(A) Mathematical model with decreasing activator production rates (left) or decreasing initial repressor numbers (right). (B) Schematic representation of the two conserved regions in the mig-1 upstream sequence. Green rectangles are exons, the gray dashed line is the upstream region, and black solid lines are introns. The orange rectangles represent the deleted regions that contain upstream motif pair #1 (Δupstream 1) and upstream motif pair #2 (Δupstream 2). (C) Single-molecule FISH (smFISH) quantification of mig-1 expression in QR and its descendants relative to the position of the seam cells H2 to V5 in control and deletion mutants of the two regions upstream of mig-1. None of the deletions affect the early phase of mig-1 expression in the QR neuroblast (hu314 Δupstream 1), p=0.59, n=22; hu315 (Δupstream 2), p=0.07, n=40; hu335 (Δupstream 1–2), p=0.89, n=22, Welch’s t-test. The upregulation of mig-1 expression in QR.pa is not significantly changed in Δupstream 1 (p=0.44, n=51) but is significantly reduced in Δupstream 2 (p<0.0001, n=83) and abolished in the double Δupstream 1–2 deletion (p<0.0001, n=29). Data are from eight independent experiments with three replicates for Δupstream 1 and 2 and three independent experiments with three replicates for the double Δupstream 1–2 deletion. The sequences of the upstream motifs in 23 different Caenorhabditis species are in Figure 3—figure supplement 1. Figure 3—figure supplement 2 shows the effect of the upstream motif pair deletions on the final position of QR.pap.