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Research Statement Supporting Figures

 
     

Figure 1. Genome-wide binding profiles for Geminin and Zic1 were used to define gene regulatory networks in neural cell fate acquisition. (A-D) Comparison of Gmnn-associated and Zic1-associated genes in NE. (A) A subset of genes is associated with both Gmnn and Zic1. p-value (Chi-square test with Yates’ correction) < 2.2X10-16. (B) z-scores for enrichment of expression in ES versus CNS tissues for all Zic and/or Gmnn associated genes. (C) Comparison of all Gmnn or Zic1 associated genes, subsets that undergo Gmnn-dependent acetylation, and transcription factors, with their relative enrichment of expression in embryonic CNS tissues. (D) GO enrichment analysis for associated genes with increased versus decreased expression in embryonic CNS, relative to ES cells. (E) Gene regulatory networks in neural cell fate acquisition. Gmnn- and Zic1-associated genes in NE that encode transcription factors and epigenetic regulatory activities were assessed for co-association with Sox2 and Sox3 in ES-derived NE (see text). The set of these genes that exhibit CNS-enriched expression (>two-fold greater expression in E14 CNS, relative to ES cells), and were associated with Gmnn and/or Zic1 plus Sox2 and/or Sox3 were used to build gene regulatory networks showing associations.

 

Figure 2. Human cortical interneuron derivation and characterization. (A) Immunocytochemistry (ICC) for ventral telencephalic/MGE, neuron, cIN and alternate cell fate (RAX/DARPP32) markers, in day 35 hESC-derived interneurons (scale bar=100µm). (B) FACs for Nkx2-1 and (C) quantitation of % ICC+ cells. Inset: FACS analysis for the SST+ cortical interneuron subtype. (D) Synapsin-GFP expressing day 35 cIN (after PSA-NCAM+ selection; scale bar=100µM) were (E) transplanted into the cortex of P2 NOD/SCID mice (20 days post-transplantation shown; B’ box in B, scale bar=100µM) or (F) transplanted into the hippocampus (CA3) of NOD/SCID adult mice (1 month post-transplantation shown, C’ box in C; scale bar=50µM). (D-E) Representative action potential firing pattern (G) evoked by injection of a square pulse, (H) spontaneous. (I) Voltage-gated currents evoked by a step from -80 to +50 mV in the absence (black) or presence (red) of the sodium channel antagonist tetrodotoxin (TTX, 0.5 µM). (J) Spontaneous synaptic currents recorded at -20 and -80 mV. Exposure to the GABAA receptor antagonist bicuculline methiodide (200 µM), indicated by the open box above each trace, blocked spontaneous Inhibitory postsynaptic currents (IPSCs). IPSCs were inward at -80 mV and outward at -20 mV, consistent with the equilibrium potential for chloride (-54 mV) with our internal and external solutions. Work performed in collaboration with Jim Huettner’s laboratory.