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1.
Adv Exp Med Biol ; 1383: 213-219, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36587160

RESUMEN

RNA-Seq, electrophysiology and optogenetics in mouse models are used to assess function, identify disease related genes and model enteric neural circuits. Lacking a comprehensive quantitative description of the murine colonic enteric nervous system (ENS) makes it difficult to most effectively use mouse data to better understand ENS function or for development of therapeutic approaches for human motility disorders. Our goal was to provide a quantitative description of mouse colon to establish the extent to which mouse colon architecture, connectivity and function is a useful surrogate for human and other mammalian ENS. Using GCaMP imaging coupled with pharmacology and quantitative confocal and 3D image reconstruction, we present quantitative and functional data demonstrating that regional structural changes and variable distribution of neurons define neural circuit dynamics and functional connectivity responsible for colonic motor patterns and regional functional differences. Our results advance utility of multispecies and gut region-specific data.


Asunto(s)
Sistema Nervioso Entérico , Neuronas , Ratones , Animales , Humanos , Neuronas/fisiología , Sistema Nervioso Entérico/fisiología , Colon , Modelos Animales de Enfermedad , Mamíferos
2.
Cell Mol Gastroenterol Hepatol ; 13(1): 309-337.e3, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-34509687

RESUMEN

BACKGROUND & AIMS: Colonic motor patterns have been described by a number of different groups, but the neural connectivity and ganglion architecture supporting patterned motor activity have not been elucidated. Our goals were to describe quantitatively, by region, the structural architecture of the mouse enteric nervous system and use functional calcium imaging, pharmacology, and electrical stimulation to show regional underpinnings of different motor patterns. METHODS: Excised colon segments from mice expressing the calcium indicator GCaMP6f or GCaMP6s were used to examine spontaneous and evoked (pharmacologic or electrical) changes in GCaMP-mediated fluorescence and coupled with assessment of colonic motor activity, immunohistochemistry, and confocal imaging. Three-dimensional image reconstruction and statistical methods were used to describe quantitatively mouse colon myenteric ganglion structure, neural and vascular network patterning, and neural connectivity. RESULTS: In intact colon, regionally specific myenteric ganglion size, architecture, and neural circuit connectivity patterns along with neurotransmitter-receptor expression underlie colonic motor patterns that define functional differences along the colon. Region-specific effects on spontaneous, evoked, and chemically induced neural activity contribute to regional motor patterns, as does intraganglionic functional connectivity. We provide direct evidence of neural circuit structural and functional regional differences that have only been inferred in previous investigations. We include regional comparisons between quantitative measures in mouse and human colon that represent an important advance in showing the usefulness and relevance of the mouse system for translation to the human colon. CONCLUSIONS: There are several neural mechanisms dependent on myenteric ganglion architecture and functional connectivity that underlie neurogenic control of patterned motor function in the mouse colon.


Asunto(s)
Sistema Nervioso Entérico , Motilidad Gastrointestinal , Animales , Colon , Ratones
3.
Front Physiol ; 12: 652714, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34408655

RESUMEN

The peristaltic contraction and relaxation of intestinal circular and longitudinal smooth muscles is controlled by synaptic circuit elements that impinge upon phenotypically diverse neurons in the myenteric plexus. While electrophysiological studies provide useful information concerning the properties of such synaptic circuits, they typically involve tissue disruption and do not correlate circuit activity with biochemically defined neuronal phenotypes. To overcome these limitations, mice were engineered to express the sensitive, fast Ca2+ indicator GCaMP6f selectively in neurons that express the acetylcholine (ACh) biosynthetic enzyme choline acetyltransfarse (ChAT) thereby allowing rapid activity-driven changes in Ca2+ fluorescence to be observed without disrupting intrinsic connections, solely in cholinergic myenteric ganglion (MG) neurons. Experiments with selective receptor agonists and antagonists reveal that most mouse colonic cholinergic (i.e., GCaMP6f+/ChAT+) MG neurons express nicotinic ACh receptors (nAChRs), particularly the ganglionic subtype containing α3 and ß4 subunits, and most express ionotropic serotonin receptors (5-HT3Rs). Cholinergic MG neurons also display small, spontaneous Ca2+ transients occurring at ≈ 0.2 Hz. Experiments with inhibitors of Na+ channel dependent impulses, presynaptic Ca2+ channels and postsynaptic receptor function reveal that the Ca2+ transients arise from impulse-driven presynaptic activity and subsequent activation of postsynaptic nAChRs or 5-HT3Rs. Electrical stimulation of axonal connectives to MG evoked Ca2+ responses in the neurons that similarly depended on nAChRs or/and 5-HT3Rs. Responses to single connective shocks had peak amplitudes and rise and decay times that were indistinguishable from the spontaneous Ca2+ transients and the largest fraction had brief synaptic delays consistent with activation by monosynaptic inputs. These results indicate that the spontaneous Ca2+ transients and stimulus evoked Ca2+ responses in MG neurons originate in circuits involving fast chemical synaptic transmission mediated by nAChRs or/and 5-HT3Rs. Experiments with an α7-nAChR agonist and antagonist, and with pituitary adenylate cyclase activating polypeptide (PACAP) reveal that the same synaptic circuits display extensive capacity for presynaptic modulation. Our use of non-invasive GCaMP6f/ChAT Ca2+ imaging in colon segments with intrinsic connections preserved, reveals an abundance of direct and modulatory synaptic influences on cholinergic MG neurons.

4.
Cell Mol Gastroenterol Hepatol ; 11(5): 1548-1592.e1, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33444816

RESUMEN

BACKGROUND AND AIMS: Bowel function requires coordinated activity of diverse enteric neuron subtypes. Our aim was to define gene expression in these neuron subtypes to facilitate development of novel therapeutic approaches to treat devastating enteric neuropathies, and to learn more about enteric nervous system function. METHODS: To identify subtype-specific genes, we performed single-nucleus RNA-seq on adult mouse and human colon myenteric plexus, and single-cell RNA-seq on E17.5 mouse ENS cells from whole bowel. We used immunohistochemistry, select mutant mice, and calcium imaging to validate and extend results. RESULTS: RNA-seq on 635 adult mouse colon myenteric neurons and 707 E17.5 neurons from whole bowel defined seven adult neuron subtypes, eight E17.5 neuron subtypes and hundreds of differentially expressed genes. Manually dissected human colon myenteric plexus yielded RNA-seq data from 48 neurons, 3798 glia, 5568 smooth muscle, 377 interstitial cells of Cajal, and 2153 macrophages. Immunohistochemistry demonstrated differential expression for BNC2, PBX3, SATB1, RBFOX1, TBX2, and TBX3 in enteric neuron subtypes. Conditional Tbx3 loss reduced NOS1-expressing myenteric neurons. Differential Gfra1 and Gfra2 expression coupled with calcium imaging revealed that GDNF and neurturin acutely and differentially regulate activity of ∼50% of myenteric neurons with distinct effects on smooth muscle contractions. CONCLUSION: Single cell analyses defined genes differentially expressed in myenteric neuron subtypes and new roles for TBX3, GDNF and NRTN. These data facilitate molecular diagnostic studies and novel therapeutics for bowel motility disorders.


Asunto(s)
Biomarcadores/análisis , Sistema Nervioso Entérico/metabolismo , Regulación de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Neurturina/metabolismo , Análisis de la Célula Individual/métodos , Proteínas de Dominio T Box/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Femenino , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Neurturina/genética , RNA-Seq/métodos , Proteínas de Dominio T Box/genética , Adulto Joven
5.
Gastroenterology ; 160(4): 1208-1223.e4, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-32980343

RESUMEN

BACKGROUND & AIMS: The colon is innervated by intrinsic and extrinsic neurons that coordinate functions necessary for digestive health. Sympathetic input suppresses colon motility by acting on intrinsic myenteric neurons, but the extent of sympathetic-induced changes on large-scale network activity in myenteric circuits has not been determined. Compounding the complexity of sympathetic function, there is evidence that sympathetic transmitters can regulate activity in non-neuronal cells (such as enteric glia and innate immune cells). METHODS: We performed anatomical tracing, immunohistochemistry, optogenetic (GCaMP calcium imaging, channelrhodopsin), and colon motility studies in mice and single-cell RNA sequencing in human colon to investigate how sympathetic postganglionic neurons modulate colon function. RESULTS: Individual neurons in each sympathetic prevertebral ganglion innervated the proximal or distal colon, with processes closely opposed to multiple cell types. Calcium imaging in semi-intact mouse colon preparations revealed changes in spontaneous and evoked neural activity, as well as activation of non-neuronal cells, induced by sympathetic nerve stimulation. The overall pattern of response to sympathetic stimulation was unique to the proximal or distal colon. Region-specific changes in cellular activity correlated with motility patterns produced by electrical and optogenetic stimulation of sympathetic pathways. Pharmacology experiments (mouse) and RNA sequencing (human) indicated that appropriate receptors were expressed on different cell types to account for the responses to sympathetic stimulation. Regional differences in expression of α-1 adrenoceptors in human colon emphasize the translational relevance of our mouse findings. CONCLUSIONS: Sympathetic neurons differentially regulate activity of neurons and non-neuronal cells in proximal and distal colon to promote distinct changes in motility patterns, likely reflecting the distinct roles played by these 2 regions.


Asunto(s)
Colon/inervación , Ganglios Simpáticos/fisiología , Motilidad Gastrointestinal/fisiología , Plexo Mientérico/fisiología , Animales , Colon/citología , Colon/efectos de los fármacos , Colon/fisiología , Femenino , Ganglios Simpáticos/efectos de los fármacos , Motilidad Gastrointestinal/efectos de los fármacos , Guanetidina/farmacología , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inervación , Mucosa Intestinal/fisiología , Masculino , Ratones , Modelos Animales , Plexo Mientérico/citología , Plexo Mientérico/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/fisiología , Optogenética , Prazosina/farmacología , RNA-Seq , Análisis de la Célula Individual , Yohimbina/farmacología
6.
Gastroenterology ; 158(8): 2221-2235.e5, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32113825

RESUMEN

BACKGROUND & AIMS: Small, 2-dimensional sections routinely used for human pathology analysis provide limited information about bowel innervation. We developed a technique to image human enteric nervous system (ENS) and other intramural cells in 3 dimensions. METHODS: Using mouse and human colon tissues, we developed a method that combines tissue clearing, immunohistochemistry, confocal microscopy, and quantitative analysis of full-thickness bowel without sectioning to quantify ENS and other intramural cells in 3 dimensions. RESULTS: We provided 280 adult human colon confocal Z-stacks from persons without known bowel motility disorders. Most of our images were of myenteric ganglia, captured using a 20× objective lens. Full-thickness colon images, viewed with a 10× objective lens, were as large as 4 × 5 mm2. Colon from 2 pediatric patients with Hirschsprung disease was used to show distal colon without enteric ganglia, as well as a transition zone and proximal pull-through resection margin where ENS was present. After testing a panel of antibodies with our method, we identified 16 antibodies that bind to molecules in neurons, glia, interstitial cells of Cajal, and muscularis macrophages. Quantitative analyses demonstrated myenteric plexus in 24.5% ± 2.4% of flattened colon Z-stack area. Myenteric ganglia occupied 34% ± 4% of myenteric plexus. Single myenteric ganglion volume averaged 3,527,678 ± 573,832 mm3 with 38,706 ± 5763 neuron/mm3 and 129,321 ± 25,356 glia/mm3. Images of large areas provided insight into why published values of ENS density vary up to 150-fold-ENS density varies greatly, across millimeters, so analyses of small numbers of thin sections from the same bowel region can produce varying results. Neuron subtype analysis revealed that approximately 56% of myenteric neurons stained with neuronal nitric oxide synthase antibody and approximately 33% of neurons produce and store acetylcholine. Transition zone regions from colon tissues of patients with Hirschsprung disease had ganglia in multiple layers and thick nerve fiber bundles without neurons. Submucosal neuron distribution varied among imaged colon regions. CONCLUSIONS: We developed a 3-dimensional imaging method for colon that provides more information about ENS structure than tissue sectioning. This approach could improve diagnosis for human bowel motility disorders and may be useful for other bowel diseases as well.


Asunto(s)
Colon/inervación , Ganglios Autónomos/patología , Enfermedad de Hirschsprung/patología , Interpretación de Imagen Asistida por Computador , Imagenología Tridimensional , Microscopía Confocal , Plexo Mientérico/patología , Plexo Submucoso/patología , Animales , Automatización , Neuronas Colinérgicas/patología , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Nitrérgicas/patología , Valor Predictivo de las Pruebas , Fijación del Tejido
7.
Front Physiol ; 11: 128, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32153427

RESUMEN

Irises isolated from the eyes of diverse species constrict when exposed to light. Depending on species this intrinsic photomechanical transduction response (PMTR) requires either melanopsin or cryptochrome (CRY) photopigment proteins, generated by their respective association with retinoid or flavin adenine dinucleotide (FAD) chromophores. Although developmentally relevant circadian rhythms are also synchronized and reset by these same proteins, the cell type, mechanism, and specificity of photomechanical transduction (PMT) and its relationship to circadian processes remain poorly understood. Here we show that PMTRs consistent with CRY activation by 430 nm blue light occur in developing chicken iris striated muscle, identify relevant mechanisms, and demonstrate that similar PMTRs occur in striated iris and pectoral muscle fibers, prevented in both cases by knocking down CRY gene transcript levels. Supporting CRY activation, iris PMTRs were reduced by inhibiting flavin reductase, but unaffected by melanopsin antagonism. The largest iris PMTRs paralleled the developmental predominance of striated over smooth muscle fibers, and shared their requirement for extracellular Ca2+ influx and release of intracellular Ca2+. Photo-stimulation of identified striated myotubes maintained in dissociated culture revealed the cellular and molecular bases of PMT. Myotubes in iris cell cultures responded to 435 nm light with increased intracellular Ca2+ and contractions, mimicking iris PMTRs and their spectral sensitivity. Interestingly PMTRs featuring contractions and requiring extracellular Ca2+ influx and release of intracellular Ca2+ were also displayed by striated myotubes derived from pectoral muscle. Consistent with these findings, cytosolic CRY1 and CRY2 proteins were detected in both iris and pectoral myotubes, and knocking down myotube CRY1/CRY2 gene transcript levels specifically blocked PMTRs in both cases. Thus CRY-mediated PMT is not unique to iris, but instead reflects a more general feature of developing striated muscle fibers. Because CRYs are core timing components of circadian clocks and CRY2 is critical for circadian regulation of myogenic differentiation CRY-mediated PMT may interact with cell autonomous clocks to influence the progression of striated muscle development.

8.
Gastroenterology ; 157(2): 522-536.e2, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31075226

RESUMEN

BACKGROUND & AIMS: Proper colon function requires signals from extrinsic primary afferent neurons (ExPANs) located in spinal ganglia. Most ExPANs express the vanilloid receptor TRPV1, and a dense plexus of TRPV1-positive fibers is found around myenteric neurons. Capsaicin, a TRPV1 agonist, can initiate activity in myenteric neurons and produce muscle contraction. ExPANs might therefore form motility-regulating synapses onto myenteric neurons. ExPANs mediate visceral pain, and myenteric neurons mediate colon motility, so we investigated communication between ExPANs and myenteric neurons and the circuits by which ExPANs modulate colon function. METHODS: In live mice and colon tissues that express a transgene encoding the calcium indicator GCaMP, we visualized levels of activity in myenteric neurons during smooth muscle contractions induced by application of capsaicin, direct colon stimulation, stimulation of ExPANs, or stimulation of preganglionic parasympathetic neuron (PPN) axons. To localize central targets of ExPANs, we optogenetically activated TRPV1-expressing ExPANs in live mice and then quantified Fos immunoreactivity to identify activated spinal neurons. RESULTS: Focal electrical stimulation of mouse colon produced phased-locked calcium signals in myenteric neurons and produced colon contractions. Stimulation of the L6 ventral root, which contains PPN axons, also produced myenteric activation and contractions that were comparable to those of direct colon stimulation. Surprisingly, capsaicin application to the isolated L6 dorsal root ganglia, which produced robust calcium signals in neurons throughout the ganglion, did not activate myenteric neurons. Electrical activation of the ganglia, which activated even more neurons than capsaicin, did not produce myenteric activation or contractions unless the spinal cord was intact, indicating that a complete afferent-to-efferent (PPN) circuit was necessary for ExPANs to regulate myenteric neurons. In TRPV1-channel rhodopsin-2 mice, light activation of ExPANs induced a pain-like visceromotor response and expression of Fos in spinal PPN neurons. CONCLUSIONS: In mice, ExPANs regulate myenteric neuron activity and smooth muscle contraction via a parasympathetic spinal circuit, linking sensation and pain to motility.


Asunto(s)
Colon/fisiopatología , Neuronas Aferentes/fisiología , Peristaltismo/fisiología , Dolor Visceral/fisiopatología , Animales , Técnicas Biosensibles/métodos , Capsaicina/administración & dosificación , Colon/efectos de los fármacos , Colon/inervación , Modelos Animales de Enfermedad , Femenino , Ganglios Espinales/citología , Humanos , Masculino , Ratones , Ratones Transgénicos , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/inervación , Músculo Liso/fisiopatología , Plexo Mientérico/citología , Plexo Mientérico/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Optogenética , Peristaltismo/efectos de los fármacos , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Dolor Visceral/inducido químicamente
9.
Dev Biol ; 444 Suppl 1: S308-S324, 2018 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-29807017

RESUMEN

Carotid body glomus cells mediate essential reflex responses to arterial blood hypoxia. They are dopaminergic and secrete growth factors that support dopaminergic neurons, making the carotid body a potential source of patient-specific cells for Parkinson's disease therapy. Like adrenal chromaffin cells, which are also hypoxia-sensitive, glomus cells are neural crest-derived and require the transcription factors Ascl1 and Phox2b; otherwise, their development is little understood at the molecular level. Here, analysis in chicken and mouse reveals further striking molecular parallels, though also some differences, between glomus and adrenal chromaffin cell development. Moreover, histology has long suggested that glomus cell precursors are 'émigrés' from neighbouring ganglia/nerves, while multipotent nerve-associated glial cells are now known to make a significant contribution to the adrenal chromaffin cell population in the mouse. We present conditional genetic lineage-tracing data from mice supporting the hypothesis that progenitors expressing the glial marker proteolipid protein 1, presumably located in adjacent ganglia/nerves, also contribute to glomus cells. Finally, we resolve a paradox for the 'émigré' hypothesis in the chicken - where the nearest ganglion to the carotid body is the nodose, in which the satellite glia are neural crest-derived, but the neurons are almost entirely placode-derived - by fate-mapping putative nodose neuronal 'émigrés' to the neural crest.


Asunto(s)
Cuerpo Carotídeo/embriología , Células Cromafines/metabolismo , Pericitos/metabolismo , Glándulas Suprarrenales/metabolismo , Glándulas Suprarrenales/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Tipificación del Cuerpo/fisiología , Diferenciación Celular , Hipoxia de la Célula/fisiología , Embrión de Pollo , Pollos/metabolismo , Ratones , Ratones Noqueados , Proteína Proteolipídica de la Mielina/fisiología , Cresta Neural/metabolismo , Neuronas/metabolismo , Pericitos/fisiología , Factores de Transcripción/metabolismo
10.
Dev Neurobiol ; 76(10): 1111-24, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-26818017

RESUMEN

The bHLH transcription factor Hand2 is essential for the acquisition and maintenance of noradrenergic properties of embryonic sympathetic neurons and controls neuroblast proliferation. Hand2 is also expressed in embryonic and postnatal parasympathetic ganglia and remains expressed in sympathetic neurons up to the adult stage. Here, we address its function in developing parasympathetic and adult sympathetic neurons. We conditionally deleted Hand2 in the parasympathetic sphenopalatine ganglion by crossing a line of floxed Hand2 mice with DbhiCre transgenic mice, taking advantage of the transient Dbh expression in parasympathetic ganglia. Hand2 elimination does not affect Dbh expression and sphenopalatine ganglion size at E12.5 and E16.5, in contrast to sympathetic ganglia. These findings demonstrate different functions for Hand2 in the parasympathetic and sympathetic lineage. Our previous Hand2 knockdown in postmitotic, differentiated chick sympathetic neurons resulted in decreased expression of noradrenergic marker genes but it was unclear whether Hand2 is required for maintaining noradrenergic neuron identity in adult animals. We now show that Hand2 elimination in adult Dbh-expressing sympathetic neurons does not decrease the expression of Th and Dbh, in contrast to the situation during development. However, gene expression profiling of adult sympathetic neurons identified 75 Hand2-dependent target genes. Interestingly, a notable proportion of down-regulated genes (15%) encode for proteins with synaptic and neurotransmission functions. These results demonstrate a change in Hand2 target genes during maturation of sympathetic neurons. Whereas Hand2 controls genes regulating noradrenergic differentiation during development, Hand2 seems to be involved in the regulation of genes controlling neurotransmission in adult sympathetic neurons. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1111-1124, 2016.


Asunto(s)
Sistema Nervioso Autónomo/crecimiento & desarrollo , Sistema Nervioso Autónomo/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neuronas/metabolismo , Animales , Sistema Nervioso Autónomo/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ganglios Parasimpáticos/citología , Ganglios Parasimpáticos/crecimiento & desarrollo , Ganglios Parasimpáticos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Antígeno Ki-67/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Ratones Transgénicos , Neuronas/citología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo , Transcriptoma
11.
Mol Biol Cell ; 26(21): 3704-18, 2015 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-26354425

RESUMEN

The extensive invasive capacity of glioblastoma (GBM) makes it resistant to surgery, radiotherapy, and chemotherapy and thus makes it lethal. In vivo, GBM invasion is mediated by Rho GTPases through unidentified downstream effectors. Mammalian Diaphanous (mDia) family formins are Rho-directed effectors that regulate the F-actin cytoskeleton to support tumor cell motility. Historically, anti-invasion strategies focused upon mDia inhibition, whereas activation remained unexplored. The recent development of small molecules directly inhibiting or activating mDia-driven F-actin assembly that supports motility allows for exploration of their role in GBM. We used the formin inhibitor SMIFH2 and mDia agonists IMM-01/-02 and mDia2-DAD peptides, which disrupt autoinhibition, to examine the roles of mDia inactivation versus activation in GBM cell migration and invasion in vitro and in an ex vivo brain slice invasion model. Inhibiting mDia suppressed directional migration and spheroid invasion while preserving intrinsic random migration. mDia agonism abrogated both random intrinsic and directional migration and halted U87 spheroid invasion in ex vivo brain slices. Thus mDia agonism is a superior GBM anti-invasion strategy. We conclude that formin agonism impedes the most dangerous GBM component-tumor spread into surrounding healthy tissue. Formin activation impairs novel aspects of transformed cells and informs the development of anti-GBM invasion strategies.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/agonistas , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Forminas , Glioblastoma/metabolismo , Glioblastoma/patología , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Humanos , Invasividad Neoplásica , Ratas , Esferoides Celulares
12.
Dev Biol ; 388(2): 149-58, 2014 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-24565998

RESUMEN

The Periostin Cre (Postn-Cre) lineage includes endocardial and neural crest derived mesenchymal cells of the cardiac cushions, neural crest-derived components of the sympathetic and enteric nervous systems, and cardiac fibroblasts. In this study, we use the Postn-Cre transgenic allele to conditionally ablate Hand2 (H2CKO). We find that Postn-Cre H2CKOs die shortly after birth despite a lack of obvious cardiac structural defects. To ascertain the cause of death, we performed a detailed comparison of the Postn-Cre lineage and Hand2 expression at mid and late stages of embryonic development. Gene expression analyses demonstrate that Postn-Cre ablates Hand2 from the adrenal medulla as well as the sphenopalatine ganglia of the head. In both cases, Hand2 loss-of-function dramatically reduces expression of Dopamine Beta Hydroxylase (Dbh), a gene encoding a crucial catecholaminergic biosynthetic enzyme. Expression of the genes Tyrosine Hydroxylase (Th) and Phenylethanolamine N-methyltransferase (Pnmt), which also encode essential catecholaminergic enzymes, were severely reduced in postnatal adrenal glands. Electrocardiograms demonstrate that 3-day postnatal Postn-Cre H2CKO pups exhibit sinus bradycardia. In conjunction with the aforementioned gene expression analyses, these results strongly suggest that the observed postnatal lethality occurs due to a catecholamine deficiency and subsequent heart failure.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Bradicardia/genética , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Linaje de la Célula , Hibridación in Situ , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa
13.
Dev Biol ; 382(1): 305-19, 2013 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-23838398

RESUMEN

We review morphogenesis of the enteric nervous system from migratory neural crest cells, and defects of this process such as Hirschsprung disease, centering on cell motility and assembly, and cell adhesion and extracellular matrix molecules, along with cell proliferation and growth factors. We then review continuum and agent-based (cellular automata) models with rules of cell movement and logistical proliferation. Both movement and proliferation at the individual cell level are modeled with stochastic components from which stereotyped outcomes emerge at the population level. These models reproduced the wave-like colonization of the intestine by enteric neural crest cells, and several new properties emerged, such as colonization by frontal expansion, which were later confirmed biologically. These models predict a surprising level of clonal heterogeneity both in terms of number and distribution of daughter cells. Biologically, migrating cells form stable chains made up of unstable cells, but this is not seen in the initial model. We outline additional rules for cell differentiation into neurons, axon extension, cell-axon and cell-cell adhesions, chemotaxis and repulsion which can reproduce chain migration. After the migration stage, the cells re-arrange as a network of ganglia. Changes in cell adhesion molecules parallel this, and we describe additional rules based on Steinberg's Differential Adhesion Hypothesis, reflecting changing levels of adhesion in neural crest cells and neurons. This was able to reproduce enteric ganglionation in a model. Mouse mutants with disturbances of enteric nervous system morphogenesis are discussed, and these suggest future refinement of the models. The modeling suggests a relatively simple set of cell behavioral rules could account for complex patterns of morphogenesis. The model has allowed the proposal that Hirschsprung disease is mostly an enteric neural crest cell proliferation defect, not a defect of cell migration. In addition, the model suggests an explanations for zonal and skip segment variants of Hirschsprung disease, and also gives a novel stochastic explanation for the observed discordancy of Hirschsprung disease in identical twins.


Asunto(s)
Sistema Nervioso Entérico/anomalías , Sistema Nervioso Entérico/embriología , Modelos Biológicos , Animales , Sistema Nervioso Entérico/patología , Ganglios/embriología , Ganglios/metabolismo , Ganglios/patología , Tracto Gastrointestinal/embriología , Tracto Gastrointestinal/inervación , Humanos , Morfogénesis , Cresta Neural/embriología , Cresta Neural/patología
14.
PLoS Genet ; 9(3): e1003405, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23555309

RESUMEN

Neural crest cells are multipotent progenitor cells that can generate both ectodermal cell types, such as neurons, and mesodermal cell types, such as smooth muscle. The mechanisms controlling this cell fate choice are not known. The basic Helix-loop-Helix (bHLH) transcription factor Twist1 is expressed throughout the migratory and post-migratory cardiac neural crest. Twist1 ablation or mutation of the Twist-box causes differentiation of ectopic neuronal cells, which molecularly resemble sympathetic ganglia, in the cardiac outflow tract. Twist1 interacts with the pro-neural factor Sox10 via its Twist-box domain and binds to the Phox2b promoter to repress transcriptional activity. Mesodermal cardiac neural crest trans-differentiation into ectodermal sympathetic ganglia-like neurons is dependent upon Phox2b function. Ectopic Twist1 expression in neural crest precursors disrupts sympathetic neurogenesis. These data demonstrate that Twist1 functions in post-migratory neural crest cells to repress pro-neural factors and thereby regulate cell fate determination between ectodermal and mesodermal lineages.


Asunto(s)
Músculo Liso , Miocardio , Cresta Neural , Neuronas , Proteínas Nucleares , Proteína 1 Relacionada con Twist , Animales , Diferenciación Celular , Linaje de la Célula , Ectodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HeLa , Proteínas de Homeodominio/metabolismo , Humanos , Mesodermo/metabolismo , Ratones , Ratones Transgénicos , Músculo Liso/citología , Músculo Liso/metabolismo , Miocardio/citología , Miocardio/metabolismo , Cresta Neural/citología , Cresta Neural/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Organogénesis/genética , Factores de Transcripción SOXE/metabolismo , Factores de Transcripción/metabolismo , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo
15.
J Neurosci ; 32(6): 2110-20, 2012 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-22323723

RESUMEN

Neural crest cell specification and differentiation to a sympathetic neuronal fate serves as an important model for neurogenesis and depends upon the function of both bHLH transcription factors, notably Hand2, and homeodomain transcription factors, including Phox2b. Here, we define a 1007 bp cis-regulatory element 5' of the Hand1 gene sufficient to drive reporter expression within the sympathetic chain of transgenic mice. Comparative genomic analyses uncovered evolutionarily conserved consensus-binding sites within this element, which chromatin immunoprecipitation and electrophoretic mobility shift assays confirm are bound by Hand2 and Phox2b. Mutational analyses revealed that the conserved Phox2 and E-box binding sites are necessary for proper cis-regulatory element activity, and expression analyses on both Hand2 conditionally null and hypomorphic backgrounds demonstrate that Hand2 is required for reporter activation in a gene dosage-dependent manner. We demonstrate that Hand2 and Hand1 differentially bind the E-boxes in this cis-regulatory element, establishing molecular differences between these two factors. Finally, we demonstrate that Hand1 is dispensable for normal tyrosine hydroxylase (TH) and dopamine ß-hydroxylase (DBH) expression in sympathetic neurons, even when Hand2 gene dosage is concurrently reduced by half. Together, these data define a tissue-specific Hand1 cis-regulatory element controlled by two factors essential for the development of the sympathetic nervous system and provide in vivo regulatory evidence to support previous findings that Hand2, rather than Hand1, is predominantly responsible for regulating TH, DBH, and Hand1 expression in developing sympathetic neurons.


Asunto(s)
Fibras Adrenérgicas/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Dosificación de Gen/genética , Proteínas de Homeodominio/fisiología , Neurogénesis/genética , Factores de Transcripción/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Secuencia Conservada/genética , Femenino , Genes Reporteros/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Unión Proteica/genética , Elementos Reguladores de la Transcripción/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
16.
Development ; 138(21): 4789-800, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21989918

RESUMEN

Targeted deletion of the bHLH DNA-binding protein Hand2 in the neural crest, impacts development of the enteric nervous system (ENS), possibly by regulating the transition from neural precursor cell to neuron. We tested this hypothesis by targeting Hand2 deletion in nestin-expressing neural precursor (NEP) cells. The mutant mice showed abnormal ENS development, resulting in lethal neurogenic pseudo-obstruction. Neurogenesis of neurons derived from NEP cells identified a second nestin non-expressing neural precursor (NNEP) cell in the ENS. There was substantial compensation for the loss of neurons derived from the NEP pool by the NNEP pool but this was insufficient to abrogate the negative impact of Hand2 deletion. Hand2-mediated regulation of proliferation affected both neural precursor and neuron numbers. Differentiation of glial cells derived from the NEP cells was significantly decreased with no compensation from the NNEP pool of cells. Our data indicate differential developmental potential of NEPs and NNEPs; NNEPs preferentially differentiate as neurons, whereas NEPs give rise to both neurons and glial cells. Deletion of Hand2 also resulted in complete loss of NOS and VIP and a significant decrease in expression of choline acetyltransferase and calretinin, demonstrating a role for Hand2 in neurotransmitter specification and/or expression. Loss of Hand2 resulted in a marked disruption of the developing neural network, exemplified by lack of a myenteric plexus and extensive overgrowth of fibers. Thus, Hand2 is essential for neurogenesis, neurotransmitter specification and neural network patterning in the developing ENS.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sistema Nervioso Entérico/anatomía & histología , Sistema Nervioso Entérico/embriología , Ganglios Autónomos/anomalías , Ganglios Autónomos/embriología , Neurogénesis/fisiología , Células Madre/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proliferación Celular , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Eliminación de Gen , Proteínas de Filamentos Intermediarios/metabolismo , Intestinos/embriología , Intestinos/inervación , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Nestina , Neuronas/química , Neuronas/citología , Neuronas/fisiología , Neurotransmisores/metabolismo , Células Madre/citología
17.
Development ; 138(11): 2249-59, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21558373

RESUMEN

Lower jaw development is a complex process in which multiple signaling cascades establish a proximal-distal organization. These cascades are regulated both spatially and temporally and are constantly refined through both induction of normal signals and inhibition of inappropriate signals. The connective tissue of the tongue arises from cranial neural crest cell-derived ectomesenchyme within the mandibular portion of the first pharyngeal arch and is likely to be impacted by this signaling. Although the developmental mechanisms behind later aspects of tongue development, including innervation and taste acquisition, have been elucidated, the early patterning signals driving ectomesenchyme into a tongue lineage are largely unknown. We show here that the basic helix-loop-helix transcription factor Hand2 plays key roles in establishing the proximal-distal patterning of the mouse lower jaw, in part through establishing a negative-feedback loop in which Hand2 represses Dlx5 and Dlx6 expression in the distal arch ectomesenchyme following Dlx5- and Dlx6-mediated induction of Hand2 expression in the same region. Failure to repress distal Dlx5 and Dlx6 expression results in upregulation of Runx2 expression in the mandibular arch and the subsequent formation of aberrant bone in the lower jaw along with proximal-distal duplications. In addition, there is an absence of lateral lingual swelling expansion, from which the tongue arises, resulting in aglossia. Hand2 thus appears to establish a distal mandibular arch domain that is conducive for lower jaw development, including the initiation of tongue mesenchyme morphogenesis.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Lengua/embriología , Animales , Tipificación del Cuerpo , Huesos/embriología , Línea Celular , Proliferación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/biosíntesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Regulación hacia Abajo , Hibridación in Situ , Maxilares/embriología , Ratones , Ratones Noqueados , Cresta Neural/citología , Cresta Neural/metabolismo , Organogénesis , Reacción en Cadena de la Polimerasa , Transducción de Señal
18.
Dev Biol ; 341(1): 291-304, 2010 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-20144608

RESUMEN

The basic helix-loop-helix DNA binding protein Hand2 has critical functions in cardiac development both in neural crest-derived and mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest has allowed us to genetically dissect Hand2-dependent defects specifically in outflow tract and cardiac cushion independent of Hand2 functions in mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest results in misalignment of the aortic arch arteries and outflow tract, contributing to development of double outlet right ventricle (DORV) and ventricular septal defects (VSD). These neural crest-derived developmental anomalies are associated with altered expression of Hand2-target genes we have identified by gene profiling. A number of Hand2 direct target genes have been identified using ChIP and ChIP-on-chip analyses. We have identified and validated a number of genes related to cell migration, proliferation/cell cycle and intracellular signaling whose expression is affected by Hand2 deletion in the neural crest and which are associated with development of VSD and DORV. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting expression of target genes associated with a number of functional interactions in neural crest-derived cells required for proper patterning of the outflow tract, generation of the appropriate number of neural crest-derived cells for elongation of the conotruncus and cardiac cushion organization. Our genetic model has made it possible to investigate the molecular genetics of neural crest contributions to outflow tract morphogenesis and cell differentiation.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Cresta Neural/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ciclo Celular , Conexinas/genética , Conexinas/metabolismo , Dosificación de Gen , Ratones , Cresta Neural/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional
19.
Dev Biol ; 329(2): 191-200, 2009 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-19254708

RESUMEN

The basic helix-loop-helix transcription factor Hand2 is essential for the proliferation and noradrenergic differentiation of sympathetic neuron precursors during development. Here we address the function of Hand2 in postmitotic, differentiated sympathetic neurons. Knockdown of endogenous Hand2 in cultured E12 chick sympathetic neurons by siRNA results in a significant (about 60%) decrease in the expression of the noradrenergic marker genes dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH). In contrast, expression of the pan-neuronal genes TuJ1, HuC and SCG10 was not affected. To analyze the in vivo role of Hand2 in differentiated sympathetic neurons we used mice harboring a conditional Hand2-null allele and excised the gene by expression of Cre recombinase under control of the DBH promotor. Mouse embryos homozygous for Hand2 gene deletion showed decreased sympathetic neuron number and TH expression was strongly reduced in the residual neuron population. The in vitro Hand2 knockdown also enhances the CNTF-induced expression of the cholinergic marker genes vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT). Taken together, these findings demonstrate that the Hand2 transcription factor plays a key role in maintaining noradrenergic properties in differentiated neurons.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Diferenciación Celular , Neuronas/fisiología , Norepinefrina/metabolismo , Sistema Nervioso Simpático/citología , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cartilla de ADN , Dopamina beta-Hidroxilasa/genética , Epistasis Genética , Femenino , Técnicas de Silenciamiento del Gen , Inmunohistoquímica , Masculino , Ratones , Neuronas/citología , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tirosina 3-Monooxigenasa/genética
20.
Dev Biol ; 319(2): 179-91, 2008 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-18501887

RESUMEN

Neural crest-derived structures that depend critically upon expression of the basic helix-loop-helix DNA binding protein Hand2 for normal development include craniofacial cartilage and bone, the outflow tract of the heart, cardiac cushion, and noradrenergic sympathetic ganglion neurons. Loss of Hand2 is embryonic lethal by E9.5, obviating a genetic analysis of its in-vivo function. We have overcome this difficulty by specific deletion of Hand2 in neural crest-derived cells by crossing our line of floxed Hand2 mice with Wnt1-Cre transgenic mice. Our analysis of Hand2 knock-out in neural crest-derived cells reveals effects on development in all neural crest-derived structures where Hand2 is expressed. In the autonomic nervous system, conditional disruption of Hand2 results in a significant and progressive loss of neurons as well as a significant loss of TH expression. Hand2 affects generation of the neural precursor pool of cells by affecting both the proliferative capacity of the progenitors as well as affecting expression of Phox2a and Gata3, DNA binding proteins important for the cell autonomous development of noradrenergic neurons. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting differentiation and cell type-specific gene expression in neural crest-derived noradrenergic sympathetic ganglion neurons. Hand2 has a pivotal function in a non-linear cross-regulatory network of DNA binding proteins that affect cell autonomous control of differentiation and cell type-specific gene expression.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Eliminación de Gen , Cresta Neural/fisiología , Neuronas/fisiología , Células Ganglionares de la Retina/fisiología , Sistema Nervioso Simpático/embriología , Animales , Regulación de la Expresión Génica , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Confocal , Sistema Nervioso Simpático/fisiología , Transcripción Genética
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