Fig. 4. Evolving a feed-forward regulatory network from a single input or a simple cascade regulatory network. (A) In the feed-forward network, factor A directly regulates each gene: sequential activation is achieved by requiring both A and B to express gene C; and both A and C to express gene D. (B) In a single-input network, factor A directly regulates the three targets B, C and D and does not have temporal patterning. (C) The simple cascade accomplishes sequential gene activation with only gene B directly activated by A. It is easy to see how generating a new single-input network might generate a selectable phenotype that could evolve feed-forward regulation. Evolving a cascade motif would require a selective advantage for each stage, but once it had evolved it could be invaded by factors such as Myod to convert it into a feed-forward network.