RESUMEN
Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.
Asunto(s)
Proteínas de Unión al ADN , Desarrollo Embrionario , Factores de Transcripción , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Mesodermo/metabolismo , Factores de Transcripción/metabolismo , Humanos , Animales , Ratones , Extremidades/crecimiento & desarrolloRESUMEN
No two fingerprint patterns are exactly alike. In this issue of Cell, Glover et al. uncover the molecular and cellular mechanisms that result in patterned skin ridges over volar digits. This study reveals that the remarkable diversity of fingerprint configurations may originate from a common patterning code.
Asunto(s)
Extremidades , HumanosRESUMEN
The fin-to-limb transition has long fascinated evolutionary biologists, but a unifying theory as to its developmental origins has remained elusive. New work by Hawkins and colleagues demonstrates the surprising potential of teleost fins to exhibit a Hox-regulated limb-like skeletal pattern, shedding new light on the evolution of proximo-distal patterning processes.
Asunto(s)
Evolución Biológica , Tipificación del Cuerpo , Aletas de Animales , Animales , Huesos , ExtremidadesRESUMEN
Lungfishes are the closest extant relatives of tetrapods and preserve ancestral traits linked with the water-to-land transition. However, their huge genome sizes have hindered understanding of this key transition in evolution. Here, we report a 40-Gb chromosome-level assembly of the African lungfish (Protopterus annectens) genome, which is the largest genome assembly ever reported and has a contig and chromosome N50 of 1.60 Mb and 2.81 Gb, respectively. The large size of the lungfish genome is due mainly to retrotransposons. Genes with ultra-long length show similar expression levels to other genes, indicating that lungfishes have evolved high transcription efficacy to keep gene expression balanced. Together with transcriptome and experimental data, we identified potential genes and regulatory elements related to such terrestrial adaptation traits as pulmonary surfactant, anxiolytic ability, pentadactyl limbs, and pharyngeal remodeling. Our results provide insights and key resources for understanding the evolutionary pathway leading from fishes to humans.
Asunto(s)
Adaptación Biológica , Evolución Biológica , Peces/genética , Secuenciación Completa del Genoma , Aletas de Animales/anatomía & histología , Aletas de Animales/fisiología , Animales , Extremidades/anatomía & histología , Extremidades/fisiología , Peces/anatomía & histología , Peces/clasificación , Peces/fisiología , Filogenia , Fenómenos Fisiológicos Respiratorios , Sistema Respiratorio/anatomía & histología , Vertebrados/genéticaRESUMEN
To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we studied neuronal networks that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. We show that a specific class of leg sensory neurons synapses directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM more accessible and affordable to the scientific community.
Asunto(s)
Envejecimiento/fisiología , Drosophila melanogaster/ultraestructura , Microscopía Electrónica de Transmisión , Neuronas Motoras/ultraestructura , Células Receptoras Sensoriales/ultraestructura , Animales , Automatización , Conectoma , Extremidades/inervación , Nervios Periféricos/ultraestructura , Sinapsis/ultraestructuraRESUMEN
Rich fossil evidence suggests that many traits and functions related to terrestrial evolution were present long before the ancestor of lobe- and ray-finned fishes. Here, we present genome sequences of the bichir, paddlefish, bowfin, and alligator gar, covering all major early divergent lineages of ray-finned fishes. Our analyses show that these species exhibit many mosaic genomic features of lobe- and ray-finned fishes. In particular, many regulatory elements for limb development are present in these fishes, supporting the hypothesis that the relevant ancestral regulation networks emerged before the origin of tetrapods. Transcriptome analyses confirm the homology between the lung and swim bladder and reveal the presence of functional lung-related genes in early ray-finned fishes. Furthermore, we functionally validate the essential role of a jawed vertebrate highly conserved element for cardiovascular development. Our results imply the ancestors of jawed vertebrates already had the potential gene networks for cardio-respiratory systems supporting air breathing.
Asunto(s)
Evolución Biológica , Peces/genética , Aletas de Animales/fisiología , Animales , Fenómenos Fisiológicos Cardiovasculares , Sistema Cardiovascular/anatomía & histología , Extremidades/fisiología , Peces/clasificación , Genoma , Pulmón/anatomía & histología , Pulmón/fisiología , Filogenia , Receptores Odorantes/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma , Vertebrados/clasificación , Vertebrados/genéticaRESUMEN
Changes in appendage structure underlie key transitions in vertebrate evolution. Addition of skeletal elements along the proximal-distal axis facilitated critical transformations, including the fin-to-limb transition that permitted generation of diverse modes of locomotion. Here, we identify zebrafish mutants that form supernumerary long bones in their pectoral fins. These new bones integrate into musculature, form joints, and articulate with neighboring elements. This phenotype is caused by activating mutations in previously unrecognized regulators of appendage patterning, vav2 and waslb, that function in a common pathway. This pathway is required for appendage development across vertebrates, and loss of Wasl in mice causes defects similar to those seen in murine Hox mutants. Concordantly, formation of supernumerary bones requires Hox11 function, and mutations in the vav2/wasl pathway drive enhanced expression of hoxa11b, indicating developmental homology with the forearm. Our findings reveal a latent, limb-like pattern ability in fins that is activated by simple genetic perturbation.
Asunto(s)
Huesos/embriología , Extremidades/embriología , Pez Cebra/embriología , Actinas/metabolismo , Aletas de Animales/embriología , Animales , Secuencia de Bases , Tipificación del Cuerpo , Sistemas CRISPR-Cas/genética , Linaje de la Célula , Epistasis Genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Genes Reporteros , Células HeLa , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Mutación/genética , Fenotipo , Filogenia , Transducción de Señal/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The molecular mechanisms underpinning vertebrate body plan evolution are beginning to be unravelled. In this issue of Cell, Kvon et al. spectacularly demonstrate how transplanting snake-specific genetic changes found uniquely in serpent enhancers leads to limb loss in mice.
Asunto(s)
Extremidades , Vertebrados , Animales , Evolución BiológicaRESUMEN
Recent advances in limb regeneration are revealing the molecular events that integrate growth control, cell fate programming, and positional information to yield the exquisite replacement of the amputated limb. Parallel progress in several invertebrate and vertebrate models has provided a broader context for understanding the mechanisms and the evolution of regeneration. Together, these discoveries provide a foundation for describing the principles underlying regeneration of complex, multi-tissue structures. As such these findings should provide a wealth of ideas for engineers seeking to reconstitute regeneration from constituent parts or to elicit full regeneration from partial regeneration events.
Asunto(s)
Extremidades/fisiología , Animales , Evolución Biológica , Epidermis/inervación , Regulación de la Expresión Génica , Humanos , Regeneración , Transducción de SeñalRESUMEN
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.
Asunto(s)
Evolución Biológica , Elementos de Facilitación Genéticos , Extremidades/crecimiento & desarrollo , Proteínas Hedgehog/genética , Serpientes/genética , Animales , Secuencia de Bases , Evolución Molecular , Técnicas de Sustitución del Gen , Ratones , Ratones Transgénicos , Mutación , Filogenia , Serpientes/clasificaciónRESUMEN
To distinguish between complex somatosensory stimuli, central circuits must combine signals from multiple peripheral mechanoreceptor types, as well as mechanoreceptors at different sites in the body. Here, we investigate the first stages of somatosensory integration in Drosophila using in vivo recordings from genetically labeled central neurons in combination with mechanical and optogenetic stimulation of specific mechanoreceptor types. We identify three classes of central neurons that process touch: one compares touch signals on different parts of the same limb, one compares touch signals on right and left limbs, and the third compares touch and proprioceptive signals. Each class encodes distinct features of somatosensory stimuli. The axon of an individual touch receptor neuron can diverge to synapse onto all three classes, meaning that these computations occur in parallel, not hierarchically. Representing a stimulus as a set of parallel comparisons is a fast and efficient way to deliver somatosensory signals to motor circuits.
Asunto(s)
Drosophila/fisiología , Vías Nerviosas , Animales , Axones/fisiología , Extremidades/inervación , Femenino , Mecanorreceptores/fisiología , Neuronas/citología , Neuronas/fisiología , Optogenética , Propiocepción , TactoRESUMEN
The selectivity with which proprioceptive sensory neurons innervate their central and peripheral targets implies that they exhibit distinctions in muscle-type identity. The molecular correlates of proprioceptor identity and its origins remain largely unknown, however. In screens to define muscle-type proprioceptor character, we find all-or-none differences in gene expression for proprioceptors that control antagonistic muscles at a single hindlimb joint. Analysis of three of these genes, cadherin13 (cdh13), semaphorin5a (sema5a), and cartilage-acidic protein-1 (crtac1), reveals expression in proprioceptor subsets that supply muscle groups located at restricted dorsoventral and proximodistal domains of the limb. Genetically altering the dorsoventral character of the limb mesenchyme elicits a change in the profile of proprioceptor cdh13, sema5a, and crtac1 expression. These findings indicate that proprioceptors acquire aspects of their muscle-type identity in response to mesenchymal signals expressed in restricted proximodistal and dorsoventral domains of the developing limb.
Asunto(s)
Extremidades/embriología , Mesodermo/metabolismo , Propiocepción , Animales , Cadherinas/genética , Proteínas de Unión al Calcio/genética , Embrión de Mamíferos/metabolismo , Extremidades/fisiología , Ratones , Músculo Esquelético/inervación , Neuronas/metabolismo , Semaforinas/genética , Transducción de Señal , TranscriptomaRESUMEN
Animals generate movement by engaging spinal circuits that direct precise sequences of muscle contraction, but the identity and organizational logic of local interneurons that lie at the core of these circuits remain unresolved. Here, we show that V1 interneurons, a major inhibitory population that controls motor output, fractionate into highly diverse subsets on the basis of the expression of 19 transcription factors. Transcriptionally defined V1 subsets exhibit distinct physiological signatures and highly structured spatial distributions with mediolateral and dorsoventral positional biases. These positional distinctions constrain patterns of input from sensory and motor neurons and, as such, suggest that interneuron position is a determinant of microcircuit organization. Moreover, V1 diversity indicates that different inhibitory microcircuits exist for motor pools controlling hip, ankle, and foot muscles, revealing a variable circuit architecture for interneurons that control limb movement.
Asunto(s)
Extremidades/fisiología , Movimiento , Células de Renshaw/química , Células de Renshaw/citología , Médula Espinal/citología , Factores de Transcripción/análisis , Animales , Ratones , Propiocepción , Células de Renshaw/clasificación , Células de Renshaw/fisiología , TranscriptomaRESUMEN
Spinal circuits can generate locomotor output in the absence of sensory or descending input, but the principles of locomotor circuit organization remain unclear. We sought insight into these principles by considering the elaboration of locomotor circuits across evolution. The identity of limb-innervating motor neurons was reverted to a state resembling that of motor neurons that direct undulatory swimming in primitive aquatic vertebrates, permitting assessment of the role of motor neuron identity in determining locomotor pattern. Two-photon imaging was coupled with spike inference to measure locomotor firing in hundreds of motor neurons in isolated mouse spinal cords. In wild-type preparations, we observed sequential recruitment of motor neurons innervating flexor muscles controlling progressively more distal joints. Strikingly, after reversion of motor neuron identity, virtually all firing patterns became distinctly flexor like. Our findings show that motor neuron identity directs locomotor circuit wiring and indicate the evolutionary primacy of flexor pattern generation.
Asunto(s)
Extremidades/fisiología , Locomoción , Neuronas Motoras/fisiología , Músculo Esquelético/inervación , Animales , Evolución Biológica , Extremidades/inervación , Técnicas In Vitro , Ratones , Médula Espinal/fisiologíaRESUMEN
Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.
Asunto(s)
Modelos Animales de Enfermedad , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Animales , Extremidades/anatomía & histología , Extremidades/crecimiento & desarrollo , Humanos , Deformidades Congénitas de las Extremidades/genética , Ratones , Regiones Promotoras Genéticas , ARN no Traducido/genética , ARN no Traducido/metabolismo , Receptor EphA4/genéticaRESUMEN
Enhancers control the location and timing of gene expression and contain the majority of variants associated with disease1-3. The ZRS is arguably the most well-studied vertebrate enhancer and mediates the expression of Shh in the developing limb4. Thirty-one human single-nucleotide variants (SNVs) within the ZRS are associated with polydactyly4-6. However, how this enhancer encodes tissue-specific activity, and the mechanisms by which SNVs alter the number of digits, are poorly understood. Here we show that the ETS sites within the ZRS are low affinity, and identify a functional ETS site, ETS-A, with extremely low affinity. Two human SNVs and a synthetic variant optimize the binding affinity of ETS-A subtly from 15% to around 25% relative to the strongest ETS binding sequence, and cause polydactyly with the same penetrance and severity. A greater increase in affinity results in phenotypes that are more penetrant and more severe. Affinity-optimizing SNVs in other ETS sites in the ZRS, as well as in ETS, interferon regulatory factor (IRF), HOX and activator protein 1 (AP-1) sites within a wide variety of enhancers, cause gain-of-function gene expression. The prevalence of binding sites with suboptimal affinity in enhancers creates a vulnerability in genomes whereby SNVs that optimize affinity, even slightly, can be pathogenic. Searching for affinity-optimizing SNVs in genomes could provide a mechanistic approach to identify causal variants that underlie enhanceropathies.
Asunto(s)
Elementos de Facilitación Genéticos , Extremidades , Polidactilia , Proteínas Proto-Oncogénicas c-ets , Humanos , Elementos de Facilitación Genéticos/genética , Extremidades/embriología , Extremidades/patología , Mutación con Ganancia de Función , Proteínas de Homeodominio/metabolismo , Factores Reguladores del Interferón/metabolismo , Especificidad de Órganos/genética , Penetrancia , Fenotipo , Polidactilia/embriología , Polidactilia/genética , Polidactilia/patología , Polimorfismo de Nucleótido Simple , Unión Proteica , Proteínas Proto-Oncogénicas c-ets/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Animal movement is controlled by motor neurons (MNs), which project out of the central nervous system to activate muscles1. MN activity is coordinated by complex premotor networks that facilitate the contribution of individual muscles to many different behaviours2-6. Here we use connectomics7 to analyse the wiring logic of premotor circuits controlling the Drosophila leg and wing. We find that both premotor networks cluster into modules that link MNs innervating muscles with related functions. Within most leg motor modules, the synaptic weights of each premotor neuron are proportional to the size of their target MNs, establishing a circuit basis for hierarchical MN recruitment. By contrast, wing premotor networks lack proportional synaptic connectivity, which may enable more flexible recruitment of wing steering muscles. Through comparison of the architecture of distinct motor control systems within the same animal, we identify common principles of premotor network organization and specializations that reflect the unique biomechanical constraints and evolutionary origins of leg and wing motor control.
Asunto(s)
Conectoma , Drosophila melanogaster , Extremidades , Neuronas Motoras , Vías Nerviosas , Sinapsis , Alas de Animales , Animales , Femenino , Masculino , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Extremidades/inervación , Extremidades/fisiología , Neuronas Motoras/fisiología , Movimiento/fisiología , Músculos/inervación , Músculos/fisiología , Red Nerviosa/anatomía & histología , Red Nerviosa/citología , Red Nerviosa/fisiología , Vías Nerviosas/anatomía & histología , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Sinapsis/fisiología , Alas de Animales/inervación , Alas de Animales/fisiologíaRESUMEN
A deep understanding of how the brain controls behaviour requires mapping neural circuits down to the muscles that they control. Here, we apply automated tools to segment neurons and identify synapses in an electron microscopy dataset of an adult female Drosophila melanogaster ventral nerve cord (VNC)1, which functions like the vertebrate spinal cord to sense and control the body. We find that the fly VNC contains roughly 45 million synapses and 14,600 neuronal cell bodies. To interpret the output of the connectome, we mapped the muscle targets of leg and wing motor neurons using genetic driver lines2 and X-ray holographic nanotomography3. With this motor neuron atlas, we identified neural circuits that coordinate leg and wing movements during take-off. We provide the reconstruction of VNC circuits, the motor neuron atlas and tools for programmatic and interactive access as resources to support experimental and theoretical studies of how the nervous system controls behaviour.