Modulation of Excitatory and Inhibitory Neuronal Balance Through Regulation of Ptf1a by Factors Binding to Zinc Finger and POU Motifs
Avila, John Manuel, Jr.
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The proper function of the nervous system depends on a delicate balance between excitatory and inhibitory neurons. Transcription factors of the basic helix-loop-helix (bHLH) family have been shown to be particularly important in generating the correct numbers of these neurons during development. One of these, Ptf1a, is required in the specification of inhibitory neurons in multiple regions of the nervous system including the dorsal spinal cord. The absence of Ptf1a in null mice disrupts the balance of excitatory and inhibitory neurons, as Ptf1a is required for generating inhibitory neurons while suppressing the excitatory phenotype. Therefore, discovering the regulators of Ptf1a expression will identify mechanisms controlling the generation of a balanced neural network required for processing somatosensory information. Using sequence conservation between divergent vertebrate species, a 1.2 kb enhancer that directs expression of a reporter gene to Ptf1a expressing domains in transgenic mice was identified approximately 11 kb 3’ of the coding region. A series of mutations across the 1.2 kb enhancer were generated to identify sequences required for activity of this enhancer. The activity of the enhancer in directing expression specifically to the developing dorsal neural tube requires at least two distinct motifs: a putative POU motif required for activity, and a zinc finger which represses activity in non-Ptf1a-expressing populations within the neural tube. The activities of these two motifs were tested by in chick and transgenic mice. Coupled with a bioinformatics approach, several candidates for the upstream transcription factors have been identified and were tested for their role in regulating the temporal and spatial specific-activity of the Ptf1a-enhancer. One factor, Zic1 was shown to repress expression of Ptf1a. Thus, a combination of transcriptional activators and repressors are required to control Ptf1a expression, which regulates the subsequent balanced generation of inhibitory and excitatory neurons in the dorsal spinal cord.