Nude Gene Research Articles

Foxn1[Cre] Expression in the Male Germline.

Foxn1 (forkhead box N1), also known as the nude gene or winged-helix nude (Whn), is a forkhead transcription factor thought to be restricted to keratinocytes in the skin and thymus. Consistent with this tissue distribution, spontaneous or targeted mutation of Foxn1 results in the absence of both hair and a thymus. Genetic manipulation of the Foxn1 locus thus represents a powerful tool for tissue specific gene control in the skin and thymus, and tools such as Cre recombinase under control of the Foxn1 locus are widely used for this purpose. Unexpectedly, we show that Foxn1[Cre] exhibits unexpected activity in male germ cells, resulting in ubiquitous targeting of loxP-flanked alleles in all tissues in offspring from Foxn1[Cre] expressing male mice. Inheritance of recombined loxP alleles occurs independently of Cre inheritance (i.e., offspring lacking Cre nonetheless exhibit recombined alleles), suggesting that Foxn1[Cre] induced recombination in male germ cells must occur prior to meiosis in diploid germ cells. Together with previously published data, our results show that Foxn1, and alleles under its control, are expressed in the pre-meiotic male germline, revealing a new tool for germline targeting of genes, and raising important concerns for gender selection when using Foxn1 regulatory elements.

NDE1 and NDEL1 from genes to (mal)functions: parallel but distinct roles impacting on neurodevelopmental disorders and psychiatric illness.

NDE1 (Nuclear Distribution Element 1, also known as NudE) and NDEL1 (NDE-Like 1, also known as NudEL) are the mammalian homologues of the fungus nudE gene, with important and at least partially overlapping roles for brain development. While a large number of studies describe the various properties and functions of these proteins, many do not directly compare the similarities and differences between NDE1 and NDEL1. Although sharing a high degree structural similarity and multiple common cellular roles, each protein presents several distinct features that justify their parallel but also unique functions. Notably both proteins have key binding partners in dynein, LIS1 and DISC1, which impact on neurodevelopmental and psychiatric illnesses. Both are implicated in schizophrenia through genetic and functional evidence, with NDE1 also strongly implicated in microcephaly, as well as other neurodevelopmental and psychiatric conditions through copy number variation, while NDEL1 possesses an oligopeptidase activity with a unique potential as a biomarker in schizophrenia. In this review, we aim to give a comprehensive overview of the various cellular roles of these proteins in a "bottom-up" manner, from their biochemistry and protein-protein interactions on the molecular level, up to the consequences for neuronal differentiation, and ultimately to their importance for correct cortical development, with direct consequences for the pathophysiology of neurodevelopmental and mental illness.

The winged-helix transcription factor JUMU regulates development, nucleolus morphology and function, and chromatin organization of Drosophila melanogaster

The PEV-modifying winged-helix/forkhead domain transcription factor JUMU of Drosophila is an essential protein of pleiotropic function. The correct gene dose of jumu is required for nucleolar integrity and correct nucleolus function. Overexpression of jumu results in bloating of euchromatic chromosome arms, displacement of the JUMU protein from the chromocenter and the nucleolus, fragile weak points, and disrupted chromocenter of polytene chromosomes. Overexpression of the acidic C terminus of JUMU alone causes nucleolus disorganization. In addition, euchromatic genes are overexpressed and HP1, which normally accumulates in the pericentric heterochromatin and spreads into euchromatic chromosome arms, although H3-K9 di-methylation remains restricted to the pericentric heterochromatin. The human winged-helix nude gene shows similarities to jumu and its overexpression in Drosophila causes bristle mutations.

T Cell Development from Kit-Negative Progenitors in the Foxn1Δ/Δ Mutant Thymus

Foxn1Delta is a hypomorphic allele of the nude gene that causes arrested thymic epithelial cell differentiation and abnormal thymic architecture lacking cortical and medullary domains. T cells develop in the Foxn1Delta/Delta adult thymus to the double- and single-positive stages, but in the apparent absence of double-negative 3 (DN3) cells; however, DN3 cells are present in the fetal thymus. To investigate the origin of this seemingly contradictory phenotype, we performed an analysis of fetal and adult DN cells in these mutants. Neither adult bone marrow-derived cells nor fetal liver cells from wild-type or Rag1-/- mice were able to differentiate to the DN2 or DN3 stage in the Foxn1Delta/Delta thymus. Our data suggest that thymopoiesis in the Foxn1Delta/Delta adult thymus proceeds from CD117- atypical progenitors, while CD117+ DN1a cells are absent or blocked in their ability to differentiate to the T lineage. Wild-type cells generated by this pathway in the postnatal thymus were exported to the periphery, demonstrating that these atypical cells contributed to the peripheral T cell pool. The Foxn1Delta/Delta adult (but not fetal) thymus also preferentially supports B cell development, specifically of the B-1 type, and this phenotype correlated with reduced Notch ligand expression in the adult stroma.

377 SPERMATOZOA RETRIEVED FROM MALE MICE FROZEN FOR 15 YEARS CAN PRODUCE NORMAL OFFSPRING

Cryopreservation of male germ cells is a strategy to conserve animal species and strains of animals valuable to biomedical research. However, to minimize damage that may occur during freezing and thawing, complex cryopreservation protocols that have been optimized for the stage and species of male germ cells are usually employed. Recently, we have found that mouse male germ cells can be cryopreserved at -80�C by freezing the whole epididymides and testes without cryoprotectant for at least one year (Ogonuki et al. 2006 Reprod. Fertil. Dev. 18, 286 abst). This study was undertaken to determine whether mouse male germ cells retrieved from the bodies of mice frozen at -20�C for 15 years could produce normal offspring by microinsemination. Mature males of BALB/c-nude and C3H/He (8 weeks of age) were euthanized by overdose of pentobarbital on February 20 and March 8, 1991, respectively, and kept in a -20�C freezer. The frozen body was thawed about 15 years after freezing (February 2006) by putting it in a water bath until the outer surface of the body was softened. The body was then removed from the water, and the testes were isolated through an abdominal incision. Testicular spermatozoa were collected from the testes and microinseminated into B6D2F1 oocytes. Within 24 h after sperm injection, over 80% of oocytes developed into 2-cell embryos. Apparently normal pups were born after embryo transfer in both strains of mice at rates of 21% (17/81) and 12% (12/97) per transfer, respectively. Two pups from the BALB/c-nude group died shortly after Caesarian section due to respiratory failure, but others grew normally and were proven to be fertile when they matured (at least 19 mice out of 20 mice tested). We further mated these F1 offspring and confirmed that the nude gene was safely propagated. The present study demonstrates that spermatozoa can retain their fertilizing ability in frozen whole bodies for longer than we anticipated. If spermatozoa of extinct mammalian species (e.g. woolly mammoth) can be retrieved from animal bodies that were kept frozen in permanent frost, live animals might be restored by injecting them into oocytes from females of closely related species.

FOXN1 Is Critical for Onycholemmal Terminal Differentiation in Nude (Foxn1nu) Mice

Nude mice have a mutation in the transcription factor Foxn1(nu), resulting in downregulation of hair keratins. Although hair follicles develop normally, the hair fibers become structurally weak, curl, and break off at the surface. Nails in nude mice are deformed, based on alterations of the onychocyte differentiation process. Elemental microanalysis of the nail plate reveals marked decreases in sulfur concentrations in the nude mouse nail plates. Immunohistochemistry shows a lack of keratin 1 expression in terminally differentiating keratinocytes of the nail matrix. Instead, the typical differentiation process of the matrix is altered toward an epidermis-like differentiation pattern, comprising the production of filaggrin-containing keratohyalin granules in cells resembling those of the stratum granulosum, which are never observed in normally haired mice. The nail plate has diffuse basophilic stippling. It is thinner than normal, weak, and in most Foxn1(nu)/Foxn1(nu) mice breaks where it separates from the hyponychium. These studies indicate that the Foxn1(nu) mutated gene has effects beyond downregulating keratin expression, including changes in filaggrin expression, and is critical for normal onycholemmal differentiation. The nails of nude mice provide new insights into the molecular controls of onychocyte differentiation, and they offer a useful model to investigate the pathogenesis of nail hypergranulosis, a common feature in human nail diseases.

Ancestral founder mutation of the nude (FOXN1) gene in congenital severe combined immunodeficiency associated with alopecia in southern Italy population.

Genetic alterations of the FOXN1 transcription factor, selectively expressed in thymic epithelia and skin, are responsible in both mice and humans for the Nude/SCID phenotype. The first described human FOXN1 mutation was a C792T transition in exon 5 resulting in the nonsense mutation R255X, and was detected in two probands originated from a small community in southern Italy. In this community, four additional children affected with congenital alopecia died in early childhood because of severe infections. In this study, we report on the screening for this mutation in 30% of the village population. This analysis led us to identify 55 heterozygous carriers (6.52%) of the R255X mutation out of 843 inhabitants screened. A genealogical study revealed that these subjects, belonging to 39 families, were linked in an extended 7-generational pedigree comprising 483 individuals. Through the archival database a single ancestral couple, born at the beginning of the 19th century, was identified. To confirm the ancestral origin of the mutation we genotyped two microsatellite markers, D17S2187 and D17S1880, flanking the FOXN1 gene on chromosome 17. The three haplotypes identified, 3/R255X/3, 3/R255X/2 and 3/R255X/1, are consistent with a single ancestral origin for the mutation R255X.

Effects of combined therapy with interleukin 2 and interleukin 12 gene-transfected tumor vaccine for head and neck carcinoma.

The biological effects of cytokines are coming to be understood. The therapeutic effects of interleukin (IL) 2, IL-12, and interferon gamma (IFN-gamma) in cancer treatment have been reported, but there are problems when these cytokines are systemically used as therapeutic agents. To examine the efficacy of IL-2 and IL-12 gene-transfected tumor cell vaccines for head and neck squamous cell carcinoma (SCC). Homozygous mice with the autosomal recessive nude gene (BALB/c nu/nu mice) were inoculated subcutaneously in the right flank with cells from a human oral floor SCC cell line (KB cells). The mice were then injected with IL-2 and IL-12 gene-transfected KB cells (KB/IL-2 and KB/IL-12 cells, respectively) irradiated with 2000 rad (20 Gy). No mice died soon after the injection of the gene immunotherapy. The treatment with either KB/human IL-2 (hIL-2) or KB/murine IL-12 (mIL-12) was not very effective. However, the treatment with both KB/hIL-2 and KB/mIL-12 cells significantly and safely inhibited the growth of established tumors (P =.04). There was no significant difference in antitumor effect between once-weekly and twice weekly injections of both KB/hIL-2 and KB/mIL-12 cells. Double gene immunotherapy is safe and effective treatment for SCC in mice.

Roles of NUDE and NUDF proteins of Aspergillus nidulans: insights from intracellular localization and overexpression effects.

The NUDF protein of the filamentous fungus Aspergillus nidulans functions in the cytoplasmic dynein pathway. It binds several proteins, including the NUDE protein. Green fluorescent protein-tagged NUDF and NUDA (dynein heavy chain) localize to linearly moving dashes ("comets") that coincide with microtubule ends. Herein, deletion of the nudE gene did not eliminate the comets of NUDF and NUDA, but affected the behavior of NUDA. Comets were also observed with the green fluorescent protein-tagged NUDE and its nonfunctional C-terminal domain. In addition, overexpressed NUDA and NUDE accumulated in specks that were either immobile or bounced randomly. Neither comets nor specks were observed with the functional N-terminal domain of NUDE, indicating that these structures are not essential for NUDE function. Furthermore, NUDF overproduction totally suppressed deletion of the nudE gene. This implies that the function of NUDE is secondary to that of NUDF. Unexpectedly, NUDF overproduction inhibited one conditional nudA mutant and all tested apsA mutants. An allele-specific interaction between the nudF and nudA genes is consistent with a direct interaction between NUDF and dynein heavy chain. Because APSA and its yeast homolog Num1p are cortical proteins, an interaction between the nudF and apsA genes suggests a role for NUDF at the cell cortex.

A zebrafish orthologue ( whnb) of the mouse nude gene is expressed in the epithelial compartment of the embryonic thymic rudiment

The cloning and characterization of the zebrafish orthologue of the mouse nude ( Whn/Foxn1) gene, whnb are reported. A previously described Whn-like gene from zebrafish, now designated as whna, is shown to be the orthologue of the mouse Foxn4 gene. The whnb gene is specifically expressed in the thymic rudiment of zebrafish embryos at day 3 after fertilization, whereas the whna gene is expressed in eye and brain structures. whnb expression is maintained in cloche mutants, where endothelial and haematopoietic cell differentiation is defective, but absent in casanova mutants where endoderm formation is impaired. In adult thymi, whnb is expressed throughout cortical and medullary areas, whereas whna expression is observed in rare cell clusters only. Our results provide the first specific marker for the epithelial compartment of the zebrafish thymus.

The Gene e.1 (nudE.1) of T4 Bacteriophage Designates a New Member of the Nudix Hydrolase Superfamily Active on Flavin Adenine Dinucleotide, Adenosine 5′-Triphospho-5′-adenosine, and ADP-ribose

The T4 bacteriophage gene e.1 was cloned into an expression vector and expressed in Escherichia coli, and the purified protein was identified as a Nudix hydrolase active on FAD, adenosine 5'-triphospho-5'-adenosine (Ap(3)A), and ADP-ribose. Typical of members of the Nudix hydrolases, the enzyme has an alkaline pH optimum (pH 8) and requires a divalent cation for activity that can be satisfied by Mg(2+) or Mn(2+). For all substrates, AMP is one of the products, and unlike most of the other enzymes active on Ap(3)A, the T4 enzyme hydrolyzes higher homologues including Ap(4-6)A. This is the first member of the Nudix hydrolase gene superfamily identified in bacterial viruses and the only one present in T4. Although the protein was predicted to be orthologous to E. coli MutT on the basis of a sequence homology search, the properties of the gene and of the purified protein do not support this notion because of the following. (a) The purified enzyme hydrolyzes substrates not acted upon by MutT, and it does not hydrolyze canonical MutT substrates. (b) The e.1 gene does not complement mutT1 in vivo. (c) The deletion of e.1 does not increase the spontaneous mutation frequency of T4 phage. The properties of the enzyme most closely resemble those of Orf186 of E. coli, the product of the nudE gene, and we therefore propose the mnemonic nudE.1 for the T4 phage orthologue.

Role of the Nude Gene in Epithelial Terminal Differentiation

Loss-of-function mutations in Whn (Hfh11, Foxn1), a winged-helix/forkhead transcription factor, cause the nude phenotype, which is characterized by the abnormal morphogenesis of the epidermis, hair follicles, and thymus. To delineate the biochemical pathway of Whn, we investigated its upstream regulation and downstream effects using primary keratinocytes from wild-type and transgenic mice. The transgenic animals express whn from the involucrin promoter, which is active in keratinocytes undergoing terminal differentiation. In wild-type cultures, as in the epidermis, Whn was induced during the early stages of terminal differentiation and declined during later stages. In transgenic keratinocytes, whn overexpression altered the terminal differentiation program, stimulating an early differentiation marker (keratin 1) and suppressing later markers (profilaggrin, loricrin, and involucrin). These results suggest a role for Whn in the stepwise or temporal regulation of differentiation, as Whn can ensure that the differentiation program is carried out in proper sequence. Before the start of differentiation, Whn levels were suppressed by the p42/p44 mitogen-activated protein kinase cascade, and this signaling pathway was rapidly inactivated as differentiation began. Thus, as keratinocytes commit to terminal differentiation, mitogen-activated protein kinase signaling decreases, which permits the induction of Whn; Whn then activates early features of the differentiation program.

The nude gene and the skin.

The nude mutation has been known for a long time. Nevertheless, the gene responsible for the defect has been identified only recently. It encodes a transcriptional activator of the family of forkhead proteins mainly expressed in thymic epithelium and distinct keratinocyte populations in the epidermis and hair follicles. The present review focuses on the molecular and functional characterization of the nude gene and its product and gives an overview as to its role in skin biology and the first identified target genes in the skin. In addition, evolutionary aspects are highlighted stressing the importance of such investigations for a comprehensive understanding of the nude gene product and the regulation of its expression. Furthermore, these studies give a hint as to when the nude gene has occurred first and how it has developed in molecular and functional terms since then.

Human equivalent of the mouse Nude/SCID phenotype: long-term evaluation of immunologic reconstitution after bone marrow transplantation

AbstractHuman Nude/SCID (severe combined immunodeficiency) is the first severe combined immunodeficiency caused by mutation of the winged–helix–nude (WHN) gene, which is expressed in the thymus but not in the hematopoietic lineage. The disease is characterized by a T-cell defect, congenital alopecia, and nail dystrophy. A Nude/SCID patient who underwent bone marrow transplantation from the human leukocyte antigen–identical heterozygote brother was studied to investigate, in this unique model, the role of the thymus in immunologic reconstitution. Despite an increase in CD3+, CD4+, and CD8+cells, CD4+ CD45 RA naive lymphocytes were not regenerated. Conversely, naive CD8+ cells were normal. After an initial recovery, lymphocyte proliferation to mitogens progressively declined compared with controls and genotypically identical donor cells grown in the WHN+/−environment. Analysis of the T-cell receptor (TCR) repertoire of CD4+ cells revealed that only 3 of 18 Vβ families had an altered CDR3 heterogeneity length profile. Conversely, CD8+lymphocytes showed an abnormal distribution in most Vβ families. These data indicate that the thymus is differentially required in the reconstitution of CD4+ and CD8+ naive subsets and in the maintenance of their TCR repertoire complexity. Taken together, these findings suggest that bone marrow transplantation is ineffective in the long-term cure of this form of SCID.

Ectopic Expression of the nude Gene Induces Hyperproliferation and Defects in Differentiation: Implications for the Self-Renewal of Cutaneous Epithelia

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 displayed 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)-α expression. The exogenous Whn protein also stimulated epithelial cell multiplication. In the epidermis, basal keratinocytes exhibited hyperproliferation, though transgene expression was restricted to suprabasal, postmitotic cells. Hair follicles failed to enter telogen (a resting period) and remained continuously in an abnormal anagen (the growth phase of the hair cycle). Ureter epithelium developed severe hyperplasia, leading to the obstruction of urine outflow and death from hydronephrosis. Though an immune infiltrate was present occasionally in transgenic skin, the infiltrate was not the primary cause of the epithelial hyperproliferation, as the immune reaction was not observed in all affected transgenics, and the transgene induced identical skin and urinary tract abnormalities in immunodeficient Rag1-null mice. Given the effects of the transgene on cell proliferation and TGFα 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.

Transcription of the Nude Gene (WHN) in Human Normal Organs and Mediastinal and Pulmonary Tumors

Mutations in the winged-helix-nude (WHN) gene in the nude mouse and rat phenotypes lead to loss of hair and athymia. The human nude gene has recently been identified, but to date, its distribution in normal human tissues and thymus tumors has not yet been investigated. We used the RT-PCR technique for the detection of WHN transcripts in normal human organs and various mediastinal tumors, including various subtypes of thymic epithelial tumors. We could show that WHN is highly transcribed in the normal thymus and skin. Unexpectedly, few transcripts were also found in the normal kidney and thyroid gland. WHN mRNA could be detected in all thymoma subtypes (n = 14) and epidermoid thymic carcinomas as well as in all squamous cell carcinomas of the lung (n = 5) investigated, but was totally absent in all other mediastinal tumors (n = 11) lacking an epidermoid differentiation. We conclude that transcription of the human WHN is not strictly specific for human thymus and skin, but is also expressed, though at very low levels, in the normal human kidney and thyroid gland. In addition, WHN gene expression may identify not only thymomas but also epidermoid differentiation in mediastinal and pulmonary tumors.

Characterization of mouse and human nude genes.

The differentiation of primitive epithelial precursor cells in the thymic primordium into subcapsular, cortical, and medullary epithelial cells of the mature thymus requires the activity of the nude gene product Whn. Whn is also required for proper keratinization of the hair shaft. We determined the nucleotide sequence of a 58 kilobase region on mouse chromosome 11 that encompasses the mouse nude gene and part of the two neighboring genes, encoding a sodium/dicarboxylate co-transporter and the retinal protein 4. Using cross-hybridization, the human orthologue of the mouse nude gene was isolated. The human WHN protein also consists of 648 amino acids, 85% of which are identical to the mouse protein. Like the mouse gene, the human gene consists of eight coding exons and utilizes two alternative first exons in a tissue-specific fashion. Sequences upstream of the two alternative first exons display promoter activity in heterologous reporter assays. Whereas both promoters appear to be active in skin (albeit at different levels), only the most upstream element is active in the thymus, indicating that transcriptional activity of the whn gene is subject to complex regulation. Nucleotide sequence database comparisons reveal that among other winged-helix genes, the HTLF and HTLFL1 genes are most closely related to whn, although the exon/intron structure of the human HTLF gene in the DNA binding domain differs from that of whn.

Transposon-induced splicing defect in the rat nude gene.

Transposon-induced splicing defect in the rat nude gene.

The product of the mouse nude locus, Whn, regulates the balance between epithelial cell growth and differentiation.

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. We show here that in skin whn is specifically expressed in epithelial cells and not the mesenchymal cells, and using a hair reconstitution assay, we demonstrate 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 revealed that these cells have an increased propensity to differentiate in an abnormal fashion, even under conditions that promote proliferation. Furthermore, nude mouse keratinocytes displayed a 100-fold increased sensitivity to the growth-inhibitory/differentiation effects of the phorbol ester TPA. In parallel with these findings, we directly show 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 will be discussed.

Rescue of the hairless phenotype in nude mice by transgenic insertion of the wild-type Hfh11 genomic locus.

Mice and rats homozygous for mutations at the nude (nu) locus exhibit the pleiotropic phenotypes of hairlessness and athymia. A recent positional cloning study identified, as a nude gene, a novel fork head transcription factor, Hfh11 (also called whn), that is expressed in skin and thymus, and is mutated in nude rodents. To obtain the direct biological proof that this gene is responsible for nude phenotype, we microinjected a cosmid clone containing the wild-type Hfh11 genomic locus into fertilized nude eggs. Two independent founder lines of transgenic mice were generated that corrected the hairless phenotype, but not the thymic defect. This partial rescue demonstrates that Hfh11 is the gene responsible for the hairless defect in the nude mouse. Taken together with previous genetic studies, this complementation result indicates that Hfh11 is indeed the nude gene and the Hfh11 locus is likely to be subject to complicated regulation.