robo4, fraunspecified double mutant embryos show a mixture of longitudinal tract phenotypes, ranging from a phenotype like that seen in the robo4 single mutant (collapsed tracts) to a phenotype like that seen in the fraunspecified single mutant (tracts positioned further away from the midline than normal) and an intermediate phenotype, although the robo-like phenotype is predominant.
Embryos mutant for both robo4 and leaX123 show defects in cardioblast and pericardial cell positioning and alignment, similar to slit2 embryos. The number of cardioblast nuclei that reach the dorsal midline in these embryos is approximately 86.2, compared to approximately 104 in wild type embryos.
Expression of leaScer\UAS.cSa in cardioblasts using Scer\GAL4Mef2.PR in a robo4 mutant background results in similar defects as when leaScer\UAS.cSa is expressed using Scer\GAL4Mef2.PR in an otherwise wild type background. Namely, a lateral shift of the row of cardioblasts away from the dorsal midline to a position normally occupied by pericardial cells while adhesion between these cells is maintained.
Embryos expressing leaScer\UAS.cSa under the control of Scer\GAL4Mef2.PR in a robo4, leaX123 double mutant background display gaps in rows of cardioblasts indicating a loss of adhesion between cardioblasts (as seen in robo4, leaX123 double mutants) as well as the mispositioning of these cells away from the dorsal midline (as seen in leaScer\UAS.cSa, Scer\GAL4Mef2.PR embryos).
Embryos expressing leaScer\UAS.cSa under the control of Scer\GAL4Mef2.PR in a robo4, leaX123 double mutant background exhibit mispositioning of cardioblasts away from the dorsal midline which is more severe as compared to expression of leaScer\UAS.cSa using Scer\GAL4Mef2.PR in an otherwise wild type background; cardioblasts are displaced even more laterally, with cardioblasts and pericardial cells even being observed in somatic muscle territory.
Expression of leaScer\UAS.cSa under the control of Scer\GAL4Mef2.PR in a robo4 mutant background results in a slight delay in dorsal closure: a slight gap between dorsal ectodermal cells is seen in late stage mutant embryos that is not observed in wild type embryos.
In RhoGAP93B1/RhoGAP93B1, robo4/+ embryos 9% of ganglionic branches cross the midline, as opposed to 1% in RhoGAP93B1 homozygotes alone. robo4 partially suppresses the ganglionic branch phenotype seen in Rac1J11, Rac2Δ embryos.
sli2/+ robo4/+ double heterozygotes have lch5 and lesA/ldaA axons misprojecting to the v'ch1 pathway in some hemisegments. No stalling of lch5 axons is seen and dorsal sensory axons have a normal morphology.
26% of segments contain Fas2-positive neurons inappropriately crossing the midline in sli1/robo4 double heterozygous embryos, in contrast to either single heterozygote which show defects in 0 or 1% of segments. 39% of segments contain Fas2-positive neurons inappropriately crossing the midline in sli2/robo4 double heterozygous embryos, in contrast to either single heterozygote which show defects in 1% of segments.