RESUMO
Antipsychotic drugs are the mainstay of treatment for schizophrenia and provide adjunct therapies for other prevalent psychiatric conditions, including bipolar disorder and major depressive disorder. However, they also induce debilitating extrapyramidal syndromes (EPS), such as Parkinsonism, in a significant minority of patients. The majority of antipsychotic drugs function as dopamine receptor antagonists in the brain while the most recent 'third'-generation, such as aripiprazole, act as partial agonists. Despite showing good clinical efficacy, these newer agents are still associated with EPS in ~ 5 to 15% of patients. However, it is not fully understood how these movement disorders develop. Here, we combine clinically-relevant drug concentrations with mutliscale model systems to show that aripiprazole and its primary active metabolite induce mitochondrial toxicity inducing robust declines in cellular ATP and viability. Aripiprazole, brexpiprazole and cariprazine were shown to directly inhibit respiratory complex I through its ubiquinone-binding channel. Importantly, all three drugs induced mitochondrial toxicity in primary embryonic mouse neurons, with greater bioenergetic inhibition in ventral midbrain neurons than forebrain neurons. Finally, chronic feeding with aripiprazole resulted in structural damage to mitochondria in the brain and thoracic muscle of adult Drosophila melanogaster consistent with locomotor dysfunction. Taken together, we show that antipsychotic drugs acting as partial dopamine receptor agonists exhibit off-target mitochondrial liabilities targeting complex I.
Assuntos
Antipsicóticos , Transtorno Depressivo Maior , Animais , Camundongos , Aripiprazol/farmacologia , Aripiprazol/uso terapêutico , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Transtorno Depressivo Maior/tratamento farmacológico , Drosophila melanogaster , Transporte de ElétronsRESUMO
Purpose: Uveal coloboma is a congenital eye malformation caused by failure of the optic fissure to close in early human development. Despite significant progress in identifying genes whose regulation is important for executing this closure, mutations are detected in a minority of cases using known gene panels, implying additional genetic complexity. We have previously shown knockdown of znf503 (the ortholog of mouse Zfp503) in zebrafish causes coloboma. Here we characterize Zfp503 knockout (KO) mice and evaluate transcriptomic profiling of mutant versus wild-type (WT) retinal pigment epithelium (RPE)/choroid. Methods: Zfp503 KO mice were generated by gene targeting using homologous recombination. Embryos were characterized grossly and histologically. Patterns and level of developmentally relevant proteins/genes were examined with immunostaining/in situ hybridization. The transcriptomic profile of E11.5 KO RPE/choroid was compared to that of WT. Results: Zfp503 is dynamically expressed in developing mouse eyes, and loss of its expression results in uveal coloboma. KO embryos exhibit altered mRNA levels and expression patterns of several key transcription factors involved in eye development, including Otx2, Mitf, Pax6, Pax2, Vax1, and Vax2, resulting in a failure to maintain the presumptive RPE, as evidenced by reduced melanin pigmentation and its differentiation into a neural retina-like lineage. Comparison of RNA sequencing data from WT and KO E11.5 embryos demonstrated reduced expression of melanin-related genes and significant overlap with genes known to be dynamically regulated at the optic fissure. Conclusions: These results demonstrate a critical role of Zfp503 in maintaining RPE fate and optic fissure closure.
Assuntos
Coloboma , Neuropeptídeos , Animais , Humanos , Camundongos , Coloboma/genética , Coloboma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melaninas/metabolismo , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/genética , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Peixe-Zebra/genéticaRESUMO
In Parkinson's disease (PD), substantia nigra (SN) dopaminergic (DA) neurons degenerate, while related ventral tegmental area (VTA) DA neurons remain relatively unaffected. Here, we present a methodology that directs the differentiation of mouse and human pluripotent stem cells toward either SN- or VTA-like DA lineage and models their distinct vulnerabilities. We show that the level of WNT activity is critical for the induction of the SN- and VTA-lineage transcription factors Sox6 and Otx2, respectively. Both WNT signaling modulation and forced expression of these transcription factors can drive DA neurons toward the SN- or VTA-like fate. Importantly, the SN-like lineage enriched DA cultures recapitulate the selective sensitivity to mitochondrial toxins as observed in PD, while VTA-like neuron-enriched cultures are more resistant. Furthermore, a proteomics approach led to the identification of compounds that alter SN neuronal survival, demonstrating the utility of our strategy for disease modeling and drug discovery.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Degeneração Neural/genética , Doença de Parkinson/genética , Células-Tronco Pluripotentes/metabolismo , Substância Negra/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Neurônios Dopaminérgicos/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Modelos Neurológicos , Células-Tronco Embrionárias Murinas/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOXD/genética , Fatores de Transcrição SOXD/metabolismo , Substância Negra/citologia , Área Tegmentar Ventral/citologiaRESUMO
Midbrain dopaminergic (DA) axons make long longitudinal projections towards the striatum. Despite the importance of DA striatal innervation, processes involved in establishment of DA axonal connectivity remain largely unknown. Here we demonstrate a striatal-specific requirement of transcriptional regulator Nolz1 in establishing DA circuitry formation. DA projections are misguided and fail to innervate the striatum in both constitutive and striatal-specific Nolz1 mutant embryos. The lack of striatal Nolz1 expression results in nigral to pallidal lineage conversion of striatal projection neuron subtypes. This lineage switch alters the composition of secreted factors influencing DA axonal tract formation and renders the striatum non-permissive for dopaminergic and other forebrain tracts. Furthermore, transcriptomic analysis of wild-type and Nolz1-/- mutant striatal tissue led to the identification of several secreted factors that underlie the observed guidance defects and proteins that promote DA axonal outgrowth. Together, our data demonstrate the involvement of the striatum in orchestrating dopaminergic circuitry formation.
Assuntos
Orientação de Axônios/fisiologia , Axônios/fisiologia , Corpo Estriado/crescimento & desenvolvimento , Neurônios Dopaminérgicos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Carbocianinas/administração & dosagem , Corpo Estriado/diagnóstico por imagem , Embrião de Mamíferos , Feminino , Corantes Fluorescentes/administração & dosagem , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microscopia Intravital , Camundongos Knockout , Técnicas Analíticas Microfluídicas , Microinjeções , Microscopia Confocal , Rede Nervosa/fisiologia , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de TecidosRESUMO
Mutations in key transcription factors SOX2 and P63 were linked with developmental defects and postnatal abnormalities such as corneal opacification, neovascularization, and blindness. The latter phenotypes suggest that SOX2 and P63 may be involved in corneal epithelial regeneration. Although P63 has been shown to be a key regulator of limbal stem cells, the expression pattern and function of SOX2 in the adult cornea remained unclear. Here, we show that SOX2 regulates P63 to control corneal epithelial stem/progenitor cell function. SOX2 and P63 were co-expressed in the stem/progenitor cell compartments of the murine cornea in vivo and in undifferentiated human limbal epithelial stem/progenitor cells in vitro. In line, a new consensus site that allows SOX2-mediated regulation of P63 enhancer was identified while repression of SOX2 reduced P63 expression, suggesting that SOX2 is upstream to P63. Importantly, knockdown of SOX2 significantly attenuated cell proliferation, long-term colony-forming potential of stem/progenitor cells, and induced robust cell differentiation. However, this effect was reverted by forced expression of P63, suggesting that SOX2 acts, at least in part, through P63. Finally, miR-450b was identified as a direct repressor of SOX2 that was required for SOX2/P63 downregulation and cell differentiation. Altogether, we propose that SOX2/P63 pathway is an essential regulator of corneal stem/progenitor cells while mutations in SOX2 or P63 may disrupt epithelial regeneration, leading to loss of corneal transparency and blindness. Stem Cells 2019;37:417-429.
Assuntos
Diferenciação Celular , Proliferação de Células , Epitélio Corneano/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Alcaloides , Animais , Camundongos , Células NIH 3T3 , Piperidinas , Fatores de Transcrição SOXB1/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Mutations in the gene encoding DJ-1 are associated with autosomal recessive forms of Parkinson's disease (PD). DJ-1 plays a role in protection from oxidative stress, but how it functions as an "upstream" oxidative stress sensor and whether this relates to PD is still unclear. Intriguingly, DJ-1 may act as an RNA binding protein associating with specific mRNA transcripts in the human brain. Moreover, we previously reported that the yeast DJ-1 homolog Hsp31 localizes to stress granules (SGs) after glucose starvation, suggesting a role for DJ-1 in RNA dynamics. Here, we report that DJ-1 interacts with several SG components in mammalian cells and localizes to SGs, as well as P-bodies, upon induction of either osmotic or oxidative stress. By purifying the mRNA associated with DJ-1 in mammalian cells, we detected several transcripts and found that subpopulations of these localize to SGs after stress, suggesting that DJ-1 may target specific mRNAs to mRNP granules. Notably, we find that DJ-1 associates with SGs arising from N-methyl-D-aspartate (NMDA) excitotoxicity in primary neurons and parkinsonism-inducing toxins in dopaminergic cell cultures. Thus, our results indicate that DJ-1 is associated with cytoplasmic RNA granules arising during stress and neurodegeneration, providing a possible link between DJ-1 and RNA dynamics which may be relevant for PD pathogenesis.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Degeneração Neural/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteína Desglicase DJ-1/metabolismo , Ribonucleoproteínas/metabolismo , Estresse Fisiológico , Animais , Grânulos Citoplasmáticos/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , N-Metilaspartato/toxicidade , Degeneração Neural/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Pressão Osmótica , Estresse Oxidativo/efeitos dos fármacos , Ligação Proteica , Ratos , Estresse Fisiológico/efeitos dos fármacosRESUMO
Distinct midbrain dopamine (mDA) neuron subtypes are found in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA), but it is mainly SNc neurons that degenerate in Parkinson's disease. Interest in how mDA neurons develop has been stimulated by the potential use of stem cells in therapy or disease modeling. However, very little is known about how specific dopaminergic subtypes are generated. Here, we show that the expression profiles of the transcription factors Sox6, Otx2, and Nolz1 define subpopulations of mDA neurons already at the neural progenitor cell stage. After cell-cycle exit, Sox6 selectively localizes to SNc neurons, while Otx2 and Nolz1 are expressed in a subset of VTA neurons. Importantly, Sox6 ablation leads to decreased expression of SNc markers and a corresponding increase in VTA markers, while Otx2 ablation has the opposite effect. Moreover, deletion of Sox6 affects striatal innervation and dopamine levels. We also find reduced Sox6 levels in Parkinson's disease patients. These findings identify Sox6 as a determinant of SNc neuron development and should facilitate the engineering of relevant mDA neurons for cell therapy and disease modeling.
Assuntos
Neurônios Dopaminérgicos/fisiologia , Fatores de Transcrição Otx/fisiologia , Fatores de Transcrição SOXD/fisiologia , Substância Negra/citologia , Área Tegmentar Ventral/citologia , Animais , Padronização Corporal , Humanos , Camundongos Transgênicos , Especificidade de Órgãos , Substância Negra/embriologia , Substância Negra/metabolismo , Área Tegmentar Ventral/embriologia , Área Tegmentar Ventral/metabolismoRESUMO
An important goal in stem cell biology is to develop methods for efficient generation of clinically interesting cell types from relevant stem cell populations. This is particularly challenging for different types of neurons of the central nervous system where hundreds of distinct neuronal cell types are generated during embryonic development. We previously used a strategy based on forced transcription factor expression in embryonic stem cell-derived neural progenitors to generate specific types of neurons, including dopamine and serotonin neurons. Here, we extend these studies and show that noradrenergic neurons can also be generated from pluripotent embryonic stem cells by forced expression of the homeobox transcription factor Phox2b under the signaling influence of fibroblast growth factor 8 (FGF8) and bone morphogenetic proteins. In neural progenitors exposed to FGF8 and sonic hedgehog both Phox2b and the related Phox2a instead promoted the generation of neurons with the characteristics of mid- and hindbrain motor neurons. The efficient generation of these neuron types enabled a comprehensive genome-wide gene expression analysis that provided further validation of the identity of generated cells. Moreover, we also demonstrate that the generated cell types are amenable to drug testing in vitro and we show that variants of the differentiation protocols can be applied to cultures of human pluripotent stem cells for the generation of human noradrenergic and visceral motor neurons. Thus, these studies provide a basis for characterization of yet an additional highly clinically relevant neuronal cell type.
Assuntos
Neurônios Adrenérgicos/citologia , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Neurônios Motores/citologia , Fatores de Transcrição/metabolismo , Neurônios Adrenérgicos/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Engenharia Genética , Genoma/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Neurônios Motores/metabolismo , Transdução de SinaisRESUMO
The severe disorders associated with a loss or dysfunction of midbrain dopamine neurons (DNs) have intensified research aimed at deciphering developmental programs controlling midbrain development. The homeodomain proteins Lmx1a and Lmx1b are important for the specification of DNs during embryogenesis, but it is unclear to what degree they may mediate redundant or specific functions. Here, we provide evidence showing that DN progenitors in the ventral midbrain can be subdivided into molecularly distinct medial and lateral domains, and these subgroups show different sensitivity to the loss of Lmx1a and Lmx1b. Lmx1a is specifically required for converting non-neuronal floor-plate cells into neuronal DN progenitors, a process that involves the establishment of Notch signaling in ventral midline cells. On the other hand, lateral DN progenitors that do not appear to originate from the floor plate are selectively ablated in Lmx1b mutants. In addition, we also reveal an unanticipated role for Lmx1b in regulating Phox2a expression and the sequential specification of ocular motor neurons (OMNs) and red nucleus neurons (RNNs) from progenitors located lateral to DNs in the midbrain. Our data therefore establish that Lmx1b influences the differentiation of multiple neuronal subtypes in the ventral midbrain, whereas Lmx1a appears to be exclusively devoted to the differentiation of the DN lineage.
Assuntos
Proteínas de Homeodomínio/metabolismo , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose , Linhagem da Célula , Dopamina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM , Mesencéfalo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
Many previous studies have focused on understanding how midbrain dopamine neurons, which are implicated in many neurological conditions, are generated during embryogenesis. One of the remaining questions concerns how different dopamine neuron subtypes are specified. A recent paper in Neural Development has revealed features of a spatial and temporal lineage map that, together with other studies, begins to elucidate the developmental origin of distinct neuronal subtypes within the developing midbrain.
Assuntos
Neurônios/classificação , Neurônios/citologia , Animais , Neurônios Dopaminérgicos/classificação , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Humanos , Mesencéfalo/citologia , Neurogênese/fisiologia , Neurônios/metabolismoRESUMO
The generation of specific types of neurons from stem cells offers important opportunities in regenerative medicine. However, future applications and proper verification of cell identities will require stringent ways to generate homogeneous neuronal cultures. Here we show that transcription factors like Lmx1a, Phox2b, Nkx2.2, and Olig2 can induce desired neuronal lineages from most expressing neural progenitor cells by a mechanism resembling developmental binary cell-fate switching. Such efficient selection of cell fate resulted in remarkable cellular enrichment that enabled global gene-expression validation of generated neurons and identification of previously unrecognized features in the studied cell lineages. Several sources of stem cells have a limited competence to differentiate into specific neuronal cell types; e.g., dopamine neurons. However, we show that the combination of factors that normally promote either regional or dedicated neuronal specification can overcome limitations in cellular competence and also promote efficient reprogramming in more remote neural contexts, including human neural progenitor cells.
Assuntos
Linhagem da Célula , Células-Tronco Neurais/citologia , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteínas Nucleares , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/genética , Proteínas de Peixe-ZebraRESUMO
Induced expression of neuroprotective genes is essential for maintaining neuronal integrity after stressful insults to the brain. Here we show that NR4A nuclear orphan receptors are induced after excitotoxic and oxidative stress in neurons, up-regulate neuroprotective genes, and increase neuronal survival. Moreover, we show that NR4A proteins are induced by cAMP response element binding protein (CREB) in neurons exposed to stressful insults and that they function as mediators of CREB-induced neuronal survival. Animals with null mutations in three of six NR4A alleles show increased oxidative damage, blunted induction of neuroprotective genes, and increased vulnerability in the hippocampus after treatment with kainic acid. We also demonstrate that NR4A and the transcriptional coactivator PGC-1alpha independently regulate distinct CREB-dependent neuroprotective gene programs. These data identify NR4A nuclear orphan receptors as essential mediators of neuroprotection after exposure to neuropathological stress.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células-Tronco Embrionárias/metabolismo , Neurônios/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/citologia , Feminino , Perfilação da Expressão Gênica , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Peróxido de Hidrogênio/farmacologia , Ionomicina/farmacologia , Ácido Caínico/farmacologia , Masculino , Camundongos , Camundongos Knockout , Neurônios/citologia , Fármacos Neuroprotetores/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Oxidantes/farmacologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
Spatially and temporally coordinated changes in gene expression are crucial to orderly progression of embryogenesis. We combine mouse genetics with experimental manipulation of signalling to analyze the kinetics by which the SHH morphogen and the BMP antagonist gremlin 1 (GREM1) control gene expression in the digit-forming mesenchyme of mouse limb buds. Although most mesenchymal cells respond rapidly to SHH signalling, the transcriptional upregulation of specific SHH target signals in the mesenchyme occurs with differential temporal kinetics and in a spatially restricted fashion. In particular, the expression of the BMP antagonist Grem1 is always upregulated in mesenchymal cells located distal to the SHH source and acts upstream of FGF signalling by the apical ectodermal ridge. GREM1/FGF-mediated feedback signalling is, in turn, required to propagate SHH and establish the presumptive digit expression domains of the Notch ligand jagged 1 (Jag1) and 5'Hoxd genes in the distal limb bud mesenchyme. Their establishment is significantly delayed in Grem1-deficient limb buds and cannot be rescued by specific restoration of SHH signalling in mutant limb buds. This shows that GREM1/FGF feedback signalling is required for regulation of the temporal kinetics of the mesenchymal response to SHH signalling. Finally, inhibition of SHH signal transduction at distinct time points reveals the differential temporal dependence of Grem1, Jag1 and 5'Hoxd gene expression on SHH signalling. In particular, the expression of Hoxd13 depends on SHH signal transduction significantly longer than does Hoxd11 expression, revealing that the reverse co-linear establishment, but not maintenance of their presumptive digit expression domains, depends on SHH signalling.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Botões de Extremidades/embriologia , Mesoderma/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas , Retroalimentação , Feminino , Genótipo , Proteínas Hedgehog/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mesoderma/citologia , Camundongos , Fenótipo , Proteínas Serrate-Jagged , Transdução de SinaisRESUMO
The Gli3 and Alx4 transcriptional regulators are expressed in the anterior limb bud mesenchyme and their disruption in mice results in preaxial polydactyly. While the polydactylous phenotype of Alx4 deficient limb buds depends on SHH, the one of Gli3 deficient limb buds is completely independent of SHH signalling, suggesting that these genes act in parallel pathways. Analysis of limb buds lacking both Gli3 and Alx4 now shows that these two genes interact during limb skeletal morphogenesis. In addition to the defects in single mutants, the stylopod is severely malformed and the anterior element of the zeugopod is lost in double mutant limbs. However, limb bud patterning in Gli3-/-; Alx4-/- double mutant embryos is not affected more than in single mutants as the expression domains of key regulators remain the same. Most interestingly, the loss of the severe preaxial polydactyly characteristic of Gli3-/- limbs in double mutant embryos establishes that this type of polydactyly requires Alx4 function.
Assuntos
Padronização Corporal/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição Kruppel-Like/genética , Botões de Extremidades/embriologia , Proteínas do Tecido Nervoso/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Polidactilia/embriologia , Polidactilia/genética , Proteína Gli3 com Dedos de ZincoRESUMO
Epithelial-mesenchymal feedback signaling is the key to diverse organogenetic processes such as limb bud development and branching morphogenesis in kidney and lung rudiments. This study establishes that the BMP antagonist gremlin (Grem1) is essential to initiate these epithelial-mesenchymal signaling interactions during limb and metanephric kidney organogenesis. A Grem1 null mutation in the mouse generated by gene targeting causes neonatal lethality because of the lack of kidneys and lung septation defects. In early limb buds, mesenchymal Grem1 is required to establish a functional apical ectodermal ridge and the epithelial-mesenchymal feedback signaling that propagates the sonic hedgehog morphogen. Furthermore, Grem1-mediated BMP antagonism is essential to induce metanephric kidney development as initiation of ureter growth, branching and establishment of RET/GDNF feedback signaling are disrupted in Grem1-deficient embryos. As a consequence, the metanephric mesenchyme is eliminated by apoptosis, in the same way as the core mesenchymal cells of the limb bud.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Epitélio/embriologia , Extremidades/embriologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Rim/embriologia , Mesoderma/metabolismo , Transdução de Sinais , Alelos , Animais , Animais Recém-Nascidos , Citocinas , Retroalimentação Fisiológica , Proteínas Hedgehog , Marcação In Situ das Extremidades Cortadas , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pulmão/embriologia , Camundongos , Modelos Genéticos , Mutação , Fases de Leitura Aberta , Fatores de Tempo , Transativadores/metabolismoRESUMO
The mouse limb deformity (ld) mutations cause limb malformations by disrupting epithelial-mesenchymal signaling between the polarizing region and the apical ectodermal ridge. Formin was proposed as the relevant gene because three of the five ld alleles disrupt its C-terminal domain. In contrast, our studies establish that the two other ld alleles directly disrupt the neighboring Gremlin gene, corroborating the requirement of this BMP antagonist for limb morphogenesis. Further doubts concerning an involvement of Formin in the ld limb phenotype are cast, as a targeted mutation removing the C-terminal Formin domain by frame shift does not affect embryogenesis. In contrast, the deletion of the corresponding genomic region reproduces the ld limb phenotype and is allelic to mutations in Gremlin. We resolve these conflicting results by identifying a cis-regulatory region within the deletion that is required for Gremlin activation in the limb bud mesenchyme. This distant cis-regulatory region within Formin is also altered by three of the ld mutations. Therefore, the ld limb bud patterning defects are not caused by disruption of Formin, but by alteration of a global control region (GCR) required for Gremlin transcription. Our studies reveal the large genomic landscape harboring this GCR, which is required for tissue-specific coexpression of two structurally and functionally unrelated genes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Deformidades Congênitas dos Membros/genética , Mutação , Sequências Reguladoras de Ácido Nucleico , Sequência de Aminoácidos , Animais , Sequência de Bases , Citocinas , Proteínas Fetais/genética , Forminas , Proteínas Hedgehog , Botões de Extremidades/fisiologia , Mesoderma , Camundongos , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Splicing de RNA , Transativadores/genética , Transativadores/metabolismoRESUMO
The vertebrate limb is one of the most relevant experimental models for analysing cell-cell signalling during patterning of embryonic fields and organogenesis. Recently, the combination of molecular and genetic studies with experimental manipulation of developing limb buds has significantly advanced our understanding of the complex molecular interactions co-ordinating limb bud outgrowth and patterning. Some of these studies have shown that there is a need to revise some of the textbook views of vertebrate limb development. In this review, we discuss how signalling by the polarizing region is established and how limb bud morphogenesis is controlled by both long-range and signal relay mechanisms. We also discuss recent results showing that differential mesenchymal responsiveness to SHH signalling is established prior to its expression by the polarizing region.