Notch signaling also inhibits CS differentiation, but in an upstream or independent way than tbx2a/b. NICD embryos were heat-shocked at 15 ss to ectopically activate Notch signaling and fixed at 24 hpf. Post heat-shock induction, NICD embryos exhibited a dramatic reduction of the CS, while siblings showed normal CS clusters. To block Notch signaling, wild-type embryos were incubated in 100 µm DAPT/E3 solution from 90% epiboly to the 24 hpf stage. DAPT treatment resulted in a significant elevation in the number of CS cells. Heat-shocked NICD tbx2a/b morphants exhibited a significant expansion in the CS that was in contrast to the reduced CS observed in uninjected heat-shocked NICD siblings. The number of cells in the these CS clusters was not significantly different to the expansion seen in wild-type embryos injected with tbx2a/b MO. DAPT-treated tbx2a/b morphants and fbyc144 mutants exhibited an expansion in the CS that was not significantly different than wild-type embryos treated with DAPT alone. Together, these results suggest that tbx2a/b are acting in the same pathway, downstream of Notch to suppress the CS. Representative images were based on thorough examination of at least 15 embryos per treatment. (*p<0.05, **p<0.001, N.S.=not significant)..