Gene regulatory networks (GRNs) ensure precise spatio-temporal control of gene expression during development. Changes in GRNs underlie the evolution of morphological novelty. We have investigated the mechanisms of GRN evolution using the dengue fever and Zika vector mosquito A. aegypti and the fruitfly Drosophila melanogaster CNS developmental paradigms. Despite substantially similar nervous system morphology, the two species show significant divergence in a set of genes coexpressed in the Drosophila ventral midline, including the master midline regulator single minded (sim) and downstream genes including short gastrulation (sog), Star, and NetrinA (NetA). In contrast to Drosophila, we find that in A. aegypti midline expression of these genes is absent or severely diminished, and instead they are co-expressed laterally in the nervous system. This suggests that in A. aegypti this “midline GRN” has been redeployed to a new location while being simultaneously lost from its previous site of activity, with a new GRN acquiring the original role. In order to characterize the GRNs, we employed SCRMshaw—a computational method we previously developed for supervised motif-blind and sequence alignment-free CRM prediction—to identify 6 novel CRMs from A. aegypti and 6 CRMs for D. melanogaster genes and validated them in vivo in transgenic D. melanogaster. Analysis of these validations and results from genetic perturbation experiments suggest that these changes are due to a trans-dependent redeployment of the GRN, potentially stemming from cis-mediated changes in the expression of sim in combination with one or more transcription factors in A. aegypti. We are continuing to identify additional relevant CRMs as well as undertaking an in-depth analysis of the expression patterns of multiple genes in this network in both species. Our results illustrate a novel “repeal, replace, and redeploy” mode of GRN evolution in which a conserved GRN undergoes neofunctionalization at a new site while its original function is co-opted by a different GRN without causing substantial changes in overall morphology and function.