Given a potential role for FURIN, TSNARE1, and CNTN4 during neurogenesis, we asked whether the decrease in head size could be attributed to changes in cell proliferation and/or apoptosis. Overexpression of CNTN4 and suppression of furin_a led to a 9.8% (P = 0.003) and a 29.8% (P < 0.001) decrease, respectively, in proliferating cells marked by phospho-histone3 (PH3), and overexpression of TSNARE1 led to a 9.5% increase (P = 0.018) in proliferating cells (N = 20 per experiment; Fig. 3C, D). Next, we wondered how more proliferating cells nevertheless resulted in a smaller head size phenotype for the case of TSNARE1. To test the possibility that cells exiting cell cycle experience a higher apoptotic index, we performed TUNEL staining on injected embryos, and determined that modulation of all three target genes led to a significant increase in apoptotic cells in the head region corresponding to our head size measurements (N = 20 per experiment; P < 0.001; Fig. 3E, F). Taken together, the data support the hypothesis that changes in FURIN, TSNARE1, and CNTN4 expression levels induce subtle neuroanatomical variation in multiple brain regions.
