beta-Catenin initiates tooth neogenesis in adult rodent incisors.

beta-Catenin signaling is required for embryonic tooth morphogenesis and promotes continuous tooth development when activated in embryos. To determine whether activation of this pathway in the adult oral cavity could promote tooth development, we induced mutation of epithelial beta-catenin to a stabilized form in adult mice. This caused increased proliferation of the incisor tooth cervical loop, outpouching of incisor epithelium, abnormal morphology of the epithelial-mesenchymal junction, and enhanced expression of genes associated with embryonic tooth development. Ectopic dental-like structures were formed from the incisor region following implantation into immunodeficient mice. Thus, forced activation of beta-catenin signaling can initiate an embryonic-like program of tooth development in adult rodent incisor teeth.

Wnt/beta-catenin signaling in oral tissue development and disease.

The Wnt/beta-catenin signaling pathway is one of several key conserved intercellular signaling pathways in animals, and plays fundamental roles in the proliferation, regeneration, differentiation, and function of many cell and tissue types. This pathway is activated in a dynamic manner during the morphogenesis of oral organs, including teeth, taste papillae, and taste buds, and is essential for these processes to occur normally. Conversely, forced activation of Wnt/beta-catenin signaling promotes the formation of ectopic teeth and taste papillae. In this review, we discuss our current understanding of the roles of Wnt/beta-catenin signaling in oral tissue development and in related human diseases, and the potential of manipulating this pathway for therapeutic purposes.

Requirement of Rac1 distinguishes follicular from interfollicular epithelial stem cells.

Epithelial stem cells in the bulge region within the hair follicle maintain the cyclic hair growth, but whether these stem cells also contribute to the epidermal renewal remains unclear. Here, we observed that the conditional deletion of the Rac1 gene in the mouse skin, including the potential follicular and epidermal stem cell compartments, results in alopecia owing to defective hair development. Surprisingly, mice lacking the expression of this Rho GTPase do not display major alterations in the interfollicular skin. Furthermore, Rac1 excision from primary epithelial keratinocytes results in the inability to reconstitute hair follicles and sebaceous glands when grafted onto mice, but epithelial cells lacking Rac1 can nonetheless form a healthy epidermis. Together, these findings support the emerging view that the epidermis and the hair follicles are maintained by different epithelial stem cells, and provide evidence that the requirement for Rac1 function can distinguish these distinct stem cells populations.

Over- and ectopic expression of Wnt3 causes progressive loss of ameloblasts in postnatal mouse incisor teeth.

Intercellular signaling is essential for the development of teeth during embryogenesis and in maintenance of the continuously growing incisor teeth in postnatal rodents. WNT intercellular signaling molecules have been implicated in the regulation of tooth development, and the Wnt3 gene shows specific expression in the enamel knot at the cap stage. We demonstrate here that Wnt3 also is expressed in specific epithelial cell layers in postnatal incisor teeth. To begin to delineate the functions of Wnt3 in developing and postnatal teeth, we determined the effects of over- and ectopic expression of Wnt3 in the tooth epithelium of mice carrying a keratin 14-Wnt3 transgene. Expression of the transgene caused a progressive loss of ameloblasts from postnatal lower incisor teeth. Loss of ameloblasts may be due to defective proliferation or differentiation of ameloblast precursors, progressive apoptosis of ameloblasts, or loss of ameloblast stem cells.

Characterization of Wnt gene expression in developing and postnatal hair follicles and identification of Wnt5a as a target of Sonic hedgehog in hair follicle morphogenesis.

Mutations in WNT effector genes perturb hair follicle morphogenesis, suggesting key roles for WNT proteins in this process. We show that expression of Wnts 10b and 10a is upregulated in placodes at the onset of follicle morphogenesis and in postnatal hair follicles beginning a new cycle of hair growth. The expression of additional Wnt genes is observed in follicles at later stages of differentiation. Among these, we find that Wnt5a is expressed in the developing dermal condensate of wild type but not Sonic hedgehog (Shh)-null embryos, indicating that Wnt5a is a target of SHH in hair follicle morphogenesis. These results identify candidates for several key follicular signals and suggest that WNT and SHH signaling pathways interact to regulate hair follicle morphogenesis.

Towards a molecular understanding of hair loss and its treatment.

Most common forms of hair loss (alopecia) are caused by aberrant hair follicle cycling and changes in hair follicle morphology. However, current treatments for alopecia do not specifically target these processes. We are now beginning to identify the molecules and molecular pathways that control normal hair follicle formation, cycling and growth. In parallel, new techniques are being developed for delivering molecules to hair follicles. Here, we outline the characteristics of common hair loss diseases, and discuss ways in which recent advances in hair follicle biology could be translated into effective therapies for these conditions.

WNT signaling in the control of hair growth and structure.

Characterization of the molecular pathways controlling differentiation and proliferation in mammalian hair follicles is central to our understanding of the regulation of normal hair growth, the basis of hereditary hair loss diseases, and the origin of follicle-based tumors. We demonstrate that the proto-oncogene Wnt3, which encodes a secreted paracrine signaling molecule, is expressed in developing and mature hair follicles and that its overexpression in transgenic mouse skin causes a short-hair phenotype due to altered differentiation of hair shaft precursor cells, and cyclical balding resulting from hair shaft structural defects and associated with an abnormal profile of protein expression in the hair shaft. A putative effector molecule for WNT3 signaling, the cytoplasmic protein Dishevelled 2 (DVL2), is normally present at high levels in a subset of cells in the outer root sheath and in precursor cells of the hair shaft cortex and cuticle which lie immediately adjacent to Wnt3-expressing cells. Overexpression of Dvl2 in the outer root sheath mimics the short-hair phenotype produced by overexpression of Wnt3, supporting the hypothesis that Wnt3 and Dvl2 have the potential to act in the same pathway in the regulation of hair growth. These experiments demonstrate a previously unrecognized role for WNT signaling in the control of hair growth and structure, as well as presenting the first example of a mammalian phenotype resulting from overexpression of a Dvl gene and providing an accessible in vivo system for analysis of mammalian WNT signaling pathways.

Expression and transgenic studies of the mouse agouti gene provide insight into the mechanisms by which mammalian coat color patterns are generated.

Expression of the agouti gene from two different promoters, one active at the midpoint of the hair cycle and the other specific for the ventrum, is responsible for generating a range of mammalian pigmentation patterns. We demonstrate that in postnatal mice transcripts from both promoters are confined to the dermal papilla of hair follicles, as predicted by classical transplantation experiments. Transcripts from the hair cycle promoter are detected in the embryonic whisker plate but not in other regions of the body before birth, whereas ventral-specific transcripts are detected in the ventral trunk of the embryo as well as ventral whisker plate. To investigate further the embryonic origins of adult pigmentation patterns, we carried out a detailed analysis of agouti expression in the embryo. The ventral-specific agouti isoform is first expressed at E10.5 in neural crest-derived ventral cells of the second branchial arch, in anterior regions of the forelimb buds and in a narrow stripe of ventral mesenchyme. By E14.5 a continuous layer of expression is observed in the upper cells of the dermis, including cells of the developing dermal papillae, and covering the entire ventral surface of the head and trunk and dorsal surfaces of the distal forelimb and hindlimb. This expression pattern reflects the domain of yellow coloration evident in adult animals and suggests that the agouti gene is regulated in part by factors responsible for establishing differences between the dorsal and ventral surfaces of the body during embryogenesis. To test the hypothesis that agouti is a paracrine signaling molecule that can influence pigment production by hair follicle melanocytes when expressed by either dermis or epidermis, as suggested by recombination and transplantation experiments, we created transgenic animals in which agouti is expressed in basal cells of the epidermis. These animals display stripes of yellow hairs corresponding to regions of epidermal agouti expression, confirming that agouti signals melanocytes to synthesize yellow pigment and providing direct evidence that it functions in a paracrine manner with a restricted radius of action.

Differences in dorsal and ventral pigmentation result from regional expression of the mouse agouti gene.

The agouti coat color gene encodes a paracrine signaling molecule that controls the production of yellow and black pigment by melanocytes within hair follicles. Some agouti alleles affect the dorsum and ventrum independently, which has provided the basis for speculation that agouti gene action in different regions of the body is controlled by distinct genetic loci that are closely linked. Using a combination of cDNA cloning and RNA expression studies, we find that alternative isoforms of agouti mRNA contain different noncoding first exons located 100 kb apart, whose patterns of expression indicate independent control by regulatory elements that are either ventral specific or hair cycle specific. These results demonstrate that the apparent genetic complexity of the agouti locus is explained by the existence of multiple regulatory elements exerting control over a single coding sequence and provide a conceptual basis for understanding differences in dorsal and ventral hair coloration in many mammalian species. The ventral-specific agouti isoform represents an example of a transcript whose expression is restricted to ventral skin and provide an approach to investigate the mechanisms by which dorsal-ventral differences in gene expression are established and maintained.

ZAP-1 DNA binding activity is first detected at the onset of zona pellucida gene expression in embryonic mouse oocytes.

ZAP-1 (zona pellucida gene activating protein-1) is a putative transcription factor controlling the oocyte-specific expression of mouse and human zona pellucida genes. The DNA binding activity of ZAP-1 first appears in oocytes from 19-day-old mouse embryos and reaches a maximum level at 10 days after birth. This developmental profile closely parallels that of mouse zona pellucida gene transcription, which is detected in oocytes at 19 days of fetal life using a sensitive RT-PCR method and is maximal in 10-day-old animals. DNA binding activity similar to that of ZAP-1 is present in ovarian extracts from rat, human, and opossum, suggesting that the ZAP-1 protein may be conserved among mammals.

Autoimmune disease of the ovary induced by a ZP3 peptide from the mouse zona pellucida.

We describe a novel experimental system in mice for the study of ovarian autoimmune disease, a condition encountered in women with premature ovarian failure. The ovarian autoimmune disease is induced in B6AF1 mice by a 15-amino acid peptide (Cys-Ser-Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln-Ile-His-Gly-Pro-Arg) from mouse ZP3, the sperm-binding component of the zona pellucida that surrounds growing and mature oocytes. Whereas the peptide induces both T cell and antibody responses, adoptive transfer of CD4+ T cell lines derived from affected animals causes oophoritis without observable antibodies to the zona pellucida peptide. The primacy of the T cell response in the pathogenesis of disease is further substantiated by defining oophoritogenic peptides as small as eight amino acids (Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln) that do not elicit an antibody response to the full-length ZP3 peptide. The identification of a well characterized peptide as a causative agent of autoimmune oophoritis should facilitate understanding of the pathogenesis of this T cell-mediated autoimmune disease. Because the proteins of the zona pellucida are conserved among mammals (the mouse and human ZP3 proteins are 67% identical), this murine model may lead to better understanding of the pathogenesis of human autoimmune oophoritis.

Oocyte-specific factors bind a conserved upstream sequence required for mouse zona pellucida promoter activity.

The zona pellucida of mouse oocytes, composed of three major glycoproteins (ZP1, ZP2, and ZP3), performs crucial functions at fertilization and in early development. The transcripts encoding mouse ZP2 and ZP3 are coordinately expressed and accumulate in oocytes during a 2-week growth phase prior to ovulation. The 5'-flanking regions of mouse Zp-2 and Zp-3 genes and their human homologs contain five short DNA sequences (4 to 12 bp) that are 60 to 100% identical and are approximately equidistant upstream of the TATAA box in the four genes. Mutation of these five elements (I, IIA, IIB, III, and IV) in Zp-luciferase constructs demonstrates that the 12-bp element IV, positioned approximately 200 bp upstream from the TATAA box, is necessary for high-level expression from the mouse Zp-2 and Zp-3 promoters after microinjection into the nuclei of 50-microns-diameter oocytes. Injection of minimal Zp-3 promoter constructs containing element IV in either orientation also resulted in high levels of reporter gene activity, suggesting that the element is not only necessary but also sufficient for expression from zona pellucida promoters. Oligonucleotides containing the conserved element from either Zp-2 or Zp-3 form DNA-protein complexes of identical mobility in gel retardation assays using extracts of oocytes but not other tissues. These data are consistent with the hypothesis that common factors binding to conserved element IV are involved in coordinate expression of the oocyte-specific Zp-2 and Zp-3 zona pellucida genes.

Vaccination with a synthetic zona pellucida peptide produces long-term contraception in female mice.

The zona pellucida surrounding mouse oocytes is an extracellular matrix composed of three sulfated glycoproteins, ZP1, ZP2, and ZP3. It has been demonstrated that a monoclonal antibody to ZP3 injected into female mice inhibits fertilization by binding to the zona pellucida and blocking sperm penetration. A complementary DNA encoding ZP3 was randomly cleaved and 200- to 1000-base pair fragments were cloned into the expression vector lambda gt11. This epitope library was screened with the aforementioned contraceptive antibody, and the positive clones were used to map the seven-amino acid epitope recognized by the antibody. Female mice were immunized with a synthetic peptide containing this B cell epitope coupled to a carrier protein to provide helper T cell epitopes. The resultant circulating antibodies to ZP3 bound to the zona pellucida of immunized animals and produced long-lasting contraception. The lack of ovarian histopathology or cellular cytotoxicity among the immunized animals may be because of the absence of zona pellucida T cell epitopes in this vaccine.

Cloning of a gene encoding an antigen associated with the centrosome in Drosophila.

The monoclonal antibody Bx63 recognizes a centrosomal antigen of Drosophila melanogaster by indirect immunofluorescence and identifies two proteins, with apparent molecular weights of 185 x 10(3) and 66 x 10(3), on Western blots. We have used this antibody to isolate five clones (lambda cs1, -2, -3, -4 and lambda j63) from lambda gt11 expression libraries of Drosophila DNA. Using polyclonal anti-centrosomal sera raised against both immunoaffinity-purified Bx63 antigen and electrophoretically purified fusion protein from clone lambda cs3, we have demonstrated that the fusion proteins encoded by four of these clones (lambda cs1-4) share at least two epitopes with the 185 x 10(3) Mr centrosomal antigen. This indicates that clones lambda cs1-4 contain DNA from the gene coding for this protein. The four clones are independent isolates from a single chromosomal site, which we show by in situ hybridization to correspond with salivary gland chromosome region 88E 4-8. A low-abundance transcript of approximately 4.0 x 10(3) bases corresponding to the cloned gene is detected in all stages of the Drosophila life-cycle.

The distribution of a 'mitosis-specific' antigen during Drosophila development.

We have used MPM-2, a monoclonal antibody raised against mitotic HeLa cells, to stain a Drosophila cell line, whole mounts of Drosophila embryos, and sectioned tissue from embryonic and larval stages of development. MPM-2 recognizes a major phosphoprotein of approximately 125 X 10(3) Mr in Drosophila tissue culture cells that, like the mammalian MPM-2 antigen, appears to be recognized only in mitotic cells. During early embryogenesis, when the embryonic nuclei divide as a syncytium with a very short nuclear division time, MPM-2 antigen is observed within the spindle compartment at all stages of the nuclear division cycle. Upon cellularization of the embryo and lengthening of the duration of the cycle, the antigen is predominantly seen in mitotic cells. Drosophila larvae contain both diploid and polytene tissues: in diploid tissue MPM-2 staining is specifically observed over mitotic cells, as expected from its distribution in cellularized embryos. Surprisingly, antigen is also detected in the nuclei of polytene cells that replicate their DNA but do not undergo mitosis.