RESUMEN
The chronologically ordered generation of distinct cell types is essential for the establishment of neuronal diversity and the formation of neuronal circuits. Recently, single-cell transcriptomic analyses of various areas of the developing vertebrate nervous system have provided evidence for the existence of a shared temporal patterning program that partitions neurons based on the timing of neurogenesis. In this review, I summarize the findings that lead to the proposal of this shared temporal program before focusing on the developing spinal cord to discuss how temporal patterning in general and this program specifically contributes to the ordered formation of neuronal circuits.
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Tipificación del Cuerpo , Regulación del Desarrollo de la Expresión Génica , Tubo Neural , Neurogénesis , Médula Espinal , Vertebrados , Animales , Tubo Neural/crecimiento & desarrollo , Neurogénesis/genética , Vertebrados/crecimiento & desarrollo , Vertebrados/genética , Vertebrados/embriología , Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica/genética , Médula Espinal/crecimiento & desarrollo , Médula Espinal/embriología , Neuronas/citología , Neuronas/metabolismo , HumanosRESUMEN
Neuronal heterogeneity has been established as a pillar of higher central nervous system function, but glial heterogeneity and its implications for neural circuit function are poorly understood. Here we show that the adult mouse dentate gyrus (DG) of the hippocampus is populated by molecularly distinct astrocyte subtypes that are associated with distinct DG layers. Astrocytes localized to different DG compartments also exhibit subtype-specific morphologies. Physiologically, astrocytes in upper DG layers form large syncytia, while those in lower DG compartments form smaller networks. Astrocyte subtypes differentially express glutamate transporters, which is associated with different amplitudes of glutamate transporter-mediated currents. Key molecular and morphological features of astrocyte diversity in the mice DG are conserved in humans. This adds another layer of complexity to our understanding of brain network composition and function, which will be crucial for further studies on astrocytes in health and disease.
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Astrocitos , Neuroglía , Adulto , Humanos , Animales , Ratones , Hipocampo , Encéfalo , Giro DentadoRESUMEN
The molecular mechanisms that produce the full array of neuronal subtypes in the vertebrate nervous system are incompletely understood. Here, we provide evidence of a global temporal patterning program comprising sets of transcription factors that stratifies neurons based on the developmental time at which they are generated. This transcriptional code acts throughout the central nervous system, in parallel to spatial patterning, thereby increasing the diversity of neurons generated along the neuraxis. We further demonstrate that this temporal program operates in stem cell-derived neurons and is under the control of the TGFß signaling pathway. Targeted perturbation of components of the temporal program, Nfia and Nfib, reveals their functional requirement for the generation of late-born neuronal subtypes. Together, our results provide evidence for the existence of a previously unappreciated global temporal transcriptional program of neuronal subtype identity and suggest that the integration of spatial and temporal patterning mechanisms diversifies and organizes neuronal subtypes in the vertebrate nervous system.
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Tipificación del Cuerpo/genética , Sistema Nervioso Central/metabolismo , Transcripción Genética , Animales , Encéfalo/citología , Regulación del Desarrollo de la Expresión Génica , Ratones , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Neuronas/fisiología , Retina/citología , Transducción de Señal , Médula Espinal/citología , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
During development, gene regulatory networks allocate cell fates by partitioning tissues into spatially organised domains of gene expression. How the sharp boundaries that delineate these gene expression patterns arise, despite the stochasticity associated with gene regulation, is poorly understood. We show, in the vertebrate neural tube, using perturbations of coding and regulatory regions, that the structure of the regulatory network contributes to boundary precision. This is achieved, not by reducing noise in individual genes, but by the configuration of the network modulating the ability of stochastic fluctuations to initiate gene expression changes. We use a computational screen to identify network properties that influence boundary precision, revealing two dynamical mechanisms by which small gene circuits attenuate the effect of noise in order to increase patterning precision. These results highlight design principles of gene regulatory networks that produce precise patterns of gene expression.
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Tipificación del Cuerpo/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Animales , Biomarcadores , Desarrollo Embrionario , Elementos de Facilitación Genéticos , Ratones , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Secuencias Reguladoras de Ácido RibonucleicoRESUMEN
The vertebrate spinal cord comprises multiple functionally distinct neuronal cell types arranged in characteristic positions. During development, these different types of neurons differentiate from transcriptionally distinct neural progenitors that are arrayed in discrete domains along the dorsal-ventral and anterior-posterior axes of the embryonic spinal cord. This organization arises in response to morphogen gradients acting upstream of a gene regulatory network, the architecture of which determines the spatial and temporal pattern of gene expression. In recent years, substantial progress has been made in deciphering the regulatory network that underlies the specification of distinct progenitor and neuronal cell identities. In this Review, we outline how distinct neuronal cell identities are established in response to spatial and temporal patterning systems, and outline novel experimental approaches to study the emergence and function of neuronal diversity in the spinal cord.
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Tipificación del Cuerpo , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Neuronas/citología , Médula Espinal/embriología , Animales , Linaje de la Célula , Proliferación Celular , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Ratones , Morfogénesis , Tubo Neural/embriología , Transducción de Señal , Células Madre/citología , Transcripción Genética , Pez CebraRESUMEN
The coordinated spatial and temporal regulation of gene expression in the vertebrate neural tube determines the identity of neural progenitors and the function and physiology of the neurons they generate. Progress has been made deciphering the gene regulatory programmes that are responsible for this process; however, the complexity of the tissue has hampered the systematic analysis of the network and the underlying mechanisms. To address this, we used single cell mRNA sequencing to profile cervical and thoracic regions of the developing mouse neural tube between embryonic days 9.5-13.5. We confirmed that the data accurately recapitulates neural tube development, allowing us to identify new markers for specific progenitor and neuronal populations. In addition, the analysis highlighted a previously underappreciated temporal component to the mechanisms that generate neuronal diversity, and revealed common features in the sequence of transcriptional events that lead to the differentiation of specific neuronal subtypes. Together, the data offer insight into the mechanisms that are responsible for neuronal specification and provide a compendium of gene expression for classifying spinal cord cell types that will support future studies of neural tube development, function and disease.
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Regulación del Desarrollo de la Expresión Génica , Análisis de la Célula Individual , Médula Espinal/embriología , Transcriptoma , Animales , Diferenciación Celular/genética , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Interneuronas/metabolismo , Masculino , Ratones , Tubo Neural/embriología , Neuronas/metabolismo , Organogénesis , ARN Mensajero/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
The morphogen Sonic Hedgehog (SHH) patterns tissues during development by directing cell fates in a concentration-dependent manner. The SHH signal is transmitted across the membrane of target cells by the heptahelical transmembrane protein Smoothened (SMO), which activates the GLI family of transcription factors through a mechanism that is undefined in vertebrates. Using CRISPR-edited null alleles and small-molecule inhibitors, we systematically analyzed the epistatic interactions between SMO and three proteins implicated in SMO signaling: the heterotrimeric G protein subunit GαS, the G protein-coupled receptor kinase 2 (GRK2), and the GαS-coupled receptor GPR161. Our experiments uncovered a signaling mechanism that modifies the sensitivity of target cells to SHH and consequently changes the shape of the SHH dose-response curve. In both fibroblasts and spinal neural progenitors, the loss of GPR161, previously implicated as an inhibitor of basal SHH signaling, increased the sensitivity of target cells across the entire spectrum of SHH concentrations. Even in cells lacking GPR161, GRK2 was required for SHH signaling, and Gαs, which promotes the activation of protein Kinase A (PKA), antagonized SHH signaling. We propose that the sensitivity of target cells to Hedgehog morphogens, and the consequent effects on gene expression and differentiation outcomes, can be controlled by signals from G protein-coupled receptors that converge on Gαs and PKA.
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Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Receptor Smoothened/metabolismo , Animales , Células Cultivadas , Cromograninas/genética , Cromograninas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Epistasis Genética , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Ratones , Células 3T3 NIH , Receptores Acoplados a Proteínas G/genética , Receptor Smoothened/genéticaRESUMEN
During tissue development, multipotent progenitors differentiate into specific cell types in characteristic spatial and temporal patterns. We addressed the mechanism linking progenitor identity and differentiation rate in the neural tube, where motor neuron (MN) progenitors differentiate more rapidly than other progenitors. Using single cell transcriptomics, we defined the transcriptional changes associated with the transition of neural progenitors into MNs. Reconstruction of gene expression dynamics from these data indicate a pivotal role for the MN determinant Olig2 just prior to MN differentiation. Olig2 represses expression of the Notch signaling pathway effectors Hes1 and Hes5. Olig2 repression of Hes5 appears to be direct, via a conserved regulatory element within the Hes5 locus that restricts expression from MN progenitors. These findings reveal a tight coupling between the regulatory networks that control patterning and neuronal differentiation and demonstrate how Olig2 acts as the developmental pacemaker coordinating the spatial and temporal pattern of MN generation.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Ciclo Celular/genética , Neuronas Motoras/citología , Neurogénesis/genética , Factor de Transcripción 2 de los Oligodendrocitos/fisiología , Proteínas Represoras/fisiología , Análisis de la Célula Individual , Factor de Transcripción HES-1/fisiología , Transcriptoma , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Colorantes Fluorescentes/metabolismo , Regulación de la Expresión Génica/fisiología , Genes Reporteros , Interneuronas/citología , Ratones Transgénicos , Factor de Transcripción 2 de los Oligodendrocitos/genética , Receptores Notch/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Represoras/genética , Transducción de Señal , Factor de Transcripción HES-1/genéticaRESUMEN
To uncover regulatory mechanisms in Hedgehog (Hh) signaling, we conducted genome-wide screens to identify positive and negative pathway components and validated top hits using multiple signaling and differentiation assays in two different cell types. Most positive regulators identified in our screens, including Rab34, Pdcl, and Tubd1, were involved in ciliary functions, confirming the central role for primary cilia in Hh signaling. Negative regulators identified included Megf8, Mgrn1, and an unannotated gene encoding a tetraspan protein we named Atthog. The function of these negative regulators converged on Smoothened (SMO), an oncoprotein that transduces the Hh signal across the membrane. In the absence of Atthog, SMO was stabilized at the cell surface and concentrated in the ciliary membrane, boosting cell sensitivity to the ligand Sonic Hedgehog (SHH) and consequently altering SHH-guided neural cell-fate decisions. Thus, we uncovered genes that modify the interpretation of morphogen signals by regulating protein-trafficking events in target cells.
Asunto(s)
Cilios/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas Hedgehog/farmacología , Neuronas/fisiología , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Cilios/efectos de los fármacos , Fluorescencia , Proteínas de la Membrana/metabolismo , Ratones , Células 3T3 NIH , Neuronas/citología , Neuronas/efectos de los fármacos , Receptor Smoothened/genéticaRESUMEN
MOTIVATION: A significant focus of biological research is to understand the development, organization and function of tissues. A particularly productive area of study is on single layer epithelial tissues in which the adherence junctions of cells form a 2D manifold that is fluorescently labeled. Given the size of the tissue, a microscope must collect a mosaic of overlapping 3D stacks encompassing the stained surface. Downstream interpretation is greatly simplified by preprocessing such a dataset as follows: (i) extracting and mapping the stained manifold in each stack into a single 2D projection plane, (ii) correcting uneven illumination artifacts, (iii) stitching the mosaic planes into a single, large 2D image and (iv) adjusting the contrast. RESULTS: We have developed PreMosa, an efficient, fully automatic pipeline to perform the four preprocessing tasks above resulting in a single 2D image of the stained manifold across which contrast is optimized and illumination is even. Notable features are as follows. First, the 2D projection step employs a specially developed algorithm that actually finds the manifold in the stack based on maximizing contrast, intensity and smoothness. Second, the projection step comes first, implying all subsequent tasks are more rapidly solved in 2D. And last, the mosaic melding employs an algorithm that globally adjusts contrasts amongst the 2D tiles so as to produce a seamless, high-contrast image. We conclude with an evaluation using ground-truth datasets and present results on datasets from Drosophila melanogaster wings and Schmidtae mediterranea ciliary components. AVAILABILITY AND IMPLEMENTATION: PreMosa is available under https://cblasse.github.io/premosa. CONTACT: blasse@mpi-cbg.de or myers@mpi-cbg.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Microscopía/métodos , Programas Informáticos , Algoritmos , Animales , Artefactos , Cilios/ultraestructura , Drosophila melanogaster/anatomía & histología , Platelmintos/ultraestructura , Alas de Animales/anatomía & histologíaRESUMEN
Tissue patterning during animal development is orchestrated by a handful of inductive signals. Most of these developmental cues act as morphogens, meaning they are locally produced secreted molecules that act at a distance to govern tissue patterning. The iterative use of the same signaling molecules in different developmental contexts demands that signal interpretation occurs in a highly context-dependent manner. Hence the interpretation of signal depends on the specific competence of the receiving cells. Moreover, it has become clear that the differential interpretation of morphogens depends not only on the level of signaling but also the signaling dynamics, particularly the duration of signaling. In this review, we outline molecular mechanisms proposed in recent studies that explain how the response to morphogens is determined by differential competence, pathway intrinsic feedback, and the interpretation of signaling dynamics by gene regulatory networks. WIREs Dev Biol 2017, 6:e271. doi: 10.1002/wdev.271 For further resources related to this article, please visit the WIREs website.
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Tipificación del Cuerpo/fisiología , Redes Reguladoras de Genes , Morfogénesis/fisiología , Transducción de Señal , Animales , Transporte Biológico , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility.
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Colesterol/metabolismo , Erizos/metabolismo , Transducción de Señal , Receptor Smoothened/agonistas , Animales , Línea Celular , Células Epiteliales/fisiología , Fibroblastos/fisiología , Humanos , RatonesRESUMEN
Membrane trafficking is key to the cell biological mechanisms underlying development. Rab GTPases control specific membrane compartments, from core secretory and endocytic machinery to less-well-understood compartments. We tagged all 27 Drosophila Rabs with YFP(MYC) at their endogenous chromosomal loci, determined their expression and subcellular localization in six tissues comprising 23 cell types, and provide this data in an annotated, searchable image database. We demonstrate the utility of these lines for controlled knockdown and show that similar subcellular localization can predict redundant functions. We exploit this comprehensive resource to ask whether a common Rab compartment architecture underlies epithelial polarity. Strikingly, no single arrangement of Rabs characterizes the five epithelia we examine. Rather, epithelia flexibly polarize Rab distribution, producing membrane trafficking architectures that are tissue- and stage-specific. Thus, the core machinery responsible for epithelial polarization is unlikely to rely on polarized positioning of specific Rab compartments.
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Membrana Celular/metabolismo , Movimiento Celular/fisiología , Drosophila melanogaster/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Epitelio/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Transporte de Proteínas/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
The core components of the planar cell polarity (PCP) signaling system, including both transmembrane and peripheral membrane associated proteins, form asymmetric complexes that bridge apical intercellular junctions. While these can assemble in either orientation, coordinated cell polarization requires the enrichment of complexes of a given orientation at specific junctions. This might occur by both positive and negative feedback between oppositely oriented complexes, and requires the peripheral membrane associated PCP components. However, the molecular mechanisms underlying feedback are not understood. We find that the E3 ubiquitin ligase complex Cullin1(Cul1)/SkpA/Supernumerary limbs(Slimb) regulates the stability of one of the peripheral membrane components, Prickle (Pk). Excess Pk disrupts PCP feedback and prevents asymmetry. We show that Pk participates in negative feedback by mediating internalization of PCP complexes containing the transmembrane components Van Gogh (Vang) and Flamingo (Fmi), and that internalization is activated by oppositely oriented complexes within clusters. Pk also participates in positive feedback through an unknown mechanism promoting clustering. Our results therefore identify a molecular mechanism underlying generation of asymmetry in PCP signaling.
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Polaridad Celular , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Endocitosis , Proteínas con Dominio LIM/metabolismo , Ubiquitinación , Animales , Análisis por Conglomerados , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas con Dominio LIM/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Alas de Animales/metabolismoRESUMEN
BACKGROUND: The conserved Fat and Core planar cell polarity (PCP) pathways work together to specify tissue-wide orientation of hairs and ridges in the Drosophila wing. Their components form intracellularly polarized complexes at adherens junctions that couple the polarity of adjacent cells and form global patterns. How Fat and Core PCP systems interact is not understood. Some studies suggest that Fat PCP directly orients patterns formed by Core PCP components. Others implicate oriented tissue remodeling in specifying Core PCP patterns. RESULTS: We use genetics, quantitative image analysis, and physical modeling to study Fat and Core PCP interactions during wing development. We show their patterns change during morphogenesis, undergoing phases of coupling and uncoupling that are regulated by antagonistic Core PCP protein isoforms Prickle and Spiny-legs. Evolving patterns of Core PCP are hysteretic: the early Core PCP pattern is modified by tissue flows and then by coupling to Fat PCP, producing sequential patterns that guide hairs and then ridges. Our data quantitatively account for altered hair and ridge polarity patterns in PCP mutants. Premature coupling between Fat and Core PCP explains altered polarity patterns in pk mutants. In other Core PCP mutants, hair polarity patterns are guided directly by Fat PCP. When both systems fail, hairs still align locally and obey signals associated with veins. CONCLUSIONS: Temporally regulated coupling between the Fat and Core PCP systems enables a single tissue to develop sequential polarity patterns that orient distinct morphological structures.
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Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas con Dominio LIM/genética , Transducción de Señal , Alas de Animales/fisiología , Animales , Polaridad Celular , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Inmunohistoquímica , Hibridación Fluorescente in Situ , Proteínas con Dominio LIM/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Larva/fisiología , Morfogénesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Pupa/genética , Pupa/crecimiento & desarrollo , Pupa/fisiología , Alas de Animales/crecimiento & desarrolloRESUMEN
Epithelial tissues develop planar polarity that is reflected in the global alignment of hairs and cilia with respect to the tissue axes. The planar cell polarity (PCP) proteins form asymmetric and polarized domains across epithelial junctions that are aligned locally between cells and orient these external structures. Although feedback mechanisms can polarize PCP proteins intracellularly and locally align polarity between cells, how global PCP patterns are specified is not understood. It has been proposed that the graded distribution of a biasing factor could guide long-range PCP. However, we recently identified epithelial morphogenesis as a mechanism that can reorganize global PCP patterns; in the Drosophila pupal wing, oriented cell divisions and rearrangements reorient PCP from a margin-oriented pattern to one that points distally. Here, we use quantitative image analysis to study how PCP patterns first emerge in the wing. PCP appears during larval growth and is spatially oriented through the activities of three organizer regions that control disc growth and patterning. Flattening morphogen gradients emanating from these regions does not reduce intracellular polarity but distorts growth and alters specific features of the PCP pattern. Thus, PCP may be guided by morphogenesis rather than morphogen gradients.
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Polaridad Celular , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/crecimiento & desarrollo , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Epitelio/embriología , Epitelio/crecimiento & desarrollo , Epitelio/metabolismo , Genes del Desarrollo , Larva/citología , Larva/crecimiento & desarrollo , Larva/metabolismo , Morfogénesis , Pupa/citología , Pupa/crecimiento & desarrollo , Pupa/metabolismo , Alas de Animales/citología , Alas de Animales/embriología , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismoRESUMEN
Planar cell polarity (PCP) proteins form polarized cortical domains that govern polarity of external structures such as hairs and cilia in both vertebrate and invertebrate epithelia. The mechanisms that globally orient planar polarity are not understood, and are investigated here in the Drosophila wing using a combination of experiment and theory. Planar polarity arises during growth and PCP domains are initially oriented toward the well-characterized organizer regions that control growth and patterning. At pupal stages, the wing hinge contracts, subjecting wing-blade epithelial cells to anisotropic tension in the proximal-distal axis. This results in precise patterns of oriented cell elongation, cell rearrangement and cell division that elongate the blade proximo-distally and realign planar polarity with the proximal-distal axis. Mutation of the atypical Cadherin Dachsous perturbs the global polarity pattern by altering epithelial dynamics. This mechanism utilizes the cellular movements that sculpt tissues to align planar polarity with tissue shape.