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1.
Nat Commun ; 11(1): 2082, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350257

RESUMO

Developmental progression depends on temporally defined changes in gene expression mediated by transient exposure of lineage intermediates to signals in the progenitor niche. To determine whether cell-intrinsic epigenetic mechanisms contribute to signal-induced transcriptional responses, here we manipulate the signalling environment and activity of the histone demethylase LSD1 during differentiation of hESC-gut tube intermediates into pancreatic endocrine cells. We identify a transient requirement for LSD1 in endocrine cell differentiation spanning a short time-window early in pancreas development, a phenotype we reproduced in mice. Examination of enhancer and transcriptome landscapes revealed that LSD1 silences transiently active retinoic acid (RA)-induced enhancers and their target genes. Furthermore, prolonged RA exposure phenocopies LSD1 inhibition, suggesting that LSD1 regulates endocrine cell differentiation by limiting the duration of RA signalling. Our findings identify LSD1-mediated enhancer silencing as a cell-intrinsic epigenetic feedback mechanism by which the duration of the transcriptional response to a developmental signal is limited.


Assuntos
Células Endócrinas/citologia , Células Endócrinas/metabolismo , Elementos Facilitadores Genéticos/genética , Inativação Gênica , Histona Desmetilases/metabolismo , Ilhotas Pancreáticas/citologia , Transdução de Sinais , Tretinoína/metabolismo , Adulto , Animais , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Células Endócrinas/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Ilhotas Pancreáticas/embriologia , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia , Adulto Jovem
2.
PLoS Biol ; 18(5): e3000719, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32421711

RESUMO

Identification of target genes that mediate required functions downstream of transcription factors is hampered by the large number of genes whose expression changes when the factor is removed from a specific tissue and the numerous binding sites for the factor in the genome. Retinoic acid (RA) regulates transcription via RA receptors bound to RA response elements (RAREs) of which there are thousands in vertebrate genomes. Here, we combined chromatin immunoprecipitation sequencing (ChIP-seq) for epigenetic marks and RNA-seq on trunk tissue from wild-type and Aldh1a2-/- embryos lacking RA synthesis that exhibit body axis and forelimb defects. We identified a relatively small number of genes with altered expression when RA is missing that also have nearby RA-regulated deposition of histone H3 K27 acetylation (H3K27ac) (gene activation mark) or histone H3 K27 trimethylation (H3K27me3) (gene repression mark) associated with conserved RAREs, suggesting these genes function downstream of RA. RA-regulated epigenetic marks were identified near RA target genes already known to be required for body axis and limb formation, thus validating our approach; plus, many other candidate RA target genes were found. Nuclear receptor 2f1 (Nr2f1) and nuclear receptor 2f2 (Nr2f2) in addition to Meis homeobox 1 (Meis1) and Meis homeobox 2 (Meis2) gene family members were identified by our approach, and double knockouts of each family demonstrated previously unknown requirements for body axis and/or limb formation. A similar epigenetic approach can be used to determine the target genes for any transcriptional regulator for which a knockout is available.


Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Estudos de Associação Genética/métodos , Código das Histonas , Tretinoína/metabolismo , Animais , Sequência de Bases , Sequenciamento de Cromatina por Imunoprecipitação , Sequência Conservada , Epigênese Genética , Camundongos , Família Multigênica , Elementos de Resposta , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo
3.
Subcell Biochem ; 95: 57-85, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32297296

RESUMO

Retinoic acid (RA), a major natural active metabolite of vitamin A (VA) is well known to play critical roles in embryonic development. The effects of RA are mediated by nuclear receptors (RARs), which regulate the expression of gene batteries involved in cell growth and differentiation. Since the early 1990s several laboratories have focused on understanding how RA-regulated genes and RAR binding sites operate by studying the differentiation of embryonal carcinoma cells and embryonic stem cells. The development of hybridization-based microarray technology and high performance software analysis programs has allowed the characterization of thousands of RA-regulated genes. During the two last decades, publication of the genome sequence of various organisms has allowed advances in massive parallel sequencing and bioinformatics analysis of genome-wide data sets. These new generation sequencing (NGS) technologies have revolutionized the field by providing a global integrated picture of RA-regulated gene networks and the regulatory programs involved in cell fate decisions during embryonal carcinoma and embryonic stem cells differentiation. Now the challenge is to reconstruct the RA-regulated gene networks at the single cell level during the development of specialized embryonic tissues.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes , Genômica , Tretinoína/metabolismo , Animais , Diferenciação Celular/genética , Humanos , Receptores do Ácido Retinoico/metabolismo
4.
Subcell Biochem ; 95: 87-117, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32297297

RESUMO

This chapter brings together data on the role of retinoic acid (RA) in the embryonic development of fins in zebrafish , limbs in amphibians , chicks , and mice, and regeneration of the amphibian limb . The intention is to determine whether there is a common set of principles by which we can understand the mode of action of RA in both embryos and adults. What emerges from this synthesis is that there are indeed commonalities in the involvement of RA in processes that ventralize, posteriorize, and proximalize the developing and regenerating limb . Different axes of the limb have historically been studied independently; as for example, the embryonic development of the anteroposterior (AP) axis of the chick limb bud versus the regeneration of the limb bud proximodistal (PD) axis . But when we take a broader view, a unifying principle emerges that explains why RA administration to embryos and regenerating limbs results in the development of multiple limbs in both cases. As might be expected, different molecular pathways govern the development of different systems and model organisms, but despite these differences, the pathways involve similar RA signaling genes, such as tbx5, meis, shh, fgfs and hox genes. Studies of developing and regenerating systems have highlighted that RA acts by being synthesized in one embryonic location while acting in another one, exactly as embryonic morphogens do, although there is no evidence for the presence of an RA gradient within the limb . What also emerges is that there is a paucity of information on the involvement of RA in development of the dorsoventral (DV) axis . A molecular explanation as to how RA establishes and alters positional information in all three axes is the most important area of study for the future.


Assuntos
Extremidades/crescimento & desenvolvimento , Regeneração , Transdução de Sinais , Tretinoína/metabolismo , Animais
5.
Subcell Biochem ; 95: 119-149, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32297298

RESUMO

As the first organ to form and function in all vertebrates, the heart is crucial to development. Tightly-regulated levels of retinoic acid (RA) are critical for the establishment of the regulatory networks that drive normal cardiac development. Thus, the heart is an ideal organ to investigate RA signaling, with much work remaining to be done in this area. Herein, we highlight the role of RA signaling in vertebrate heart development and provide an overview of the field's inception, its current state, and in what directions it might progress so that it may yield fruitful insight for therapeutic applications within the domain of regenerative medicine.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Transdução de Sinais , Tretinoína/metabolismo , Animais , Humanos , Medicina Regenerativa , Vertebrados/embriologia , Vertebrados/genética
6.
Subcell Biochem ; 95: 151-174, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32297299

RESUMO

Retinoic acid (RA), the bioactive metabolite of vitamin A (VA), has long been recognized as a critical regulator of the development of the respiratory system. During embryogenesis, RA signaling is involved in the development of the trachea, airways, lung, and diaphragm. During postnatal life, RA continues to impact respiratory health. Disruption of RA activity during embryonic development produces dramatic phenotypes in animal models and human diseases, including tracheoesophageal fistula, tracheomalacia, congenital diaphragmatic hernia (CDH), and lung agenesis or hypoplasia. Several experimental methods have been used to target RA pathways during the formation of the embryonic lung. These have been performed in different animal models using gain- and loss-of-function strategies and dietary, pharmacologic, and genetic approaches that deplete retinoid stores or disrupt retinoid signaling. Experiments utilizing these methods have led to a deeper understanding of RA's role as an important signaling molecule that influences all stages of lung development. Current research is uncovering RA cross talk interactions with other embryonic signaling factors, such as fibroblast growth factors, WNT, and transforming growth factor-beta.


Assuntos
Sistema Respiratório/embriologia , Sistema Respiratório/metabolismo , Transdução de Sinais , Tretinoína/metabolismo , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pneumopatias/embriologia , Pneumopatias/metabolismo
7.
Subcell Biochem ; 95: 175-196, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32297300

RESUMO

Explaining how the extensive diversity in form of vertebrate teeth arose in evolution and the mechanisms by which teeth are made during embryogenesis are intertwined questions that can merit from a better understanding of the roles of retinoic acid (RA) in tooth development. Pioneering studies in rodents showed that dietary vitamin A (VA), and eventually RA (one of the major active metabolites of VA), are required for proper tooth formation and that dentin-forming odontoblast cells seem to be especially sensitive to changes in RA levels. Later, rodent studies further indicated that RA signaling interactions with other cell-signaling pathways are an important part of RA's actions in odontogenesis. Recent investigations employing zebrafish and other teleost fish continued this work in an evolutionary context, and specifically demonstrated that RA is required for the initiation of tooth development. RA is also sufficient in certain circumstances to induce de novo tooth formation. Both effects appear to involve cranial-neural crest cells, again suggesting that RA signaling has a particular influence on odontoblast development. These teleost studies have also highlighted both evolutionary conservation and change in how RA is employed during odontogenesis in different vertebrate lineages, and thus raises the possibility that developmental changes to RA signaling has led to some of the diversity of form seen across vertebrate dentitions. Future progress in this area will come at least in part from expanding the species examined to get a better picture of how often RA signaling has changed in evolution and how this relates to the evolution of dental form.


Assuntos
Evolução Biológica , Dentição , Odontogênese , Transdução de Sinais , Dente/embriologia , Tretinoína/metabolismo , Peixe-Zebra/embriologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Dente/metabolismo
8.
Subcell Biochem ; 95: 197-225, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32297301

RESUMO

Fetal Alcohol Spectrum Disorder (FASD) is a complex set of developmental malformations, neurobehavioral anomalies and mental disabilities induced by exposing human embryos to alcohol during fetal development. Several experimental models and a series of developmental and biochemical approaches have established a strong link between FASD and reduced retinoic acid (RA) signaling. RA signaling is involved in the regulation of numerous developmental decisions from patterning of the anterior-posterior axis, starting at gastrulation, to the differentiation of specific cell types within developing organs, to adult tissue homeostasis. Being such an important regulatory signal during embryonic development, mutations or environmental perturbations that affect the level, timing or location of the RA signal can induce multiple and severe developmental malformations. The evidence connecting human syndromes to reduced RA signaling is presented here and the resulting phenotypes are compared to FASD. Available data suggest that competition between ethanol clearance and RA biosynthesis is a major etiological component in FASD.


Assuntos
Desenvolvimento Embrionário , Transtornos do Espectro Alcoólico Fetal/metabolismo , Transdução de Sinais , Tretinoína/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Etanol/efeitos adversos , Transtornos do Espectro Alcoólico Fetal/genética , Humanos
9.
World Neurosurg ; 138: 461-467, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32200015

RESUMO

Caudal regression syndrome (CRS) represents a spectrum of clinical phenotypes with varying degrees of malformation of the lower body with involvement of structures deriving from all 3 layers of the trilaminar embryo. We review areas of active investigation in the diagnosis, etiology, epidemiology, and treatment of the disease with a focus on underlying genetics. CRS pathobiology is complex and multifactorial with a significant contribution from environmental factors as evidenced in twin studies. Contemporary genomic and genetic investigations in both human primary tissue and murine in vitro and in vivo models implicate various genes associated with caudal differentiation and neural cell migration in embryogenesis. A large number of identified targets center around the metabolic regulation of retinoic acid and its derivatives. Dysregulation of retinoic acid homeostasis has been associated with abnormal embryonic cell migration, differentiation, and organogenesis with resulting malformations and agenesis in both a laboratory and a clinical setting. There appears to be a significant overlap in potential genetic targets with CRS and other developmental syndromes with similar presentations, such as VACTERL (vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and limb abnormalities) association. CRS represents a spectrum of caudal developmental abnormalities with treatment options limited to mild and moderate expressions of disease. Continued research is necessary to further clarify mechanisms of disease pathobiology and complex polygenetic and environmental interaction. Despite this, progress has been made in identifying genetic targets and downstream effectors contributing to preclinical and clinical progression.


Assuntos
Anormalidades Múltiplas/genética , Genômica , Deformidades Congênitas dos Membros/genética , Malformações do Sistema Nervoso/genética , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/diagnóstico por imagem , Anormalidades Múltiplas/patologia , Animais , Humanos , Deformidades Congênitas dos Membros/diagnóstico , Deformidades Congênitas dos Membros/diagnóstico por imagem , Deformidades Congênitas dos Membros/patologia , Malformações do Sistema Nervoso/diagnóstico , Malformações do Sistema Nervoso/diagnóstico por imagem , Malformações do Sistema Nervoso/patologia , Tretinoína/metabolismo
10.
PLoS One ; 15(2): e0228436, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32027669

RESUMO

BACKGROUND: Cytochrome P450 1b1 (Cyp1b1) deletion and dietary retinol deficiency during pregnancy (GVAD) affect perinatal liver functions regulated by Srebp. Cyp1b1 is not expressed in perinatal liver but appears in the E9.5 embryo, close to sites of retinoic acid (RA) signaling. HYPOTHESIS: Parallel effects of Cyp1b1 and retinol on postnatal Srebp derive from effects in the developing liver or systemic signaling. APPROACH: Cluster postnatal increases in hepatic genes in relation to effects of GVAD or Cyp1b1 deletion. Sort expression changes in relation to genes regulated by Srebp1 and Srebp2.Test these treatments on embryos at E9.5, examining changes at the site of liver initiation. Use in situ hybridization to resolve effects on mRNA distributions of Aldh1a2 and Cyp26a1 (RA homeostasis); Hoxb1 and Pax6 (RA targets). Assess mice lacking Lrat and Rbp4 (DKO mice) that severely limits retinol supply to embryos. RESULTS: At birth, GVAD and Cyp1b1 deletion stimulate gene markers of hepatic stellate cell (HSC) activation but also suppress Hamp. These treatments then selectively prevent the postnatal onset of genes that synthesize cholesterol (Hmgcr, Sqle) and fatty acids (Fasn, Scd1), but also direct cholesterol transport (Ldlr, Pcsk9, Stard4) and retinoid synthesis (Aldh1a1, Rdh11). Extensive support by Cyp1b1 is implicated, but with distinct GVAD interventions for Srebp1 and Srebp2. At E9.5, Cyp1b1 is expressed in the septum transversum mesenchyme (STM) with ß-carotene oxygenase (Bco1) that generates retinaldehyde. STM provides progenitors for the HSC and supports liver expansion. GVAD and Cyp1b1-/- do not affect RA-dependent Hoxb1 and Pax6. In DKO embryos, RA-dependent Cyp26a1 is lost but Hoxb1 is sustained with Cyp1b1 at multiple sites. CONCLUSION: Cyp1b1-/- suppresses genes supported by Srebp. GVAD effects distinguish Srebp1 and Srebp2 mediation. Srebp regulation overlaps appreciably in cholesterol and retinoid homeostasis. Bco1/Cyp1b1 partnership in the STM may contribute to this later liver regulation.


Assuntos
Colesterol/biossíntese , Citocromo P-450 CYP1B1/fisiologia , Desenvolvimento Fetal , Fígado/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/fisiologia , Tretinoína/metabolismo , Animais , Animais Recém-Nascidos , Citocromo P-450 CYP1B1/genética , Embrião de Mamíferos , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Desenvolvimento Fetal/genética , Fígado/efeitos dos fármacos , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Proteínas Plasmáticas de Ligação ao Retinol/genética , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Tretinoína/farmacologia
11.
Endocrinology ; 161(2)2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31913463

RESUMO

Dietary vitamin A is metabolized into bioactive retinoic acid (RA) in vivo and regulates the development of many embryonic tissues. RA signaling is active in the oral ectoderm-derived tissues of the neuroendocrine system, but its role there has not yet been fully explored. We show here that RA signaling is active during pituitary organogenesis and dependent on the pituitary transcription factor Prop1. Prop1-mutant mice show reduced expression of the aldehyde dehydrogenase gene Aldh1a2, which metabolizes the vitamin A-intermediate retinaldehyde into RA. To elucidate the specific function of RA signaling during neuroendocrine development, we studied a conditional deletion of Aldh1a2 and a dominant-negative mouse model of inhibited RA signaling during pituitary organogenesis. These models partially phenocopy Prop1-mutant mice by exhibiting embryonic pituitary dysmorphology and reduced hormone expression, especially thyrotropin. These findings establish the role of RA in embryonic pituitary stem cell progression to differentiated hormone cells and raise the question of gene-by-environment interactions as contributors to pituitary development and disease.


Assuntos
Aldeído Desidrogenase 1/metabolismo , Proteínas de Homeodomínio/metabolismo , Hipófise/embriologia , Retinal Desidrogenase/metabolismo , Tretinoína/metabolismo , Aldeído Desidrogenase 1/genética , Animais , Camundongos Endogâmicos C57BL , Organogênese , Hipófise/metabolismo , Retinal Desidrogenase/genética , Transdução de Sinais , Células-Tronco/metabolismo , Tireotropina/metabolismo
12.
Arch Biochem Biophys ; 681: 108256, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31923393

RESUMO

Aldehyde dehydrogenases catalyze the NAD(P)+-dependent oxidation of aldehydes to their corresponding carboxylic acids. The three-dimensional structures of the human ALDH1A enzymes were recently obtained, while a complete kinetic characterization of them, under the same experimental conditions, is lacking. We show that the three enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, have similar topologies, although with decreasing volumes in their substrate-binding pockets. The activity with aliphatic and retinoid aldehydes was characterized side-by-side, using an improved HPLC-based method for retinaldehyde. Hexanal was the most efficient substrate. ALDH1A1 displayed lower Km values with hexanal, trans-2-hexenal and citral, compared to ALDH1A2 and ALDH1A3. ALDH1A2 was the best enzyme for the lipid peroxidation product, 4-hydroxy-2-nonenal, in terms of kcat/Km. The catalytic efficiency towards all-trans and 9-cis-retinaldehyde was in general lower than for alkanals and alkenals. ALDH1A2 and ALDH1A3 showed higher catalytic efficiency for all-trans-retinaldehyde. The lower specificity of ALDH1A3 for 9-cis-retinaldehyde against the all-trans- isomer might be related to the smaller volume of its substrate-binding pocket. Magnesium inhibited ALDH1A1 and ALDH1A2, while it activated ALDH1A3, which is consistent with cofactor dissociation being the rate-limiting step for ALDH1A1 and ALDH1A2, and deacylation for ALDH1A3, with hexanal as a substrate. We mutated both ALDH1A1 (L114P) and ALDH1A2 (N475G, A476V, L477V, N478S) to mimic their counterpart substrate-binding pockets. ALDH1A1 specificity for citral was traced to residue 114 and to residues 458 to 461. Regarding retinaldehyde, the mutants did not show significant differences with their respective wild-type forms, suggesting that the mutated residues are not critical for retinoid specificity.


Assuntos
Aldeído Desidrogenase 1/metabolismo , Aldeído Oxirredutases/metabolismo , Células-Tronco Neoplásicas/metabolismo , Retinal Desidrogenase/metabolismo , Tretinoína/metabolismo , Humanos , Magnésio/metabolismo , Modelos Moleculares , Células-Tronco Neoplásicas/patologia , Retinaldeído/metabolismo , Especificidade por Substrato
13.
Nat Commun ; 11(1): 63, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896743

RESUMO

Each vestibular sensory epithelium in the inner ear is divided morphologically and physiologically into two zones, called the striola and extrastriola in otolith organ maculae, and the central and peripheral zones in semicircular canal cristae. We found that formation of striolar/central zones during embryogenesis requires Cytochrome P450 26b1 (Cyp26b1)-mediated degradation of retinoic acid (RA). In Cyp26b1 conditional knockout mice, formation of striolar/central zones is compromised, such that they resemble extrastriolar/peripheral zones in multiple features. Mutants have deficient vestibular evoked potential (VsEP) responses to jerk stimuli, head tremor and deficits in balance beam tests that are consistent with abnormal vestibular input, but normal vestibulo-ocular reflexes and apparently normal motor performance during swimming. Thus, degradation of RA during embryogenesis is required for formation of highly specialized regions of the vestibular sensory epithelia with specific functions in detecting head motions.


Assuntos
Membrana dos Otólitos/embriologia , Ácido Retinoico 4 Hidroxilase/metabolismo , Tretinoína/metabolismo , Animais , Potenciais Evocados/genética , Potenciais Evocados/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Cabeça/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteopontina/metabolismo , Membrana dos Otólitos/citologia , Membrana dos Otólitos/metabolismo , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Ácido Retinoico 4 Hidroxilase/genética , Sáculo e Utrículo/citologia , Sáculo e Utrículo/embriologia , Tremor/genética , Tremor/fisiopatologia , Testes de Função Vestibular , Vestíbulo do Labirinto/embriologia , Vestíbulo do Labirinto/metabolismo
15.
Hum Genomics ; 13(1): 61, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31796115

RESUMO

Retinoic acid (RA) is a potent morphogen required for embryonic development. RA is formed in a multistep process from vitamin A (retinol); RA acts in a paracrine fashion to shape the developing eye and is essential for normal optic vesicle and anterior segment formation. Perturbation in RA-signaling can result in severe ocular developmental diseases-including microphthalmia, anophthalmia, and coloboma. RA-signaling is also essential for embryonic development and life, as indicated by the significant consequences of mutations in genes involved in RA-signaling. The requirement of RA-signaling for normal development is further supported by the manifestation of severe pathologies in animal models of RA deficiency-such as ventral lens rotation, failure of optic cup formation, and embryonic and postnatal lethality. In this review, we summarize RA-signaling, recent advances in our understanding of this pathway in eye development, and the requirement of RA-signaling for embryonic development (e.g., organogenesis and limb bud development) and life.


Assuntos
Olho/metabolismo , Transdução de Sinais/genética , Tretinoína/metabolismo , Animais , Olho/embriologia , Regulação da Expressão Gênica , Humanos , Fenótipo
16.
Sci Adv ; 5(12): eaax5933, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31844664

RESUMO

Despite its importance in central nervous system development, development of the human neural tube (NT) remains poorly understood, given the challenges of studying human embryos, and the developmental divergence between humans and animal models. We report a human NT development model, in which NT-like tissues, neuroepithelial (NE) cysts, are generated in a bioengineered neurogenic environment through self-organization of human pluripotent stem cells (hPSCs). NE cysts correspond to the neural plate in the dorsal ectoderm and have a default dorsal identity. Dorsal-ventral (DV) patterning of NE cysts is achieved using retinoic acid and/or sonic hedgehog and features sequential emergence of the ventral floor plate, P3, and pMN domains in discrete, adjacent regions and a dorsal territory progressively restricted to the opposite dorsal pole. This hPSC-based, DV patterned NE cyst system will be useful for understanding the self-organizing principles that guide NT patterning and for investigations of neural development and neural disease.


Assuntos
Padronização Corporal/genética , Tubo Neural/crescimento & desenvolvimento , Neurogênese/genética , Células-Tronco Pluripotentes/citologia , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Ectoderma/crescimento & desenvolvimento , Ectoderma/metabolismo , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Hedgehog/genética , Humanos , Tubo Neural/metabolismo , Células-Tronco Pluripotentes/metabolismo , Tretinoína/metabolismo
17.
PLoS One ; 14(11): e0224628, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31682623

RESUMO

In murine fetal germ cells, retinoic acid (RA) is an extrinsic cue for meiotic initiation that stimulates transcriptional activation of the Stimulated by retinoic acid gene 8 (Stra8), which is required for entry of germ cells into meiotic prophase I. Canonically, the biological activities of RA are mediated by nuclear RA receptors. Recent studies in somatic cells found that RA noncanonically stimulates intracellular signal transduction pathways to regulate multiple cellular processes. In this study, using a germ cell culture system, we investigated (1) whether RA treatment activates any mitogen-activated protein kinase (MAPK) pathways in fetal germ cells at the time of sex differentiation, and (2) if this is the case, whether the corresponding RA-stimulated signaling pathway regulates Stra8 expression in fetal germ cells and their entry into meiosis. When XX germ cells at embryonic day (E) 12.5 were cultured with RA, the extracellular-signal-regulated kinase (ERK) 1/2 pathway was predominantly activated. MEK1/2 inhibitor (U0126) treatment suppressed the mRNA expressions of RA-induced Stra8 and meiotic marker genes (Rec8, Spo11, Dmc1, and Sycp3) in both XX and XY fetal germ cells. Furthermore, U0126 treatment dramatically reduced STRA8 protein levels and numbers of meiotic cells among cultured XX and XY fetal germ cells even in the presence of RA. Taken together, our results suggest the novel concept that the RA functions by stimulating the ERK1/2 pathway and that this activity is critical for Stra8 expression and meiotic progression in fetal germ cells.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Germinativas/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Meiose/fisiologia , Tretinoína/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Butadienos/farmacologia , Células Cultivadas , Meios de Cultura/metabolismo , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células Germinativas/efeitos dos fármacos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Meiose/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Nitrilos/farmacologia , Cultura Primária de Células , Diferenciação Sexual/efeitos dos fármacos , Diferenciação Sexual/fisiologia
18.
J Fluoresc ; 29(6): 1277-1283, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31755049

RESUMO

Tretinoin or All-trans retinoic acid (ATRA) is an efficient medication in leukemia treatment. Arsenic trioxide (ATO) significantly improves the effectiveness of ATRA. In this study, the effect of ATO on ATRA binding to human serum albumin (HSA) was investigated. Fluorescence and UV-Vis spectroscopy and equilibrium dialysis technique were used to determine ATRA binding to HSA in the presence and absence of ATO and of two compounds, warfarin and ibuprofen, specific for binding to HSA sites I and II, respectively ("site markers"). The association constants for ATRA binding and the number of binding sites as well as the thermodynamic parameters of complex formation, were obtained at different temperatures. Fluorescence results showed a static quenching mechanism for ATRA binding to HSA. The calculated thermodynamic parameters revealed that the binding reaction is a spontaneous and exothermic process and also that hydrogen bonds and van der Waals forces have a central role in the binding of ATRA to HSA. Competitive experiments showed that none of markers seriously prevents ATRA binding to HSA. Interestingly, the fluorescence and equilibrium dialysis data showed that ATO increases the binding of ATRA to HSA, and converts the binding mode of ATRA from mainly hydrogen bonding to include hydrophobic interactions as well. These results suggest that ATO can prevent the metabolism of ATRA and keep it in the blood for longer by increasing the binding of ATRA to HSA.


Assuntos
Trióxido de Arsênio/química , Albumina Sérica Humana/química , Tretinoína/química , Sítios de Ligação , Transferência de Energia , Humanos , Albumina Sérica Humana/metabolismo , Espectrometria de Fluorescência , Termodinâmica , Tretinoína/metabolismo
19.
Int J Mol Sci ; 20(20)2019 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-31635381

RESUMO

A wide variety of peptides not only interact with the cell surface, but govern complex signaling from inside the cell. This has been referred to as an "intracrine" action, and the orchestrating molecules as "intracrines". Here, we review the intracrine action of dynorphin B, a bioactive end-product of the prodynorphin gene, on nuclear opioid receptors and nuclear protein kinase C signaling to stimulate the transcription of a gene program of cardiogenesis. The ability of intracrine dynorphin B to prime the transcription of its own coding gene in isolated nuclei is discussed as a feed-forward loop of gene expression amplification and synchronization. We describe the role of hyaluronan mixed esters of butyric and retinoic acids as synthetic intracrines, controlling prodynorphin gene expression, cardiogenesis, and cardiac repair. We also discuss the increase in prodynorphin gene transcription and intracellular dynorphin B afforded by electromagnetic fields in stem cells, as a mechanism of cardiogenic signaling and enhancement in the yield of stem cell-derived cardiomyocytes. We underline the possibility of using the diffusive features of physical energies to modulate intracrinergic systems without the needs of viral vector-mediated gene transfer technologies, and prompt the exploration of this hypothesis in the near future.


Assuntos
Diferenciação Celular/genética , Encefalinas/genética , Encefalinas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Animais , Butiratos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Peptídeos Opioides/genética , Peptídeos Opioides/metabolismo , Organogênese/genética , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Tretinoína/metabolismo
20.
Food Funct ; 10(11): 7152-7163, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31596288

RESUMO

This study demonstrated different effects of bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) signaling on the induction of germ cell formation in chickens. In vitro, BMP4 significantly promoted primordial germ cell (PGC) formation, while RA promoted spermatogonial stem cell (SSC) formation. Hematoxylin-Eosin (HE) staining of reproductive ridge and testicular slices showed that BMP4 signaling was activated during PGC formation but was inhibited during PGC differentiation into SSC. In contrast, RA signaling was significantly activated during PGC differentiation to SSC. Mechanistically, elevated expression of phosphorylated mothers against decapentaplegic homolog 5 (p-Smad5) activated BMP4 signaling, while inhibition of p-Smad5 significantly reduced the PGC formation. Additionally, BMP4 regulated the PGC formation through histone acetylation and DNA methylation in deleted in azoospermia-like (DAZL) gene. Luciferase report showed RA binding to RARα regulated stimulated by RA 8 (Stra8) promoter activity during SSC formation, while mutations in RAR binding sites inhibited the Stra8 expression and SSC formation. Further, both HAT and HDAC regulated the RARα isoform, and HAT binding to RARα activated the Stra8 transcription. RNA-seq of embryonic stem cells (ESC), PGC, and SSC showed inverse expression of genes related to the BMP4 and RA pathways during PGC and SSC formation. Additionally, Smad5 and Smurf were critical for the interactions between the two pathways. Specifically, through Smurf promotion of Smad5 ubiquitination, RA could inhibit the BMP4 signal transduction. In conclusion, the BMP4 and RA signaling pathways play opposing roles in germ cell formation, driven by epigenetic processes such as phosphorylation, ubiquitination, and histone acetylation.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Células Germinativas/metabolismo , Tretinoína/metabolismo , Células-Tronco Germinativas Adultas/citologia , Animais , Diferenciação Celular , Galinhas , Feminino , Regulação da Expressão Gênica , Células Germinativas/citologia , Masculino , Receptor alfa de Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico/metabolismo , Transdução de Sinais , Proteína Smad5/genética , Proteína Smad5/metabolismo
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