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1.
Dev Biol ; 493: 17-28, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36279927

RESUMO

Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 â€‹kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 â€‹kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.


Assuntos
Pronefro , Tretinoína , Animais , Xenopus laevis/genética , Xenopus laevis/metabolismo , Tretinoína/farmacologia , Tretinoína/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Pronefro/metabolismo , Rim/metabolismo , Família Aldeído Desidrogenase 1 , Retinal Desidrogenase/metabolismo
2.
Dis Model Mech ; 14(5)2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33737325

RESUMO

Heterozygous mutations in HNF1B cause the complex syndrome renal cysts and diabetes (RCAD), characterized by developmental abnormalities of the kidneys, genital tracts and pancreas, and a variety of renal, pancreas and liver dysfunctions. The pathogenesis underlying this syndrome remains unclear as mice with heterozygous null mutations have no phenotype, while constitutive/conditional Hnf1b ablation leads to more severe phenotypes. We generated a novel mouse model carrying an identified human mutation at the intron-2 splice donor site. Unlike heterozygous mice previously characterized, mice heterozygous for the splicing mutation exhibited decreased HNF1B protein levels and bilateral renal cysts from embryonic day 15, originated from glomeruli, early proximal tubules (PTs) and intermediate nephron segments, concurrently with delayed PT differentiation, hydronephrosis and rare genital tract anomalies. Consistently, mRNA sequencing showed that most downregulated genes in embryonic kidneys were primarily expressed in early PTs and the loop of Henle and involved in ion/drug transport, organic acid and lipid metabolic processes, while the expression of previously identified targets upon Hnf1b ablation, including cystic disease genes, was weakly or not affected. Postnatal analyses revealed renal abnormalities, ranging from glomerular cysts to hydronephrosis and, rarely, multicystic dysplasia. Urinary proteomics uncovered a particular profile predictive of progressive decline in kidney function and fibrosis, and displayed common features with a recently reported urine proteome in an RCAD pediatric cohort. Altogether, our results show that reduced HNF1B levels lead to developmental disease phenotypes associated with the deregulation of a subset of HNF1B targets. They further suggest that this model represents a unique clinical/pathological viable model of the RCAD disease.


Assuntos
Doenças do Sistema Nervoso Central/genética , Esmalte Dentário/anormalidades , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/genética , Genes Controladores do Desenvolvimento , Haploinsuficiência/genética , Fator 1-beta Nuclear de Hepatócito/genética , Doenças Renais Císticas/genética , Animais , Animais Recém-Nascidos , Polaridade Celular , Doenças do Sistema Nervoso Central/patologia , Cílios/patologia , Esmalte Dentário/patologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Embrião de Mamíferos/patologia , Dosagem de Genes , Perfilação da Expressão Gênica , Heterozigoto , Humanos , Hidronefrose/complicações , Doenças Renais Císticas/patologia , Glomérulos Renais/patologia , Túbulos Renais/patologia , Camundongos Endogâmicos C57BL , Mutação/genética , Néfrons/patologia , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença
3.
Sci Rep ; 9(1): 2225, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30778115

RESUMO

Renal Cysts and Diabetes Syndrome (RCAD) is an autosomal dominant disorder caused by mutations in the HNF1B gene encoding for the transcriptional factor hepatocyte nuclear factor-1B. RCAD is characterized as a multi-organ disease, with a broad spectrum of symptoms including kidney abnormalities (renal cysts, renal hypodysplasia, single kidney, horseshoe kidneys, hydronephrosis), early-onset diabetes mellitus, abnormal liver function, pancreatic hypoplasia and genital tract malformations. In the present study, using capillary electrophoresis coupled to mass spectrometry (CE-MS), we investigated the urinary proteome of a pediatric cohort of RCAD patients and different controls to identify peptide biomarkers and obtain further insights into the pathophysiology of this disorder. As a result, 146 peptides were found to be associated with RCAD in 22 pediatric patients when compared to 22 healthy age-matched controls. A classifier based on these peptides was generated and further tested on an independent cohort, clearly discriminating RCAD patients from different groups of controls. This study demonstrates that the urinary proteome of pediatric RCAD patients differs from autosomal dominant polycystic kidney disease (PKD1, PKD2), congenital nephrotic syndrome (NPHS1, NPHS2, NPHS4, NPHS9) as well as from chronic kidney disease conditions, suggesting differences between the pathophysiology behind these disorders.


Assuntos
Biomarcadores , Doenças do Sistema Nervoso Central/metabolismo , Esmalte Dentário/anormalidades , Diabetes Mellitus Tipo 2/metabolismo , Doenças Renais Císticas/metabolismo , Proteoma , Proteômica , Adolescente , Biomarcadores/urina , Doenças do Sistema Nervoso Central/diagnóstico , Doenças do Sistema Nervoso Central/urina , Criança , Pré-Escolar , Esmalte Dentário/metabolismo , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/urina , Diagnóstico Diferencial , Feminino , Humanos , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/urina , Masculino , Espectrometria de Massas , Peptídeos/urina , Fenótipo , Proteômica/métodos , Reprodutibilidade dos Testes
4.
J Cell Biochem ; 119(12): 10338-10350, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30171710

RESUMO

Transient receptor potential cation channel-2 (TRPP2) is a nonspecific Ca2+ -dependent cation channel with versatile functions including control of extracellular calcium entry at the plasma membrane, release of intracellular calcium ([Ca2+ ]i) from internal stores of endoplasmic reticulum, and calcium-dependent mechanosensation in the primary cilium. In early Xenopus embryos, TRPP2 is expressed in cilia of the gastrocoel roof plate (GRP) involved in the establishment of left-right asymmetry, and in nonciliated kidney field (KF) cells, where it plays a central role in early specification of nephron tubule cells dependent on [Ca2+ ]i signaling. Identification of proteins binding to TRPP2 in embryo cells can provide interesting clues about the mechanisms involved in its regulation during these various processes. Using mass spectrometry, we have therefore characterized proteins from late gastrula/early neurula stage embryos coimmunoprecipitating with TRPP2. Binding of three of these proteins, golgin A2, protein kinase-D1, and disheveled-2 has been confirmed by immunoblotting analysis of TRPP2-coprecipitated proteins. Expression analysis of the genes, respectively, encoding these proteins, golga2, prkd1, and dvl2 indicates that they are likely to play a role in these two regions. Golga2 and prkd1 are expressed at later stage in the developing pronephric tubule where golgin A2 and protein kinase-D1 might also interact with TRPP2. Colocalization experiments using exogenously expressed fluorescent versions of TRPP2 and dvl2 in GRP and KF reveal that these two proteins are generally not coexpressed, and only colocalized in discrete region of cells. This was observed in KF cells, but does not appear to occur in the apical ciliated region of GRP cells.


Assuntos
Proteínas Desgrenhadas/genética , Canais de Cátion TRPP/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animais , Cálcio/metabolismo , Cílios/genética , Retículo Endoplasmático/genética , Células Epiteliais/metabolismo , Gástrula/embriologia , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Rim/embriologia , Rim/metabolismo , Transdução de Sinais/genética , Xenopus laevis/embriologia
5.
Int J Dev Biol ; 62(4-5): 325-333, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29877572

RESUMO

The POU (Pit-Oct-Unc) genes encode a large transcription factor family comprising 6 classes (pou1f to pou6f ) involved in many developmental processes, such as cell commitment and differentiation. The pou3f class contains four members (pou3f1, pou3f2, pou3f3, pou3f4) characterized by expression in ectodermal tissue derivatives, such as nervous system and otic vesicle, during mammalian development. In order to obtain insights into the potential conservation of this class of transcription factors in vertebrates, we carried out a phylogenetic analysis and a comprehensive comparative study of pou3f expression in the frog Xenopus laevis. All vertebrates examined possessed members of the four pou3f subfamilies, excepting the zebrafish, which lacked a pou3f4 gene. Whole mount in situ hybridization and real-time quantitative polymerase chain reaction (RT-qPCR) analyses revealed that Xenopus pou3f genes were expressed in the forming neural tube and their expression was maintained in the brain, mostly in the dorsal part, at tailbud stages. The pou3f2, pou3f3, and pou3f4 genes were also expressed in the developing otic vesicle, and pou3f1 in some cells of the epidermis. Besides ectodermal derivatives, pou3f3 and pou3f4 were expressed in the developing kidney. Their expression started at the early tailbud stage in the pronephric anlage and partly overlapped. In the mature pronephric tubule, pou3f3 was restricted to the intermediate tubule, while pou3f4 was also expressed in the distal and connecting tubule. Together, our results highlight a significant conservation of pou3f gene expression in vertebrates and indicate that they may have distinct but also redundant functions during neural and renal development.


Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Rim/metabolismo , Fatores do Domínio POU/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Desenvolvimento Embrionário/fisiologia , Rim/embriologia , Organogênese/fisiologia , Fatores do Domínio POU/genética , Proteínas de Xenopus/genética , Xenopus laevis/embriologia
6.
Genes Dev ; 31(13): 1325-1338, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28794185

RESUMO

Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.


Assuntos
Linhagem da Célula/genética , Coração/embriologia , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Organogênese/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Linhagem Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento/genética , Cardiopatias Congênitas/genética , Humanos , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Mutação , Sementes , Xenopus laevis/embriologia
7.
J Cell Sci ; 128(5): 888-99, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25588842

RESUMO

In Xenopus laevis embryos, kidney field specification is dependent on retinoic acid (RA) and coincides with a dramatic increase of Ca(2+) transients, but the role of Ca(2+) signaling in the kidney field is unknown. Here, we identify TRPP2, a member of the transient receptor potential (TRP) superfamily of channel proteins encoded by the pkd2 gene, as a central component of Ca(2+) signaling in the kidney field. TRPP2 is strongly expressed at the plasma membrane where it might regulate extracellular Ca(2+) entry. Knockdown of pkd2 in the kidney field results in the downregulation of pax8, but not of other kidney field genes (lhx1, osr1 and osr2). We further show that inhibition of Ca(2+) signaling with an inducible Ca(2+) chelator also causes downregulation of pax8, and that pkd2 knockdown results in a severe inhibition of Ca(2+) transients in kidney field explants. Finally, we show that disruption of RA results both in an inhibition of intracellular Ca(2+) signaling and of TRPP2 incorporation into the plasma membrane of kidney field cells. We propose that TRPP2-dependent Ca(2+) signaling is a key component of pax8 regulation in the kidney field downstream of RA-mediated non-transcriptional control of TRPP2.


Assuntos
Sinalização do Cálcio/fisiologia , Embrião não Mamífero/embriologia , Rim/embriologia , Fatores de Transcrição Box Pareados/metabolismo , Canais de Cátion TRPP/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Embrião não Mamífero/citologia , Rim/citologia , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados/genética , Canais de Cátion TRPP/genética , Proteínas de Xenopus/genética , Xenopus laevis
8.
Dev Biol ; 397(2): 175-90, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25446030

RESUMO

The respective role of Pax2 and Pax8 in early kidney development in vertebrates is poorly understood. In this report, we have studied the roles of Pax8 and Pax2 in Xenopus pronephros development using a loss-of-function approach. Our results highlight a differential requirement of these two transcription factors for proper pronephros formation. Pax8 is necessary for the earliest steps of pronephric development and its depletion leads to a complete absence of pronephric tubule. Pax2 is required after the establishment of the tubule pronephric anlage, for the expression of several terminal differentiation markers of the pronephric tubule. Neither Pax2 nor Pax8 is essential to glomus development. We further show that Pax8 controls hnf1b, but not lhx1 and Osr2, expression in the kidney field as soon as the mid-neurula stage. Pax8 is also required for cell proliferation of pronephric precursors in the kidney field. It may exert its action through the wnt/beta-catenin pathway since activation of this pathway can rescue MoPax8 induced proliferation defect and Pax8 regulates expression of the wnt pathway components, dvl1 and sfrp3. Finally, we observed that loss of pronephros in Pax8 morphants correlates with an expanded vascular/blood gene expression domain indicating that Pax8 function is important to delimit the blood/endothelial genes expression domain in the anterior part of the dorso-lateral plate.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Fator de Transcrição PAX2/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Pronefro/embriologia , Via de Sinalização Wnt/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Animais , Bromodesoxiuridina , Primers do DNA/genética , Hibridização In Situ , Fator de Transcrição PAX8 , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Via de Sinalização Wnt/genética , Xenopus/genética
9.
Int J Dev Biol ; 58(5): 363-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25354457

RESUMO

Arid5b belongs to the ARID family of transcription factors characterised by a helix-turn-helix motif- based DNA-binding domain called ARID (A-T Rich Interaction Domain). In human, alternative splicing leads to long and short isoforms (isoform1 and 2, respectively) which differ in their N-terminal part. In this study, we report the cloning and expression pattern of Xenopus laevis arid5b. We have isolated a full length cDNA that shows homology with the human arid5b isoform1. Furthermore, 5'RACE experiments revealed the presence of a shorter isoform equivalent to the human isoform2. Temporal expression analysis by RT-qPCR indicated that X. laevis arid5b isoform1 and isoform2 are differentially expressed during development. Isoform1 is strongly expressed maternally, while isoform2 expression is essentially restricted to tailbud stages. Spatial expression analysis by whole mount in situ showed that arid5b is predominantly expressed in the developing pronephros. Arid5b mRNAs are detected in the antero-dorsal part of the pronephros anlage at the early tailbud stage and later on, in the proximal part of the pronephric tubule. RT-qPCR analyses with primers that allow to discriminate isoform1 from isoform2 showed that the latter is enriched in the pronephros anlage. In agreement with a specific pronephric signature of the isoform2, we also observed that isoform2 but not isoform1 is upregulated in animal caps induced to form pronephric tissue in response to activin A and retinoic acid. These results indicate that the two arid5b isoforms are differentially expressed and likely play different roles during early Xenopus development.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Pronefro/metabolismo , Isoformas de Proteínas/genética , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Embrião não Mamífero/metabolismo , Pronefro/embriologia , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis
10.
J Biol Chem ; 288(23): 16655-16670, 2013 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-23592779

RESUMO

Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity.


Assuntos
Fator 8 de Crescimento de Fibroblasto/metabolismo , Cabeça/embriologia , Sistema de Sinalização das MAP Quinases/fisiologia , Crista Neural/embriologia , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Sobrevivência Celular/fisiologia , Endocitose/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 8 de Crescimento de Fibroblasto/genética , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Inativação Gênica , Ligantes , Camundongos , Camundongos Transgênicos , Crista Neural/citologia , Ligação Proteica , Receptores de Superfície Celular/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética
11.
Development ; 140(4): 873-85, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23362348

RESUMO

The nephron is a highly specialised segmented structure that provides essential filtration and resorption renal functions. It arises by formation of a polarised renal vesicle that differentiates into a comma-shaped body and then a regionalised S-shaped body (SSB), with the main prospective segments mapped to discrete domains. The regulatory circuits involved in initial nephron patterning are poorly understood. We report here that HNF1B, a transcription factor known to be involved in ureteric bud branching and initiation of nephrogenesis, has an additional role in segment fate acquisition. Hnf1b conditional inactivation in murine nephron progenitors results in rudimentary nephrons comprising a glomerulus connected to the collecting system by a short tubule displaying distal fates. Renal vesicles develop and polarise normally but fail to progress to correctly patterned SSBs. Major defects are evident at late SSBs, with altered morphology, reduction of a proximo-medial subdomain and increased apoptosis. This is preceded by strong downregulation of the Notch pathway components Lfng, Dll1 and Jag1 and the Irx1/2 factors, which are potential regulators of proximal and Henle's loop segment fates. Moreover, HNF1B is recruited to the regulatory sequences of most of these genes. Overexpression of a HNF1B dominant-negative construct in Xenopus embryos causes downregulation specifically of proximal and intermediate pronephric segment markers. These results show that HNF1B is required for the acquisition of a proximo-intermediate segment fate in vertebrates, thus uncovering a previously unappreciated function of a novel SSB subcompartment in global nephron segmentation and further differentiation.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fator 1-beta Nuclear de Hepatócito/metabolismo , Néfrons/embriologia , Organogênese/fisiologia , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Animais , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas Histológicas , Proteínas de Homeodomínio/metabolismo , Imageamento Tridimensional , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Camundongos , Néfrons/metabolismo , Organogênese/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tomografia Óptica , Fatores de Transcrição/metabolismo
12.
Biol Cell ; 104(9): 516-32, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22548301

RESUMO

BACKGROUND INFORMATION: In Xenopus, the functional kidney of the tadpole, the pronephros, forms from the kidney field, which is specified at completion of gastrulation. Specification of the kidney field requires retinoic acid (RA) signalling during gastrulation, while fibroblast growth factor (FGF) signals inhibit should be inhibit this process. RESULTS: We have analysed the functional interactions taking place during gastrulation between RA and FGF signals in the lateral marginal zone (LMZ), that is in the environment of unspecified pronephric mesoderm precursors. Inhibition of FGF receptor (FGFR) signalling with SU5402 does not significantly affect expression of genes encoding RA metabolism enzymes and RA receptor-α in LMZ explants. Furthermore, SU5402 has no effect on the expression of hoxa1, a major RA target in the LMZ, showing that FGF is not antagonising RA in the LMZ. Disruption of RA signalling affects FGF ligand production to some extent, especially FGF8b, but the strongest effect is the down-regulation of the mitogen-activated protein kinase phosphatase-3 (MKP3)-encoding gene, mkp3. A strong up-regulation of mkp3 occurs in response to exogenous RA. This effect is reduced in a context of FGFR inhibition, suggesting that RA and FGF signals are co-operating upstream of mkp3. Mkp3 knockdown results in an inhibition of the kidney field markers pax8 and lhx1 and in a defective development of the pronephros. CONCLUSIONS: FGF is not negatively influencing pronephric specification by antagonising RA signalling. Functional interactions between RA and FGF rather take place at the level of the transcriptional regulation of mkp3, indicating that RA may antagonise FGF signalling at the level of the extracellular signal-regulated kinase (Erk) pathway. A fine tuning of Erk signalling by MKP3 is important for the proper establishment of the kidney field.


Assuntos
Rim/embriologia , Rim/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Tretinoína/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Fosfatase 6 de Especificidade Dupla , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Rim/metabolismo , Masculino , Fosfoproteínas Fosfatases/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/genética
14.
Dev Biol ; 320(2): 351-65, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18614163

RESUMO

Although FGFs are known to affect mesoderm patterning, their influence on intermediate mesoderm specification during gastrulation is ignored. Here, we show that pronephros precursors are exposed to FGF, but a strict control of FGF signals is necessary to allow pronephros development. We provide evidence that this control is mediated by the paired-like homeobox genes Mix.1 and Mix.2. Morpholino-based Mix.1/2 knockdown, or repression of Mix.1 target genes with an enRMix.1 construct, causes an expansion of FGF4 and FGF8 expression in the lateral marginal zone at gastrula stage, together with an inhibition of pronephros development at neurula and tailbud stages. Expression of the nephrogenic mesoderm markers Xlim-1 and XPax-8 can be rescued in Mix.1/2 morphants by intrablastocoelic injections of the FGFR inhibitor SU5402 at mid-gastrula stage, showing that inhibition of pronephros development results from an increase of FGF signalling. We further show that Mix.1 overexpression results in the down-regulation of FGF3, 4, 8 and XmyoD, in addition to Xbra. However, cells overexpressing Mix.1 can normally populate somites, indicating that Mix.1 does not affect their fate cell autonomously. These data support the idea that Mix.1/2 regulates levels and/or duration of FGF signals to which pronephros precursors are exposed during gastrulation.


Assuntos
Fatores de Crescimento de Fibroblastos/genética , Gastrulação , Proteínas de Homeodomínio/fisiologia , Rim/embriologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/embriologia , Animais , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Rim/crescimento & desenvolvimento , Organogênese
15.
Dev Biol ; 299(1): 35-51, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16979153

RESUMO

The mechanisms by which a subset of mesodermal cells are committed to a nephrogenic fate are largely unknown. In this study, we have investigated the role of retinoic acid (RA) signalling in this process using Xenopus laevis as a model system and Raldh2 knockout mice. Pronephros formation in Xenopus embryo is severely impaired when RA signalling is inhibited either through expression of a dominant-negative RA receptor, or by expressing the RA-catabolizing enzyme XCyp26 or through treatment with chemical inhibitors. Conversely, ectopic RA signalling expands the size of the pronephros. Using a transplantation assay that inhibits RA signalling specifically in pronephric precursors, we demonstrate that this signalling is required within this cell population. Timed antagonist treatments show that RA signalling is required during gastrulation for expression of Xlim-1 and XPax-8 in pronephric precursors. Moreover, experiments conducted with a protein synthesis inhibitor indicate that RA may directly regulate Xlim-1. Raldh2 knockout mouse embryos fail to initiate the expression of early kidney-specific genes, suggesting that implication of RA signalling in the early steps of kidney formation is evolutionary conserved in vertebrates.


Assuntos
Linhagem da Célula , Néfrons/citologia , Néfrons/embriologia , Transdução de Sinais , Tretinoína/metabolismo , Aldeído Oxirredutases/deficiência , Animais , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/fisiologia , Linhagem da Célula/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Gástrula/citologia , Gástrula/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes Reporter , Proteínas de Homeodomínio/genética , Humanos , Proteínas com Homeodomínio LIM , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Camundongos , Néfrons/efeitos dos fármacos , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/genética , Ácido Retinoico 4 Hidroxilase , Receptor alfa de Ácido Retinoico , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição , Tretinoína/farmacologia , Proteínas de Xenopus/genética , Xenopus laevis
16.
PLoS Genet ; 2(7): e102, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16839186

RESUMO

Understanding the role of gene duplications in establishing vertebrate innovations is one of the main challenges of Evo-Devo (evolution of development) studies. Data on evolutionary changes in gene expression (i.e., evolution of transcription factor-cis-regulatory elements relationships) tell only part of the story; protein function, best studied by biochemical and functional assays, can also change. In this study, we have investigated how gene duplication has affected both the expression and the ligand-binding specificity of retinoic acid receptors (RARs), which play a major role in chordate embryonic development. Mammals have three paralogous RAR genes--RAR alpha, beta, and gamma--which resulted from genome duplications at the origin of vertebrates. By using pharmacological ligands selective for specific paralogues, we have studied the ligand-binding capacities of RARs from diverse chordates species. We have found that RAR beta-like binding selectivity is a synapomorphy of all chordate RARs, including a reconstructed synthetic RAR representing the receptor present in the ancestor of chordates. Moreover, comparison of expression patterns of the cephalochordate amphioxus and the vertebrates suggests that, of all the RARs, RAR beta expression has remained most similar to that of the ancestral RAR. On the basis of these results together, we suggest that while RAR beta kept the ancestral RAR role, RAR alpha and RAR gamma diverged both in ligand-binding capacity and in expression patterns. We thus suggest that neofunctionalization occurred at both the expression and the functional levels to shape RAR roles during development in vertebrates.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Receptores do Ácido Retinoico/metabolismo , Sequência de Aminoácidos , Animais , Evolução Molecular , Lampreias , Ligantes , Dados de Sequência Molecular , Mutação , Filogenia , Retinoides/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Ativação Transcricional , Xenopus
17.
Blood ; 108(9): 2989-97, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16835375

RESUMO

STAT5 regulates definitive (adult stage) erythropoiesis through its ability to transduce signals from the erythropoietin receptor. A function for STAT-dependent signaling during primitive (embryonic) erythropoiesis has not been analyzed. We tested this in the Xenopus system, because STAT5 is expressed at the right time and place to regulate development of the embryonic primitive ventral blood island. Depletion of STAT5 activity results in delayed accumulation of the first globin-expressing cells, indicating that the gene does regulate primitive erythropoiesis. Our results suggest that in this context STAT5 functions as a repressor, since forced expression of an activator isoform blocks erythropoiesis, while embryos expressing a repressor isoform develop normally. The erythroid phenotype caused by the activator isoform of STAT5 resembles that caused by overexpression of fibroblast growth factor (FGF). We show that STAT5 isoforms can function epistatic to FGF and can be phosphorylated in response to hyperactivated FGF signaling in Xenopus embryos. Therefore, our data indicate that STAT5 functions in both primitive and definitive erythropoiesis, but by different mechanisms.


Assuntos
Eritropoese/fisiologia , Fator de Transcrição STAT5/genética , Proteínas de Xenopus/genética , Xenopus/embriologia , Xenopus/genética , Animais , Embrião não Mamífero , Desenvolvimento Embrionário/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/metabolismo
18.
J Cell Biol ; 169(2): 227-31, 2005 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-15837801

RESUMO

Vertebrate oocytes arrest in metaphase of the second meiotic division (MII), where they maintain a high cdc2/cyclin B activity and a stable, bipolar spindle because of cytostatic factor (CSF) activity. The Mos-MAPK pathway is essential for establishing CSF. Indeed, oocytes from the mos-/- strain do not arrest in MII and activate without fertilization, as do Xenopus laevis oocytes injected with morpholino oligonucleotides directed against Mos. In Xenopus oocytes, p90Rsk (ribosomal S6 kinase), a MAPK substrate, is the main mediator of CSF activity. We show here that this is not the case in mouse oocytes. The injection of constitutively active mutant forms of Rsk1 and Rsk2 does not induce a cell cycle arrest in two-cell mouse embryos. Moreover, these two mutant forms do not restore MII arrest after their injection into mos-/- oocytes. Eventually, oocytes from the triple Rsk (1, 2, 3) knockout present a normal CSF arrest. We demonstrate that p90Rsk is not involved in the MII arrest of mouse oocytes.


Assuntos
Blastômeros/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Meiose/fisiologia , Oócitos/fisiologia , Proteínas Proto-Oncogênicas c-mos/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Feminino , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Gravidez , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Xenopus laevis
19.
J Cell Sci ; 116(Pt 12): 2541-50, 2003 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-12734397

RESUMO

Wnt signaling has an important role in cell-fate determination, tissue patterning and tumorigenesis. Wnt proteins signal through seven-pass transmembrane receptors of the frizzled family to activate beta-catenin-dependent transcription of target genes. Using early Xenopus embryos, we show that frizzled receptors can dimerize and that dimerization is correlated with activation of the Wnt/beta-catenin pathway. Co-immunoprecipitation studies revealed that the receptor Xfz3 exists as a dimer when expressed in Xenopus embryos, and it has been shown to activate the Wnt/beta-catenin pathway as revealed by expression of the target gene siamois. Xfz3 dimerization requires intramolecular and/or intermolecular disulfide linkages, and the N-terminal extracellular region of the receptor, including the cysteine-rich domain (CRD), is sufficient for dimerization. The receptor Xfz7 behaves differently from Xfz3 when overexpressed in the embryo as Xfz7 is monomeric and is unable to directly activate the Wnt/beta-catenin pathway. However, activation of this pathway can be achieved by artificially forcing Xfz7 dimerization. These results provide the first direct evidence for the dimerization of frizzled receptors and suggest that dimerization contributes to transducing the Wnt/beta-catenin signal.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transativadores/metabolismo , Proteínas de Xenopus , Proteínas de Peixe-Zebra , Animais , Dimerização , Embrião não Mamífero/citologia , Feminino , Receptores Frizzled , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mercaptoetanol/farmacologia , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Wnt , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , beta Catenina
20.
Dev Biol ; 257(2): 302-14, 2003 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-12729560

RESUMO

Wnt-11/Xfz7 signaling plays a major role in the regulation of convergent extension movements affecting the dorsal marginal zone (DMZ) of gastrulating Xenopus embryos. In order to provide data concerning the molecular targets of Wnt-11/Xfz7 signals, we have analyzed the regulation of the Rho GTPase Cdc42 by Wnt-11. In animal cap ectoderm, Cdc42 activity increases as a response to Wnt-11 expression. This increase is inhibited by pertussis toxin, or sequestration of free Gbetagamma subunits by exogenous Galphai2 or Galphat. Activation of Cdc42 is also produced by the expression of bovine Gbeta1 and Ggamma2. This process is abolished by a PKC inhibitor, while phorbol esther treatment of ectodermal explants activates Cdc42 in a PKC-dependent way, implicating PKC downstream of Gbetagamma. In activin-treated animal caps and in the embryo, interference with Gbetagamma signaling rescues morphogenetic movements inhibited by Wnt-11 hyperactivation, thus phenocopying the dominant negative version of Cdc42 (N(17)Cdc42). Conversely, expression of Gbeta1gamma2 blocks animal cap elongation. This effect is reversed by N(17)Cdc42. Together, our results strongly argue for a role of Gbetagamma signaling in the regulation of Cdc42 activity downstream of Wnt-11/Xfz7 in mesodermal cells undergoing convergent extension. This idea is further supported by the observation that expression of Galphat in the DMZ causes severe gastrulation defects.


Assuntos
Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Gástrula/metabolismo , Glicoproteínas/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Xenopus laevis/embriologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Ativinas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Desgrenhadas , Ectoderma/metabolismo , Embrião não Mamífero , Inibidores Enzimáticos/farmacologia , Gástrula/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Toxina Pertussis/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Transdução de Sinais , Proteínas Wnt , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Proteína cdc42 de Ligação ao GTP/genética
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