RESUMEN

The enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract that controls essential functions such as motility, secretion and blood flow, comprises a vast number of neurons and glial cells that are organized into complex networks of interconnected ganglia distributed throughout the entire length of the gut wall. Enteric neurons and glia are derived from neural crest cells that undergo extensive migration, proliferation, differentiation and survival in order to form a functional ENS. Investigations of the developmental processes that underlie ENS formation in animal models, and of the common human congenital ENS abnormality Hirschsprung's disease, have been intimately related and recently led to major advances in the field. This review touches on some of these advances and introduces two topics that are elaborated upon in this journal issue: (i) genome wide approaches for profiling gene expression in wild type and mutant ENS that have been used to identify novel molecules with important roles in enteric neurogenesis, and (ii) the use of multilineage ENS progenitors isolated from embryonic or postnatal gut as novel cell replacement therapies for Hirschsprung's disease. Such studies will not only unravel the mechanisms underlying ENS development, but will also shed light on the pathogenesis of ENS developmental disorders and help to establish novel therapeutic strategies for restoring or repairing malfunctioning enteric neural circuits prevalent in numerous gastrointestinal diseases.

Asunto(s)

Sistema Nervioso Entérico/embriología , Sistema Nervioso Entérico/crecimiento & desarrollo , Animales , Congresos como Asunto , Sistema Nervioso Entérico/anatomía & histología , Sistema Nervioso Entérico/fisiología , Enfermedad de Hirschsprung/fisiopatología , Humanos

RESUMEN

Mice lacking both alleles of the LIM-homeobox gene Lhx7 display dramatically reduced number of forebrain cholinergic neurons. Given the fact that sex differences are consistently observed in forebrain cholinergic function, in the present study we investigated whether the absence of LHX7 differentially affects water maze performance in the two sexes. Herein we demonstrate that LHX7 null mutants display a sex-dependent impairment in water maze, with females appearing more affected than males. Moreover, neurotrophin assessment revealed a compensatory increase of brain-derived neurotrophic factor and neurotrophin 3 in the neocortex of both male and female mutants and an increase of nerve growth factor levels only in the females. Nevertheless, the compensatory increase of cortical neurotrophin levels did not restore cognitive abilities of Lhx7 homozygous mutants. Finally, our analysis revealed that cortical neurotrophin levels correlate negatively with water maze proficiency, indicating that there is an optimal neurotrophin level for successful cognitive performance.

Asunto(s)

Corteza Cerebral/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Aprendizaje por Laberinto/fisiología , Factores de Crecimiento Nervioso/metabolismo , Caracteres Sexuales , Análisis de Varianza , Animales , Corteza Cerebral/citología , Femenino , Regulación de la Expresión Génica/genética , Proteínas con Homeodominio LIM , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Tiempo de Reacción/genética , Factores de Tiempo , Factores de Transcripción

RESUMEN

It has been reported recently that mice lacking both alleles of the LIM-homeobox gene Lhx7, display dramatically reduced number of forebrain cholinergic neurons. In the present study, we investigated whether the Lhx7 mutation affects male and female mice differently, given the fact that gender differences are consistently observed in forebrain cholinergic function. Our results show that in adult male as well as female Lhx7 homozygous mutants there is a dramatic loss of choline acetyltransferase immunoreactive forebrain neurons, both projection and interneurons. The reduction of forebrain choline acetyltransferase immunoreactive neurons in Lhx7 homozygous mutants is accompanied by a decrease of acetylcholinesterase histochemical staining in all forebrain cholinergic neuron target areas of both male and female homozygous mutants. Furthermore, there was an increase of M1-, but not M2-, muscarinic acetylcholine receptor binding site density in the somatosensory cortex and basal ganglia of only the female homozygous mutant mice. Such an increase can be regarded as a mechanism acting to compensate for the dramatically reduced cholinergic input, raising the possibility that the forebrain cholinergic system in female mice may be more plastic and responsive to situations of limited neurotransmitter availability. Finally, our study provides additional data for the sexual dimorphism of the forebrain cholinergic system, as female mice appear to have a lower density of M1-muscarinic acetylcholine receptors in the striatal areas of the basal ganglia and a higher density of M2-muscarinic acetylcholine receptors, in a number of cortical areas, as well as the striatal areas of the basal ganglia.

Asunto(s)

Colina O-Acetiltransferasa/metabolismo , Proteínas de Homeodominio/genética , Prosencéfalo/fisiología , Animales , Colina O-Acetiltransferasa/deficiencia , Femenino , Homocigoto , Proteínas con Homeodominio LIM , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Prosencéfalo/enzimología , Receptores Muscarínicos/fisiología , Caracteres Sexuales , Factores de Transcripción

RESUMEN

Although a normal respiratory rhythm is vital at birth, little is known about the genetic factors controlling the prenatal maturation of the respiratory network in mammals. In Phox2a mutant mice, which do not express A6 neurons, we previously hypothesized that the release of endogenous norepinephrine by A6 neurons is required for a normal respiratory rhythm to occur at birth. Here we investigated the role of the Ret gene, which encodes a transmembrane tyrosine kinase receptor, in the maturation of norepinephrine and respiratory systems. As Ret-null mutants (Ret-/-) did not survive after birth, our experiments were performed in wild-type (wt) and Ret-/- fetuses exteriorized from pregnant heterozygous mice at gestational day 18. First, in wt fetuses, quantitative in situ hybridization revealed high levels of Ret transcripts in the pontine A5 and A6 areas. Second, in Ret-/- fetuses, high-pressure liquid chromatography showed significantly reduced norepinephrine contents in the pons but not the medulla. Third, tyrosine hydroxylase immunocytochemistry revealed a significantly reduced number of pontine A5 and A6 neurons but not medullary norepinephrine neurons in Ret-/- fetuses. Finally, electrophysiological and pharmacological experiments performed on brainstem 'en bloc' preparations demonstrated impaired resting respiratory activity and abnormal responses to central hypoxia and norepinephrine application in Ret-/- fetuses. To conclude, our results show that Ret gene contributes to the prenatal maturation of A6 and A5 neurons and respiratory system. They support the hypothesis that the normal maturation of the respiratory network requires afferent activity corresponding to the A6 excitatory and A5 inhibitory input balance.

Asunto(s)

Neuronas/fisiología , Proteínas Proto-Oncogénicas c-ret/fisiología , Mecánica Respiratoria/fisiología , Sistema Respiratorio/crecimiento & desarrollo , Sistema Respiratorio/inervación , Animales , Monoaminas Biogénicas/metabolismo , Tronco Encefálico/citología , Tronco Encefálico/crecimiento & desarrollo , Femenino , Genotipo , Proteínas de Homeodominio/genética , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Red Nerviosa/embriología , Red Nerviosa/crecimiento & desarrollo , Red Nerviosa/fisiología , Norepinefrina/fisiología , Puente/metabolismo , Embarazo , Proteínas Proto-Oncogénicas c-ret/biosíntesis , Proteínas Proto-Oncogénicas c-ret/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Sistema Respiratorio/embriología , Rombencéfalo/enzimología , Rombencéfalo/crecimiento & desarrollo

RESUMEN

Normal intestinal motility requires orderly development of the complex nerve plexuses and smooth muscular layers in the gut wall. Organization of these structures results, in part, from cell autonomous programmes directed by transcription factors, which orchestrate appropriate temporal and spatial expression of specific target genes. Hox proteins appear to function in combination to dictate regional codes that establish major structural landmarks in the gut such as sphincters and muscle layers. These codes are translated in part by intercellular signals, which allow populations of cells in the embryonic gut wall to alter the developmental fate of their neighbours. Some of the best characterized intercellular signalling pathways involved in enteric neurodevelopment are mediated by GDNF/GFRa1/RET, EDN3/ENDRB, and NETRINS/DCC. These signals affect enteric neural precursors as they colonize the gut, and perturbations of these molecules are associated with various types of intestinal neuropathology.

Asunto(s)

Sistema Nervioso Entérico/fisiología , Genes Homeobox , Transducción de Señal/fisiología , Animales , Sistema Nervioso Entérico/embriología , Motilidad Gastrointestinal/fisiología , Regulación del Desarrollo de la Expresión Génica , Humanos , Intestinos/inervación

RESUMEN

RET forms the catalytic component within the receptor complex that transmits signals from the GDNF family of neurotrophic factors. To study the mechanisms regulating the cell-type specific expression of this gene, we have cloned and characterised the murine c-ret locus. A cosmid contig comprising approximately 60 kb of the mouse genome encompassing the entire structural gene and flanking sequences have been isolated and the transcription initiation site identified and promoter characterised. The murine c-ret promoter lacks a TATA initiation motif and has GC enriched DNA sequences reminiscent of CpG islands. Analysis of transgenic mice lines bearing the Lac Z (beta-galactosidase) reporter gene under the control of 5' flanking sequences show modularity in the organisation of cis-regulatory domains within the locus. Cloned 5' flanking sequences comprise a distal regulatory domain directing Lac Z expression at the primitive streak, lateral mesoderm and facial ganglia and a proximal sensory neurones specific regulatory domain inducing Lac Z expression primarily within the developing somatosensory system. The spatial and temporal progression of transgene expression precisely recapitulates endogenous gene expression in developing sensory ganglia including its induction in postnatal Isolectin B4 binding nociceptive neurones.

Asunto(s)

Mapeo Cromosómico , Proteínas de Drosophila , Ganglios Sensoriales/embriología , Expresión Génica , Genes Reguladores/genética , Operón Lac/genética , Ratones/embriología , Ratones/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Animales Recién Nacidos/metabolismo , Secuencia de Bases/genética , Clonación Molecular , Secuencia de Consenso/genética , Embrión de Mamíferos/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Lectinas/metabolismo , Ratones Transgénicos/genética , Neuronas Aferentes/metabolismo , Nociceptores/metabolismo , Regiones Promotoras Genéticas/genética , Estructura Terciaria de Proteína/fisiología , Proteínas Proto-Oncogénicas c-ret , Médula Espinal/embriología , Sitio de Iniciación de la Transcripción/fisiología

RESUMEN

The RET receptor tyrosine kinase has a critical role in kidney organogenesis and the development of the enteric nervous system. Two major isoforms, RET9 and RET51, differ in the amino acid sequence of the C-terminal tail as a result of alternative splicing. To determine the roles of these isoforms in vivo, we used targeted mutagenesis to generate mice that express either RET9 or RET51. Monoisoformic RET9 mice, which lack RET51, are viable and appear normal. In contrast, monoisoformic RET51 animals, which lack RET9, have kidney hypodysplasia and lack enteric ganglia from the colon. To study the differential activities of the two RET isoforms further, we generated transgenic mice expressing ligand-dependent and constitutively active forms of RET9 or RET51 under the control of the Hoxb7 regulatory sequences. Such RET9 transgenes are capable of rescuing the kidney agenesis in RET-deficient mice or causing kidney hypodysplasia in wild-type animals. In contrast, similar RET51 transgenes fail to rescue the kidney agenesis or cause hypodysplasia. Our findings show that RET9 and RET51 have different signaling properties in vivo and define specific temporal and spatial requirements of c-Ret function during renal development and histogenesis of the enteric nervous system.

Asunto(s)

Proteínas de Drosophila , Desarrollo Embrionario y Fetal , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Empalme Alternativo , Animales , Genes Homeobox , Ratones , Ratones Transgénicos , Isoformas de Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/genética , Transgenes

RESUMEN

Mox1 and Mox2 homeobox genes have been shown to be critical in axial skeleton and in limb muscle development respectively. Pax1 and Pax3 gene products are also implicated in these processes. Mox and Pax expression patterns are highly overlapping both spatially and temporally during embryonic development. We show here for the first time that Mox proteins physically interact with Pax1 and Pax3 using the yeast two-hybrid protein interaction assay as well as in vitro biochemical assays. There is a strong preference of Mox1 to associate with Pax1 rather than Pax3 and of Mox2 to associate with Pax3 rather than Pax1. The observed interactions are mediated through the homeodomain of Mox.

Asunto(s)

Antígenos de Superficie/metabolismo , Proteínas de Unión al ADN/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas , Factores de Transcripción/metabolismo , Animales , Antígenos CD , Escherichia coli , Genes Homeobox/fisiología , Ratones , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Proteínas Recombinantes/metabolismo , Técnicas del Sistema de Dos Híbridos

RESUMEN

The neuropeptide galanin is expressed developmentally in the dorsal root ganglion (DRG) and is rapidly up-regulated 120-fold after peripheral nerve section in the adult. Here we report that adult mice carrying a loss-of-function mutation in the galanin gene have a 13% reduction in the number of cells in the DRG associated with a 24% decrease in the percentage of neurons that express substance P. These deficits are associated with a 2.8- and 2.6-fold increase in the number of apoptotic cells in the DRG at postnatal days 3 and 4, respectively. After crush injury to the sciatic nerve, the rate of peripheral nerve regeneration is reduced by 35% with associated long-term functional deficits. Cultured DRG neurons from adult mutant mice demonstrate similar deficits in neurite number and length. These results identify a critical role for galanin in the development and regeneration of sensory neurons.

Asunto(s)

Galanina/fisiología , Regeneración Nerviosa , Neuronas Aferentes/citología , Neuronas Aferentes/fisiología , Animales , Axones , Galanina/genética , Ratones , Ratones Noqueados

RESUMEN

The principal neuronal types of the mammalian cerebral cortex are the excitatory pyramidal cells and the inhibitory interneurons, the non-pyramidal cells. It is thought that these neurons arise in the ventricular zone surrounding the telencephalic ventricles. From there, newly generated neurons migrate outward along the processes of radial glial cells to reach the cortical plate where they accumulate in an 'inside-out' sequence to form the six-layered structure of the neocortex. Here we review emerging evidence that pyramidal neurons are generated in the cortical ventricular zone, whereas the majority of the non-pyramidal cells arise in the ganglionic eminences of the ventral telencephalon. These neurons follow tangential migratory routes to reach their positions in the developing cortex.

Asunto(s)

Corteza Cerebral/fisiología , Ganglios/fisiología , Neuronas/fisiología , Animales , Corteza Cerebral/citología

RESUMEN

During development of the neocortex, the marginal zone (layer I) and the subplate (layer VII) are the first layers to form from a primordial plexiform neoropil. The cortical plate (layers II-VI) is subsequently established between these superficial and deep components of the primordial plexiform neuropil. Neurons in the early zones are thought to play important roles in the formation of the cortex: the Cajal-Retzius cells of the marginal zone are instrumental in neuronal migration and laminar formation, and cells of the subplate are involved in the formation of cortical connections. Using the fluorescent tracer 1,1'-dioctodecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), we have shown here that a substantial proportion of neurons of the marginal zone, including cells with features of Cajal-Retzius cells, and of the subplate and lower intermediate zone are not born in the ventricular neuroepithelium but instead originate in the medial ganglionic eminence (MGE), the pallidal primordium. These neurons follow a tangential migratory route to their positions in the developing cortex. They express the neurotransmitter GABA but seem to lack the calcium binding protein calretinin; some migrating cells found in the marginal zone express reelin. In addition, migrating cells express the LIM-homeobox gene Lhx6, a characteristic marker of the MGE. It is suggested that this gene uniquely or in combination with other transcription factors may be involved in the decision of MGE cells to differentiate in situ or migrate to the neocortex.

Asunto(s)

Encéfalo/embriología , Corteza Cerebral/embriología , Desarrollo Embrionario y Fetal , Proteínas del Tejido Nervioso , Neuronas/citología , Animales , Encéfalo/citología , Carbocianinas , Movimiento Celular , Corteza Cerebral/citología , Colorantes Fluorescentes , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Morfogénesis , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Proteína Reelina , Dedos de Zinc

RESUMEN

The skeletal muscles of the limbs develop from myogenic progenitors that originate in the paraxial mesoderm and migrate into the limb-bud mesenchyme. Among the genes known to be important for muscle development in mammalian embryos are those encoding the basic helix-loop-helix (bHLH) myogenic regulatory factors (MRFs; MyoD, Myf5, myogenin and MRF4) and Pax3, a paired-type homeobox gene that is critical for the development of limb musculature. Mox1 and Mox2 are closely related homeobox genes that are expressed in overlapping patterns in the paraxial mesoderm and its derivatives. Here we show that mice homozygous for a null mutation of Mox2 have a developmental defect of the limb musculature, characterized by an overall reduction in muscle mass and elimination of specific muscles. Mox2 is not needed for the migration of myogenic precursors into the limb bud, but it is essential for normal appendicular muscle formation and for the normal regulation of myogenic genes, as demonstrated by the downregulation of Pax3 and Myf5 but not MyoD in Mox2-deficient limb buds. Our findings show that the MOX2 homeoprotein is an important regulator of vertebrate limb myogenesis.

Asunto(s)

Antígenos de Superficie/genética , Extremidades/embriología , Genes Homeobox , Músculo Esquelético/embriología , Animales , Antígenos CD , Antígenos de Superficie/fisiología , Desarrollo Embrionario y Fetal/genética , Desarrollo Embrionario y Fetal/fisiología , Expresión Génica , Marcación de Gen , Genes Reporteros , Masculino , Mesodermo/fisiología , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Músculo Esquelético/anomalías , Mutación , Miogenina/genética

RESUMEN

The mammalian enteric nervous system is derived from neural crest cells which invade the foregut and hindgut mesenchyme. It has been established that signalling molecules produced by the mesenchyme of the gut wall play a critical role in the development of the mammalian enteric nervous system. Recent studies have characterised further the role of such molecules and have identified novel extracellular and intracellular signals that are critical for enteric ganglia formation.

Asunto(s)

Sistema Nervioso Entérico/embriología , Animales , Desarrollo Embrionario y Fetal/fisiología , Sistema Nervioso Entérico/citología , Mamíferos , Transducción de Señal , Células Madre/citología

RESUMEN

RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN. Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14. 5-15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives.

Asunto(s)

Proteínas de Drosophila , Sistema Nervioso Entérico/embriología , Sistema Nervioso Entérico/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Muerte Celular/genética , Diferenciación Celular/genética , Supervivencia Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Factor Neurotrófico Ciliar , Sistema Digestivo/embriología , Sistema Digestivo/inervación , Dopamina/metabolismo , Embrión de Mamíferos/citología , Edad Gestacional , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Homocigoto , Mesencéfalo/embriología , Mesencéfalo/metabolismo , Ratones , Ratones Transgénicos , Mutación , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/farmacología , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/metabolismo , Neurotrofina 3 , Neurturina , Proteínas Proto-Oncogénicas c-ret , Ratas , Ratas Wistar , Transducción de Señal

RESUMEN

In mammals, rostral ectomesenchyme cells of the mandibular arch give rise to odontogenic cells, while more caudal cells form the distal skeletal elements of the lower jaw. Signals from the epithelium are required for the development of odontogenic and skeletogenic mesenchyme cells. We show that rostral-caudal polarity is first established in mandibular branchial arch ectomesenchymal cells by a signal, Fgf-8, from the rostral epithelium. All neural crest-derived ectomesenchymal cells are equicompetent to respond to Fgf-8. The restriction into rostral (Lhx-7-expressing) and caudal (Gsc-expressing) domains is achieved by cells responding differently according to their proximity to the source of the signal. Once established, spatial expression domains and cell fates are fixed and maintained by Fgf-8 in conjunction with another epithelial signal, endothelin-1, and by positional changes in ectomesenchymal cell competence to respond to the signal.

Asunto(s)

Región Branquial/embriología , Polaridad Celular/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas de Homeodominio/genética , Proteínas Represoras , Factores de Transcripción , Animales , Región Branquial/citología , Inducción Embrionaria/genética , Endotelina-1/genética , Endotelina-1/metabolismo , Factor 8 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Proteína Goosecoide , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM , Esbozos de los Miembros , Mandíbula/embriología , Mesodermo , Ratones , Ratones Endogámicos , Odontogénesis/genética

RESUMEN

The enteric nervous system of vertebrates is derived from neural crest cells that invade the gut wall and generate a highly organised network of enteric ganglia. Among the genes that play an important role in ENS development is c-Ret, mutations of which result in failure of formation of enteric ganglia (intestinal aganglionosis). To further understand the development of the mammalian ENS in general and the mechanism of action of the RET RTK in particular, we have developed and used an organotypic culture system of mouse fetal gut. At the stage of culture initiation, the gut is partially populated by undifferentiated ENS progenitors, but culture for several days results in extensive neuronal and glial differentiation. Using this organ culture system, we have compared the development of the ENS in wild-type and RET-deficient gut and showed that the aganglionic phenotype observed in vivo is consistently reproduced under the in vitro culture conditions. Microinjection of RET+ cells isolated from E11.5 mouse bowel into wild-type or RET-deficient aganglionic gut in organ culture, results in extensive repopulation of their wall by exogenously derived neurons and glia. Finally, using a similar approach, we demonstrate that single RET+ cells introduced into the wall of wild-type gut generate both cell lineages of the ENS, i.e. neurons and glia. Our data show the NC-derived RET+ population of fetal gut in mammalian embryos consists of multipotential progenitors capable of colonising efficiently both wild-type and RET-deficient aganglionic bowel in organ culture.

Asunto(s)

Sistema Digestivo/embriología , Proteínas de Drosophila , Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/embriología , Esófago/embriología , Técnicas de Cultivo de Órganos/métodos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Diferenciación Celular , Sistema Digestivo/inervación , Esófago/inervación , Ratones , Mutación , Neuroglía , Neuronas , Proteínas Proto-Oncogénicas c-ret , Células Madre

RESUMEN

The neuropeptide galanin is predominantly expressed by the lactotrophs (the prolactin secreting cell type) in the rodent anterior pituitary and in the median eminence and paraventricular nucleus of the hypothalamus. Prolactin and galanin colocalize in the same secretory granule, the expression of both proteins is extremely sensitive to the estrogen status of the animal. The administration of estradiol-17beta induces pituitary hyperplasia followed by adenoma formation and causes a 3,000-fold increase in the galanin mRNA content of the lactotroph. To further study the role of galanin in prolactin release and lactotroph growth we now report the generation of mice carrying a loss-of-function mutation of the endogenous galanin gene. There is no evidence of embryonic lethality and the mutant mice grow normally. The specific endocrine abnormalities identified to date, relate to the expression of prolactin. Pituitary prolactin message levels and protein content of adult female mutant mice are reduced by 30-40% compared with wild-type controls. Mutant females fail to lactate and pups die of starvation/dehydration unless fostered onto wild-type mothers. Prolactin secretion in mutant females is markedly reduced at 7 days postpartum compared with wild-type controls with an associated failure in mammary gland maturation. There is an almost complete abrogation of the proliferative response of the lactotroph to high doses of estrogen, with a failure to up-regulate prolactin release, STAT5 expression or to increase pituitary cell number. These data further support the hypothesis that galanin acts as a paracrine regulator of prolactin expression and as a growth factor to the lactotroph.

Asunto(s)

División Celular/fisiología , Galanina/fisiología , Adenohipófisis/metabolismo , Prolactina/metabolismo , Alelos , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Células Cultivadas , Cartilla de ADN , Estrógenos/fisiología , Femenino , Galanina/genética , Ratones , Ratones Mutantes , Adenohipófisis/citología

RESUMEN

The enteric nervous system (ENS) in vertebrates is derived from the neural crest and constitutes the most complex part of the peripheral nervous system. Natural and induced mutagenesis in mammals has shown that the tyrosine kinase receptor RET and its functional ligand glial cell line-derived neurotrophic factor (GDNF) play key roles in the development of the ENS in humans and mice. We have developed and briefly describe here a number of assays that analyze the specific function of the RET receptor and its ligand. Our data suggest that the RET signal transduction pathway has multiple roles in the development of the mammalian ENS.

Asunto(s)

Sistema Digestivo/inervación , Proteínas de Drosophila , Factores de Crecimiento Nervioso , Sistema Nervioso Periférico/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Animales , Factor Neurotrófico Derivado de la Línea Celular Glial , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Humanos , Mamíferos , Ratones , Proteínas del Tejido Nervioso/fisiología , Sistema Nervioso Periférico/embriología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas Receptoras/genética , Vertebrados

RESUMEN

LIM-homeobox containing (Lhx) genes encode trascriptional regulators which play critical roles in a variety of developmental processes. We have identified two genes belonging to a novel subfamily of mammalian Lhx genes, designated Lhx6 and Lhx7. Whole-mount in situ hybridisation showed that Lhx6 and Lhx7 were expressed during mouse embryogenesis in overlapping domains of the first branchial arch and the basal forebrain. More specifically, expression of Lhx6 and Lhx7 was detected prior to initiation of tooth formation in the presumptive oral and odontogenic mesenchyme of the maxillary and mandibular processes. During tooth formation, expression was restricted to the mesenchyme of individual teeth. Using explant cultures, we have shown that expression of Lhx6 and Lhx7 in mandibular mesenchyme was under the control of signals derived from the overlying epithelium; such signals were absent from the epithelium of the non-odontogenic second branchial arch. Furthermore, expression studies and bead implantation experiments in vitro have provided strong evidence that Fgf8 is primarily responsible for the restricted expression of Lhx6 and Lhx7 in the oral aspect of the maxillary and mandibular processes. In the telencephalon, expression of both genes was predominantly localised in the developing medial ganglionic eminences, flanking a Fgf8-positive midline region. We suggest that Fgf8 and Lhx6 and Lhx7 are key components of signalling cascades which determine morphogenesis and differentiation in the first branchial arch and the basal forebrain.

Asunto(s)

Genes Homeobox , Cabeza/embriología , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso , Secuencia de Aminoácidos , Animales , Región Branquial/embriología , ADN Complementario/genética , Factor 8 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Ganglios/embriología , Regulación de la Expresión Génica , Biblioteca de Genes , Proteínas de Homeodominio/biosíntesis , Proteínas con Homeodominio LIM , Mandíbula/embriología , Maxilar/embriología , Ratones , Datos de Secuencia Molecular , Prosencéfalo/embriología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Diente/embriología , Factores de Transcripción

RESUMEN

The 29 amino acid neuropeptide galanin is widely distributed in the nervous and endocrine systems; highest levels of galanin synthesis and storage occur within the hypothalamus in the median eminence, but it is also abundantly expressed in the basal forebrain, the peripheral nervous system, and gut. To further define the role played by galanin in the peripheral nervous and endocrine systems, a mouse strain carrying a loss-of-function germ-line mutation of the galanin locus, engineered by targeted mutagenesis in embryonic stem cells, has been generated. The mutation removes the first five exons containing the entire coding region for the galanin peptide. Germ-line transmission of the disrupted galanin locus has been obtained, and the mutation has been bred to homozygosity on the inbred 129O1aHsd background. Phenotypic analysis of mice lacking a functional galanin gene demonstrate that these animals are viable, grow normally, and can reproduce. A marked reduction in both the anterior pituitary prolactin content and in circulating plasma levels of the hormone is evident. Lactation is abolished along with abrogation of the proliferative response of the lactotroph to estrogen. The responses of sensory neurons to injury in the mutants are markedly impaired. Peripheral nerve regeneration is reduced with associated long-term functional deficits. There is a striking reduction in the development of chronic neuropathic pain. These two phenotypic changes may be explained, in part, by the observation that a subset of dorsal root ganglion neurons is lost in the mutant animals, implying a role for galanin as a trophic cell survival factor. These initial findings have important implications for our understanding and potential therapeutic treatment of (a) sensory nerve regeneration and neuropathic pain and (b) disordered pituitary proliferation and the development of prolactinoma.

Asunto(s)

Galanina/fisiología , Regulación de la Expresión Génica/fisiología , Regeneración Nerviosa/fisiología , Dolor , Secuencia de Aminoácidos , Animales , Marcación de Gen , Humanos , Ratones , Datos de Secuencia Molecular , Fenómenos Fisiológicos del Sistema Nervioso , Ratas , Receptores de Galanina , Receptores de Neuropéptido/fisiología , Eliminación de Secuencia