Branching Gene Regulatory Network for Neurotransmitter versus Axonal Pathfinding

In previous studies we found that the two transcription factors collier (FB:knot, kn) [vertebrate Ebf1] and ladybird early (lbe) [vertebrate Lbx1/2], when co-misexpressed from elav-Gal4, generate ectopic Nplp1 neuropeptidergic cells in the entire ventral nerve cord during Dmel. embryonic development. We also found that the axon generated by the ectopic Nplp1 cells, seem to follow the same fascicle as the wild type Nplp1 neurons. Therefore we used RNA-seq on control and UAS‑col,lbe co-misexpression to find candidate factors important in axon pathfinding of the Nplp1 neurons. We also included single misexpression of UAS-col and UAS-lbe, together with co-misexpression of UAS-ap, eya, since we know that col and lbe activate those two factors during the cell fate specification of Nplp1 neurons. We generated single misexpression lines UAS-col-HA (ChrIII) and UAS-myc-lbe (ChrIII). Then we generated the double misexpression lines UAS-col, lbe (UAS-col-HA, UAS-myc-lbe) and UAS-ap, eya (UAS-Flag-ap; UAS-eya-HPC4). We misexpressed these constructs from elav-Gal4, a pan neuronal driver mostly active in postmitotic neurons, and compared the TPMs of the misexpression to the TPMs of the control (in our case elav-Gal4 heterozygous over a wildtype chromosome from the OregonR stock). We performed the same experiments at two different time points in embryonic development i.e. stage16 and at the late stage AFT (air filled trachea). Within each experiment, we have three biological replicates per genotype (control and misexpressions). In total, we have had 30 samples (3 samples over 5 groups per experiment, at two different time points).