Fig 1.
Comparative gene expression analysis of SFTA3 and NKX2.1.
A) SFTA3 and NKX2.1 pairwise gene expression analysis indicates significant levels of enrichment in the NKX2.1-positve progenitor population in comparison to the NKX2.1-negative cohort. Fold change differences of 2, 4, and 8 between samples are represented by central diagonal lines. Orange and blue points signify enriched and depleted differentially expressed genes (FDR<0.05) comparing NKX2.1-positive to NKX2.1-negative progenitor populations with the total number of genes indicated for each category. B) RT-PCR analysis of SFTA3 and NKX2.1 gene expression at specific time points during the hESC differentiation timeline. In the RT-PCR time course, the data were normalized to the housekeeping gene GAPDH. C) In vitro day 26 immunocytochemistry and quantification of neural progenitors for NKX2.1 and SP-H. Data represented as ± SEM. Scale bar = 100 μm. D) RNA-seq comparison of FACS isolated NKX2.1-positive and NKX2.1-negative cell populations in comparison to the developmental transcriptome of various structures of the human brain at 8 and 9 pcw. Brain structure legend: DTH, dorsal thalamus; DFC, dorsolateral prefrontal cortex; HIP, hippocampus; OFC, orbital frontal cortex; Ocx, occipital neocortex; MFC, anterior cingulate cortex; PCx, parietal neocortex; URL. upper rhombic lip; VFC, ventrolateral prefrontal cortex; STC, posterior superior temporal cortex; M1C-S1C, primary motor-sensory cortex; ITC, inferolateral temporal cortex; AMY, amygaloid complex; CGE, caudal ganglionic eminence; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence.
Fig 2.
Characterization of day 25 SFTA3 and NKX2.1 KO neural progenitors from hESCs.
A) RT-PCR data comparing gene expression levels between NKX2.1 control and KO cell progenitors. Data represented as mean ± SEM. * = p<0.05. B) RT-PCR data comparing gene expression levels between SFTA3 control and KO cell progenitors. Data represented as mean ± SEM. * = p<0.05. C) Composite confocal microscopy images that show day 25 immunolabeling of control and knockout hESNPs following differentiation to MGE-like fate. All hESC-derived cells (HuNu, green) and other markers in red are indicated in each panel. Markers label progenitors of the ventral forebrain (NKX2.1, DLX2, Olig2), immature neurons (DCX), neural stem cells (nestin), and telencephalic progenitors (FOXG1). Scale bar = 20 μm. D) Quantification of day 25 immunocytochemistry. Data represented as mean ± SEM. *** = p<0.001, ** = p<0.01, * = p<0.05 (ANOVA).
Fig 3.
Characterization of mature hESC-derived neurons from day 45 SFTA3 and NKX2.1 KO lines.
A) Composite micrographs representing day 45 immunocytochemistry of mature inhibitory interneurons differentiated from control and KO hESNPs. Immunolabeling for markers of immature neuronal precursor cells (DCX), mature neurons (MAP2) and GABAergic neurons (GABA). Scale bar = 100 μm. B) Day 45 immunocytochemistry quantification analysis of GABA, DCX, and MAP2 expression. Data represented as ± SEM. *** = p<0.001, ** = p<0.01, * = p<0.05 (ANOVA).