The Impact of the Williams Syndrome Mutations on Neural
James S. McDonnell Foundation Collaborative Activity Award:
Project 5: Where are the Williams syndrome genes expressed in the nonhuman primate brain? (Amaral and Korenberg).
The development of a non-human primate model for human behavior and genetic dissection is greatly aided by the high homology existing between the human and rhesus DNA sequences, both at the cDNA and the genomic levels. While it is currently not feasible to produce Williams syndrome knock-outs in nonhuman primates, we can use a second strategy to determine the regional expression patterns of Williams syndrome related genes, particularly CYLN2, GTF2IRD1 and GTF2I. We will use advanced neuroanatomical techniques, such as in situ hybridization and immunocytochemistry in the monkey to identify regional and developmental expression of genes in the Williams syndrome region that may be related to the specific social phenotype. With the recent finding of varied deletions in humans with Williams Syndrome and their concomitant alterations in the typical social profile of the disorder, we are in an unprecedented position to use these few genetic markers to probe for regions in the nonhuman primate brain that may be intimately associated with the mediation of social behavior. High levels of, or differential, expression in particular brain areas or particular cell types may indicate candidate structures involved in social behavior. This knowledge will also pave the way to molecular lesioning techniques that hold the promise of producing a brain alteration reminiscent of the alterations involved in Williams syndrome.
Analysis of gene expression in rhesus monkey brain will be performed by tissue in situ hybridization (to detect gene specific RNA) using RNA probes derived from rhesus specific genes encoding the human homologs for CYLN2, GTF2IRD1, and GTF2I. It will be important to determine that the sequences of the genes defined in humans are highly homologous in the rhesus monkey and do not differ in critical domains, splice variants or expression. Therefore, gene sequences will be determined in the rhesus monkey by using reverse transcription PCR (RTPCR) based sequence analyses templated on RNA from normal rhesus brain tissues. Northern analyses will contain the RNA species corresponding to humans. This is important in establishing the rhesus monkey as an appropriate model in that variation of sequence or domain structures may affect developmental processes.
To evaluate the potential consequences of exon-specific variants known for GTF2I, and their potential importance in developmental processes, exon specific probes or antibodies generated to the novel junction fragments resulting from the splice variants will be utilized. Differential usage has already been established and the gamma form of the gene for GTF2I (containing both exons) established as most highly expressed in the central nervous system. Further, fleeting and specific phosphorylation has been established to occur during B cell antigen crosslinking and may also be important during unique events in brain development such as sulcal formation or gyrification. This will therefore be studied with immunohistochemistry using domain and phosphorylation product specific antibodies, and the results compared to neural structures employing appropriate specific neuronal and glial markers. Northern analyses of brain regions with rhesus during specific developmental stages will be used to establish use of splice variants during development. Tissue will be obtained from fetal material obtained through Caesarian section at the California Regional Primate Research Center throughout fetal life. Material will be available for regional analysis in the adult brain from a library of over 80 cases of adult macaque monkeys maintained in the Amaral laboratory.
Thus, the specific aim of this project is to determine whether the genes associated with Williams hypersociability are differentially expressed in certain regions of the central nervous system. We anticipate carrying out these studies both in mature and in embryonic and fetal tissue in order to determine whether the genes are developmentally regulated.
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