RESUMO
During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.
Assuntos
Estruturas da Membrana Celular , Miosinas , Tubo Neural , Transdução de Sinais , Animais , Camundongos , Transporte Biológico , Estruturas da Membrana Celular/metabolismo , Proteínas Hedgehog/metabolismo , Miosinas/metabolismo , Pseudópodes/metabolismo , Tubo Neural/citologia , Tubo Neural/metabolismoRESUMO
Ligands of the Hedgehog (HH) pathway are paracrine signaling molecules that coordinate tissue development in metazoans. A remarkable feature of HH signaling is the repeated use of cholesterol in steps spanning ligand biogenesis, secretion, dispersal, and reception on target cells. A cholesterol molecule covalently attached to HH ligands is used as a molecular baton by transfer proteins to guide their secretion, spread, and reception. On target cells, a signaling circuit composed of a cholesterol transporter and sensor regulates transmission of HH signals across the plasma membrane to the cytoplasm. The repeated use of cholesterol in signaling supports the view that the HH pathway likely evolved by coopting ancient systems to regulate the abundance or organization of sterol-like lipids in membranes.
Assuntos
Colesterol , Proteínas Hedgehog , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ligantes , Colesterol/metabolismo , Transdução de Sinais , Esteróis/metabolismoRESUMO
Hedgehog pathway components and select G protein-coupled receptors (GPCRs) localize to the primary cilium, an organelle specialized for signal transduction. We investigated whether cells distinguish between ciliary and extraciliary GPCR signaling. To test whether ciliary and extraciliary cyclic AMP (cAMP) convey different information, we engineered optogenetic and chemogenetic tools to control the subcellular site of cAMP generation. Generating equal amounts of ciliary and cytoplasmic cAMP in zebrafish and mammalian cells revealed that ciliary cAMP, but not cytoplasmic cAMP, inhibited Hedgehog signaling. Modeling suggested that the distinct geometries of the cilium and cell body differentially activate local effectors. The search for effectors identified a ciliary pool of protein kinase A (PKA). Blocking the function of ciliary PKA, but not extraciliary PKA, activated Hedgehog signal transduction and reversed the effects of ciliary cAMP. Therefore, cells distinguish ciliary and extraciliary cAMP using functionally and spatially distinct pools of PKA, and different subcellular pools of cAMP convey different information.
Assuntos
Cílios/metabolismo , AMP Cíclico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Neurônios/metabolismo , Optogenética/métodos , Transdução de Sinais/fisiologia , Peixe-Zebra/metabolismoRESUMO
Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.
Assuntos
Elementos Facilitadores Genéticos/genética , Ensaios de Triagem em Larga Escala/métodos , Polidactilia/genética , Animais , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Introdução de Genes/métodos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Mutação , Fenótipo , Polidactilia/metabolismo , RNA não Traduzido/genéticaRESUMO
Understanding transcription factor navigation through the nucleus remains critical for developing targeted therapeutics. The GLI1 transcription factor must maintain maximal Hedgehog pathway output in basal cell carcinomas (BCCs), and we have previously shown that resistant BCCs increase GLI1 deacetylation through atypical protein kinase Cι/λ (aPKC) and HDAC1. Here we identify a lamina-associated polypeptide 2 (LAP2) isoform-dependent nuclear chaperoning system that regulates GLI1 movement between the nuclear lamina and nucleoplasm to achieve maximal activation. LAP2ß forms a two-site interaction with the GLI1 zinc-finger domain and acetylation site, stabilizing an acetylation-dependent reserve on the inner nuclear membrane (INM). By contrast, the nucleoplasmic LAP2α competes with LAP2ß for GLI1 while scaffolding HDAC1 to deacetylate the secondary binding site. aPKC functions to promote GLI1 association with LAP2α, promoting egress off the INM. GLI1 intranuclear trafficking by LAP2 isoforms represents a powerful signal amplifier in BCCs with implications for zinc finger-based signal transduction and therapeutics.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Células 3T3 , Animais , Carcinoma Basocelular/metabolismo , Linhagem Celular , Cromatina , Proteínas de Ligação a DNA/fisiologia , Células HEK293 , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/fisiologia , Histona Desacetilase 1/metabolismo , Humanos , Proteínas de Membrana/fisiologia , Camundongos , Chaperonas Moleculares/metabolismo , Lâmina Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de Zinco/fisiologia , Dedos de ZincoRESUMO
The gastrointestinal tract is enveloped by concentric and orthogonally aligned layers of smooth muscle; however, an understanding of the mechanisms by which these muscles become patterned and aligned in the embryo has been lacking. We find that Hedgehog acts through Bmp to delineate the position of the circumferentially oriented inner muscle layer, whereas localized Bmp inhibition is critical for allowing formation of the later-forming, longitudinally oriented outer layer. Because the layers form at different developmental stages, the muscle cells are exposed to unique mechanical stimuli that direct their alignments. Differential growth within the early gut tube generates residual strains that orient the first layer circumferentially, and when formed, the spontaneous contractions of this layer align the second layer longitudinally. Our data link morphogen-based patterning to mechanically controlled smooth muscle cell alignment and provide a mechanistic context for potentially understanding smooth muscle organization in a wide variety of tubular organs.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mucosa Intestinal/crescimento & desenvolvimento , Desenvolvimento Muscular/genética , Músculo Liso/crescimento & desenvolvimento , Miócitos de Músculo Liso/metabolismo , Animais , Padronização Corporal/fisiologia , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Embrião de Galinha , Embrião de Mamíferos , Feminino , Proteínas Hedgehog/metabolismo , Masculino , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Transdução de Sinais/fisiologiaRESUMO
The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition.
Assuntos
Proteínas Hedgehog/metabolismo , Receptor Smoothened/química , Proteínas de Xenopus/química , Regulação Alostérica , Animais , Sítios de Ligação , Linhagem Celular , Colesterol/química , Colesterol/metabolismo , Cristalografia por Raios X , Citometria de Fluxo , Proteínas Hedgehog/genética , Humanos , Camundongos , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química , Alcaloides de Veratrum/metabolismo , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.
Assuntos
Colesterol/metabolismo , Proteínas Hedgehog/metabolismo , Receptor Patched-1/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Microscopia Crioeletrônica , Dimerização , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Células HEK293 , Proteínas Hedgehog/química , Proteínas Hedgehog/genética , Humanos , Camundongos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Receptor Patched-1/química , Receptor Patched-1/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Transdução de SinaisRESUMO
Aberrant tissue-immune interactions are the hallmark of diverse chronic lung diseases. Here, we sought to define these interactions in emphysema, a progressive disease characterized by infectious exacerbations and loss of alveolar epithelium. Single-cell analysis of human emphysema lungs revealed the expansion of tissue-resident lymphocytes (TRLs). Murine studies identified a stromal niche for TRLs that expresses Hhip, a disease-variant gene downregulated in emphysema. Stromal-specific deletion of Hhip induced the topographic expansion of TRLs in the lung that was mediated by a hyperactive hedgehog-IL-7 axis. 3D immune-stem cell organoids and animal models of viral exacerbations demonstrated that expanded TRLs suppressed alveolar stem cell growth through interferon gamma (IFNγ). Finally, we uncovered an IFNγ-sensitive subset of human alveolar stem cells that was preferentially lost in emphysema. Thus, we delineate a stromal-lymphocyte-epithelial stem cell axis in the lung that is modified by a disease-variant gene and confers host susceptibility to emphysema.
Assuntos
Enfisema , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Humanos , Camundongos , Animais , Enfisema Pulmonar/genética , Pulmão , Linfócitos , Células-TroncoRESUMO
Signaling receptors dynamically exit cilia upon activation of signaling pathways such as Hedgehog. Here, we find that when activated G protein-coupled receptors (GPCRs) fail to undergo BBSome-mediated retrieval from cilia back into the cell, these GPCRs concentrate into membranous buds at the tips of cilia before release into extracellular vesicles named ectosomes. Unexpectedly, actin and the actin regulators drebrin and myosin 6 mediate ectosome release from the tip of cilia. Mirroring signal-dependent retrieval, signal-dependent ectocytosis is a selective and effective process that removes activated signaling molecules from cilia. Congruently, ectocytosis compensates for BBSome defects as ectocytic removal of GPR161, a negative regulator of Hedgehog signaling, permits the appropriate transduction of Hedgehog signals in Bbs mutants. Finally, ciliary receptors that lack retrieval determinants such as the anorexigenic GPCR NPY2R undergo signal-dependent ectocytosis in wild-type cells. Our data show that signal-dependent ectocytosis regulates ciliary signaling in physiological and pathological contexts.
Assuntos
Cílios/metabolismo , Vesículas Extracelulares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Humanos , Rim/citologia , Rim/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Receptores de Somatostatina/metabolismo , Transdução de SinaisRESUMO
Injured skeletal muscle regenerates, but with age or in muscular dystrophies, muscle is replaced by fat. Upon injury, muscle-resident fibro/adipogenic progenitors (FAPs) proliferated and gave rise to adipocytes. These FAPs dynamically produced primary cilia, structures that transduce intercellular cues such as Hedgehog (Hh) signals. Genetically removing cilia from FAPs inhibited intramuscular adipogenesis, both after injury and in a mouse model of Duchenne muscular dystrophy. Blocking FAP ciliation also enhanced myofiber regeneration after injury and reduced myofiber size decline in the muscular dystrophy model. Hh signaling through FAP cilia regulated the expression of TIMP3, a secreted metalloproteinase inhibitor, that inhibited MMP14 to block adipogenesis. A pharmacological mimetic of TIMP3 blocked the conversion of FAPs into adipocytes, pointing to a strategy to combat fatty degeneration of skeletal muscle. We conclude that ciliary Hh signaling by FAPs orchestrates the regenerative response to skeletal muscle injury.
Assuntos
Adipogenia , Proteínas Hedgehog/metabolismo , Músculo Esquelético/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Adipócitos/metabolismo , Animais , Cílios/metabolismo , Distrofina/genética , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Desenvolvimento Muscular , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Regeneração , Inibidor Tecidual de Metaloproteinase-3/metabolismoRESUMO
Liver cancer has the second highest worldwide cancer mortality rate and has limited therapeutic options. We analyzed 363 hepatocellular carcinoma (HCC) cases by whole-exome sequencing and DNA copy number analyses, and we analyzed 196 HCC cases by DNA methylation, RNA, miRNA, and proteomic expression also. DNA sequencing and mutation analysis identified significantly mutated genes, including LZTR1, EEF1A1, SF3B1, and SMARCA4. Significant alterations by mutation or downregulation by hypermethylation in genes likely to result in HCC metabolic reprogramming (ALB, APOB, and CPS1) were observed. Integrative molecular HCC subtyping incorporating unsupervised clustering of five data platforms identified three subtypes, one of which was associated with poorer prognosis in three HCC cohorts. Integrated analyses enabled development of a p53 target gene expression signature correlating with poor survival. Potential therapeutic targets for which inhibitors exist include WNT signaling, MDM4, MET, VEGFA, MCL1, IDH1, TERT, and immune checkpoint proteins CTLA-4, PD-1, and PD-L1.
Assuntos
Carcinoma Hepatocelular/genética , Genômica , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/virologia , Metilação de DNA , Humanos , Isocitrato Desidrogenase/genética , Neoplasias Hepáticas/virologia , MicroRNAs/genética , MutaçãoRESUMO
The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP.
Assuntos
Evolução Biológica , Elementos Facilitadores Genéticos , Extremidades/crescimento & desenvolvimento , Proteínas Hedgehog/genética , Serpentes/genética , Animais , Sequência de Bases , Evolução Molecular , Técnicas de Introdução de Genes , Camundongos , Camundongos Transgênicos , Mutação , Filogenia , Serpentes/classificaçãoRESUMO
Communication between cells pervades the development and physiology of metazoans. In animals, this process is carried out by a relatively small number of signaling pathways, each consisting of a chain of biochemical events through which extracellular stimuli control the behavior of target cells. One such signaling system is the Hedgehog pathway, which is crucial in embryogenesis and is implicated in many birth defects and cancers. Although Hedgehog pathway components were identified by genetic analysis more than a decade ago, our understanding of the molecular mechanisms of signaling is far from complete. In this review, we focus on the biochemistry and cell biology of the Hedgehog pathway. We examine the unique biosynthesis of the Hedgehog ligand, its specialized release from cells into extracellular space, and the poorly understood mechanisms involved in ligand reception and pathway activation at the surface of target cells. We highlight several critical questions that remain open.
Assuntos
Proteínas Hedgehog/metabolismo , Transdução de Sinais , Animais , Retroalimentação Fisiológica , Humanos , Ligantes , Modelos BiológicosRESUMO
The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
Assuntos
Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/metabolismo , Acilação , Aciltransferases/genética , Aciltransferases/ultraestrutura , Regulação Alostérica , Animais , Células COS , Domínio Catalítico , Chlorocebus aethiops , Microscopia Crioeletrônica , Células HEK293 , Heme/metabolismo , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Simulação de Dinâmica Molecular , Palmitoil Coenzima A/metabolismo , Conformação Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
The direction of left-right visceral asymmetry is conserved in vertebrates. Deviations of the standard asymmetric pattern are rare, and the underlying mechanisms are not understood. Here, we use the teleost Astyanax mexicanus, consisting of surface fish with normal left-oriented heart asymmetry and cavefish with high levels of reversed right-oriented heart asymmetry, to explore natural changes in asymmetry determination. We show that Sonic Hedgehog (Shh) signaling is increased at the posterior midline, Kupffer's vesicle (the teleost left-right organizer) is enlarged and contains longer cilia, and the number of dorsal forerunner cells is increased in cavefish. Furthermore, Shh increase in surface fish embryos induces asymmetric changes resembling the cavefish phenotype. Asymmetric expression of the Nodal antagonist Dand5 is equalized or reversed in cavefish, and Shh increase in surface fish mimics changes in cavefish dand5 asymmetry. Shh decrease reduces the level of right-oriented heart asymmetry in cavefish. Thus, naturally occurring modifications in cavefish heart asymmetry are controlled by the effects of Shh signaling on left-right organizer function.
Assuntos
Padronização Corporal , Coração , Proteínas Hedgehog , Transdução de Sinais , Animais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Padronização Corporal/genética , Coração/embriologia , Characidae/embriologia , Characidae/genética , Regulação da Expressão Gênica no Desenvolvimento , Cílios/metabolismo , Embrião não Mamífero/metabolismoRESUMO
A growing wealth of data suggest that reactive oxygen species (ROS) signalling might be crucial in conferring embryonic or adult stem cells their specific properties. However, how stem cells control ROS production and scavenging, and how ROS in turn contribute to stemness, remain poorly understood. Using the Xenopus retina as a model system, we first investigated the redox status of retinal stem cells (RSCs). We discovered that they exhibit higher ROS levels compared with progenitors and retinal neurons, and express a set of specific redox genes. We next addressed the question of ROS functional involvement in these cells. Using pharmacological or genetic tools, we demonstrate that inhibition of NADPH oxidase-dependent ROS production increases the proportion of quiescent RSCs. Surprisingly, this is accompanied by an apparent acceleration of the mean division speed within the remaining proliferating pool. Our data further unveil that such impact on RSC cell cycling is achieved by modulation of the Wnt/Hedgehog signalling balance. Altogether, we highlight that RSCs exhibit distinctive redox characteristics and exploit NADPH oxidase signalling to limit quiescence and fine-tune their proliferation rate.
Assuntos
Células-Tronco Adultas , Células-Tronco Neurais , Animais , Xenopus laevis/metabolismo , Espécies Reativas de Oxigênio , Proliferação de Células , Proteínas Hedgehog , Retina/metabolismo , Células-Tronco Adultas/metabolismo , NADPH Oxidases/genética , Via de Sinalização WntRESUMO
Ciliopathies are characterized by the absence or dysfunction of primary cilia. Despite the fact that cognitive impairments are a common feature of ciliopathies, how cilia dysfunction affects neuronal development has not been characterized in detail. Here, we show that primary cilium-mediated signaling is required cell-autonomously by neurons during neural circuit formation. In particular, a functional primary cilium is crucial during axonal pathfinding for the switch in responsiveness of axons at a choice point, or intermediate target. Utilizing different animal models and in vivo, ex vivo, as well as in vitro experiments, we provide evidence for a critical role of primary cilium-mediated signaling in long-range axon guidance. The primary cilium on the cell body of commissural neurons transduces long-range guidance signals sensed by growth cones navigating an intermediate target. In extension of our finding that Shh is required for the rostral turn of post-crossing commissural axons, we suggest a model implicating the primary cilium in Shh signaling upstream of a transcriptional change of axon guidance receptors, which in turn mediate the repulsive response to floorplate-derived Shh shown by post-crossing commissural axons.
RESUMO
Enhancers can regulate the promoters of their target genes over very large genomic distances. It is widely assumed that mechanisms of enhancer action involve the reorganization of three-dimensional chromatin architecture, but this is poorly understood. The predominant model involves physical enhancer-promoter interaction by looping out the intervening chromatin. However, studying the enhancer-driven activation of the Sonic hedgehog gene (Shh), we have identified a change in chromosome conformation that is incompatible with this simple looping model. Using super-resolution 3D-FISH and chromosome conformation capture, we observe a decreased spatial proximity between Shh and its enhancers during the differentiation of embryonic stem cells to neural progenitors. We show that this can be recapitulated by synthetic enhancer activation, is impeded by chromatin-bound proteins located between the enhancer and the promoter, and appears to involve the catalytic activity of poly (ADP-ribose) polymerase. Our data suggest that models of enhancer-promoter communication need to encompass chromatin conformations other than looping.
Assuntos
Montagem e Desmontagem da Cromatina , Elementos Facilitadores Genéticos , Proteínas Hedgehog/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Neurogênese , Neurônios/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional , Animais , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Camundongos , Modelos Genéticos , Neurogênese/genética , Conformação de Ácido Nucleico , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Animals use a small number of morphogens to pattern tissues, but it is unclear how evolution modulates morphogen signaling range to match tissues of varying sizes. Here, we used single-molecule imaging in reconstituted morphogen gradients and in tissue explants to determine that Hedgehog diffused extracellularly as a monomer, and rapidly transitioned between membrane-confined and -unconfined states. Unexpectedly, the vertebrate-specific protein SCUBE1 expanded Hedgehog gradients by accelerating the transition rates between states without affecting the relative abundance of molecules in each state. This observation could not be explained under existing models of morphogen diffusion. Instead, we developed a topology-limited diffusion model in which cell-cell gaps create diffusion barriers, which morphogens can only overcome by passing through a membrane-unconfined state. Under this model, SCUBE1 promoted Hedgehog secretion and diffusion by allowing it to transiently overcome diffusion barriers. This multiscale understanding of morphogen gradient formation unified prior models and identified knobs that nature can use to tune morphogen gradient sizes across tissues and organisms.