RESUMEN
Hedgehog (HH) signaling is important for embryonic pattering and stem cell differentiation. The G protein-coupled receptor (GPCR) Smoothened (SMO) is the key HH signal transducer modulating both transcription-dependent and transcription-independent responses. We show that SMO protects naive mouse embryonic stem cells (ESCs) from dissociation-induced cell death. We exploited this SMO dependency to perform a genetic screen in haploid ESCs where we identify the Golgi proteins TMED2 and TMED10 as factors for SMO regulation. Super-resolution microscopy shows that SMO is normally retained in the endoplasmic reticulum (ER) and Golgi compartments, and we demonstrate that TMED2 binds to SMO, preventing localization to the plasma membrane. Mutation of TMED2 allows SMO accumulation at the plasma membrane, recapitulating early events after HH stimulation. We demonstrate the physiologic relevance of this interaction in neural differentiation, where TMED2 functions to repress HH signal strength. Identification of TMED2 as a binder and upstream regulator of SMO opens the way for unraveling the events in the ER-Golgi leading to HH signaling activation.
Asunto(s)
Proteínas Hedgehog , Receptores Acoplados a Proteínas G , Animales , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de la Membrana , Ratones , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/genética , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteínas de Transporte VesicularRESUMEN
Three central cell populations play roles in morphogen action, the cells that produce, the cells that distribute, and the cells that respond to the morphogen. Taking advantage of the properties of embryonic stem cell to aggregate and readily differentiate into neural progenitor tissue, we describe an approach using genetically modified murine stem cell lines to individually address the contribution of these cells in the establishment and response to a morphogenetic gradient in mosaic spinal cord organoids.
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Células Madre Embrionarias de Ratones , Organoides , Animales , Ratones , Transducción de Señal , Médula EspinalRESUMEN
BACKGROUND: Sonic Hedgehog (Shh) has a catalytic cleft characteristic for zinc metallopeptidases and has significant sequence similarities with some bacterial peptidoglycan metallopeptidases defining a subgroup within the M15A family that, besides having the characteristic zinc coordination motif, can bind two calcium ions. Extracellular matrix (ECM) components in animals include heparan-sulfate proteoglycans, which are analogs of bacterial peptidoglycan and are involved in the extracellular distribution of Shh. RESULTS: We found that the zinc-coordination center of Shh is required for its association to the ECM as well as for non-cell autonomous signaling. Association with the ECM requires the presence of at least 0.1 µM zinc and is prevented by mutations affecting critical conserved catalytical residues. Consistent with the presence of a conserved calcium binding domain, we find that extracellular calcium inhibits ECM association of Shh. CONCLUSIONS: Our results indicate that the putative intrinsic peptidase activity of Shh is required for non-cell autonomous signaling, possibly by enzymatically altering ECM characteristics.
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Matriz Extracelular/metabolismo , Proteínas Hedgehog/metabolismo , Zinc/química , Secuencias de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Calcio , Células Cultivadas , Colesterol , Células HEK293 , Proteínas Hedgehog/química , Proteínas Hedgehog/genética , Humanos , Ratones , Mutación , Péptido Hidrolasas/química , Dominios Proteicos , Transducción de SeñalRESUMEN
As a morphogen, Sonic Hedgehog (Shh) mediates signaling at a distance from its sites of synthesis. After secretion, Shh must traverse a distance through the extracellular matrix (ECM) to reach the target cells and activate the Hh response. ECM proteins, in particular, the heparan sulfate proteoglycans (HSPGs) of the glypican family, have both negative and positive effects on Shh signaling, all attributed to their ability to bind Shh. Using mouse embryonic stem cell-derived mosaic tissues with compartments that lack the glycosyltransferases Exostosin1 and Exostosin2, or the HSPG core protein Glypican5, we show that Shh accumulates around its source cells when they are surrounded by cells that have a mutated ECM. This accumulation of Shh is correlated with an increased noncell autonomous Shh response. Our results support a model in which Shh presented on the cell surface accumulates at or near ECM that lacks HSPGs, possibly due to the absence of these Shh sequestering molecules. Stem Cells 2019;37:899-909.
Asunto(s)
Glipicanos/genética , Proteínas Hedgehog/genética , Células Madre Embrionarias de Ratones/metabolismo , N-Acetilglucosaminiltransferasas/genética , Transducción de Señal/genética , Animales , Diferenciación Celular , Linaje de la Célula/genética , Movimiento Celular , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Edición Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Glipicanos/deficiencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , N-Acetilglucosaminiltransferasas/deficiencia , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Transporte de Proteínas , Transfección , Proteína Fluorescente RojaRESUMEN
Sonic Hedgehog (Shh) signaling is characterized by non-cell autonomy; cells expressing Shh do not respond to the ligand. Here, we identify several Shh mutations that can activate the Hedgehog (Hh) pathway cell-autonomously. Cell-autonomous pathway activation requires the extracellular cysteine rich domain of Smoothened, but is otherwise independent of the Shh receptors Patched1 and -2. Many of the Shh mutants that gain activity fail to undergo auto processing resulting in the perdurance of the Shh pro-peptide, a form of Shh that is sufficient to activate the Hh response cell-autonomously. Our results demonstrate that Shh is capable of activating the Hh pathway via Smoothened, independently of Patched1/2, and that it harbors an intrinsic mechanism that prevents cell-autonomous activation of the Shh response.
Asunto(s)
Mutación con Ganancia de Función/genética , Proteínas Hedgehog/genética , Receptor Patched-1/metabolismo , Receptor Patched-2/metabolismo , Receptor Smoothened/metabolismo , Secuencia de Aminoácidos , Animales , Pollos , Holoprosencefalia/genética , Holoprosencefalia/patología , Luciferasas/metabolismo , Ratones , Modelos Biológicos , Tubo Neural/embriología , Tubo Neural/metabolismo , Dominios Proteicos , Receptor Smoothened/química , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
Sonic Hedgehog (Shh) coordinates Zn2+ in a manner that resembles that of peptidases. The ability of Shh to undergo autoproteolytic processing is impaired in mutants that affect the Zn2+ coordination, while mutating residues essential for catalytic activity results in more stable forms of Shh. The residues involved in Zn2+ coordination in Shh are found to be mutated in some individuals with the congenital birth defect holoprosencephaly, demonstrating their importance in development. Highly conserved Shh domains are found in parts of some bacterial proteins that are members of the larger family of DD-peptidases, supporting the notion that Shh acts as a peptidase. Whereas this Hh/DD-peptidase motif is present in Hedgehog (Hh) proteins of nearly all animals, it is not present in Drosophila Hh, indicating that Hh signaling in fruit flies is derived, and perhaps not a good model for vertebrate Shh signaling. A sequence analysis of Hh proteins and their possible evolutionary precursors suggests that the evolution of modern Hh might have involved horizontal transfer of a bacterial gene coding of a Hh/DD-peptidase into a Cnidarian ancestor, recombining to give rise to modern Hh.
RESUMEN
UVR in sunlight causes mutations that drive basal cell carcinomas. However, the incidence of these tumors plateaus with prolonged exposure, but the incidence of other skin cancers increases. Makarova et al. now show that vitamin D3 produced in the skin by UVR protects against its oncogenic effects by inhibiting Hedgehog signaling, whereas dietary vitamin D3 does not.
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Carcinoma Basocelular , Neoplasias Cutáneas/epidemiología , Animales , Carcinogénesis , Colecalciferol , Ratones , Piel , Luz SolarRESUMEN
Smoothened (Smo) inhibition by Patched (Ptch) is central to Hedgehog (Hh) signaling. Ptch, a proton driven antiporter, is required for Smo inhibition via an unknown mechanism. Hh ligand binding to Ptch reverses this inhibition and activated Smo initiates the Hh response. To determine whether Ptch inhibits Smo strictly in the same cell or also mediates non-cell-autonomous Smo inhibition, we generated genetically mosaic neuralized embryoid bodies (nEBs) from mouse embryonic stem cells (mESCs). These experiments utilized novel mESC lines in which Ptch1, Ptch2, Smo, Shh and 7dhcr were inactivated via gene editing in multiple combinations, allowing us to measure non-cell autonomous interactions between cells with differing Ptch1/2 status. In several independent assays, the Hh response was repressed by Ptch1/2 in nearby cells. When 7dhcr was targeted, cells displayed elevated non-cell autonomous inhibition. These findings support a model in which Ptch1/2 mediate secretion of a Smo-inhibitory cholesterol precursor.
Asunto(s)
Receptor Patched-1/metabolismo , Receptor Patched-2/metabolismo , Receptor Smoothened/antagonistas & inhibidores , Animales , Técnicas de Inactivación de Genes , Proteínas Hedgehog/metabolismo , Ratones , Células Madre Embrionarias de Ratones/fisiología , Receptor Patched-1/genética , Receptor Patched-2/genética , Transducción de Señal , Receptor Smoothened/genéticaRESUMEN
The distribution of Sonic Hedgehog (Shh) is a highly regulated and critical process for development. Several negative feedback mechanisms are in place, including the Shh-induced upregulation of Hedgehog-interacting protein (Hhip). Hhip sequesters Shh, leading to a non-cell autonomous inhibition of the pathway. Hhip overexpression has a severe effect on neural tube development, raising the question why normal sites of Hhip expression have a seemingly unimpaired response to Shh. Here we show that although Hhip is able to leave its sites of synthesis to inhibit Shh non-cell autonomously, activation of Smoothened (Smo) drastically increases Hhip internalization and degradation cell autonomously. Although Hhip is unable to cell autonomously inhibit the consequences of Smo activation, it can inhibit the Shh response non-cell autonomously. Our data provide a mechanism by which the Shh ligand can activate the response and negate cell autonomous effects of Hhip, while Hhip can still induce non-cell autonomous inhibition.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Hedgehog/metabolismo , Glicoproteínas de Membrana/metabolismo , Tubo Neural/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Animales , Proteínas Portadoras/genética , Embrión de Pollo , Perros , Electroporación , Retroalimentación Fisiológica , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Células de Riñón Canino Madin Darby , Glicoproteínas de Membrana/genética , Tubo Neural/citología , Tubo Neural/embriología , Plásmidos/química , Plásmidos/metabolismo , Proteolisis , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusión/genética , Receptor Smoothened , Transfección , CigotoRESUMEN
The Hedgehog (Hh) signaling response is regulated by the interaction of three key components that include the sonic hedgehog (Shh) ligand, its receptor patched 1 (Ptch1) and the pathway activator smoothened (Smo). Under the prevailing model of Shh pathway activation, the binding of Shh to Ptch1 (the key Shh receptor) results in the release of Ptch1-mediated inhibition of Smo, leading to Smo activation and subsequent cell-autonomous activation of the Shh response. Consistent with this model, Ptch1(-/-) cells show a strong upregulation of the Shh response. Our finding that this response can be inhibited by the Shh-blocking antibody 5E1 indicates that the Shh response in Ptch1(-/-) cells remains ligand dependent. Furthermore, we find that Shh induces a strong response in Ptch1(-/-);Shh(-/-) cells, and that Ptch1(-/-) fibroblasts retain their ability to migrate towards Shh, demonstrating that Ptch1(-/-) cells remain sensitive to Shh. Expression of a dominant-negative Ptch1 mutant in the developing chick neural tube had no effect on Shh-mediated patterning, but expression of a dominant-negative form of patched 2 (Ptch2) caused an activation of the Shh response. This indicates that, at early developmental stages, Ptch2 functions to suppress Shh signaling. We found that Ptch1(-/-);Ptch2(-/-) cells cannot further activate the Shh response, demonstrating that Ptch2 mediates the response to Shh in the absence of Ptch1.
Asunto(s)
Proteínas Hedgehog/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Antiportadores/metabolismo , Moléculas de Adhesión Celular/metabolismo , Quimiotaxis , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HEK293 , Proteínas Hedgehog/deficiencia , Humanos , Inmunoglobulina G/metabolismo , Ratones , Mutación/genética , Neuronas/citología , Neuronas/metabolismo , Receptores Patched , Receptor Patched-1 , Receptor Patched-2 , Unión Proteica , Estructura Secundaria de Proteína , Protones , Receptores de Superficie Celular/química , Receptores de Superficie Celular/deficiencia , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Receptor SmoothenedRESUMEN
Emerging evidence points to an unexpected diversification of core promoter recognition complexes that serve as important regulators of cell-type specific gene transcription. Here, we report that the orphan TBP-associated factor TAF9B is selectively up-regulated upon in vitro motor neuron differentiation, and is required for the transcriptional induction of specific neuronal genes, while dispensable for global gene expression in murine ES cells. TAF9B binds to both promoters and distal enhancers of neuronal genes, partially co-localizing at binding sites of OLIG2, a key activator of motor neuron differentiation. Surprisingly, in this neuronal context TAF9B becomes preferentially associated with PCAF rather than the canonical TFIID complex. Analysis of dissected spinal column from Taf9b KO mice confirmed that TAF9B also regulates neuronal gene transcription in vivo. Our findings suggest that alternative core promoter complexes may provide a key mechanism to lock in and maintain specific transcriptional programs in terminally differentiated cell types.DOI: http://dx.doi.org/10.7554/eLife.02559.001.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Neuronas Motoras/metabolismo , Regiones Promotoras Genéticas , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/metabolismo , Animales , Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Ratones Noqueados , Neuronas Motoras/citología , Complejos Multiproteicos/metabolismo , Unión Proteica , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. It is typically detected at an advanced stage, at which the therapeutic options are very limited. One remarkable feature of PDAC that contributes to its resilience to treatment is the extreme stromal activation seen in these tumors. Often, the vast majority of tumor bulk consists of non-tumor cells that together provide a tumor-promoting environment. One of the signals that maintains and activates the stroma is the developmental protein Sonic Hedgehog (SHH). As the disease progresses, tumor cells produce increasing amounts of SHH, which activates the surrounding stroma to aid in tumor progression. To better understand this response and identify targets for inhibition, we aimed to elucidate the proteins that mediate the SHH-driven stromal response in PDAC. For this a novel mixed-species coculture model was set up in which the cancer cells are human, and the stroma is modeled by mouse fibroblasts. In conjunction with next-generation sequencing we were able to use the sequence difference between these species to genetically distinguish between the epithelial and stromal responses to SHH. The stromal SHH-dependent genes from this analysis were validated and their relevance for human disease was subsequently determined in two independent patient cohorts. In non-microdissected tissue from PDAC patients, in which a large amount of stroma is present, the targets were confirmed to associate with tumor stroma versus normal pancreatic tissue. Patient survival analysis and immunohistochemistry identified CDA, EDIL3, ITGB4, PLAUR and SPOCK1 as SHH-dependent stromal factors that are associated with poor prognosis in PDAC patients. Summarizing, the presented data provide insight into the role of the activated stroma in PDAC, and how SHH acts to mediate this response. In addition, the study has yielded several candidates that are interesting therapeutic targets for a disease for which treatment options are still inadequate.
Asunto(s)
Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidad , Proteínas Hedgehog/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Técnicas de Cocultivo , Supervivencia sin Enfermedad , Femenino , Proteínas Hedgehog/genética , Humanos , Masculino , Ratones , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Células del Estroma/metabolismo , Células del Estroma/patología , Tasa de SupervivenciaRESUMEN
Regulation of the Hedgehog (Hh) pathway relies on an interaction of two receptors. In the absence of Hh, Patched1 (Ptch1) inhibits the pathway. Binding of the ligand Hh to Ptch1 stimulates the localization of the activating receptor Smoothened (Smo) to the primary cilium, which is required for the transcriptional Hh response. Hh can also induce chemotaxis through a nontranscriptional pathway. We assessed the effects of defective ciliary localization of Smo on its subcellular trafficking and ability to mediate chemotactic signaling. Cells expressing mutants of Smo that could not localize to the primary cilium or cells lacking the primary cilium showed altered intracellular trafficking of Smo and, in response to Hh or Smo agonists, decreased transcriptional signaling and enhanced chemotactic responsiveness. Thus, the ciliary localization machinery appears to transport Smo to subcellular sites where it can mediate transcriptional signaling and away from locations where it can mediate chemotactic signaling. The subcellular localization of Smo is thus a crucial determinant of its signaling characteristics and implies the existence of a pool of Smo dedicated to chemotaxis.
Asunto(s)
Quimiotaxis , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Western Blotting , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Proteínas Hedgehog/genética , Cinesinas/genética , Cinesinas/metabolismo , Ratones , Ratones Noqueados , Microscopía Fluorescente , Mutación , Transporte de Proteínas , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Receptor SmoothenedRESUMEN
The cranial part of the amniote neural tube is formed by folding and fusion of the ectoderm-derived neural plate (primary neurulation). After posterior neuropore closure, however, the caudal neural tube is formed by cavitation of tail bud mesenchyme (secondary neurulation). In mouse embryos, the secondary neural tube expresses several genes important in early patterning and induction, in restricted domains similar to the primary neural tube, yet it does not undergo neuronal differentiation, but subsequently degenerates. Although the secondary neural tube, isolated from surrounding tissues, is responsive to exogenous Sonic Hedgehog proteins in vitro, motor neuron differentiation is never observed. This cannot be attributed to the properties of the secondary notochord, since it is able to induce motor neuron differentiation in naive chick neural plate explants. Taken together, these results support that the lack of motor neuron differentiation is an intrinsic property of the mouse secondary neural tube.
Asunto(s)
Neuronas Motoras/citología , Tubo Neural/embriología , Animales , Diferenciación Celular/fisiología , Femenino , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos ICR , Neuronas Motoras/metabolismo , Tubo Neural/citología , Ratas , Ratas Sprague-DawleyRESUMEN
IMPORTANCE OF THE FIELD: The Hedgehog (Hh) pathway is required during many developmental events; in adults the Hedgehog pathway is involved in the maintenance of several stem cell niches. It is therefore not surprising that aberrantly regulated Hh pathway activity can cause birth defects in the developing organism, as well as neoplastic disease later in life. AREAS COVERED IN THIS REVIEW: As a consequence of the involvement in pathogenesis, the Hh pathway components are subject to an intense scrutiny as potential targets for therapeutic agents. We aim to provide an overview of the biology of the Hh proteins and the cellular response, in conjunction with potential therapeutic interventions. WHAT THE READER WILL GAIN: Specifically, we focus on the recently discovered non-cell-autonomous Shh signaling used by tumors and the implications of this for the design of treatment strategies. This should provide the reader with up-to-date knowledge on the role of the Hh pathway in tumor progression and the options to treat these malignancies. TAKE HOME MESSAGE: An important concept that we advocate in this review is the need to recognize the need to target both the stromal and the tumor compartment in malignancies that rely on paracrine Shh signaling.
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Proteínas Hedgehog/fisiología , Neoplasias/tratamiento farmacológico , Neoplasias/fisiopatología , Comunicación Paracrina , Acilación/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Endocitosis/efectos de los fármacos , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/fisiología , Ligandos , Comunicación Paracrina/efectos de los fármacos , Comunicación Paracrina/fisiología , Procesamiento Proteico-Postraduccional , Vías Secretoras/efectos de los fármacos , Nicho de Células Madre/efectos de los fármacos , Nicho de Células Madre/fisiopatología , Células del Estroma/efectos de los fármacos , Células del Estroma/fisiologíaRESUMEN
Dispatched 1 (Disp1) encodes a twelve transmembrane domain protein that is required for long-range sonic hedgehog (Shh) signaling. Inhibition of Disp1 function, both by RNAi or dominant-negative constructs, prevents secretion and results in the accumulation of Shh in source cells. Measuring the Shh response in neuralized embryoid bodies (EBs) derived from embryonic stem (ES) cells, with or without Disp1 function, demonstrates an additional role for Disp1 in cells transporting Shh. Co-cultures with Shh-expressing cells revealed a significant reduction in the range of the contact-dependent Shh response in Disp1(-/-) neuralized EBs. These observations support a dual role for Disp1, not only in the secretion of Shh from the source cells, but also in the subsequent transport of Shh through tissue.
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Proteínas Hedgehog/metabolismo , Transducción de Señal , Animales , Western Blotting , Línea Celular , Células Cultivadas , Perros , Células Madre Embrionarias , Proteínas Hedgehog/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Interferencia de ARN , Ratas , Transducción de Señal/genética , Transducción de Señal/fisiología , Vertebrados/fisiologíaRESUMEN
Sonic hedgehog (Shh) is a morphogen involved in many developmental processes. Injection of cells (5E1) that produce a Shh-blocking antibody causes an attenuation of the Shh response, and this causes vascular malformations and impaired remodeling characterized by hemorrhages and protrusions of the anterior cardinal vein and outflow tract, delayed fusion of the dorsal aortae, impaired branching of the internal carotid artery, and delayed remodeling of the aortic arches. Distribution of smooth muscle cells in the vessel wall is unchanged. In 5E1-injected embryos, we also observed impaired assembly of endothelial cells into vascular tubes, particularly in the sixth branchial arch, around the anterior cardinal vein and around the dorsal aorta. In 5E1-treated embryos, increased numbers of macrophage-like cells, apoptotic cells, and a decreased level of proliferation were observed in head mesenchyme. Together, these observations show that Shh signaling is required at multiple stages for proper vessel formation and remodeling.
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Proteínas Aviares/metabolismo , Vasos Sanguíneos/metabolismo , Región Branquial/metabolismo , Proteínas Hedgehog/metabolismo , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Proteínas Aviares/genética , Vasos Sanguíneos/embriología , Región Branquial/embriología , Trasplante de Células , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/inmunología , Hibridomas , Inmunohistoquímica , Hibridación in Situ , Ratones , CodornizRESUMEN
The hedgehog (Hh) pathway is required for many developmental processes, as well as for adult homeostasis. Although all known effects of Hh signaling affecting patterning and differentiation are mediated by members of the Gli family of zinc finger transcription factors, we demonstrate that the Hh-dependent formation of neurites from motor neurons, like migration of fibroblasts, requires leukotriene synthesis and is different from the Gli-mediated Hh response. Smoothened activity is required for the use of the leukotriene metabolism, and inversely, the leukotriene metabolism is required for mediating the Hh effects on neurite projection. These data establish a function for the previously described arachidonic acid-dependent Hh pathway in a developmentally relevant model system.
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Diferenciación Celular , Embrión de Mamíferos/citología , Proteínas Hedgehog/metabolismo , Leucotrienos/biosíntesis , Neuronas Motoras/citología , Neuritas/metabolismo , Animales , Ácido Araquidónico/farmacología , Diferenciación Celular/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/enzimología , Inhibidores Enzimáticos/farmacología , Proteínas Hedgehog/agonistas , Técnicas In Vitro , Indoles/farmacología , Factores de Transcripción de Tipo Kruppel/metabolismo , Inhibidores de la Lipooxigenasa , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/enzimología , Neuritas/efectos de los fármacos , Neuritas/enzimología , Codorniz , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Tubulina (Proteína)/metabolismo , Proteína con Dedos de Zinc GLI1RESUMEN
Sonic hedgehog (Shh) is a morphogen pivotal for development and tissue maintenance. Biological effects of Shh are mediated through a pathway that involves binding to patched1 (Ptch1), thereby releasing Smoothened (Smo) from inhibition resulting in the activation of Gli transcription factors, which mediate the induction of Shh target genes. Here, we describe a novel signal transduction pathway for Shh, which is transcription/translation-independent, SuFu insensitive, and consequently independent of Gli-mediated induction of transcription. Through this alternative pathway Shh, transduced via Smo, induced altered cell morphology together with lamellipodia formation. Migration assays demonstrate that this cytoskeletal rearrangement mediates the migratory response to Shh. This Shh-induced, Smo mediated migration utilizes and requires the metabolism of arachidonic acid through the 5-lipoxygenase pathway. These data provide a link between a seemingly novel Gli-independent Hh signaling pathway and the leukotriene metabolism, and might explain the developmental abnormalities observed in both patients with defective leukotriene metabolism as well as in rodent models of defective Rho family GTPase signaling.
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Ácido Araquidónico/metabolismo , Movimiento Celular , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Transducción de Señal , Actinas/metabolismo , Animales , Araquidonato 5-Lipooxigenasa/metabolismo , Línea Celular , Forma de la Célula , Quimiotaxis , Fibroblastos/enzimología , Cinética , Leucotrienos/metabolismo , Ratones , Ratones Endogámicos C3H , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Seudópodos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Receptor Smoothened , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética , Transfección , Proteína con Dedos de Zinc GLI1RESUMEN
During development of the neural tube, inhibition of the Notch response as well as the activation of the Sonic Hedgehog (Shh) response results in the formation of neuronal cell types. To determine whether Shh and Notch act independently, we tested the effects of the Notch inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) on neuralized, embryonic stem (ES) cell-derived embryoid bodies (EBs), while varying the levels of Shh pathway activation. Shh-resistant EBs were derived from Smo null ES cells, while EBs with constitutive high level of Shh pathway activation were derived from Ptc1 null ES cells. Intermediate levels of Shh pathway activation was achieved by the addition of ShhN to the EB culture medium. It was found that DAPT-mediated inhibition of the Notch response resulted in enhanced neuronal differentiation. In the absence of Shh, more interneurons were detected, while the main effect of DAPT on EBs with an activated Shh response was the precocious loss of ventral neuronal precursor-specific markers.