The penetrance of the Fas2-positive axon crossover phenotype is increased in robo¹ embryos that also carry Khc::Ggal\MLCK^KA.ftz compared to either single mutant alone. A loss of integrity of the longitudinal connectives is seen in the robo¹ Khc::Ggal\MLCK^KA.ftz double mutant embryos and only remnants of the pCC/MP2 pathway are evident. In robo⁸ homozygous embryos that also carry Khc::Ggal\MLCK^KA.ftz, small gaps in the longitudinal connectives start to appear in addition to the robo⁸ phenotype. The penetrance of the Fas2-positive axon crossover phenotype is increased in sli² embryos that also carry Khc::Ggal\MLCK^KA.ftz compared to either single mutant alone. In embryos heterozygous for robo¹ and carrying two copies of Khc::Ggal\MLCK^KA.ftz, pioneer neurons of the pCC/MP2 and MP1 pathways will periodically cross the midline. Khc::Ggal\MLCK^KA.ftz mediated stalls are still evident, but in addition, the pCC neuron will occasionally cross the midline and pioneer neurons within the MP cluster extend axons across the midline as observed in robo mutants. In double homozygous Sos^e49 Khc::Ggal\MLCK^KA.ftz embryos the pCC/MP2 pathway freely crosses the midline in almost every segment. Sos^JC2 suppresses the axon crossing over phenotype seen in embryos carrying Khc::Ggal\MLCK^KA.ftz. Sos^JC2 partially suppresses the interaction between robo¹ and Khc::Ggal\MLCK^KA.ftz. The penetrance of the Fas2-positive axon crossover phenotype seen in embryos carrying Khc::Ggal\MLCK^KA.ftz is increased if they are also homozygous for drk^e0A. Homozygous comm⁸ embryos that also carry four copies of Khc::Ggal\MLCK^KA.ftz show several Fas2-positive axons abnormally crossing the midline, but commissures do not form and the connectives become slightly disorganised. Fas2-positive axons are seen to cross the midline in comm⁶ embryos which also carry Khc::Ggal\MLCK^KA.ftz. The penetrance of the phenotype is increased as the number of copies of Khc::Ggal\MLCK^KA.ftz is increased.

Pioneer neurons of the pCC/MP2 pathway stall less often and form the pathway with fewer errors in Khc::Ggal\MLCK^KA.ftz chic^sand-1 double mutant embryos compared to Khc::Ggal\MLCK^KA.ftz single mutant embryos; only 15% of hemisegments have gaps in the pCC/MP2 pathway in double homozygous embryos and only 18% of hemisegments have gaps in the pCC/MP2 pathway in Khc::Ggal\MLCK^KA.ftz chic^sand-1/Khc::Ggal\MLCK^KA.ftz + embryos.

Only 17% of hemisegments have gaps in the pCC/MP2 pathway in Khc::Ggal\MLCK^KA.ftz chic^gdh-5 double homozygous embryos.

In Khc::Ggal\MLCK^KA.ftz chic^sand-1 embryos, all branches of the motor neuron projection are at least partially restored to wild type. Innervation to muscle 12 by the ISNb branch is restored from 24% of hemisegments in chic^sand-1 single mutants to 96% of hemisegments in the Khc::Ggal\MLCK^KA.ftz chic^sand-1 double mutants. Similarly, in 88% of double mutant hemisegments, the ISN branch extends beyond the third branch point to innervate the dorsal-most muscles (1 and 2), compared to only 48% in chic^sand-1 single mutants. The subtle overextension phenotype observed at muscle 12 of Khc::Ggal\MLCK^KA.ftz single mutants is still seen in the double mutants.

The frequency of motor neuron axon stalls seen in chic²²¹ or chic^gdh-5 embryos is increased by Khc::Ggal\MLCK^KA.ftz.

Only 16% of double homozygous Khc::Ggal\MLCK^KA.ftz chic^sand-1 embryos and 18% of Khc::Ggal\MLCK^KA.ftz chic^sand-1/Khc::Ggal\MLCK^KA.ftz + embryos show abnormal midline crossing of axons within the pCC/MP2 pathway (compared to 51% of Khc::Ggal\MLCK^KA.ftz single homozygotes).

One copy of chic²²¹ suppresses the frequency of midline crossovers seen in Khc::Ggal\MLCK^KA.ftz embryos. However, Khc::Ggal\MLCK^KA.ftz chic²²¹ double mutant embryos show major defects in the formation of the central nervous system axon scaffold, with gaps or thinning of the longitudinal connectives, as axon bundles appear to preferentially cross the midline.

The number of embryos displaying abnormal midline crossing of axons increases from 5% in Khc::Ggal\MLCK^KA.ftz heterozygotes to just over 60% if the embryos are also heterozygous for either robo¹ or robo⁸.