RESUMO
BACKGROUND: During mammalian preimplantation development, as the fertilized egg develops and differentiates, three cell lineages become specified: trophectoderm (TE), epiblast, and primitive endoderm (PrE). Through two steps of cell fate decisions, 16-cell blastomeres develop into TE and an inner cell mass (ICM), and thereafter, the latter differentiates into pluripotent epiblast and PrE. Although bromodomain and extra-terminal domain (BET) proteins, such as BRD4, are necessary for the transcriptional activation of genes involved in the maintenance of mouse embryonic stem cells by occupying their enhancers, their roles in the development of mouse preimplantation are unknown. RESULTS: To evaluate the effect of BET protein deficiency on cell lineage formation, we cultured preimplantation embryos in the presence of JQ1, which blocks the binding of BET bromodomains to acetylated-histones. We found BET inhibition blocked the transcriptional activation of genes, such as Nanog, Otx2, and Sox2, important for the formation of the epiblast lineage in blastocysts. Expression studies with lineage-specific markers in morulae and blastocysts revealed BET proteins were essential for the specification and maintenance of the epiblast lineage but were dispensable for the formation of primarily extraembryonic TE and PrE lineages. Additional Ingenuity Pathway Analysis and expression studies with a transcriptionally active form of signal transducer and activator of the transcription 3 (STAT3) suggested BET-dependent activation was partly associated with the STAT3-dependent pathway to maintain the epiblast lineage. To identify BET proteins involved in the formation of the epiblast lineage, we analyzed mutant embryos deficient in Brd4, Brd2, and double mutants. Abolishment of NANOG-positive epiblast cells was only evident in Brd4/Brd2 double-deficient morulae. Thus, the phenotype of JQ1-treated embryos is reproduced not by a Brd4- or Brd2-single deficiency, but only Brd4/Brd2-double deficiency, demonstrating the redundant roles of BRD2 and BRD4 in the specification of the epiblast lineage. CONCLUSIONS: BET proteins are essential to the specification and maintenance of the epiblast lineage by activating lineage-specific core transcription factors during mouse preimplantation development. Among BET proteins, BRD4 plays a central role and BRD2 a complementary role in the specification and maintenance of epiblast lineages. Additionally, BET-dependent maintenance of the epiblast lineage may be partly associated with the STAT3-dependent pathway.
Assuntos
Blastocisto , Regulação da Expressão Gênica no Desenvolvimento , Animais , Linhagem da Célula , Camadas Germinativas/metabolismo , Mamíferos/genética , Camundongos , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Acquisition of cis-regulatory elements is a major driving force of evolution, and there are several examples of developmental enhancers derived from transposable elements (TEs). However, it remains unclear whether one enhancer element could have been produced via cooperation among multiple, yet distinct, TEs during evolution. Here we show that an evolutionarily conserved genomic region named AS3_9 comprises three TEs (AmnSINE1, X6b_DNA and MER117), inserted side-by-side, and functions as a distal enhancer for wnt5a expression during morphogenesis of the mammalian secondary palate. Functional analysis of each TE revealed step-by-step retroposition/transposition and co-option together with acquisition of a binding site for Msx1 for its full enhancer function during mammalian evolution. The present study provides a new perspective suggesting that a huge variety of TEs, in combination, could have accelerated the diversity of cis-regulatory elements involved in morphological evolution.
Assuntos
Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos/genética , Fator de Transcrição MSX1/genética , Sequências Reguladoras de Ácido Nucleico , Proteína Wnt-5a/biossíntese , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Evolução Molecular , Regulação da Expressão Gênica , Humanos , Fator de Transcrição MSX1/metabolismo , Mamíferos , Camundongos , Camundongos Knockout , Palato/crescimento & desenvolvimento , Transgenes , Proteína Wnt-5a/genéticaRESUMO
To understand genetic programs controlling mammalian central nervous system (CNS) development, we have identified one transgene-inserted mutation, which showed embryonic lethality during neurulation. Determination of the transgene integration site and rescue experiments revealed that the Brd2 gene, whose products specifically bind acetylated histone H4 and can mediate transcription, was the cause of this mutation. Expression studies with specific markers demonstrated that cell cycle progression was accelerated and neuronal differentiation as well as cell cycle exit were impaired in Brd2-deficient neruoepithelial cells. To investigate whether Brd2 regulates neuronal differentiation through a E2F1 transcriptional factor, which directly binds Brd2 and controls genes expression for cell cycle progression and exit, we analyzed Brd2;E2F1 double mutant phenotypes and, consequently found that abnormalities in neuronal differentiation and cell cycle progression due to Brd2-deficiency were restored by removing the E2F1 gene. These findings suggest that Brd2 is required for cell cycle exit and neuronal differentiation of neuroepithelial cells through the E2F1 pathway during mouse CNS development.
Assuntos
Ciclo Celular/fisiologia , Sistema Nervoso Central/crescimento & desenvolvimento , Fator de Transcrição E2F1/metabolismo , Células Neuroepiteliais/citologia , Neurogênese/fisiologia , Neurônios/citologia , Proteínas Serina-Treonina Quinases/fisiologia , Alelos , Animais , Ciclo Celular/genética , Diferenciação Celular , Sistema Nervoso Central/metabolismo , Proteínas Cromossômicas não Histona , Fator de Transcrição E2F1/genética , Camundongos , Camundongos Transgênicos , Neurogênese/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de TranscriçãoRESUMO
Cell surface-tethered heparan sulfate glycosaminoglycan chains primarily function in a cell autonomous manner, while extracellular matrix-associated heparan sulfate glycosaminoglycan chains function in a non-cell autonomous manner. In addition, the cleaved forms of cell surface-tethered heparan sulfate chains enzymatically released by proteases and heparanases, called shedding, can contribute to non-cell autonomous mechanisms. The movement of heparan sulfate chains to surrounding cells mediated by transcytosis or filopodia also involves another non-cell autonomous mechanism. To determine cell autonomous or non-cell autonomous roles of heparan sulfate glycosaminoglycan chains during early embryogenesis, direct conclusions can be drawn by analyzing chimeric embryos which are composed of wild-type and heparan sulfate glycosaminoglycan chain-deficient cells. Here, we describe methods of production of these chimeric embryos and analysis of their cellular phenotypes with immunohistochemistry at a single-cell level.
Assuntos
Glicosaminoglicanos/química , Animais , Membrana Celular , Embrião de Mamíferos , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato , CamundongosRESUMO
Embryonic development and growth in placental mammals proceeds in utero with the support of exchanges of gases, nutrients and waste products between maternal tissues and offspring. Murine embryos are surrounded by several extraembryonic membranes, parietal and visceral yolk sacs, and amnion in the uterus. Notably, the parietal yolk sac is the most outer membrane, consists of three layers, trophoblasts and parietal endoderm (PaE) cells, and is separated by a thick basal lamina termed Reichert's membrane (RM). RM is composed of extracellular matrix (ECM) initially formed as the basement membrane of the trophectoderm of pre-implanted embryos and followed by the heavy deposition of ECM mainly produced in PaE cells of post-implanted embryos. In addition to the physiological roles of RM, such as gas and nutrient exchange, it also plays a crucial role in cushioning and dispersing intrauterine pressures exerted on embryos for normal egg-cylinder morphogenesis. Mechanistically, such intrauterine pressures generated by uterine smooth muscle contractions appear to be involved in the elongation of the egg-cylinder shape, along with primary axis formation, as an important biomechanical element in utero. This review focuses on our current views of the roles of RM in properly buffering intrauterine mechanical forces for mouse egg-cylinder morphogenesis. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.
Assuntos
Placenta , Saco Vitelino , Animais , Membrana Basal , Endoderma , Feminino , Gases , Mamíferos , Camundongos , Gravidez , ResíduosRESUMO
Previously, we have shown that the translocation of Grainyhead-like 3 (GRHL3) transcription factor from the nucleus to the cytoplasm triggers the switch from canonical Wnt signaling for epidermal differentiation to non-canonical Wnt signaling for epithelial morphogenesis. However, the molecular mechanism that underlies the cytoplasmic localization of GRHL3 protein and that activates non-canonical Wnt signaling is not known. Here, we show that ubiquitin-specific protease 39 (USP39), a deubiquitinating enzyme, is involved in the subcellular localization of GRHL3 as a potential GRHL3-interacting protein and is necessary for epithelial morphogenesis to up-regulate expression of planar cell polarity (PCP) components. Notably, mouse Usp39-deficient embryos display early embryonic lethality due to a failure in primitive streak formation and apico-basal polarity in epiblast cells, resembling those of mutant embryos of the Prickle1 gene, a crucial PCP component. Current findings provide unique insights into how differentiation and morphogenesis are coordinated to construct three-dimensional complex structures via USP39.
Assuntos
Polaridade Celular , Fatores de Transcrição , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Diferenciação Celular , Polaridade Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas com Domínio LIM , Mamíferos , Camundongos , Morfogênese , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Retroposons, such as short interspersed elements (SINEs) and long interspersed elements (LINEs), are the major constituents of higher vertebrate genomes. Although there are many examples of retroposons' acquiring function, none has been implicated in the morphological innovations specific to a certain taxonomic group. We previously characterized a SINE family, AmnSINE1, members of which constitute a part of conserved noncoding elements (CNEs) in mammalian genomes. We proposed that this family acquired genomic functionality or was exapted after retropositioning in a mammalian ancestor. Here we identified 53 new AmnSINE1 loci and refined 124 total loci, two of which were further analyzed. Using a mouse enhancer assay, we demonstrate that one SINE locus, AS071, 178 kbp from the gene FGF8 (fibroblast growth factor 8), is an enhancer that recapitulates FGF8 expression in two regions of the developing forebrain, namely the diencephalon and the hypothalamus. Our gain-of-function analysis revealed that FGF8 expression in the diencephalon controls patterning of thalamic nuclei, which act as a relay center of the neocortex, suggesting a role for FGF8 in mammalian-specific forebrain patterning. Furthermore, we demonstrated that the locus, AS021, 392 kbp from the gene SATB2, controls gene expression in the lateral telencephalon, which is thought to be a signaling center during development. These results suggest important roles for SINEs in the development of the mammalian neuronal network, a part of which was initiated with the exaptation of AmnSINE1 in a common mammalian ancestor.
Assuntos
Encéfalo/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Elementos Nucleotídeos Curtos e Dispersos , Animais , Encéfalo/embriologia , Embrião de Galinha , Cromossomos Humanos/genética , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica , Humanos , Mamíferos/embriologia , Camundongos , Camundongos Transgênicos , FilogeniaRESUMO
The mouse embryonic axis is initially formed with a proximal-distal orientation followed by subsequent conversion to a prospective anterior-posterior (A-P) polarity with directional migration of visceral endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this developmental process. However, the genetic regulatory mechanism governing axis conversion is poorly understood. Here, defective axis conversion due to Otx2 deficiency can be rescued by expression of Dkk1, a Wnt antagonist, or following removal of one copy of the beta-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of beta-catenin localization is impaired in the Otx2-deficient and Wnt-misexpressing visceral endoderm. Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive guidance cues, respectively, in the migration of visceral endoderm cells. We propose that Wnt/beta-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo.
Assuntos
Padronização Corporal/fisiologia , Endoderma/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Fatores de Transcrição Otx/genética , Transdução de Sinais/fisiologia , Proteínas Wnt/fisiologia , Animais , Padronização Corporal/genética , Movimento Celular/fisiologia , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Endoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Vísceras/citologia , Vísceras/embriologia , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/fisiologiaRESUMO
Mammalian embryogenesis proceeds in utero with the support of nutrients and gases from maternal tissues. However, the contribution of the mechanical environment provided by the uterus to embryogenesis remains unaddressed. Notably, how intrauterine pressures are produced, accurately adjusted, and exerted on embryos are completely unknown. Here, we find that Reichert's membrane, a specialized basement membrane that wraps around the implanted mouse embryo, plays a crucial role as a shock absorber to protect embryos from intrauterine pressures. Notably, intrauterine pressures are produced by uterine smooth muscle contractions, showing the highest and most frequent periodic peaks just after implantation. Mechanistically, such pressures are adjusted within the sealed space between the embryo and uterus created by Reichert's membrane and are involved in egg-cylinder morphogenesis as an important biomechanical environment in utero. Thus, we propose the buffer space sealed by Reichert's membrane cushions and disperses intrauterine pressures exerted on embryos for egg-cylinder morphogenesis.
Assuntos
Membrana Basal/metabolismo , Animais , Feminino , Camundongos , Morfogênese , GravidezRESUMO
In many tissues, homeostasis is maintained by physical contact between stem cells and an anatomically defined niche. However, how stem cell homeostasis is achieved in environments where cells are motile and dispersed among their progeny remains unknown. Using murine spermatogenesis as a model, we find that spermatogenic stem cell density is tightly regulated by the supply of fibroblast growth factors (FGFs) from lymphatic endothelial cells. We propose that stem cell homeostasis is achieved through competition for a limited supply of FGFs. We show that the quantitative dependence of stem cell density on FGF dosage, the biased localization of stem cells toward FGF sources, and stem cell dynamics during regeneration following injury can all be predicted and explained within the framework of a minimal theoretical model based on "mitogen competition." We propose that this model provides a generic and robust mechanism to support stem cell homeostasis in open, or facultative, niche environments.
Assuntos
Fator 5 de Crescimento de Fibroblastos/fisiologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Homeostase , Mitógenos/farmacologia , Espermatogênese , Espermatozoides/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Autorrenovação Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espermatozoides/fisiologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologiaRESUMO
Epithelial cell shape change is a pivotal driving force for morphogenesis of complex three-dimensional architecture. However, molecular mechanisms triggering shape changes of epithelial cells in the course of growth and differentiation have not been entirely elucidated. Grhl3 plays a crucial role as a downstream transcription factor of Wnt/ß-catenin in epidermal differentiation. Here, we show Grhl3 induced large, mature epidermal cells, enriched with actomyosin networks, from embryoid bodies in vitro. Such epidermal cells were apparently formed by the simultaneous activation of canonical and non-canonical Wnt signaling pathways. A nuclear transcription factor, GRHL3 is localized in the cytoplasm and cell membrane during epidermal differentiation. Subsequently, such extranuclear GRHL3 is essential for the membrane-associated expression of VANGL2 and CELSR1. Cytoplasmic GRHL3, thereby, allows epidermal cells to acquire mechanical properties for changes in epithelial cell shape. Thus, we propose that cytoplasmic localization of GRHL3 upon epidermal differentiation directly triggers epithelial morphogenesis.
Assuntos
Diferenciação Celular , Forma Celular , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Epiderme/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Morfogênese , Fatores de Transcrição/metabolismo , Alelos , Animais , Biomarcadores/metabolismo , Polaridade Celular , Citoesqueleto/metabolismo , Corpos Embrioides/metabolismo , Células Epidérmicas/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Mutação/genética , Células NIH 3T3 , Neurulação , Via de Sinalização WntRESUMO
The original version of this Article contained an error in the labelling of Fig. 4. In panel i, the sixth column was incorrectly labelled as NSC23766 negative, and should have been NSC23766 positive. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMO
During primary neurulation, the separation of a single-layered ectodermal sheet into the surface ectoderm (SE) and neural tube specifies SE and neural ectoderm (NE) cell fates. The mechanisms underlying fate specification in conjunction with neural tube closure are poorly understood. Here, by comparing expression profiles between SE and NE lineages, we observed that uncommitted progenitor cells, expressing stem cell markers, are present in the neural plate border/neural fold prior to neural tube closure. Our results also demonstrated that canonical Wnt and its antagonists, DKK1/KREMEN1, progressively specify these progenitors into SE or NE fates in accord with the progress of neural tube closure. Additionally, SE specification of the neural plate border via canonical Wnt signaling is directed by the grainyhead-like 3 (Grhl3) transcription factor. Thus, we propose that the fate specification of uncommitted progenitors in the neural plate border by canonical Wnt signaling and its downstream effector Grhl3 is crucial for neural tube closure. This study implicates that failure in critical genetic factors controlling fate specification of progenitor cells in the neural plate border/neural fold coordinated with neural tube closure may be potential causes of human neural tube defects.
Assuntos
Proteínas de Ligação a DNA/genética , Crista Neural/embriologia , Placa Neural/embriologia , Fatores de Transcrição/genética , Via de Sinalização Wnt/fisiologia , Animais , Padronização Corporal/fisiologia , Diferenciação Celular , Ectoderma/embriologia , Técnicas de Cultura Embrionária , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Células-Tronco Neurais/citologia , Neurulação/fisiologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição SOX9/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMO
During mouse embryogenesis, diffusible growth factors, i.e. fibroblast growth factors, Wnt, bone morphogenetic protein and Hedgehog family members, emanating from localized areas can travel through the extracellular space and reach their target cells to specify the cell fate and form tissue architectures in coordination. However, the mechanisms by which these growth factors travel great distances to their target cells and control the signalling activity as morphogens remain an enigma. Recent studies in mice and other model animals have revealed that heparan sulfate proteoglycans (HSPGs) located on the cell surface (e.g. syndecans and glypicans) and in the extracellular matrix (ECM; e.g. perlecan and agrin) play crucial roles in the extracellular distribution of growth factors. Principally, the function of HSPGs depends primarily on the fine features and localization of their heparan sulfate glycosaminoglycan chains. Cell-surface-tethered HSPGs retain growth factors as co-receptors and/or endocytosis mediators, and enzymatic release of HSPGs from the cell membrane allows HSPGs to transport or move multiple growth factors. By contrast, ECM-associated HSPGs function as a reservoir or barrier in a context-dependent manner. This review is focused on our current understanding of the extracellular distribution of multiple growth factors controlled by HSPGs in mammalian development.
Assuntos
Desenvolvimento Embrionário/fisiologia , Espaço Extracelular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transdução de Sinais/fisiologia , Animais , Transporte Biológico/fisiologia , Proteoglicanas de Heparan Sulfato/biossíntese , Proteoglicanas de Heparan Sulfato/química , Camundongos , Modelos BiológicosRESUMO
Fibroblast Growth Factor (FGF) signaling plays crucial roles in multiple cellular processes including cell proliferation, differentiation, survival, and migration during mammalian embryogenesis. In the extracellular matrix, as well as at the cell surface, the movement of FGF ligands to target cells and the subsequent complex formations with their receptors are positively and negatively controlled extracellularly by heparan sulfate proteoglycans (HSPGs) such as syndecans, glypicans, and perlecan. Additionally, spreading of HSPGs by cleavage with sheddases such as proteinases and heparanases, and the overall length and sulfation level of specific heparan sulfate structures further generate a great diversity of FGF signaling outcomes. This review presents our current understanding of the regulatory mechanisms of FGF signaling in extracellular spaces through HSPGs in mammalian development.
Assuntos
Diferenciação Celular/genética , Fatores de Crescimento de Fibroblastos/genética , Mamíferos/crescimento & desenvolvimento , Proteoglicanas/metabolismo , Animais , Proliferação de Células , Matriz Extracelular/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , Mamíferos/genética , Proteoglicanas/genética , Transdução de Sinais/genéticaRESUMO
Mouse anterior-posterior axis polarization is preceded by formation of the distal visceral endoderm (DVE) by unknown mechanisms. Here, we show by in vitro culturing of embryos immediately after implantation in microfabricated cavities that the external mechanical cues exerted on the embryo are crucial for DVE formation, as well as the elongated egg cylinder shape, without affecting embryo-intrinsic transcriptional programs except those involving DVE-specific genes. This implies that these developmental events immediately after implantation are not simply embryo-autonomous processes but require extrinsic factors from maternal tissues. Moreover, the mechanical forces induce a breach of the basement membrane barrier at the distal portion locally, and thereby the transmigrated epiblast cells emerge as the DVE cells. Thus, we propose that external mechanical forces exerted by the interaction between embryo and maternal uterine tissues directly control the location of DVE formation at the distal tip and consequently establish the mammalian primary body axis.
Assuntos
Membrana Basal/metabolismo , Padronização Corporal/genética , Implantação do Embrião , Embrião de Mamíferos/metabolismo , Animais , Movimento Celular , Técnicas de Cultura Embrionária , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Transdução de Sinais/genética , Transcrição GênicaRESUMO
Heparan sulfate (HS) proteoglycans modulate the activity of multiple growth factors on the cell surface and extracellular matrix. However, it remains unclear how the HS chains control the movement and reception of growth factors into targeted receiving cells during mammalian morphogenetic processes. Here, we found that HS-deficient Ext2 null mutant mouse embryos fail to respond to fibroblast growth factor (FGF) signaling. Marker expression analyses revealed that cell surface-tethered HS chains are crucial for local retention of FGF4 and FGF8 ligands in the extraembryonic ectoderm. Fine chimeric studies with single-cell resolution and expression studies with specific inhibitors for HS movement demonstrated that proteolytic cleavage of HS chains can spread FGF signaling to adjacent cells within a short distance. Together, the results show that spatiotemporal expression of cell surface-tethered HS chains regulate the local reception of FGF-signaling activity during mammalian embryogenesis.
Assuntos
Embrião de Mamíferos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Heparitina Sulfato/metabolismo , Transdução de Sinais/fisiologia , Animais , Dissacarídeos/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Fator 4 de Crescimento de Fibroblastos/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Knockout , Modelos Biológicos , Mutação/genética , N-Acetilglucosaminiltransferases/genética , Técnicas de Cultura de Órgãos , Ligação Proteica , Transdução de Sinais/genéticaRESUMO
Anterior visceral endoderm (AVE) plays essential roles with respect to anterior-posterior axis development in the early mouse embryo. To assess the genetic cascade involved in AVE formation, the cis-regulatory elements directing expression of vertebrate Otx2 genes in the AVE were analyzed via generation of transgenic mice. Otx2 expression in AVE is regulated directly by the forkhead transcription factor, Foxa2. Moreover, Foxa2 is essential for expression of the Wnt antagonists, Dkk1 and Cerl, in visceral endoderm during the pre- to early streak stages; however, Foxa2 appears to be dispensable for subsequent Dkk1 expression associated with forebrain induction. Thus, we propose that Foxa2 is crucial in early anterior-posterior axis polarization in terms of regulation of expression of AVE-specific genes. These findings provide profound insights into conserved roles of Foxa2 transcription factors in anterior specification throughout the evolution of the chordate body plan.
Assuntos
Padronização Corporal , Endoderma/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Fator 3-beta Nuclear de Hepatócito/genética , Vísceras/embriologia , Animais , Endoderma/citologia , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Vísceras/citologia , beta-Galactosidase/metabolismoRESUMO
Otx2 expression in the forebrain and midbrain was found to be regulated by two distinct enhancers (FM and FM2) located at 75 kb 5' upstream and 115 kb 3' downstream. The activities of these two enhancers were absent in anterior neuroectoderm earlier than E8.0; however, at E9.5 their regions of activity spanned the entire mesencephalon and diencephalon with their caudal limits at the boundary with the metencephalon or isthmus. In telencephalon, activities were found only in the dorsomedial aspect. Potential binding sites of OTX and TCF were essential to FM activity, and TCF sites were also essential to FM2 activity. The FM2 enhancer appears to be unique to rodent; however, the FM enhancer region is deeply conserved in gnathostomes. Studies of mutants lacking FM or FM2 enhancer demonstrated that these enhancers indeed regulate Otx2 expression in forebrain and midbrain. Development of mesencephalic and diencephalic regions was differentially regulated in a dose-dependent manner by the cooperation between Otx1 and Otx2 under FM and FM2 enhancers: the more caudal the structure the higher the OTX dose requirement. At E10.5 Otx1-/-Otx2DeltaFM/DeltaFM mutants, in which Otx2 expression under the FM2 enhancer remained, exhibited almost complete loss of the entire diencephalon and mesencephalon; the telencephalon did, however, develop.