RESUMO
Kruppel-like transcription factor 5 (Klf5) was detected in the developing and mature murine bladder urothelium. Herein we report a critical role of KLF5 in the formation and terminal differentiation of the urothelium. The Shh(GfpCre) transgene was used to delete the Klf5(floxed) alleles from bladder epithelial cells causing prenatal hydronephrosis, hydroureter, and vesicoureteric reflux. The bladder urothelium failed to stratify and did not express terminal differentiation markers characteristic of basal, intermediate, and umbrella cells including keratins 20, 14, and 5, and the uroplakins. The effects of Klf5 deletion were unique to the developing bladder epithelium since maturation of the epithelium comprising the bladder neck and urethra was unaffected by the lack of KLF5. mRNA analysis identified reductions in Pparγ, Grhl3, Elf3, and Ovol1expression in Klf5 deficient fetal bladders supporting their participation in a transcriptional network regulating bladder urothelial differentiation. KLF5 regulated expression of the mGrhl3 promoter in transient transfection assays. The absence of urothelial Klf5 altered epithelial-mesenchymal signaling leading to the formation of an ectopic alpha smooth muscle actin positive layer of cells subjacent to the epithelium and a thinner detrusor muscle that was not attributable to disruption of SHH signaling, a known mediator of detrusor morphogenesis. Deletion of Klf5 from the developing bladder urothelium blocked epithelial cell differentiation, impaired bladder morphogenesis and function causing hydroureter and hydronephrosis at birth.
Assuntos
Diferenciação Celular/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Bexiga Urinária/citologia , Urotélio/embriologia , Animais , Proliferação de Células , Primers do DNA/genética , Imuno-Histoquímica , Camundongos , Análise em Microsséries , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Bexiga Urinária/embriologia , Microtomografia por Raio-XRESUMO
Here we describe the first detailed catalog of gene expression in the developing lower urinary tract (LUT), including epithelial and mesenchymal portions of the developing bladder, urogenital sinus, urethra, and genital tubercle (GT) at E13 and E14. Top compartment-specific genes implicated by the microarray data were validated using whole-mount in situ hybridization (ISH) over the entire LUT. To demonstrate the potential of this resource to implicate developmentally critical features, we focused on gene expression patterns and pathways in the sexually indeterminate, androgen-independent GT. GT expression patterns reinforced the proposed similarities between development of GT, limb, and craniofacial prominences. Comparison of spatial expression patterns predicted a network of Wnt7a-associated GT-enriched epithelial genes, including Gjb2, Dsc3, Krt5, and Sostdc1. Known from other contexts, these genes are associated with normal epidermal differentiation, with disruptions in Dsc3 and Gjb2 showing palmo-plantar keratoderma in the limb. We propose that this gene network contributes to normal foreskin, scrotum, and labial development. As several of these genes are known to be regulated by, or contain cis elements responsive to retinoic acid, estrogen, or androgen, this implicates this pathway in the later androgen-dependent development of the GT.
Assuntos
Expressão Gênica , Redes Reguladoras de Genes , Sistema Urogenital/embriologia , Androgênios/genética , Animais , Diferenciação Celular/genética , Embrião de Mamíferos , Epiderme , Extremidades , Genitália Masculina/embriologia , Masculino , Camundongos , Organogênese/genética , Uretra/embriologiaRESUMO
The forkhead box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) is expressed in a variety of tissues during embryogenesis, including vascular, airway, and intestinal smooth muscle cells (SMCs). Although global deletion of Foxm1 in Foxm1(-/-) mice is lethal in the embryonic period due to multiple abnormalities in the liver, heart, and lung, the specific role of Foxm1 in SMC remains unknown. In the present study, Foxm1 was deleted conditionally in the developing SMC (smFoxm1(-/-) mice). The majority of smFoxm1(-/-) mice died immediately after birth due to severe pulmonary hemorrhage and structural defects in arterial wall and esophagus. Although Foxm1 deletion did not influence SMC differentiation, decreased proliferation of SMC was found in smFoxm1(-/-) blood vessels and esophagus. Depletion of Foxm1 in cultured SMC caused G(2) arrest and decreased numbers of cells undergoing mitosis. Foxm1-deficiency in vitro and in vivo was associated with reduced expression of cell cycle regulatory genes, including cyclin B1, Cdk1-activator Cdc25b phosphatase, Polo-like 1 and JNK1 kinases, and cMyc transcription factor. Foxm1 is critical for proliferation of smooth muscle cells and is required for proper embryonic development of blood vessels and esophagus.
Assuntos
Vasos Sanguíneos/embriologia , Esôfago/embriologia , Fatores de Transcrição Forkhead/fisiologia , Músculo Liso/embriologia , Animais , Proliferação de Células , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/genética , Genes Letais , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso/citologia , RNA Interferente PequenoRESUMO
Actin, a major component of the cytoplasm, is also abundant in the nucleus. Nuclear actin is involved in a variety of nuclear processes including transcription, chromatin remodeling, and intranuclear transport. Nevertheless, the regulation of nuclear actin by posttranslational modifications has not been investigated. We now show that nuclear actin is modified by SUMO2 and SUMO3 and that computational modeling and site-directed mutagenesis identified K68 and K284 as critical sites for SUMOylating actin. We also present a model for the actin-SUMO complex and show that SUMOylation is required for the nuclear localization of actin.
Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Simulação por Computador , Ácidos Graxos Insaturados/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Ubiquitinas/genéticaRESUMO
Skeletal muscle alpha-actin (ACTA1) is the major actin in postnatal skeletal muscle. Mutations of ACTA1 cause mostly fatal congenital myopathies. Cardiac alpha-actin (ACTC) is the major striated actin in adult heart and fetal skeletal muscle. It is unknown why ACTC and ACTA1 expression switch during development. We investigated whether ACTC can replace ACTA1 in postnatal skeletal muscle. Two ACTC transgenic mouse lines were crossed with Acta1 knockout mice (which all die by 9 d after birth). Offspring resulting from the cross with the high expressing line survive to old age, and their skeletal muscles show no gross pathological features. The mice are not impaired on grip strength, rotarod, or locomotor activity. These findings indicate that ACTC is sufficiently similar to ACTA1 to produce adequate function in postnatal skeletal muscle. This raises the prospect that ACTC reactivation might provide a therapy for ACTA1 diseases. In addition, the mouse model will allow analysis of the precise functional differences between ACTA1 and ACTC.
Assuntos
Actinas/fisiologia , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Actinas/genética , Animais , Perfilação da Expressão Gênica , Coração/embriologia , Camundongos , Camundongos Knockout , Músculo Esquelético/ultraestrutura , Miocárdio/ultraestrutura , Isoformas de Proteínas/fisiologiaRESUMO
In late 2004, an International Consortium of research groups were charged with the task of producing a high-quality molecular anatomy of the developing mammalian urogenital tract (UGT). Given the importance of these organ systems for human health and reproduction, the need for a systematic molecular and cellular description of their developmental programs was deemed a high priority. The information obtained through this initiative is anticipated to enable the highest level of basic and clinical research grounded on a 21st-century view of the developing anatomy. There are three components to the Genitourinary Developmental Molecular Anatomy Project GUDMAP; all of these are intended to provide resources that support research on the kidney and UGT. The first provides ontology of the cell types during UGT development and the molecular hallmarks of those cells as discerned by a variety of procedures, including in situ hybridization, transcriptional profiling, and immunostaining. The second generates novel mouse strains. In these strains, cell types of particular interest within an organ are labeled through the introduction of a specific marker into the context of a gene that exhibits appropriate cell type or structure-specific expression. In addition, the targeting construct enables genetic manipulation within the cell of interest in many of the strains. Finally, the information is annotated, collated, and promptly released at regular intervals, before publication, through a database that is accessed through a Web portal. Presented here is a brief overview of the Genitourinary Developmental Molecular Anatomy Project effort.
Assuntos
Sistema Urogenital/anatomia & histologia , Sistema Urogenital/crescimento & desenvolvimento , Animais , Bases de Dados Genéticas , Camundongos , Biologia MolecularRESUMO
Actin is abundant in the nucleus and has been implicated in transcription; however, the nature of this involvement has not been established. Here we demonstrate that beta-actin is critically involved in transcription because antibodies directed against beta-actin, but not muscle actin, inhibited transcription in vivo and in vitro. Chromatin immunoprecipitation assays demonstrated the recruitment of actin to the promoter region of the interferon-gamma-inducible MHC2TA gene as well as the interferon-alpha-inducible G1P3 gene. Further investigation revealed that actin and RNA polymerase II co-localize in vivo and also co-purify. We employed an in vitro system with purified nuclear components to demonstrate that antibodies to beta-actin block the initiation of transcription. This assay also demonstrates that beta-actin stimulates transcription by RNA polymerase II. Finally, DNA-binding experiments established the presence of beta-actin in pre-initiation complexes and also showed that the depletion of actin prevented the formation of pre-initiation complexes. Together, these data suggest a fundamental role for actin in the initiation of transcription by RNA polymerase II.
Assuntos
Actinas/fisiologia , RNA Polimerase II/fisiologia , Transcrição Gênica/fisiologia , Sequência de Bases , Primers do DNA , Células HeLa , Humanos , Proteínas Mitocondriais , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas/genética , Transativadores/genéticaRESUMO
PURPOSE: Our understanding of urogenital tract development and its response to disease or injury is hindered by complex interactions between epithelial and mesenchymal cells, and the difficulties in studying either component in isolation. We investigated whether transgenic mice could be generated to express enhanced green fluorescent protein (EGFP) in smooth muscle cells (SMCs) and whether such cells could then be purified using flow cytometric sorting to isolate RNA to be used in future gene expression assays. MATERIALS AND METHODS: A 13.7 kb mouse smooth muscle gamma-actin promoter fragment was ligated to an EGFP reporter gene and microinjected into male mouse pronuclei. Adult transgenic mice were sacrificed and urogenital tissues were removed for histological and immunohistochemical studies. In other animals conditions were determined for dissociating bladder cells and the subsequent purification of bladder SMCs by sorting. RESULTS: Six lines of transgenic mice were generated (transgene copy numbers 1 to 30). EGFP was expressed in all smooth muscle beds examined except those associated with small blood vessels. EGFP levels appeared to correlate with transgene copy number. Histological and immunohistochemical analysis confirmed that reporter gene expression was restricted to SMCs of all tissues examined. Parameters for generating bladder cell suspensions were established and EGFP labeled bladder SMCs were identified by flow cytometric analysis. CONCLUSIONS: Several lines of transgenic mice have been generated in which SMCs of urogenital tissues have been labeled with EGFP and pure populations of SMCs have been obtained. The methods established for the rapid dissociation and purification of bladder SMCs should minimize degradative changes. These approaches may enable us to address issues involving bladder SMC development and differentiation as well as the response to injury and disease by performing transcriptome wide analyses on purified SMC populations.
Assuntos
Actinas/fisiologia , Proteínas Luminescentes/biossíntese , Músculo Liso/citologia , Músculo Liso/metabolismo , Sistema Urogenital/citologia , Sistema Urogenital/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Fluorescência Verde , Proteínas Luminescentes/análise , Camundongos/genética , Bexiga Urinária/citologia , Bexiga Urinária/metabolismoRESUMO
Mice that lack cardiac muscle alpha-actin die during the perinatal period. Approximately 56% of mice that are homozygous null (-/-) for a functional cardiac alpha-actin gene do not survive to term, and the remainder generally die within 2 wk of birth. We found that there were neither morphologic differences nor differences in the extent of apoptosis between the mutant and normal hearts on embryonic day (E) 12 and E14 of development. However, apoptosis was greater in the hearts of homozygous null mice on E17 and postnatal day 1 when compared with wild-type hearts. The antiapoptotic factor Bcl-x/(L) was localized in regions adjacent to where apoptosis was detected. The distribution patterns of the apoptosis triggering protein p53 were similar to those of apoptotic cells. The growth of the prenatal and postnatal hearts of the cardiac alpha-actin-deficient mice was retarded, and the cytoplasmic filaments were disorganized. Although apoptotic cells were observed in both the atria and ventricles in the hearts of the homozygous null animals, the frequency was greater in the ventricles than in the atria. Our results indicate that the functional and structural disturbances in the mice with a homozygous lack of cardiac alpha-actin seem to be due to disorganized development of acto-myosin filaments in the affected cardiomyocytes. Other actin isoforms cannot compensate for the lack of cardiac alpha-actin, and this seems to induce apoptosis in defective cardiac myocytes, which are not able to cope with the increased workload in the perinatal phase.
Assuntos
Actinas/genética , Apoptose , Cardiopatias Congênitas/patologia , Miocárdio/patologia , Citoesqueleto de Actina/patologia , Animais , Causas de Morte , Feminino , Coração/embriologia , Cardiopatias Congênitas/mortalidade , Masculino , Camundongos , Camundongos Mutantes , GravidezRESUMO
Tropomyosin is an actin-binding protein responsible for stabilizing the actin microfilament system in the cytoskeleton of nonmuscle cells and is involved in processes such as growth, differentiation, and polarity of neuronal cells. From the gamma gene, at least 11 different isoforms have been described, with three different C-terminal exons used (9a, 9c, 9d). The precise roles that the different isoforms play are unknown. To examine the localization and hence determine the function of these isoforms in developing mouse, specific antibodies to exons 9a and 9c were made. These were used with previously developed 9d and N-terminal 1b antibodies on Western blots and immunohistochemical analysis of mouse brains. We were able to show that all three C-termini are used in the brain. 9c isoforms are highly enriched in brain and neural cells, and we also detected significant amounts of 9a-containing isoforms in brain. gamma gene activity is relatively constant in the brain, but the choice of C-terminus is developmentally regulated. A more detailed study of the brain revealed regional expression differences. The hippocampus, cerebellum, and cortex were analyzed in depth and revealed that different isoforms could be sorted into different neuronal compartments, which change with development for 9d. Furthermore, a comparison with a homologous exon to 9c from the alpha-tropomyosin gene showed that expression from these exons is related to the maturational state of the neuron, even though both are sorted differently intracellularly. These data suggest that the large numbers of tropomyosin isoforms are likely to have specific roles in microfilament dynamics and neural cell function.
Assuntos
Encéfalo/citologia , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/fisiologia , Tropomiosina/genética , Animais , Western Blotting , Encéfalo/embriologia , Embrião de Mamíferos , Éxons , Imuno-Histoquímica , Camundongos , Neurônios/citologia , Isoformas de Proteínas/genéticaRESUMO
To determine the significance of actin isoforms in chemomechanical coupling, we compared tension and ATPase rate in heart myofilaments from nontransgenic (NTG) and transgenic (TG) mice in which enteric gamma-actin replaced >95% of the cardiac alpha-actin. Enteric gamma-actin was expressed against three backgrounds: mice expressing cardiac alpha-actin, heterozygous null cardiac alpha-actin mice, and homozygous null cardiac alpha-actin mice. There were no differences in maximum Ca(2+) activated tension or maximum rate of tension redevelopment after a quick release and rapid restretch protocol between TG and NTG skinned fiber bundles. However, compared with NTG controls, Ca(2+) sensitivity of tension was significantly decreased and economy of tension development was significantly increased in myofilaments from all TG hearts. Shifts in myosin isoform population could not fully account for this increase in the economy of force production of TG myofilaments. Our results indicate that an exchange of cardiac alpha-actin with an actin isoform differing in only five amino acids has a significant impact on both Ca(2+) regulation of cardiac myofilaments and the cross-bridge cycling rate.