Mutations at the nude locus of mice and rats disrupt normal hair growth and thymus development, causing nude mice and rats to be immune-deficient. The mouse nude locus has been localized on chromosome 11 within a region of less than 1 megabase. One of the genes from this critical region, designated whn (winged-helix nude), encodes a new member of the winged-helix domain family of transcription factors, and it is disrupted on mouse nu and rat rnuN alleles. Mutant transcripts do not encode the characteristic DNA-binding domain, strongly suggesting that the whn gene is the nude gene. Mutations in winged-helix domain genes cause homeotic transformations in Drosophila and distort cell-fate decisions during vulval development in Caenorhabditis elegans. The whn gene is thus the first member of this class of genes to be implicated in a specific developmental defect in vertebrates (Nehls, 1994).

The development of the thymus depends initially on epithelial-mesenchymal interactions, and subsequently on reciprocal lympho-stromal interactions. The genetic steps governing development and differentiation of the thymic microenvironment are unknown. By a targeted disruption of the whn gene, which recapitulates the phenotype of the athymic nude mouse, the Whn transcription factor has been shown to be the product of the nude locus. Formation of the thymic epithelial primordium before the entry of lymphocyte progenitors does not require the activity of Whn. However, subsequent differentiation of primitive precursor cells into subcapsular, cortical, and medullary epithelial cells of the postnatal thymus depends on activity of the whn gene. These results define the first genetically separable steps during thymic epithelial differentiation (Nehls, 1996).

Mutations in the winged-helix nude (whn) gene are associated with the phenotype of congenital athymia and hairlessness in mouse and rat. The whn gene encodes a presumptive transcription factor with a DNA binding domain of the forkhead/winged-helix class. Two previously described null alleles encode truncated whn proteins lacking the characteristic DNA binding domain. In the rat rnu allele described here, a nonsense mutation in exon 8 of the whn gene was identified. The truncated whnrnu protein contains the DNA binding domain but lacks the 175 C-terminal amino acids of the wild-type protein. To facilitate the identification of functionally important regions in this region, a whn homolog from the pufferfish Fugu rubripes was isolated. Comparison of derived protein sequences with the mouse whn gene reveals the presence of a conserved acidic protein domain in the C terminus, in addition to the highly conserved DNA binding domain. Using fusions with a heterologous DNA binding domain, a strong transcriptional activation domain was localized to the C-terminal cluster of acidic amino acids. Since the whnrnu mutant protein lacks this domain, these results indicate that a transactivation function is essential for the activity of the whn transcription factor (Schuddekopf, 1996).

Mutations in the winged-helix nude (whn) gene result in the nude mouse and rat phenotypes. The pleiotropic nude phenotype, which affects the hair, skin, and thymus, suggests that whn plays a pivotal role in the development and/or maintenance of these organs. However, little is known about whn function in these organs. In skin whn is specifically expressed in epithelial cells and not the mesenchymal cells: using a hair reconstitution assay, it has been demonstrated that the abnormal nude mouse hair development is attributable to a functional defect of the epithelial cells. Examination of nude mouse primary keratinocytes in culture reveals that these cells have an increased propensity to differentiate in an abnormal fashion, even under conditions that promote proliferation. Furthermore, nude mouse keratinocytes display a 100-fold increased sensitivity to the growth-inhibitory/differentiation effects of the phorbol ester TPA. In parallel with these findings, it has been directly shown that Whn functions as a transcription factor that can specifically suppress expression of differentiation/TPA-responsive genes. The region of Whn responsible for these effects was mapped to the carboxy-terminal transactivating domain. These results establish whn as a key regulatory factor involved in maintaining the balance between keratinocyte growth and differentiation. The general implications of these findings for an epithelial self-renewal model are discussed (Brissette, 1996).

In the mouse, the product of the nude locus, Whn, is required for the keratinization of the hair shaft and the differentiation of epithelial progenitor cells in the thymus. A bacterially expressed peptide representing the presumptive DNA binding domain of the mouse whn gene in vitro specifically binds to an 11-bp consensus sequence containing the invariant tetranucleotide 5'-ACGC. In transient transfection assays, such binding sites stimulate reporter gene expression about 30- to 40-fold, when positioned upstream of a minimal promotor. Whn homologs from humans, bony fish (Danio rerio), cartilaginous fish (Scyliorhinus caniculus), agnathans (Lampetra planeri), and cephalochordates (Branchiostoma lanceolatum) share at least 80% of amino acids in the DNA binding domain. In agreement with this remarkable structural conservation, the DNA binding domains from zebrafish, which possesses a thymus but no hair, and amphioxus, which possesses neither thymus nor hair, recognize the same target sequence as the mouse DNA binding domain in vitro and in vivo. The genomes of vertebrates and cephalochordates contain only a single whn-like gene, suggesting that the primordial whn gene was not subject to gene-duplication events. Although the role of whn in cephalochordates and agnathans is unknown, its requirement in the development of the thymus gland and the differentiation of skin appendages in the mouse suggests that changes in the transcriptional control regions of whn genes accompanied their functional reassignments during evolution (Schlake, 1997).

Nude mice are characterized by the absence of visible hair, epidermal defects, and the failure to develop a thymus. This phenotype results from loss-of-function mutations in Whn (Hfh11), a winged-helix transcription factor. In murine epidermis and hair follicles, endogenous whn expression is induced as epithelial cells initiate terminal differentiation. Using the promoter for the differentiation marker involucrin, transgenic mice that ectopically express whn in stratified squamous epithelia, hair follicles, and the transitional epithelium of the urinary tract were generated. Transgenic epidermis and hair follicles display impaired terminal differentiation and a subset of hair defects, such as delayed growth, a waved coat, and curly whiskers, correlated with decreased transforming growth factor (TGF)-alpha expression. The exogenous Whn protein also stimulates epithelial cell multiplication. In the epidermis, basal keratinocytes exhibits hyperproliferation, though transgene expression is restricted to suprabasal, postmitotic cells. Hair follicles fail to enter telogen (a resting period) and remain continuously in an abnormal anagen (the growth phase of the hair cycle). Ureter epithelium develop severe hyperplasia, leading to the obstruction of urine outflow and death from hydronephrosis. Though an immune infiltrate is present occasionally in transgenic skin, the infiltrate is not the primary cause of the epithelial hyperproliferation, because the immune reaction is not observed in all affected transgenics, and the transgene induces identical skin and urinary tract abnormalities in immunodeficient Rag1-null mice. Given the effects of the transgene on cell proliferation and TGFalpha expression, the results suggest that Whn modulates growth factor production by differentiating epithelial cells, thereby regulating the balance between proliferative and postmitotic populations in self-renewing epithelia (Prowse, 1999).

Loss-of-function mutations in Whn (wing-helix nude; Hfh 11), a winged-helix/forkhead transcription factor, result in the nude mouse phenotype. To determine the whn expression pattern during development, mice were used in which a beta-galactosidase reporter gene was placed under the control of the wild-type whn promoter by homologous recombination. Sites of reporter expression were confirmed by immunohistochemical staining for Whn protein or by in situ hybridization for whn mRNA. At all developmental stages, whn expression is restricted to epithelial cells. In addition to the skin and thymus, whn is expressed in the developing nails, nasal passages, tongue, palate, and teeth. In embryonic epidermis, suprabasal cells induce whn expression at the same time that terminal differentiation markers first appear. As the epidermis matures, whn promoter activity is found primarily in the first suprabasal layer, which contains keratinocytes in the early stages of terminal differentiation. In developing and mature anagen hair follicles, whn is expressed at high levels in the postmitotic precursor cells of the hair shaft and inner root sheath. Though principally associated with terminal differentiation, whn expression is also detected in progenitor cell compartments: in the hair bulb matrix and basal epidermal layer, a small subclass of cells expresses whn, while in the outer root sheath, whn promoter activity is induced as the follicle completes its elongation. Within these compartments, rare cells exhibit both whn expression and the nuclear proliferation marker Ki-67. The results suggest that whn expression encompasses the transition from a proliferative to a postmitotic state and that whn regulates the initiation of terminal differentiation. During thymus development, whn expression first appears in epithelial cells of the thymic primordium, and in the mature thymus, whn expressing epithelial cells are present throughout the medulla, cortex, and subcapsular region. Given the sites of whn expression in the mouse, as well as the presence of homologs in lower vertebrates and cephalochordates, the whn gene may influence either fundamental or common features of epithelial cell differentiation (Lee, 1999).

The nude locus encodes Whn, a transcription factor of the forkhead/winged-helix class. Mutations in Whn cause failure of differentiation of thymic epithelium with a corresponding lack of intrathymic T-cell development; in the skin, differentiation of follicular keratinocytes is disturbed resulting, in the formation of fragile hair shafts. A novel nude allele, nu(StL), has been identified and characterized. nu(StL) encodes a truncated Whn transcription factor protein, designated Whn(StL), lacking the activation domain but retaining the characteristic DNA binding domain. In contrast, the previously described Whn(nu) mutant protein lacks both domains. nu(StL)/nu(StL) mice show an alymphoid thymic rudiment and lack of peripheral T cells, similar to nu/nu mice. In the skin, impaired expression of hair keratin genes mHa1, mHa2, mHa3 and mHa4, mHb3, mHb4, mHb5, and mHb6 is observed in a pattern that parallels that of nu/nu mice: both mutant alleles behave as hypomorphs with respect to the expression of these hair keratin genes. However, a significant difference between these two alleles exists for mHa5 expression, which is reduced in nu(StL)/nu(StL) but not in nu/nu mice. The mutant Whn protein in nu/nu mice cannot enter the nucleus, whereas the mutant Whn protein in nu(StL)/nu(StL) mice is present in the nucleus. The antimorphic characteristic of the activation-deficient Whn(StL) protein with respect to mHa5 expression is therefore most likely caused by its non-productive interaction with other proteins at cis-regulatory regions of the mHa5 gene. These results indicate that the molecular consequences of mutations of the Whn gene can be different and demonstrate an unexpected complexity of transcriptional control mechanisms of hair keratin genes (Schorpp, 2000).

The molecular characteristics of the nude phenotype (alopecia and thymic aplasia) in humans and rodents are unknown. The nude locus encodes Whn, a transcription factor of the forkhead/winged-helix class. Expression of Whn in HeLa cells induces expression of human hair keratin genes Ha3-II and Hb5. Correspondingly, in nude mice, which are homozygous for a loss-of-function mutation of Whn, expression of mouse mHa3 and mHb5 hair keratin genes is severely reduced. Characterization of a previously identified nude allele, nu(Y), reveals a mis-sense mutation (R320C) in the DNA binding domain of Whn. This mutant protein is unable to activate hair keratin gene expression in HeLa cells. When the Whn transcription factor is expressed in two parts, one containing the N-terminal DNA binding domain and the other the C-terminal activation domain, no activation of hair keratin genes in HeLa cells is observed. However, when these two proteins are noncovalently linked by means of synthetic dimerizers, hair keratin gene expression is induced. This finding suggests that target gene activation by Whn depends on the structural integrity and physical proximity of DNA binding and activation domains, providing a molecular framework to explain the loss-of-function phenotypes of all previously characterized nude mutations. These results implicate Whn as a transcriptional regulator of hair keratin genes and reveal the nude phenotype as the first example of an inherited skin disorder that is caused by loss of expression rather than mutation of keratin genes (Schlake, 2000).

During vertebrate retinogenesis, seven classes of cells are specified from multipotent progenitors. To date, the mechanisms underlying multipotent cell fate determination by retinal progenitors remain poorly understood. The Foxn4 winged helix/forkhead transcription factor is shown to be expressed in a subset of mitotic progenitors during mouse retinogenesis. Targeted disruption of Foxn4 largely eliminates amacrine neurons and completely abolishes horizontal cells, while overexpression of Foxn4 strongly promotes an amacrine cell fate. These results indicate that Foxn4 is both necessary and sufficient for commitment to the amacrine cell fate and is nonredundantly required for the genesis of horizontal cells. Furthermore, evidence is provided that Foxn4 controls the formation of amacrine and horizontal cells by activating the expression of the retinogenic factors Math3, NeuroD1, and the Prospero-like transcription factor Prox1. These data suggest a model in which Foxn4 cooperates with other key retinogenic factors to mediate the multipotent differentiation of retinal progenitors (Li, 2004).