Asunto(s)
Animales Modificados Genéticamente/embriología , Aves/embriología , Técnicas de Laboratorio Clínico , Esbozos de los Miembros/cirugía , Animales , Aves/genética , Aves/cirugía , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Transferencia de Gen , Esbozos de los Miembros/crecimiento & desarrollo , Microesferas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Transducción de Señal/genética , Coloración y Etiquetado/métodosRESUMEN
The proper development of the musculoskeletal system in the tetrapod limb requires the coordinated development of muscle, tendon and cartilage. This paper examines the morphogenesis of muscle and tendon in the developing avian hind limb. Based on a developmental series of embryos labeled with myosin and tenascin antibodies in whole mount, an integrative description of the temporal sequence and spatial pattern of muscle and tendon morphogenesis and their relationship to cartilage throughout the chick hind limb is presented for the first time. Anatomically distinct muscles arise by the progressive segregation of muscle: differentiated myotubes first appear as a pair of dorsal and ventral muscle masses; these masses subdivide into dorsal and ventral thigh, shank and foot muscle masses; and finally these six masses segregate into individual muscles. From their initial appearance, most myotubes are precisely oriented and their pattern presages the pattern of future, individual muscles. Anatomically distinct tendons emerge from three tendon primordia associated with the major joints of the limb. Contrary to previous reports, comparison of muscle and tendon reveals that much of their morphogenesis is temporally and spatially closely associated. To test whether reciprocal muscle-tendon interactions are necessary for correct muscle-tendon patterning or whether morphogenesis of each of these tissues is autonomous, two sets of experiments were conducted: (1) tendon development was examined in muscleless limbs produced by coelomic grafting of early limb buds and (2) muscle development was analyzed in limbs where tendon had been surgically altered. These experiments demonstrate that in the avian hind limb the initial morphogenetic events, formation of tendon primordia and initial differentiation of myogenic precursors, occur autonomously with respect to one another. However, later morphogenetic events, such as subdivision of muscle masses and segregation of tendon primordia into individual tendons, do require to various degrees reciprocal interactions between muscle and tendon. The dependence of these later morphogenetic events on tissue interactions differs between different proximodistal regions of the limb.
Asunto(s)
Esbozos de los Miembros/embriología , Músculo Esquelético/embriología , Tendones/embriología , Animales , Cartílago/anatomía & histología , Cartílago/embriología , Comunicación Celular , Diferenciación Celular , Embrión de Pollo , Ectodermo , Pie/anatomía & histología , Pie/embriología , Miembro Posterior/embriología , Inmunohistoquímica , Articulaciones/anatomía & histología , Articulaciones/citología , Articulaciones/embriología , Esbozos de los Miembros/anatomía & histología , Esbozos de los Miembros/inervación , Esbozos de los Miembros/cirugía , Modelos Biológicos , Morfogénesis , Músculo Esquelético/anatomía & histología , Músculo Esquelético/citología , Miosinas/análisis , Miosinas/inmunología , Células Madre , Tenascina/análisis , Tenascina/inmunología , Tendones/anatomía & histología , Tendones/citología , Tendones/inervación , Muslo/anatomía & histología , Muslo/embriologíaRESUMEN
Unilateral limb-bud removal (LBR) before the outgrowth of sensory or motor neurons to the leg of chick embryos was used to examine the role of limb (target)-derived signals in the development and survival of lumbar motoneurons and sensory neurons in the dorsal root ganglia (DRG). After LBR, motor and sensory neurons underwent normal initial histological differentiation, and cell growth in both populations was unaffected. Before their death, target-deprived motoneurons also expressed a cell-specific marker, the homeodomain protein islet-1. Proliferation of sensory and motor precursor cells was also unaffected by LBR, and the migration of neural crest cells to the DRG and of motoneurons into the ventral horn occurred normally. During the normal period of programmed cell death (PCD), increased numbers of both sensory and motor neurons degenerated after LBR. However, whereas motoneuron loss increased by 40-50% (90% total), only approximately 25% more sensory neurons degenerated after LBR. A significant number of the surviving sensory neurons projected to aberrant targets in the tail after LBR, and many of these were lost after ablation of both the limb and tail. Treatment with neurotrophic factors (or muscle extract) rescued sensory and motor neurons from cell death after LBR without affecting precursor proliferation of either population. Activity blockade with curare failed to rescue motoneurons after LBR, and combined treatment with curare plus muscle extract was no more effective than muscle extract alone. Treatment with the antioxidant N-acetylcysteine rescued motoneurons from normal cell death but not after LBR. Two specific inhibitors of the interleukin beta1 converting enzyme (ICE) family of cysteine proteases also failed to prevent motoneuron death after LBR. Taken together these data provide definitive evidence that the loss of spinal neurons after LBR cannot be attributed to altered proliferation, migration, or differentiation. Rather, in the absence of limb-derived trophic signals, the affected neurons fail to survive and undergo PCD. Although normal cell death and cell death after target deprivation share many features in common, the intracellular pathways of cell death in the two may be distinct.
Asunto(s)
Neuronas Motoras/citología , Neuronas Aferentes/citología , Médula Espinal/citología , Médula Espinal/embriología , Acetilcisteína/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Axones/fisiología , Factor Neurotrófico Derivado del Encéfalo/farmacología , Recuento de Células , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Embrión de Pollo , Inhibidores de Cisteína Proteinasa/farmacología , Depuradores de Radicales Libres/farmacología , Ganglios Espinales/citología , Esbozos de los Miembros/citología , Esbozos de los Miembros/cirugía , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/ultraestructura , Factores de Crecimiento Nervioso/farmacología , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/ultraestructura , Neurotrofina 3 , Oligopéptidos/farmacologíaRESUMEN
The neurons of the dorsal root ganglia (DRG) that supply muscle spindles require target-derived factors for survival. One necessary factor for these neurons is neurotrophin-3 (NT3). To determine whether NT3 can promote the survival of these neurons in the absence of other target-derived factors, we analyzed the effects of exogenous NT3 after early limb bud deletion in the chick. In control embryos, limb bud deletion eliminated approximately 90% of the trkC-positive (trkC+) neurons in lumbar DRG on the deleted side. In addition, the deletion led to a dramatic loss of collateral sensory projections to motoneurons. Exogenous NT3 restored a normal population of trkC+ neurons in lumbar DRG on the deleted side and increased the number of trkC+ neurons in DRG with normal targets (contralateral lumbar and thoracic). The effect was highly selective; NT3 increased the number of trkC+ neurons without significantly changing the number of either trkA+ or trkB+ neurons. The effect of NT3 was attributable to the rescue of DRG neurons from cell death, because exogenous NT3 reduced the number of pyknotic nuclei without significantly altering proliferation. Analysis of spinal projections showed further that many of the trkC+ neurons rescued by NT3 projected to the ventral spinal cord. These neurons thus had central projections characteristic of muscle spindle afferents. Together, our results indicate that NT3 signaling is both necessary and sufficient for the development of the proprioceptive phenotype, even in the absence of other signals from limb muscle.
Asunto(s)
Husos Musculares/citología , Factores de Crecimiento Nervioso/farmacología , Neuronas Aferentes/citología , Animales , Especificidad de Anticuerpos , Diferenciación Celular/efectos de los fármacos , Línea Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Embrión de Pollo , Ganglios Espinales/citología , Humanos , Riñón/citología , Esbozos de los Miembros/cirugía , Husos Musculares/efectos de los fármacos , Husos Musculares/fisiología , Neuronas Aferentes/química , Neuronas Aferentes/efectos de los fármacos , Neurotrofina 3 , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/inmunología , Proteínas Tirosina Quinasas Receptoras/análisis , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptor de Factor Neurotrófico Ciliar , Receptor trkA , Receptor trkC , Receptores de Factor de Crecimiento Nervioso/análisis , Receptores de Factor de Crecimiento Nervioso/inmunología , Médula Espinal/citología , Factores de Tiempo , TransfecciónRESUMEN
We have manipulated the chick limb bud by dorsoventrally inverting the ectoderm, by grafting the AER to the dorsal or ventral ectoderm and by insertion of an FGF-4 soaked heparin bead into the mesoderm. After dorso-ventral reversal of the ectoderm, Wnt-7a expression is autonomous from an early stage of limb development in the original dorsal ectoderm. Exogenous FGF-4 causes ectopic Wnt-7a expression and induces ectopic Shh. In addition, exogenous FGF-4 increases the thickness of cartilages and also shortens them, and both Bmp-2 and Bmp-4 may mediate this effect. The ectoderm outside the AER can regulate not only the dorso-ventral polarity of the underlying mesenchyme cells but also the cartilage formation, and both Bmp-2 and Bmp-4 may mediate this control.
Asunto(s)
Proteínas Aviares , Proteínas Morfogenéticas Óseas/genética , Factores de Crecimiento de Fibroblastos/farmacología , Esbozos de los Miembros/embriología , Esbozos de los Miembros/metabolismo , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/farmacología , Transactivadores , Factor de Crecimiento Transformador beta , Animales , Tipificación del Cuerpo/efectos de los fármacos , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Cartílago/efectos de los fármacos , Cartílago/embriología , Embrión de Pollo , Ectodermo/efectos de los fármacos , Ectodermo/metabolismo , Factor 4 de Crecimiento de Fibroblastos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas Hedgehog , Hibridación in Situ , Esbozos de los Miembros/efectos de los fármacos , Esbozos de los Miembros/cirugía , Factores de Tiempo , Proteínas WntRESUMEN
In previous studies we have observed that the interdigital mesenchyme of the chick leg bud, in the stages preceding the onset of cell death, retains a significant regulatory potential, forming ectopic extra digits under a variety of surgical manipulations. Most evidence suggests that interdigital extra digits are caused by the abolition of local antichondrogenic effects operating in the interdigital spaces under normal conditions rather than by modifications of the signalling mechanisms accounting for the normal patterning of the digits in early stages of development. The interdigital spaces exhibit a complex scaffold of extracellular matrix with well-defined domains of spatial distribution of type I and type VI collagens, tenascin, fibronectin, laminin and elastic matrix components that have been proposed to play a role in the establishment of the non-chondrogenic fate of the interdigital tissue in situ. In an attempt to analyze this possible role of the interdigital extracellular matrix (ECM), in the present work we have studied changes in the pattern of ECM distribution associated with the formation of extra digits. Extra digits were induced by making a T-cut in the third interdigital space of the leg but of stage 29 HH chick embryos. Subsequent modifications of the ECM were detected immunohistochemically in whole-mount specimens using laser confocal microscopy. Our results reveal that in the first hours after the operation, changes in the ECM apparently related to the healing of the wound cause a significant reorganization of the normal ECM scaffold of the interdigit. In addition, chondrogenesis of the interdigital tissue is preceded by disappearance of elastin fibers in the interdigital mesenchyme subjacent to the wound and by an intense deposition of tenascin. Tenascin deposition and loss of the elastin fibrillar scaffold were also observed preceding chondrogenesis in fragments of interdigital tissue explanted to culture conditions. The significance of these observations in relation to the establishment of the skeletal elements of the autopodium is discussed.