EXPRESSION OF ENGRAILED IN IDENTIFIED NEURONS OF CNS

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The present work is part of a general effort to determine the role of homeobox genes in the development and maintenance of neuronal specificity in the central nervous system. Health related aspects of the work are readily recognized in the growing number of studies that show specific defects in brain and spinal cord, and in a number of non-neuronal tissues, when homeobox genes have been disrupted during development. Although the CNS defects resulting from homeobox disruption are typically described in rather broadly, the defects must ultimately reside in alterations in the phenotype of individual neurons, and further, result from the altered expression of particular structural genes. Homeobox genes are commonly regarded as developmental genes. Nonetheless, some continue expression into adulthood, and it is therefore speculated that they have a role in maintaining the integrity of neuronal structure, connectivity, or function. The first specific aim is to characterize the pattern of Engrailed expression in the adult CNS of Schistocerca. Engrailed is the protein product of the engrailed gene and will be detected using a monoclonal antibody (MAb 4D9) that recognizes the protein's homeodomain. Immunolabeled ganglia of the CNS will be examined to assess the stereotypy of the labeling, its extent and its location relative to landmarks. The second specific aim is to test the hypothesis that Engrailed expression is type-specific, with expression occurring interneurons but not efferents of lineally related neuronal groups. We will examine segmentally homologous midline groups of neurons which each comprise the progeny of an identified embryonic stem cell. A direct test of the hypothesis will be made by double labeling experiments: MAb 4D9 will be used together with retrograde labeling or intracellular injection of dye into identified neurons in the midline groups. We will assess the overlap between labels in different neuronal types. Corroborating evidence for type-speCificity will be obtained by a quantitative analysis of Engrailed-positive and -negative neurons within the lineage groups. The third specific aim is to characterize the Engrailed expression during embryonic development. We hypothesize that Engrailed expression evolves in a definable spatial and temporal pattern during embryogenesis to yield the mature pattern. Two levels of inquiry are proposed, one at population level: We will construct a population profile by counting immunolabeled and unlabeled cells in the midline groups at 5% increments from 30% to 100% of embryonic development. Our second level of inquiry will characterize expression with cellular resolution. We will trace expression within the stem cell and in a distinct anatomical figure that contains the most recently born progeny. These data together with estimates of cell cycle time will allow a characterization of the onset and offset of expression relative to time of birth.