RESUMEN
The contraction of skeletal muscle is dependent on synaptic transmission through acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). The lack of an AChR subunit causes a fetal akinesia in humans, leading to death in the first trimester and characteristic features of Fetal Akinesia Deformation Sequences (FADS). A corresponding null mutation of the delta-subunit in zebrafish (sofa potato; sop) leads to the death of embryos around 5 d postfertilization (dpf). In sop(-/-) mutants, we expressed modified delta-subunits, with one (delta1YFP) or two yellow fluorescent protein (delta2YFP) molecules fused at the intracellular loop, under the control of an alpha-actin promoter. AChRs containing these fusion proteins are fluorescent, assemble on the plasma membrane, make clusters under motor neuron endings, and generate synaptic current. We screened for germ-line transmission of the transgene and established a line of sop(-/-) fish stably expressing the delta2YFP. These delta2YFP/sop(-/-) embryos can mount escape behavior close to that of their wild-type siblings. Synaptic currents in these embryos had a smaller amplitude, slower rise time, and slower decay when compared with wild-type fish. Remarkably, these embryos grow to adulthood and display complex behaviors such as feeding and breeding. To the best of our knowledge, this is the first case of a mutant animal corresponding to first trimester lethality in human that has been rescued by a transgene and survived to adulthood. In the rescued fish, a foreign promoter drove the transgene expression and the NMJ had altered synaptic strength. The survival of the transgenic animal delineates requirements for gene therapies of NMJ.
Asunto(s)
Longevidad/genética , Mutación/genética , Enfermedades de la Unión Neuromuscular/genética , Receptores Colinérgicos/genética , Pez Cebra/crecimiento & desarrollo , Pez Cebra/genética , Acetilcolina/metabolismo , Animales , Animales Modificados Genéticamente , Conducta Alimentaria/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Luminiscentes/genética , Masculino , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiopatología , Enfermedades de la Unión Neuromuscular/metabolismo , Enfermedades de la Unión Neuromuscular/fisiopatología , Subunidades de Proteína/química , Subunidades de Proteína/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Conducta Sexual Animal/fisiología , Maduración Sexual/genética , Membranas Sinápticas/genética , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestructura , Transmisión Sináptica/genética , Transgenes/genética , Pez Cebra/metabolismoRESUMEN
Numerous animals use chemical cues within their environments to execute various behaviors. One of these behaviors is orientation to an odor source. Crayfish, in particular, can orient to food sources under a number of different conditions. It has not been determined, however, what kind of search strategy these animals employ to successfully locate a food source. To determine the role of antennae and antennules in this behavior and to investigate different modes of orientation behavior, the orientation patterns of crayfish with complete and partial antennal lesions were examined. Detailed analysis of orientation paths confirmed that crayfish could not locate odor sources with either bilateral or unilateral lesions. This suggests that crayfish are using the spatial information obtained from these appendages to successfully orient. Animals using information from the bilaterally paired appendages in the control group exhibited increased walking speed, increased speed to source and decreased heading angles towards the source compared to these measurements taken from lesioned groups. There was no significant difference in any parameters between animals with unilateral or bilateral lesions. This strongly suggests that these animals are reliant on the spatial comparison of differences between bilaterally paired olfactory appendages for successful orientation.