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Daniel Metzger

Publications and source records attributed to Daniel Metzger.

At least 37 records · Page 2Linked to original sources
PubMed2005

Temporally controlled targeted somatic mutagenesis in embryonic surface ectoderm and fetal epidermal keratinocytes unveils two distinct developmental functions of BRG1 in limb morphogenesis and skin barrier formation.

Animal SWI2/SNF2 protein complexes containing either the brahma (BRM) or brahma-related gene 1 (BRG1) ATPase are involved in nucleosome remodelling and may control the accessibility of sequence-specific transcription factors to DNA. In vitro studies have indicated that BRM and BRG1 could regulate the expression of distinct sets of genes. However, as mice lacking BRM are viable and fertile, BRG1 might efficiently compensate for BRM loss. By contrast, as Brg1-null fibroblasts are viable but Brg1-null embryos die during the peri-implantation stage, BRG1 might exert cell-specific functions. To further investigate the in vivo role of BRG1, we selectively ablated Brg1 in keratinocytes of the forming mouse epidermis. We show that BRG1 is selectively required for epithelial-mesenchymal interactions in limb patterning, and during keratinocyte terminal differentiation, in which BRM can partially substitute for BRG1. By contrast, neither BRM nor BRG1 are essential for the proliferation and early differentiation of keratinocytes, which may require other ATP-dependent nucleosome-remodelling complexes. Finally, we demonstrate that cell-specific targeted somatic mutations can be created at various times during the development of mouse embryos cell-specifically expressing the tamoxifen-activatable Cre-ER(T2) recombinase.

Alleles↗
PubMed2005

Targeted somatic mutagenesis in the mouse epidermis.

The efficient introduction of somatic mutations in a given gene at a given time and in specific cell types of the skin will greatly facilitate the studies of a number of genes expressed in the skin and the production of animal models for skin diseases. We describe here strategies and techniques to create spatiotemporally controlled somatic mutations of target genes in the skin using a conditional Cre/LoxP system. They are based on cell-specific expression of the chimeric Cre recombinase Cre-ERT2, whose activity is induced by antiestrogens such as Tamoxifen (Tam), and which is obtained by fusing the Cre recombinase with a mutated ligand binding domain of the human estrogen receptor ERalpha. As an example, we present ablation of the retinoid receptor RXRalpha in epidermal basal keratinocytes of adult mice.

Animals↗
PubMed2004

Specific deletion of focal adhesion kinase suppresses tumor formation and blocks malignant progression.

We have generated mice with a floxed fak allele under the control of keratin-14-driven Cre fused to a modified estrogen receptor (CreER(T2)). 4-Hydroxy-tamoxifen treatment induced fak deletion in the epidermis, and suppressed chemically induced skin tumor formation. Loss of fak induced once benign tumors had formed inhibited malignant progression. Although fak deletion was associated with reduced migration of keratinocytes in vitro, we found no effect on wound re-epithelialization in vivo. However, increased keratinocyte cell death was observed after fak deletion in vitro and in vivo. Our work provides the first experimental proof implicating FAK in tumorigenesis, and this is associated with enhanced apoptosis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2004

Altered growth in male peroxisome proliferator-activated receptor gamma (PPARgamma) heterozygous mice: involvement of PPARgamma in a negative feedback regulation of growth hormone action.

The peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in fat tissue development and physiology. Mutations in the gene encoding this receptor have been associated to disorders in lipid metabolism. A thorough investigation of mice in which one PPARgamma allele has been mutated reveals that male PPARgamma heterozygous (PPARgamma +/-) mice exhibit a reduced body size associated with decreased body weight, reflecting lean mass reduction. This phenotype is reproduced when treating the mice with a PPARgamma- specific antagonist. Monosodium glutamate treatment, which induces weight gain and alters body growth in wild-type mice, further aggravates the growth defect of PPARgamma +/- mice. The levels of circulating GH and that of its downstream effector, IGF-I, are not altered in mutant mice. However, the IGF-I mRNA level is decreased in white adipose tissue (WAT) of PPARgamma +/- mice and is not changed by acute administration of recombinant human GH, suggesting an altered GH action in the mutant animals. Importantly, expression of the gene encoding the suppressor of cytokine signaling-2, which is an essential negative regulator of GH signaling, is strongly increased in the WAT of PPARgamma +/- mice. Although the relationship between the altered GH signaling in WAT and reduced body size remains unclear, our results suggest a novel role of PPARgamma in GH signaling, which might contribute to the metabolic disorder affecting insulin signaling in PPARgamma mutant mice.

Animals↗
PubMed2004

In vivo activation of PPAR target genes by RXR homodimers.

The ability of a retinoid X receptor (RXR) to heterodimerize with many nuclear receptors, including LXR, PPAR, NGF1B and RAR, underscores its pivotal role within the nuclear receptor superfamily. Among these heterodimers, PPAR:RXR is considered an important signalling mediator of both PPAR ligands, such as fatty acids, and 9-cis retinoic acid (9-cis RA), an RXR ligand. In contrast, the existence of an RXR/9-cis RA signalling pathway independent of PPAR or any other dimerization partner remains disputed. Using in vivo chromatin immunoprecipitation, we now show that RXR homodimers can selectively bind to functional PPREs and induce transactivation. At the molecular level, this pathway requires stabilization of the homodimer-DNA complexes through ligand-dependent interaction with the coactivator SRC1 or TIF2. This pathway operates both in the absence and in the presence of PPAR, as assessed in cells carrying inactivating mutations in PPAR genes and in wild-type cells. In addition, this signalling pathway via PPREs is fully functional and can rescue the severe hypothermia phenotype observed in fasted PPARalpha-/- mice. These observations have important pharmacological implications for the development of new rexinoid-based treatments.

Alitretinoin↗
PubMed2004

Epithelial Bmpr1a regulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development.

Bone morphogenetic protein (BMP) signaling is thought to perform multiple functions in the regulation of skin appendage morphogenesis and the postnatal growth of hair follicles. However, definitive genetic evidence for these roles has been lacking. Here, we show that Cre-mediated mutation of the gene encoding BMP receptor 1A in the surface epithelium and its derivatives causes arrest of tooth morphogenesis and lack of external hair. The hair shaft and hair follicle inner root sheath (IRS) fail to differentiate, and expression of the known transcriptional regulators of follicular differentiation Msx1, Msx2, Foxn1 and Gata3 is markedly downregulated or absent in mutant follicles. Lef1 expression is maintained, but nuclear beta-catenin is absent from the epithelium of severely affected mutant follicles, indicating that activation of the WNT pathway lies downstream of BMPR1A signaling in postnatal follicles. Mutant hair follicles fail to undergo programmed regression, and instead continue to proliferate, producing follicular cysts and matricomas. These results provide definitive genetic evidence that epithelial Bmpr1a is required for completion of tooth morphogenesis, and regulates terminal differentiation and proliferation in postnatal hair follicles.

Animals↗
PubMed2004

Peroxisome proliferator-activated receptor gamma is required in mature white and brown adipocytes for their survival in the mouse.

The peroxisome proliferator-activated receptor gamma (PPARgamma) mediates the activity of the insulin-sensitizing thiazolidinediones and plays an important role in adipocyte differentiation and fat accretion. The analysis of PPARgamma functions in mature adipocytes is precluded by lethality of PPARgamma(-/-) fetuses and tetraploid-rescued pups. Therefore we have selectively ablated PPARgamma in adipocytes of adult mice by using the tamoxifen-dependent Cre-ER(T2) recombination system. We show that mature PPARgamma-null white and brown adipocytes die within a few days and are replaced by newly formed PPARgamma-positive adipocytes, demonstrating that PPARgamma is essential for the in vivo survival of mature adipocytes, in addition to its well established requirement for their differentiation. Our data suggest that potent PPARgamma antagonists could be used to acutely reduce obesity.

Adipocytes↗
PubMed2004

Friedreich ataxia mouse models with progressive cerebellar and sensory ataxia reveal autophagic neurodegeneration in dorsal root ganglia.

Friedreich ataxia (FRDA), the most common recessive ataxia, is characterized by degeneration of the large sensory neurons of the spinal cord and cardiomyopathy. It is caused by severely reduced levels of frataxin, a mitochondrial protein involved in iron-sulfur cluster (ISC) biosynthesis. Through a spatiotemporally controlled conditional gene-targeting approach, we have generated two mouse models for FRDA that specifically develop progressive mixed cerebellar and sensory ataxia, the most prominent neurological features of FRDA. Histological studies showed both spinal cord and dorsal root ganglia (DRG) anomalies with absence of motor neuropathy, a hallmark of the human disease. In addition, one line revealed a cerebellar granule cell loss, whereas both lines had Purkinje cell arborization defects. These lines represent the first FRDA models with a slowly progressive neurological degeneration. We identified an autophagic process as the causative pathological mechanism in the DRG, leading to removal of mitochondrial debris and apparition of lipofuscin deposits. These mice therefore represent excellent models for FRDA to unravel the pathological cascade and to test compounds that interfere with the degenerative process.

Animals↗
PubMed2004

Brain masculinization requires androgen receptor function.

Testicular testosterone produced during a critical perinatal period is thought to masculinize and defeminize the male brain from the inherent feminization program and induce male-typical behaviors in the adult. These actions of testosterone appear to be exerted not through its androgenic activity, but rather through its conversion by brain aromatase into estrogen, with the consequent activation of estrogen receptor (ER)-mediated signaling. Thus, the role of androgen receptor (AR) in perinatal brain masculinization underlying the expression of male-typical behaviors remains unclear because of the conversion of testosterone into estrogen in the brain. Here, we report a null AR mutation in mice generated by the Cre-loxP system. The AR-null mutation in males (AR(L-/Y)) resulted in the ablation of male-typical sexual and aggressive behaviors, whereas female AR-null homozygote (AR(L-/L-)) mice exhibited normal female sexual behaviors. Treatment with nonaromatizable androgen (5alpha-dihydrotestosterone, DHT) was ineffective in restoring the impaired male sexual behaviors, but it partially rescued impaired male aggressive behaviors in AR(L-/Y) mice. Impaired male-typical behaviors in ERalpha(-/-) mice were restored on DHT treatment. The role of AR function in brain masculinization at a limited perinatal stage was studied in AR(L-/L-) mice. Perinatal DHT treatment of females led to adult females sensitive to both 17beta-estradiol and DHT in the induction of male-typical behaviors. However, this female brain masculinization was abolished by AR inactivation. Our results suggested that perinatal brain masculinization requires AR function and that expression of male-typical behaviors in adults is mediated by both AR-dependent and -independent androgen signaling.

Androgen Receptor Antagonists↗
PubMed2004

Tissue-specific and inducible Cre-mediated recombination in the gut epithelium.

We generated two complementary systems for Cre-mediated recombination of target genes in the mouse digestive epithelium and tested them with a Cre-reporter mouse strain. Cre was expressed under the control of a 9 kb regulatory region of the murine villin gene (vil-Cre). Genetic recombination was initiated at embryonic day (E) 9 in the visceral endoderm, and by E12.5 in the entire intestinal epithelium, but not in other tissues. Cre expression was maintained throughout adulthood. Furthermore, transgenic mice bearing a tamoxifen-dependent Cre recombinase (vil-Cre-ERT2) expressed under the control of the villin promoter were created to perform targeted spatiotemporally controlled somatic recombination. After tamoxifen treatment, recombination was detectable throughout the digestive epithelium. The recombined locus persisted for 60 days after tamoxifen administration, despite rapid intestinal cell renewal, indicating that epithelial progenitor cells had been targeted. The villin-Cre and villin-Cre-ERT2 mice provide valuable tools for studies of cell lineage allocation and gene function in the developing and adult intestine.

Animals↗
PubMed2004

Retinal dystrophy resulting from ablation of RXR alpha in the mouse retinal pigment epithelium.

Vitamin A (retinol) actions in eye development are mediated by retinoic acid receptors (RARs and RXRs). Using the Cre/loxP system, we have selectively ablated RXR alpha in the retinal pigment epithelium (RPE), a cell monolayer critically involved in visual retinoid renewal and phagocytosis of photoreceptor outer segments. In the mutant (RXR alpha (rpe-/-)) mice, RPE cells are morphologically and functionally abnormal and display decreased expression of proteins involved in the visual retinoid cycle, namely RPE65, CRALBP, and RGR. RXR alpha (rpe-/-) mice also show alterations of photoreceptor cells including: 1) decrease in their number; 2) outer segment shortening and disorganization, and 3) reduced light responses in electroretinograms. These results indicate that RXR alpha is required for normal maturation of the RPE, which is known to play essential roles in photoreceptor cell function and survival, and point to a possible involvement of RXR alpha signaling pathways in the RPE in human retinal diseases.

Animals↗
PubMed2004

Peroxisome-proliferator-activated receptor (PPAR)-gamma activation stimulates keratinocyte differentiation.

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.

Animals↗
PubMed2003

Suppressive function of androgen receptor in bone resorption.

As locally converted estrogen from testicular testosterone contributes to apparent androgen activity, the physiological significance of androgen receptor (AR) function in the beneficial effects of androgens on skeletal tissues has remained unclear. We show here that inactivation of AR in mice using a Cre-loxP system-mediated gene-targeting technique caused bone loss in males but not in females. Histomorphometric analyses of 8-week-old male AR knockout (ARKO) mice showed high bone turnover with increased bone resorption that resulted in reduced trabecular and cortical bone mass without affecting bone shape. Bone loss in orchidectomized male ARKO mice was only partially prevented by treatment with aromatizable testosterone. Analysis of primary osteoblasts and osteoclasts from ARKO mice revealed that AR function was required for the suppressive effects of androgens on osteoclastogenesis supporting activity of osteoblasts but not on osteoclasts. Furthermore, expression of the receptor activator of NF-kappaB ligand (RANKL) gene, which encodes a major osteoclastogenesis inducer, was found to be up-regulated in osteoblasts from AR-deficient mice. Our results indicate that AR function is indispensable for male-type bone formation and remodeling.

Androgen-Insensitivity Syndrome↗
PubMed2003

Hedgehog signaling regulates sebaceous gland development.

Epithelial progenitor cells in skin give rise to multiple lineages, comprising the hair follicle, an associated sebaceous gland, and overlying epidermis; however, the signals that regulate sebocyte development are poorly understood. We tested the potential involvement of the Hedgehog pathway in sebaceous gland development using transgenes designed to either block or stimulate Hedgehog signaling in cutaneous keratinocytes in vivo. Whereas inhibition of the Hedgehog pathway selectively suppressed sebocyte development, Hedgehog pathway activation led to a striking increase both in size and number of sebaceous glands. Remarkably, ectopic Hedgehog signaling also triggered the formation of sebaceous glands from footpad epidermis, in regions normally devoid of hair follicles and associated structures. These ectopic sebaceous glands expressed molecular markers of sebocyte differentiation and were functional, secreting their contents directly onto the skin's surface instead of into a hair canal. The Hedgehog pathway thus plays a key role in sebocyte cell fate decisions and is a potential target for treatment of skin disorders linked to abnormal sebaceous gland function, such as acne.

Animals↗
PubMed2003

Tamoxifen-inducible glia-specific Cre mice for somatic mutagenesis in oligodendrocytes and Schwann cells.

Inducible transgenesis provides a valuable technique for the analysis of gene function in vivo. We report the generation and characterization of mouse lines carrying glia lineage-specific transgenes expressing an improved variant of the tamoxifen-inducible Cre recombinase, CreERT2, where the recombinase is fused to a mutated ligand binding domain of the human estrogen receptor. Using a PLP-CreERT2 transgene, we have generated mice that show specific inducible Cre function, as analyzed by cross-breeding experiments into the Rosa26 Cre-LacZ reporter line, in developing and adult Schwann cells, in mature myelinating oligodendrocytes, and in undifferentiated NG2-positive oligodendrocyte precursors in the adult. Using a P0Cx-CreERT2 transgene, we have also established mouse lines with inducible Cre function specifically in the Schwann cell lineage. These tamoxifen-inducible CreERT2 lines will allow detailed spatiotemporally controlled analysis of gene functions in loxP-based conditional mutant mice in both developing and adult Schwann cells and in the oligodendrocyte lineage.

Animals↗
PubMed2003

Sequential roles of Brg, the ATPase subunit of BAF chromatin remodeling complexes, in thymocyte development.

T cells develop through distinct stages directed by a series of signals. We explored the roles of SWI/SNF-like BAF chromatin remodeling complexes in this process by progressive deletion of the ATPase subunit, Brg, through successive stages of early T cell development. Brg-deficient cells were blocked at each of the developmental transitions examined. Bcl-xL overexpression suppressed cell death without relieving the developmental blockades, leading to the accumulation of Brg-deleted cells that were unexpectedly cell cycle arrested. These defects resulted partly from the disruptions of pre-TCR and potentially Wnt signaling pathways controlling the expression of genes such as c-Kit and c-Myc critical for continued development. Our studies indicate that BAF complexes dynamically remodel chromatin to propel sequential developmental transitions in response to external signals.

Adenosine Triphosphatases↗