The transcriptional activator retinoic acid is one of the most teratogenic substances known, and through accidental exposure of human embryos to the acne drug Acutane(R), cerebellar development was found to be particularly vulnerable. A high susceptibility to systemically applied retinoic acid could indicate that the developing cerebellum is naturally disposed to respond to endogenously synthesized retinoic acid and that a precisely regulated spatial and temporal distribution of the endogenous compound is essential for normal development. Our preliminary observations are consistent with this notion: with a novel technique for the detection of retinoic-acid synthesizing enzymes we find very little synthesis in the cerebellum proper, but high levels in the closely apposed choroid plexus. Moreover, enzyme levels in the choroid plexus shown two maxima that seem to parallel two processes in cerebellar morphogenesis: the first during Purkinje cell development at late fetal stages and the second during the postnatal stage of granule cell development; cultures of the developing cerebellum reveal a potent growth-promoting effect secreted by the choroid plexus. Here we propose to test the hypothesis that the metencephalic choroid plexus represents a paracrine organ for the developing cerebellum, influencing neuronal differentiation through spatially and temporally regulated retinoic acid secretion. In detail, we plan to address to following topics: (1) We will characterize the distribution of retinoic acid synthesis in the developing metencephalon by functional assay, and by in situ hybridization with a probe to the choroid plexus enzyme that we recently isolated for the mouse. (2) We will assay the extent to which retinoic acid accounts for the choroid-plexus neurite inducing activity, and what isomer is synthesized in vivo. (3) We will test for effects of thyroid hormone, a factor known to influence cerebellar development and which interacts with retinoic acid through the direct cooperation between the thyroid hormone receptor and the cerebellar retinoic acid receptor RXR. (4) We will attempt to perturb the distribution of retinoic acid in the developing cerebellum in vivo by unilateral lesioning of the choroid plexus and by application of retinoic-acid soaked beads. (5) We will try to isolate the human homolog of the murine retinoic-acid generating enzyme, in order to facilitate future work on cerebellar abnormalities in humans. This is exemplified by the Acutane(R)-induced cerebellar malformation which resembles the sporadically occurring Dandy-Walker syndrome and Joubert syndrome, in which ataxia and mental retardation are observed.
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