RESUMO
BACKGROUND: The Fgf2 gene is expressed in the brain neuroepithelium during embryonic development and in astroglial cells throughout life. Previous knockout studies suggested that FGF2 plays a role in the proliferation of neural progenitors in the embryonic cerebral cortex. These studies exclusively used knockout alleles lacking the Fgf2 exon 1. However, the description of putative alternative exons located downstream from the canonical exon 1 raised the possibility that alternatively spliced transcripts may compensate for the lack of the canonical exon 1 in the Fgf2 -/- mice. RESULTS: We generated and characterized a new line of Fgf2 knockout mice lacking the expression of exon 3, which is conserved in all Fgf2 transcripts and contains essential heparin and receptor binding interfaces. The expression of Fgf2 exon 3 was prevented by inserting a transcriptional STOP cassette in the Fgf2 genomic locus. These mice demonstrate a phenotype in the adult neocortex characterized by decreased density and number of cortical excitatory neurons and astrocytes, which is virtually identical to that of the Fgf2 -/- mice lacking exon 1. In addition, we also show that the Fgf2 exon 3 knockout mice have decreased proliferation of precursors in the adult cerebral cortex, which had not been previously investigated in the other mutant lines. CONCLUSION: The results demonstrate that the phenotype of two completely different Fgf2 KO mouse lines, lacking exon 1 or exon 3, is remarkably similar. The combined results from these KO models clearly indicate that FGF2 plays a role in cortical cell genesis during embryonic development as well as in adulthood. Thus, FGF2 may be required for the maintenance of the pool of adult cortical progenitor cells.
Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Neurônios/metabolismo , Células-Tronco/metabolismo , Animais , Astrócitos/citologia , Western Blotting , Diferenciação Celular/genética , Proliferação de Células , Córtex Cerebral/citologia , Éxons , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Neurônios/citologia , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologiaRESUMO
Guanylin and uroguanylin, peptides synthesized in the intestine and kidney, have been postulated to have both paracrine and endocrine functions, forming a potential enteric-renal link to coordinate salt ingestion with natriuresis. To explore the in vivo role of uroguanylin in the regulation of sodium excretion, we created gene-targeted mice in which uroguanylin gene expression had been ablated. Northern and Western analysis confirmed the absence of uroguanylin message and protein in knockout mice, and cGMP levels were decreased in the mucosa of the small intestine. Ussing chamber analysis of jejunum revealed that Na+/H+ exchanger-mediated Na+ absorption and tissue conductance was not altered in the knockout animals, but short-circuit current, an index of electrogenic anion secretion, was reduced. Renal clearance measurements showed that uroguanylin deficiency results in impaired ability to excrete an enteral load of NaCl, primarily due to an inappropriate increase in renal Na+ reabsorption. Finally, telemetric recordings of blood pressure demonstrated increased mean arterial pressure in uroguanylin knockout animals that was independent of the level of dietary salt intake. Together, these findings establish a role for uroguanylin in an enteric-renal communication axis as well as a fundamental principle of this axis in the maintenance of salt homeostasis in vivo.
Assuntos
Pressão Sanguínea , Natriurese , Peptídeos/fisiologia , Cloreto de Sódio/metabolismo , Animais , GMP Cíclico/análise , Genótipo , Jejuno/metabolismo , Rim/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Peptídeos NatriuréticosRESUMO
The ligand specificity of transforming growth factor beta (TGFbeta) in vivo in mouse cardiac cushion epithelial-to-mesenchymal transition (EMT) is poorly understood. To elucidate the function of TGFbeta in cushion EMT, we analyzed Tgfb1(-/-), Tgfb2(-/-), and Tgfb3(-/-) mice between embryonic day (E) 9.5 and E14.5 using both in vitro and in vivo approaches. Atrioventricular (AV) canal collagen gel assays at E9.5 indicated normal EMT in both Tgfb1(-/-) and Tgfb3(-/-) mice. However, analysis of Tgfb2(-/-) AV explants at E9.5 and E10.5 indicated that EMT, but not cushion cell proliferation, was initially delayed but later remained persistent. This was concordant with the observation that Tgfb2(-/-) embryos, and not Tgfb1(-/-) or Tgfb3(-/-) embryos, develop enlarged cushions at E14.5 with elevated levels of well-validated indicators of EMT. Collectively, these data indicate that TGFbeta2, and not TGFbeta1 or TGFbeta3, mediates cardiac cushion EMT by promoting both the initiation and cessation of EMT.
Assuntos
Células Epiteliais/fisiologia , Coração/embriologia , Mesoderma/embriologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Epiteliais/citologia , Coração/fisiologia , Ligantes , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/fisiologia , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/fisiologia , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/fisiologiaRESUMO
Fibroblast growth factor-2 (FGF2) is produced as high molecular weight isoforms (HMW) and a low molecular weight isoform (LMW) by means of alternative usage of translation start sites in a single Fgf2 mRNA. Although the physiological function of FGF2 and FGF2 LMW has been investigated in myocardial capillarogenesis during normal cardiac growth, the role of FGF2 HMW has not been determined. Here, we report the generation of FGF2 HMW-deficient mice in which FGF2 HMW isoforms are ablated by the Tag-and-Exchange gene targeting technique. These mice are normal and fertile with normal fecundity, and have a normal life span. Histological, immunohistochemical, and morphometric analyses indicate normal myocardial architecture, blood vessel, and cardiac capillary density in young adult FGF2 HMW-deficient mice. These mice along with the FGF2- and FGF2 LMW-deficient mice that we have generated previously will be very useful for elucidating the differential functions of FGF2 isoforms in pathophysiology of cardiovascular diseases.
Assuntos
Vasos Coronários/metabolismo , Fator 2 de Crescimento de Fibroblastos/fisiologia , Miocárdio/metabolismo , Animais , Capilares/fisiologia , Fator 2 de Crescimento de Fibroblastos/genética , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologiaRESUMO
Loss of one copy of the human ATP2C1 gene, encoding SPCA1 (secretory pathway Ca(2+)-ATPase isoform 1), causes Hailey-Hailey disease, a skin disorder. We performed targeted mutagenesis of the Atp2c1 gene in mice to analyze the functions of this Golgi membrane Ca(2+) pump. Breeding of heterozygous mutants yielded a normal Mendelian ratio among embryos on gestation day 9.5; however, null mutant (Spca1(-/-)) embryos exhibited growth retardation and did not survive beyond gestation day 10.5. Spca1(-/-) embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1(-/-) embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca(2+) pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer.
Assuntos
ATPases Transportadoras de Cálcio/deficiência , Carcinoma de Células Escamosas/metabolismo , Perda do Embrião/metabolismo , Neoplasias Esofágicas/metabolismo , Complexo de Golgi/metabolismo , Perda de Heterozigosidade , Neoplasias Cutâneas/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Apoptose/genética , Membrana Basal/metabolismo , Membrana Basal/ultraestrutura , ATPases Transportadoras de Cálcio/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Sistema Cardiovascular/embriologia , Invaginações Revestidas da Membrana Celular/genética , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/ultraestrutura , Desmossomos/genética , Desmossomos/metabolismo , Desmossomos/ultraestrutura , Perda do Embrião/genética , Perda do Embrião/patologia , Retículo Endoplasmático Rugoso/genética , Retículo Endoplasmático Rugoso/metabolismo , Retículo Endoplasmático Rugoso/ultraestrutura , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Feminino , Predisposição Genética para Doença , Complexo de Golgi/ultraestrutura , Hematopoese/genética , Heterozigoto , Homozigoto , Humanos , Endogamia , Perda de Heterozigosidade/genética , Masculino , Camundongos , Camundongos Knockout , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/patologia , Pênfigo Familiar Benigno/genética , Pênfigo Familiar Benigno/metabolismo , Pênfigo Familiar Benigno/patologia , Gravidez , Transporte Proteico/genética , Ribossomos/metabolismo , Vesículas Secretórias/genética , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologiaRESUMO
The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and HCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon.
Assuntos
Acidose/genética , Colo/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Aldosterona/metabolismo , Animais , Ânions , AMP Cíclico/metabolismo , Mucosa Intestinal/metabolismo , Rim/metabolismo , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Fenótipo , Fosforilação , Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/fisiologiaRESUMO
The NHE4 Na+/H+ exchanger is abundantly expressed on the basolateral membrane of gastric parietal cells. To test the hypothesis that it is required for normal acid secretion, NHE4-null mutant (NHE4-/-) mice were prepared by targeted disruption of the NHE4 (Slc9a4) gene. NHE4-/- mice survived and appeared outwardly normal. Analysis of stomach contents revealed that NHE4-/- mice were hypochlorhydric. The reduction in acid secretion was similar in 18-day-old, 9-week-old, and 6-month-old mice, indicating that the hypochlorhydria phenotype did not progress over time, as was observed in mice lacking the NHE2 Na+/H+ exchanger. Histological abnormalities were observed in the gastric mucosa of 9-week-old NHE4-/- mice, including sharply reduced numbers of parietal cells, a loss of mature chief cells, increased numbers of mucous and undifferentiated cells, and an increase in the number of necrotic and apoptotic cells. NHE4-/- parietal cells exhibited limited development of canalicular membranes and a virtual absence of tubulovesicles, and some of the microvilli had centrally bundled actin. We conclude that NHE4, which may normally be coupled with the AE2 Cl-/HCO3- exchanger, is important for normal levels of gastric acid secretion, gastric epithelial cell differentiation, and development of secretory canalicular and tubulovesicular membranes.
Assuntos
Ácido Gástrico/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Acloridria/patologia , Alelos , Processamento Alternativo , Animais , Apoptose , Northern Blotting , Western Blotting , Diferenciação Celular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Éxons , Gastrinas/metabolismo , Concentração de Íons de Hidrogênio , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Modelos Biológicos , Modelos Genéticos , Mutação , Necrose , Células Parietais Gástricas/citologia , Células Parietais Gástricas/ultraestrutura , Fenótipo , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trocadores de Sódio-Hidrogênio/metabolismo , Fatores de TempoRESUMO
Transforming growth factors beta (TGF-betas) are peptides involved in autocrine and paracrine control of cell growth and differentiation. In the kidneys, TGF-beta(2) has been shown to localize specifically in renin-producing cells in various conditions stimulating the renin response. To test in vivo the functional role of TGF-beta(2), the renin response was investigated in mice heterozygous for a null mutation of the TGF-beta(2) gene, which had a twofold reduction in the amount of TGF-beta(2) mRNA. Although the increase in plasma renin concentration triggered by dehydration was not different from wild-type mice, renal renin mRNA and protein levels were higher in mutant mice under hydrated or dehydrated conditions. These data suggest that TGF-beta(2) exerts an inhibitory effect on renin synthesis and release from the juxtaglomerular apparatuses.
Assuntos
Deleção de Genes , Heterozigoto , Rim/metabolismo , Mutação/fisiologia , Renina/metabolismo , Fator de Crescimento Transformador beta/genética , Animais , Arteríolas/metabolismo , Água Corporal/metabolismo , Desidratação/metabolismo , Feto/metabolismo , Genótipo , Sistema Justaglomerular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , RNA Mensageiro/metabolismo , Circulação Renal , Renina/genética , Fator de Crescimento Transformador beta2 , Privação de Água/fisiologiaRESUMO
The AE2 Cl-/HCO3- exchanger is expressed in numerous cell types, including epithelial cells of the kidney, respiratory tract, and alimentary tract. In gastric epithelia, AE2 is particularly abundant in parietal cells, where it may be the predominant mechanism for HCO3- efflux and Cl- influx across the basolateral membrane that is needed for acid secretion. To investigate the hypothesis that AE2 is critical for parietal cell function and to assess its importance in other tissues, homozygous null mutant (AE2(-/-)) mice were prepared by targeted disruption of the AE2 (Slc4a2) gene. AE2(-/-) mice were emaciated, edentulous (toothless), and exhibited severe growth retardation, and most of them died around the time of weaning. AE2(-/-) mice exhibited achlorhydria, and histological studies revealed abnormalities of the gastric epithelium, including moderate dilation of the gastric gland lumens and a reduction in the number of parietal cells. There was little evidence, however, that parietal cell viability was impaired. Ultrastructural analysis of AE2(-/-) gastric mucosa revealed abnormal parietal cell structure, with severely impaired development of secretory canaliculi and few tubulovesicles but normal apical microvilli. These results demonstrate that AE2 is essential for gastric acid secretion and for normal development of secretory canalicular and tubulovesicular membranes in mouse parietal cells.
Assuntos
Proteínas de Transporte de Ânions , Antiporters , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Acloridria/genética , Alelos , Animais , Northern Blotting , Western Blotting , Sobrevivência Celular , Antiportadores de Cloreto-Bicarbonato , Células Epiteliais/metabolismo , Epitélio/metabolismo , Mucosa Gástrica/metabolismo , Vetores Genéticos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Mutação , Células Parietais Gástricas/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Proteínas SLC4A , TransgenesRESUMO
BACKGROUND & AIMS: A genome-level understanding of the molecular basis of segmental gene expression along the anterior-posterior (A-P) axis of the mammalian gastrointestinal (GI) tract is lacking. We hypothesized that functional patterning along the A-P axis of the GI tract could be defined at the molecular level by analyzing expression profiles of large numbers of genes. METHODS: Incyte GEM1 microarrays containing 8638 complementary DNAs (cDNAs) were used to define expression profiles in adult mouse stomach, duodenum, jejunum, ileum, cecum, proximal colon, and distal colon. Highly expressed cDNAs were classified based on segmental expression patterns and protein function. RESULTS: 571 cDNAs were expressed 2-fold higher than reference in at least 1 GI tissue. Most of these genes displayed sharp segmental expression boundaries, the majority of which were at anatomically defined locations. Boundaries were particularly striking for genes encoding proteins that function in intermediary metabolism, transport, and cell-cell communication. Genes with distinctive expression profiles were compared with mouse and human genomic sequence for promoter analysis and gene discovery. CONCLUSIONS: The anatomically defined organs of the GI tract (stomach, small intestine, colon) can be distinguished based on a genome-level analysis of gene expression profiles. However, distinctions between various regions of the small intestine and colon are much less striking. We have identified novel genes not previously known to be expressed in the adult GI tract. Identification of genes coordinately regulated along the A-P axis provides a basis for new insights and gene discovery relevant to GI development, differentiation, function, and disease.