SHH mediated human dopaminergic neuron differentiation is facilitated by transcription factor ZIC3

Distinct neuronal subtype specification involves a complex network of transcription factors (TF). Previous studies from our laboratory showed Zinc finger transcription factor of cerebellum (ZIC) 3 to regulate the expression of Tyrosine Hydroxylase (TH), a proxy marker for dopaminergic (DA) neurons, in mouse olfactory bulb (OB) and mid brain (MB) neurons by distinct mechanisms. In absence of ZIC3 consensus binding site in proximal region of mouse TH promoter, ZIC3 interacts with ER81 in OB and regulates TH expression whereas in absence of ER81 in midbrain, ZIC3 regulates TH by enhancing the expression of PITX3. To analyze whether this observation is evolutionarily conserved in humans, different human stem cell model systems were utilized to understand the role of ZIC3 in human DA generation. Differentiation of dental pulp stem cells (DPSCs) with proper cues resulted in the expression of TH. Gain and loss of function demonstrated ZIC3 to be essential for the expression of TH. ZIC3 activates the transcription of TH gene by binding to response element within the region of TH promoter. Addition of SHH, a known morphogen that facilitates dopaminergic differentiation, increased the expression of TH, however, this regulation was suppressed in the absence of ZIC3. Mechanistic insight demonstrated SHH influences ZIC3 expression by GLI protein binding to ZIC3 promoter. Similar role of ZIC3 in TH expression was observed in hiPSCs differentiated to DA like neurons. Conclusively, the present study for the first time demonstrates the undetermined importance of ZIC3 in SHH mediated TH specification in cells of human origin.