Fig. 3. Drosophila Cas and integrin mutants exhibit ISNb motor axon defasciculation defects. (A-I) Abdominal segments of filleted stage 16/17 Drosophila embryos immunostained with the monoclonal antibody 1D4 to visualize all motor axons (Anterior, left, dorsal, up). The intersegmental nerve (ISN), intersegmental nerve b (ISNb), intersegmental nerve d (ISNd), segmental nerve c (SNc), segmental nerve a (SNa) and ventral muscles 6, 7, 12 and 13 are indicated. (A) ISNb motor axons in DCas^{Df(3L)Exel6083}/+ embryos defasciculate normally from the ISN and grow dorsally to innervate muscles 6/7 and 12/13 (open arrows, used similarly in D,E,H,I). (B-E) DCas loss-of-function mutant embryos often fail to innervate their muscle targets (black arrows) and exhibit a range of ISNb guidance phenotypes indicative of an increase in axon-axon fasciculation. These defects include ISNb axons remaining abnormally fasciculated with the ISN (B,C, arrowheads), and ISNb axons abnormally bundled together (C, broad arrow). ISNd axons also do not defasciculate normally to their muscle targets (B, white arrow). (D) ISNb axons in DCas mutants are also abnormally fasciculated with the ISN (arrowhead), where they often separate at inappropriate locations and extend abnormally (small arrows). (E) ISNb axons in a DCas mutant are abnormally fasciculated such that they extend past their muscle targets (arrowhead, data not shown) and eventually take abnormal routes to reach their muscle targets (large black arrow). (F,G) Axons in 1 integrin (mew^M6) (F, 1Int) and 2 integrin (if^K27E) (G, 2Int) LOF mutants exhibit axon guidance defects resembling DCas LOF mutants, including abnormal fasciculation of ISNb axons with the ISN (arrowheads), abnormal fasciculation of ISNd axons with the ISN (white arrow, F) and failure of ISNb axons to innervate their muscle targets (black arrows). (H,I) Expression of DCas rescues ISNb defects observed in DCas LOF mutants. Expression of either ^MycDCas under the DCas^P1-GAL4 driver (H, UAS-^MycDCas/+; Df(3L)ED201/DCas^P1) or ^MycDCas in all neurons using the ELAV-GAL4 driver (I, ELAV-GAL4/UAS-^MycDCas; DCas^{Df(3L)Exel6083}/DCas^{Df(3L)Exel6083}) restores the normal ISNb innervation pattern in DCas mutants (open arrows). Scale bar: 15 µm. (J) Schematic summarizing ISNb motor axon guidance phenotypes.