 and Dlx5 can bind to the B-TAAT site as well as to a fragment containing the D- and E-TAAT sites in the Msx2 AER enhancer sequences. (c)2002 Elsevier Science (USA).     

The role of activins and follistatins in skin and hair follicle development and function

Investigations of the signalling between epithelial and mesenchymal compartments of skin during hair follicle initiation in utero and hair cycling have revealed the importance of the TGFβ superfamily in ectodermal organogenesis and morphogenesis. In particular the activins, their receptors and binding proteins such as follistatin, have been shown to be important regulators of cell proliferation, differentiation and apoptosis in hair follicle initiation, hair cycling, normal skin homeostasis and wound healing. Transgenic mice lacking various components of the activin signalling pathways display varying ectodermal pathologies including altered pelage hair follicle initiation. This review summarises the activin signal transduction pathways and the interactions between activins and other TGFβ signalling systems during hair follicle formation, hair growth cycling, skin function and wound healing.