Effects of Zmiz1 deletion in ETPs were inconsistent with Notch1 loss of function. (A) Representative thymuses of VavCre control, Zmiz1ΔVavCre, and Notch1ΔVavCre mice. (B-C) Representative FACS plots of the ETP-DN2 transition (CD44⁺Lin⁻) (B) and relative cell numbers (C) showing shift to DN2 cells (red line) (N = 11 per group). (D-E) LSK BM precursors from VavCre or Zmiz1ΔVavCre mice were transduced with vector control or ICN1 and cultured on OP9-DL1 cells for 7 days. Representative fluorescence-activated cell sorting (FACS) plots (D) and relative cell numbers (E) of transduced DAPI⁻CD45⁺GFP⁻CD44⁺Lin⁻ cells showing shift to DN2 cells (red line) similar to Notch1 gain of function (panel D rightmost FACS plot) (N = 3 per group). (F-J) RNA-Seq analysis on sorted ETPs was performed using similar strategy and analysis as described in Figure 3 and supplemental Figure 6. Target genes were defined as FC > 1.25, P < .01. Overlaps of Notch1- and Zmiz1-regulated genes are shown in Venn (F) or column (G-H) formats with cooperativity or antagonism of shared genes shown in panel I. (J) Fold change of known direct Notch1 target genes during T-cell development. Red arrows identify Myc. (K) Working model of possible Zmiz1 functions in ETPs. (L) Microarray data (www.immgen.org)⁶⁵  showing that the Notch1 target genes in panel J, except Myc and Lef1 (which are not regulated by Zmiz1 in ETPs), increase >2× at DN3a and then decrease >2× at DN3b. Two sample Student t test. *P < .05; **P < .01; ****P < .0001.