RESUMO
During vertebrate embryogenesis, the neuroectoderm differentiates into neural tissues and also into non-neural tissues such as the choroid plexus in the brain and the retinal pigment epithelium in the eye. The molecular mechanisms that pattern neural and non-neural tissues within the neuroectoderm remain unknown. We report that FGF9 is normally expressed in the distal region of the optic vesicle that is destined to become the neural retina, suggesting a role in neural patterning in the optic neuroepithelium. Ectopic expression of FGF9 in the proximal region of the optic vesicle extends neural differentiation into the presumptive retinal pigment epithelium, resulting in a duplicate neural retina in transgenic mice. Ectopic expression of constitutively active Ras is also sufficient to convert the retinal pigment epithelium to neural retina, suggesting that Ras-mediated signaling may be involved in neural differentiation in the immature optic vesicle. The original and the duplicate neural retinae differentiate and laminate with mirror-image polarity in the absence of an RPE, suggesting that the program of neuronal differentiation in the retina is autonomously regulated. In mouse embryos lacking FGF9, the retinal pigment epithelium extends into the presumptive neural retina, indicating a role of FGF9 in defining the boundary of the neural retina.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Cristalino/embriologia , Epitélio Pigmentado Ocular/embriologia , Retina/embriologia , Proteínas ras/biossíntese , Animais , Indução Embrionária , Fator 9 de Crescimento de Fibroblastos , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Fenótipo , Proteínas Recombinantes/biossíntese , Transdução de Sinais , Fatores de Transcrição/biossíntese , Proteínas ras/genéticaRESUMO
PURPOSE: The choroidal vasculature is essential for normal retinal function. However, mechanisms that control choroid development are unknown. In the present study, we provide evidence that the retinal pigment epithelium (RPE) plays an essential role in regulation of the choroid development in the mouse eye. METHODS: Transgenic mice that transiently express FGF9 in the embryonic RPE were generated. Postnatal eyes were analyzed by histology and in situ hybridizations. RESULTS: In the transgenic mice, most of the RPE was converted to neural retina. The choroid formed only in regions where patches of RPE were present. The choroid failed to develop in the absence of the RPE. CONCLUSIONS: The presence of the RPE appears to be required for choroid development, suggesting that molecular interactions between RPE and periocular mesenchyme are essential for melanocyte differentiation and vascular development in the choroid.
Assuntos
Corioide/crescimento & desenvolvimento , Epitélio Pigmentado Ocular/fisiologia , Animais , Diferenciação Celular , Corioide/irrigação sanguínea , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Fator 9 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Hibridização In Situ , Linfocinas/genética , Linfocinas/metabolismo , Masculino , Melanócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Several families of growth factors have been identified as regulators of cell fate in the developing lens. Members of the fibroblast growth factor family are potent inducers of lens fiber differentiation. Members of the transforming growth factor beta (TGFbeta) family, particularly bone morphogenetic proteins, have also been implicated in various stages of lens and ocular development, including lens induction and lens placode formation. However, at later stages of lens development, TGFbeta family members have been shown to induce pathological changes in lens epithelial cells similar to those seen in forms of human subcapsular cataract. Previous studies have shown that type I and type II TGFbeta receptors, in addition to being expressed in the epithelium, are also expressed in patterns consistent with a role in lens fiber differentiation. In this study we have investigated the consequences of disrupting TGFbeta signaling during lens fiber differentiation by using the mouse alphaA-crystallin promoter to overexpress mutant (kinase deficient), dominant-negative forms of either type I or type II TGFbeta receptors in the lens fibers of transgenic mice. Mice expressing these transgenes had pronounced bilateral nuclear cataracts. The phenotype was characterized by attenuated lens fiber elongation in the cortex and disruption of fiber differentiation, culminating in fiber cell apoptosis and degeneration in the lens nucleus. Inhibition of TGFbeta signaling resulted in altered expression patterns of the fiber-specific proteins, alpha-crystallin, filensin, phakinin and MIP. In addition, in an in vitro assay of cell migration, explanted lens cells from transgenic mice showed impaired migration on laminin and a lack of actin filament assembly, compared with cells from wild-type mice. These results indicate that TGFbeta signaling is a key event during fiber differentiation and is required for completion of terminal differentiation.
Assuntos
Receptores de Ativinas Tipo I/fisiologia , Cristalino/embriologia , Glicoproteínas de Membrana , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Actinas/metabolismo , Receptores de Ativinas Tipo I/genética , Animais , Apoptose , Aquaporinas , Catarata/embriologia , Catarata/genética , Catarata/metabolismo , Diferenciação Celular , Divisão Celular , Movimento Celular , Cristalinas/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Proteínas de Filamentos Intermediários/genética , Cristalino/citologia , Cristalino/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de SinaisRESUMO
Cardiac muscle regeneration after injury is limited by "irreversible" cell cycle exit. Telomere shortening is one postulated basis for replicative senescence, via down-regulation of telomerase reverse transcriptase (TERT); telomere dysfunction also is associated with greater sensitivity to apoptosis. Forced expression of TERT in cardiac muscle in mice was sufficient to rescue telomerase activity and telomere length. Initially, the ventricle was hypercellular, with increased myocyte density and DNA synthesis. By 12 wk, cell cycling subsided; instead, cell enlargement (hypertrophy) was seen, without fibrosis or impaired function. Likewise, viral delivery of TERT was sufficient for hypertrophy in cultured cardiac myocytes. The TERT virus and transgene also conferred protection from apoptosis, in vitro and in vivo. Hyperplasia, hypertrophy, and survival all required active TERT and were not seen with a catalytically inactive mutation. Thus, TERT can delay cell cycle exit in cardiac muscle, induce hypertrophy in postmitotic cells, and promote cardiac myocyte survival.
Assuntos
Miocárdio/citologia , Miocárdio/enzimologia , Telomerase/fisiologia , Animais , Apoptose/fisiologia , Sequência de Bases , Cardiomegalia/enzimologia , Cardiomegalia/etiologia , Divisão Celular/fisiologia , Tamanho Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Primers do DNA/genética , Proteínas de Ligação a DNA , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Transgênicos , Ratos , Telomerase/genética , Telômero/ultraestruturaRESUMO
Alterations in the ocular vasculature are associated with retinal diseases such as retinopathy of prematurity and diabetic retinopathy. Vascular endothelial growth factor (VEGF) as a potent stimulator for normal and abnormal vascular growth has been extensively studied. However, little is known about secreted factors that negatively regulate vascular growth in ocular tissues. We now report that expression of a self-activating TGFbeta1 in the ocular lens of transgenic mice results in inhibition of retinal angiogenesis followed by retinal degeneration. Transgenic TGFbeta1 can rescue the hyperplasic hyaloid tissue and reverse the corneal deficiency in TGFbeta2-null embryos. These results demonstrate that TGFbeta signaling modulates development of ocular vasculature and cornea in a dosage-dependent manner and that TGFbeta1 can substitute for TGFbeta2 in ocular tissues.
Assuntos
Olho/irrigação sanguínea , Neovascularização Fisiológica , Fator de Crescimento Transformador beta/fisiologia , Animais , Fatores de Crescimento Endotelial/análise , Fatores de Crescimento Endotelial/genética , Linfocinas/análise , Linfocinas/genética , Camundongos , Camundongos Transgênicos , Receptores Proteína Tirosina Quinases/análise , Receptores de Fatores de Crescimento/análise , Receptores de Fatores de Crescimento do Endotélio Vascular , Degeneração Retiniana/etiologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
A distinctive feature of gonadal maturation in mammals is the movement to an extraabdominal location. Testicular descent is a complex, multistage process whereby the embryonic gonads migrate from their initial abdominal position to the scrotum. Failure in this process results in cryptorchidism, a frequent congenital birth defect in humans. We report here a new mouse transgenic insertional mutation, cryptorchidism with white spotting (crsp). Males homozygous for crsp exhibit a high intraabdominal position of the testes, associated with complete sterility. Heterozygous males have a wild-type phenotype, and homozygous females are fertile. Surgically descended testes in crsp/crsp males show normal spermatogenesis. Using FISH and genetic analyses, the transgenic insert causing the crsp mutation has been mapped to the distal part of mouse chromosome 5. Transgene integration resulted in a 550-kb deletion located upstream of the Brca2 gene. A candidate gene encoding a novel G protein-coupled receptor (Great) with an expression pattern suggesting involvement in testicular descent has been identified.
Assuntos
Criptorquidismo/genética , Camundongos Transgênicos , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Mapeamento de Sequências Contíguas , Modelos Animais de Doenças , Feminino , Deleção de Genes , Homozigoto , Hibridização in Situ Fluorescente , Infertilidade , Masculino , Meiose , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Fenótipo , Pigmentação/genética , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Espermatogênese/genética , Testículo/fisiologia , Distribuição Tecidual , TransgenesRESUMO
The vertebrate lens has a distinct polarity with cuboidal epithelial cells on the anterior side and differentiated fiber cells on the posterior side. It has been proposed that the anterior-posterior polarity of the lens is imposed by factors present in the ocular media surrounding the lens (aqueous and vitreous humor). The differentiation factors have been hypothesized to be members of the fibroblast growth factor (FGF) family. Though FGFs have been shown to be sufficient for induction of lens differentiation both in vivo and in vitro, they have not been demonstrated to be necessary for endogenous initiation of fiber cell differentiation. To test this possibility, we have generated transgenic mice with ocular expression of secreted self-dimerizing versions of FGFR1 (FR1) and FGFR3 (FR3). Expression of FR3, but not FR1, leads to an expansion of proliferating epithelial cells from the anterior to the posterior side of the lens due to a delay in the initiation of fiber cell differentiation. This delay is most apparent postnatally and correlates with appropriate changes in expression of marker genes including p57(KIP2), Maf and Prox1. Phosphorylation of Erk1 and Erk2 was reduced in the lenses of FR3 mice compared with nontransgenic mice. Though differentiation was delayed in FR3 mice, the lens epithelial cells still retained their intrinsic ability to respond to FGF stimulation. Based on these results we propose that the initiation of lens fiber cell differentiation in mice requires FGF receptor signaling and that one of the lens differentiation signals in the vitreous humor is a ligand for FR3, and is therefore likely to be an FGF or FGF-like factor.
Assuntos
Cristalino/citologia , Cristalino/embriologia , Proteínas Tirosina Quinases , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Padronização Corporal , Diferenciação Celular , Cristalinas/biossíntese , Inibidor de Quinase Dependente de Ciclina p57 , Proteínas de Ligação a DNA/isolamento & purificação , Indução Embrionária , Células Epiteliais , Fatores de Crescimento de Fibroblastos , Proteínas de Homeodomínio/isolamento & purificação , Cristalino/irrigação sanguínea , Camundongos , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Proteínas Nucleares/isolamento & purificação , Fosforilação , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Proto-Oncogênicas c-maf , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos , Transgenes , Proteínas Supressoras de TumorRESUMO
Insulin-like growth factor-I (IGF-I) has been implicated as a regulator of lens development. Experiments performed in the chick have indicated that IGF-I can stimulate lens fiber cell differentiation and may be involved in controlling lens polarization. To assess IGF-I activity on mammalian lens cells in vivo, we generated transgenic mice in which this factor was overexpressed from the alphaA-crystallin promoter. Interestingly, we observed no premature differentiation of lens epithelial cells. The pattern of lens polarization was perturbed, with an apparent expansion of the epithelial compartment towards the posterior lens pole. The distribution of immunoreactivity for MIP26 and p57(KIP2) and a modified pattern of proliferation suggested that this morphological change was best described as an expansion of the germinative and transitional zones. The expression of IGF-I signaling components in the normal transitional zone and expansion of the transitional zone in the transgenic lens both suggest that endogenous IGF-I may provide a spatial cue that helps to control the normal location of this domain.
Assuntos
Fator de Crescimento Insulin-Like I/biossíntese , Cristalino/embriologia , Cristalino/metabolismo , Animais , Catarata/genética , Diferenciação Celular , Divisão Celular , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Modelos Genéticos , Fenótipo , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Transdução de Sinais , TransgenesRESUMO
The molecular basis of X-linked recessive anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has remained elusive. Here we report hypomorphic mutations in the gene IKBKG in 12 males with EDA-ID from 8 kindreds, and 2 patients with a related and hitherto unrecognized syndrome of EDA-ID with osteopetrosis and lymphoedema (OL-EDA-ID). Mutations in the coding region of IKBKG are associated with EDA-ID, and stop codon mutations, with OL-EDA-ID. IKBKG encodes NEMO, the regulatory subunit of the IKK (IkappaB kinase) complex, which is essential for NF-kappaB signaling. Germline loss-of-function mutations in IKBKG are lethal in male fetuses. We show that IKBKG mutations causing OL-EDA-ID and EDA-ID impair but do not abolish NF-kappaB signaling. We also show that the ectodysplasin receptor, DL, triggers NF-kappaB through the NEMO protein, indicating that EDA results from impaired NF-kappaB signaling. Finally, we show that abnormal immunity in OL-EDA-ID patients results from impaired cell responses to lipopolysaccharide, interleukin (IL)-1beta, IL-18, TNFalpha and CD154. We thus report for the first time that impaired but not abolished NF-kappaB signaling in humans results in two related syndromes that associate specific developmental and immunological defects.
Assuntos
Displasia Ectodérmica/genética , Displasia Ectodérmica/imunologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/genética , Adolescente , Criança , Pré-Escolar , Códon de Terminação/genética , Displasia Ectodérmica/metabolismo , Ectodisplasinas , Ligação Genética , Humanos , Quinase I-kappa B , Imunidade Celular , Síndromes de Imunodeficiência/metabolismo , Lactente , Masculino , Proteínas de Membrana/metabolismo , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Síndrome , Cromossomo X/genéticaRESUMO
Members of the tumour-necrosis factor receptor (TNFR) family that contain an intracellular death domain initiate signalling by recruiting cytoplasmic death domain adapter proteins. Edar is a death domain protein of the TNFR family that is required for the development of hair, teeth and other ectodermal derivatives. Mutations in Edar-or its ligand, Eda-cause hypohidrotic ectodermal dysplasia in humans and mice. This disorder is characterized by sparse hair, a lack of sweat glands and malformation of teeth. Here we report the identification of a death domain adapter encoded by the mouse crinkled locus. The crinkled mutant has an hypohidrotic ectodermal dysplasia phenotype identical to that of the edar (downless) and eda (Tabby) mutants. This adapter, which we have called Edaradd (for Edar-associated death domain), interacts with the death domain of Edar and links the receptor to downstream signalling pathways. We also identify a missense mutation in its human orthologue, EDARADD, that is present in a family affected with hypohidrotic ectodermal dysplasia. Our findings show that the death receptor/adapter signalling mechanism is conserved in developmental, as well as apoptotic, signalling.
Assuntos
Displasia Ectodérmica/genética , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Receptor Edar , Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , NF-kappa B/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores da Ectodisplasina , Receptores do Fator de Necrose Tumoral/química , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
In most mammals, male development is triggered by the transient expression of the Y-chromosome gene, Sry, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Several genes, in particular Sox9, have a crucial role in this pathway. Despite this, the direct downstream targets of Sry and the nature of the pathway itself remain to be clearly established. We report here a new dominant insertional mutation, Odsex (Ods), in which XX mice carrying a 150-kb deletion (approximately 1 Mb upstream of Sox9) develop as sterile XX males lacking Sry. During embryogenesis, wild-type XX fetal gonads downregulate Sox9 expression, whereas XY and XX Ods/+ fetal gonads upregulate and maintain its expression. We propose that Ods has removed a long-range, gonad-specific regulatory element that mediates the repression of Sox9 expression in XX fetal gonads. This repression would normally be antagonized by Sry protein in XY embryos. Our data are consistent with Sox9 being a direct downstream target of Sry and provide genetic evidence to support a general repressor model of sex determination in mammals.
Assuntos
Transtornos do Desenvolvimento Sexual , Proteínas de Grupo de Alta Mobilidade/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA/genética , Feminino , Genes Dominantes , Hibridização In Situ , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese Insercional , Linhagem , Fenótipo , Fatores de Transcrição SOX9 , Deleção de SequênciaRESUMO
PURPOSE: Previous studies have shown that inactivation of the retinoblastoma tumor suppressor protein (pRb) can cause lens fiber cell proliferation and apoptosis. Because pRb is thought to block cell cycle progression by inhibition of E2F transcription factors, experiments were conducted to test whether overexpression of different E2F family members would be sufficient to induce fiber cell proliferation and subsequent apoptosis. The in vivo functions of the transcription factor E2F2 have not previously been analyzed or described in transgenic mice. METHODS: Human E2F1 and E2F2 cDNAs were linked to the alphaA-crystallin promoter. Transgenic mice were generated by microinjection. Changes in cell cycle regulation were assayed by immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) incorporation and by in situ hybridization. Cell death was assayed using the TdT-dUTP terminal nick-end labeling (TUNEL) assay. RESULTS: At embryonic day (E)15.5, strong expression of the E2F1 and E2F2 transgenes was detected in lens fiber cells with little or no expression in epithelial cells. BrdU incorporation and TUNEL assays showed that overexpression of either E2F1 or E2F2 in lens fiber cells was sufficient to cause cell cycle entry and subsequent apoptosis. Expression of either E2F1 or E2F2 was sufficient to induce the transcription of cyclins (A2, B1, and E), as well as p53 and Bax in the lens fibercells. CONCLUSIONS: Expression of either E2F1 or E2F2 can induce postmitotic lens fiber cells to re-enter the cell cycle. Inappropriate cell cycle entry is recognized by p53 in each case, and programmed cell death ensues.
Assuntos
Apoptose , Proteínas de Transporte , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ligação a DNA , Cristalino/citologia , Proteínas Proto-Oncogênicas c-bcl-2 , Fatores de Transcrição/metabolismo , Animais , Bromodesoxiuridina/metabolismo , Divisão Celular , Ciclinas/metabolismo , Primers do DNA/química , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F2 , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Cristalino/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Gravidez , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 de Ligação ao Retinoblastoma , Fase S , Fator de Transcrição DP1 , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2RESUMO
FGF-10, a member of the fibroblast growth factor family, is expressed in mesodermally derived cell populations during embryogenesis. During normal ocular development, FGF-10 is expressed in the perioptic mesenchyme adjacent to the Harderian and lacrimal gland primordia. In this report, we provide evidence that FGF-10 is both necessary and sufficient to initiate glandular morphogenesis. Lens-specific expression of FGF-10 was sufficient to induce ectopic ocular glands within the cornea. In addition, lacrimal and Harderian glands were not seen in FGF-10 null fetuses. Based on these results we propose that FGF-10 is an inductive signal that initiates ocular gland morphogenesis.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Glândula de Harder/embriologia , Animais , Indução Embrionária , Desenvolvimento Embrionário e Fetal , Fator 10 de Crescimento de Fibroblastos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glândula de Harder/fisiologia , Camundongos , Camundongos TransgênicosRESUMO
Vascular endothelial growth factor (VEGF) is a secreted mitogen which specifically stimulates proliferation of vascular endothelial cells in vitro and in vivo. Its expression pattern is consistent with it being an important regulator of vasculogenesis and angiogenesis, and targeted disruption of VEGF-A has demonstrated that it is essential for vascular development. To determine if VEGF-A was sufficient to alter vascularization in the eye we generated transgenic mice which express human VEGF-A(165) specifically in the lens. Expression of transgenic VEGF-A led to excessive proliferation and accumulation of disorganized angioblasts and endothelial cells around the lens. The results support the hypothesis that VEGF-A can initiate the process of vascularization by stimulating chemoattraction and proliferation of angioblasts and endothelial cells and that VEGF-A expression can stimulate angiogenic remodeling. However, VEGF-A alone was not sufficient to direct blood vessel organization or maturation.
Assuntos
Fatores de Crescimento Endotelial/genética , Olho/irrigação sanguínea , Cristalino/irrigação sanguínea , Linfocinas/genética , Neovascularização Fisiológica , Animais , Membrana Basal/metabolismo , Colágeno/biossíntese , Endotélio Vascular/citologia , Olho/embriologia , Olho/crescimento & desenvolvimento , Humanos , Hiperplasia , Laminina/biossíntese , Cristalino/embriologia , Cristalino/crescimento & desenvolvimento , Mesoderma/citologia , Camundongos , Camundongos Transgênicos , Células-Tronco , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We investigated the mechanism of tissue induction and specification using the lacrimal gland as a model system. This structure begins its morphogenesis as a bud-like outgrowth of the conjunctival epithelium and ultimately forms a branched structure with secretory function. Using a reporter transgene as a specific marker for gland epithelium, we show that the transcription factor Pax6 is required for normal development of the gland and is probably an important competence factor. In investigating the cell-cell signaling required, we show that fibroblast growth factor (FGF) 10 is sufficient to stimulate ectopic lacrimal bud formation in ocular explants. Expression of FGF10 in the mesenchyme adjacent to the presumptive lacrimal bud and absence of lacrimal gland development in FGF10-null mice strongly suggest that it is an endogenous inducer. This was supported by the observation that inhibition of signaling by a receptor for FGF10 (receptor 2 IIIb) suppressed development of the endogenous lacrimal bud. In explants of mesenchyme-free gland epithelium, FGF10 stimulated growth but not branching morphogenesis. This suggested that its role in induction is to stimulate proliferation and, in turn, that FGF10 combines with other factors to provide the instructive signals required for lacrimal gland development.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Proteínas de Homeodomínio , Aparelho Lacrimal/embriologia , Animais , Epitélio/embriologia , Proteínas do Olho/fisiologia , Fator 10 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos/fisiologia , Fator 7 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/deficiência , Fatores de Crescimento de Fibroblastos/genética , Substâncias de Crescimento/fisiologia , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Camundongos Knockout , Morfogênese , Técnicas de Cultura de Órgãos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Proteínas Repressoras , Transdução de SinaisRESUMO
The transforming-growth-factor-beta-activated kinase TAK1 is a member of the mitogen-activated protein kinase kinase kinase family, which couples extracellular stimuli to gene transcription. The in vivo function of TAK1 is not understood. Here, we investigated the potential involvement of TAK1 in cardiac hypertrophy. In adult mouse myocardium, TAK1 kinase activity was upregulated 7 days after aortic banding, a mechanical load that induces hypertrophy and expression of transforming growth factor beta. An activating mutation of TAK1 expressed in myocardium of transgenic mice was sufficient to produce p38 mitogen-activated protein kinase phosphorylation in vivo, cardiac hypertrophy, interstitial fibrosis, severe myocardial dysfunction, 'fetal' gene induction, apoptosis and early lethality. Thus, TAK1 activity is induced as a delayed response to mechanical stress, and can suffice to elicit myocardial hypertrophy and fulminant heart failure.
Assuntos
Pressão Sanguínea , Baixo Débito Cardíaco/etiologia , Cardiomegalia/etiologia , MAP Quinase Quinase Quinases/biossíntese , Fator 6 Ativador da Transcrição , Animais , Aorta/cirurgia , Proteínas de Ligação a DNA/metabolismo , Diástole , Regulação para Baixo , MAP Quinase Quinase Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Nucleares/metabolismo , Fator de Resposta Sérica , Transdução de Sinais , Sístole , Fatores de Transcrição , Fator de Crescimento Transformador beta/biossíntese , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The anterior segment of the vertebrate eye is constructed by proper spatial development of cells derived from the surface ectoderm, which become corneal epithelium and lens, neuroectoderm (posterior iris and ciliary body) and cranial neural crest (corneal stroma, corneal endothelium and anterior iris). Although coordinated interactions between these different cell types are presumed to be essential for proper spatial positioning and differentiation, the requisite intercellular signals remain undefined. We have generated transgenic mice that express either transforming growth factor (alpha) (TGF(alpha)) or epidermal growth factor (EGF) in the ocular lens using the mouse (alpha)A-crystallin promoter. Expression of either growth factor alters the normal developmental fate of the innermost corneal mesenchymal cells so that these cells often fail to differentiate into corneal endothelial cells. Both sets of transgenic mice subsequently manifest multiple anterior segment defects, including attachment of the iris and lens to the cornea, a reduction in the thickness of the corneal epithelium, corneal opacity, and modest disorganization in the corneal stroma. Our data suggest that formation of a corneal endothelium during early ocular morphogenesis is required to prevent attachment of the lens and iris to the corneal stroma, therefore permitting the normal formation of the anterior segment.
Assuntos
Câmara Anterior/anormalidades , Câmara Anterior/embriologia , Ectoderma/fisiologia , Endotélio Corneano/embriologia , Fator de Crescimento Epidérmico/fisiologia , Cristalino/embriologia , Fator de Crescimento Transformador alfa/fisiologia , Animais , Caderinas/análise , Caderinas/genética , Cristalinas/genética , Modelos Animais de Doenças , Desenvolvimento Embrionário e Fetal , Endotélio Corneano/anormalidades , Fator de Crescimento Epidérmico/genética , Receptores ErbB/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Fator de Crescimento Transformador alfa/genéticaRESUMO
Maf is a basic domain/leucine zipper domain protein originally identified as a proto-oncogene whose consensus target site in vitro, the T-MARE, is an extended version of an AP-1 site normally recognized by Fos and Jun. Maf and the closely related family members Neural retina leucine zipper (Nrl), L-Maf, and Krml1/MafB have been implicated in a wide variety of developmental and physiologic roles; however, mutations in vivo have been described only for Krml1/MafB, in which a loss-of-function causes abnormalities in hindbrain development due to failure to activate the Hoxa3 and Hoxb3 genes. We have used gene targeting to replace Maf coding sequences with those of lacZ, and have carried out a comprehensive analysis of embryonic expression and the homozygous mutant phenotype in the eye. Maf is expressed in the lens vesicle after invagination, and becomes highly upregulated in the equatorial zone, the site at which self-renewing anterior epithelial cells withdraw from the cell cycle and terminally differentiate into posterior fiber cells. Posterior lens cells in Maf(lacZ) mutant mice exhibit failure of elongation at embryonic day 11.5, do not express (&agr;)A- and all of the (beta)-crystallin genes, and display inappropriately high levels of DNA synthesis. This phenotype partially overlaps with those reported for gene targeting of Prox1 and Sox1; however, expression of these genes is grossly normal, as is expression of Eya1, Eya2, Pax6, and Sox2. Recombinant Maf protein binds to T-MARE sites in the (alpha)A-, (beta)B2-, and (beta)A4-crystallin promoters but fails to bind to a point mutation in the (alpha)A-crystallin promoter that has been shown previously to be required for promoter function. Our results indicate that Maf directly activates many if not all of the (beta)-crystallin genes, and suggest a model for coordinating cell cycle withdrawal with terminal differentiation.
Assuntos
Proteínas de Ligação a DNA/genética , Cristalino/embriologia , Proteínas Proto-Oncogênicas/genética , Animais , Sítios de Ligação , Diferenciação Celular/genética , Cristalinas/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Marcação de Genes , Histocitoquímica , Hibridização In Situ , Óperon Lac , Cristalino/anormalidades , Cristalino/metabolismo , Camundongos , Camundongos Knockout , Mutação , Fenótipo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-maf , Ativação Transcricional/genéticaRESUMO
In an effort to identify a promoter suitable for studying early ocular development, we generated transgenic mice carrying the lacZ reporter gene linked to the tyrosinase-related protein 2 (TRP2) promoter. TRP2-lacZ was expressed in early retinal pigment epithelium (RPE) and early neural crest cells in embryos. The promoter activity was robust and consistent in independent transgenic lines. The transgene was also expressed in the optic nerve and neural crest-derived neuronal cells in which the endogenous TRP2 gene is not expressed. This suggests that repressor elements may be missing in the promoter used in this study. To test whether this promoter can be used to study melanocyte development, we cross-mated TRP2-lacZ transgenic mice with mice heterozygous for the Patch (Ph) mutation. The pattern of beta-galactosidase activity in the embryos correlates well with the pigmentation phenotype in postnatal and adult Ph/+ mice. We also generated transgenic mice expressing fibroblast growth factor 9 (FGF9) directed by the TRP2 promoter and examined the effect on ocular development. Ectopic expression of FGF9 in the early embryonic RPE switched its differentiation pathway to a neuronal fate, resulting in formation of a duplicated neural retina in transgenic mice. These studies demonstrate that the TRP2 promoter is valuable for transgenic studies of ocular differentiation and development of neural crest cells.
Assuntos
Olho/embriologia , Fatores de Crescimento de Fibroblastos , Oxirredutases Intramoleculares/genética , Crista Neural/embriologia , Regiões Promotoras Genéticas/genética , Animais , Diferenciação Celular , Olho/crescimento & desenvolvimento , Fator 9 de Crescimento de Fibroblastos , Regulação da Expressão Gênica no Desenvolvimento , Substâncias de Crescimento/genética , Histocitoquímica , Imuno-Histoquímica , Hibridização In Situ , Óperon Lac , Melanócitos/metabolismo , Camundongos , Camundongos Transgênicos , Crista Neural/crescimento & desenvolvimento , Epitélio Pigmentado Ocular/embriologiaRESUMO
Mice homozygous for the transgenic insertion in line OVE250 exhibit severe progressive glomerulonephritis. Ultrastructural changes in the glomerular basement membrane (GBM) at 2 weeks of age resemble those in Alport syndrome. The transgenic insertion site was mapped by FISH to mouse chromosome 1 close to Pax3. Genetic and molecular analyses identified a deletion of genomic DNA at the transgene insertion site. Exons 1 through 12 of the collagen IV gene Col4a4, exons 1 and 2 of the adjacent Col4a3 gene, and the intergenic promoter region are deleted. Transcripts of Col4a3 and Col4a4 are undetectable in mutant kidney, and both proteins are missing from the GBM. Persistent cellular proliferation in mutant kidneys suggests that interaction with the extracellular matrix may be important for cell maturation. Evolutionarily conserved sequence elements in the promoter regions of human and mouse Col4a3 and Col4a4 include a 19-bp element that was tandemly duplicated in the human lineage and a CTC box element common to several genes encoding extracellular matrix proteins. This new animal model of Alport syndrome, Col4Delta3-4, lacks both alpha3 and alpha4 chains of collagen IV and exhibits an earlier disease onset than mice lacking alpha3 only.