UC San Diego

Neurotransmitter specification, which is vital for appropriate communication between neurons, is developmentally regulated by the combinatorial expression of genes and calcium spike activity. In order to identify molecules that may be involved in activity-dependent neurotransmitter specification, I performed a genetic screen using 1156 non-redundant full-length cDNAs from the Emb7 library. The Emb7 library is a good starting point for such a screen because these clones are expressed during stages 20-27 when calcium spiking occurs. For both the pilot screen and the main screen, clones were combined in pools of 8 and transcribed into mRNA. The pools of mRNA along with mRNA encoding hKir2.1 were then injected into Xenopus tropicalis embryos at the 1 cell stage. Larvae were fixed, sectioned and stained with GABA antibodies. The GABA-IR cells were counted per 100 [mu]M of the embryo spinal cord. The pilot screen of transcription factors identified 3 individual clones that appeared to occlude the effect of hKir2.1 on calcium spike suppression. Morf4l2, which is expressed in the Xenopus spinal cord and related to the MRG family of transcription factors was of particular interest. MRG15 has been shown to be involved in synaptic plasticity and in guiding other transcription factors. In the main, unbiased, screen, 6 clones were of high interest because they are expressed in Xenopus spinal cord and have known functions. Of the 6 clones, Brain acid soluble protein 1 (Basp1) and Tetraspanin 7 (Tspan7) are potential candidates for further study. Identifying these types of molecules is vital to understand how neurotransmitter specification is regulated by both gene expression and calcium signaling