Fig. 7. Dorsal-Twist patterning activities. (A) Previous studies identified a cluster of three low affinity Dorsal operator sites in a distal region of the snail 5' cis-regulatory region. The proximal regulatory region contains two Twist binding sites. The diagrams above the representation of the snail gene depict cross sections of embryos with Dorsal (left) and Twist (right) protein gradients (there are peak levels in ventral regions). The diagram to the far right summarizes a mutant embryo derived from Toll^rm9/Toll^rm10 females that contains a twist-bcd transgene. Normally, these embryos lack snail expression, but the twist-bcd transgene leads to the activation of snail in anterior regions. These results suggest that Twist plays a critical role in the on/off regulation of snail expression since the mutant embryos lack a Dorsal gradient. (B) Summary of Dorsal/Twist-regulated expression of snail and sim. We find that high amounts of Twist-bcd support ectopic sim expression (blue) that requires at least low levels of nuclear Dorsal and is repressed by Snail. High levels of ectopic nuclear Dorsal supported by the Toll^10b-bcd transgene produces only very low levels of snail expression (red) and no sim expression (data not shown) in the absence of Twist. We conclude that Dorsal and Twist transcription factors function interdependently in order to generate multiple thresholds of gene expression. (sna^-: no Snail protein; gd^-: no nuclear Dorsal protein; twi^–: no Twist protein; Toll^rm9/rm10: low levels of nuclear Dorsal protein).