In cultured neurons from stage 11 shot3 homozygous embryos, there are significantly more microtubules in axon shaft filopodia, as compared to controls. In both shot3 hetero- and homozygous conditions, the axonal microtubules are significantly more disorganized than in controls.
The medulla in shot3 heterozygous adults shows axon swelling with microtubule disorganization.
The microtubule organization in shot3 mutant follicle cell clones is perturbed as their apical enrichment is lost.
Cultured primary neurons derived from homozygous embryos have a significantly reduced axon length compared to wild-type controls.
shot3/shotVV third instar larvae show overgrowth (increase in bouton number) at the neuromuscular junction.
shot3/shotVV axons show enhanced regenerative axonal sprouting compared to controls after a crushing injury.
In shot3 embryos, the pCC axon aberrantly crosses the midline in 16% of CNS segments.
Homozygous germ-line clones produce egg chambers, but they do not progress beyond stage 7 of oogenesis. Each mutant egg chamber contains 16 nuclei, and the pattern of divisions is also normal. Fusome structure and persistence appear normal. In addition the fusome/spectrosome is still distributed asymmetrically in early stem cell/cystoblast division, with the larger portion being found in the stem cell after division. In mutant cysts all of the nuclei, including the two at the posterior of the egg chamber appear polyploid: All of the germ-line cells adopt a nurse cell fate, no oocyte is specified. The synaptonemal complex accumulates in none or two or more cells all throughout region 2a of the cyst, but this accumulation fails to refine onto one cell only and is always lost region 2b. In wild-type ovaries, microtubules are highly concentrated around the fusome from region 2a on, and in region 2b and 3 a microtubule organising centre (MTOC) is visible in the oocyte. This organisation and concentration of microtubules is lost in mutant germ-line clones; microtubules fail to concentrate around the fusome., and an MTOC does not form in region 2b or 3. the migration of the centrosomes into the oocyte normally seen in wild-type ovaries fails to occur.
Mutant mushroom body neuroblast clones examined in the adult show abnormal processes projecting out from the calyx. Mutant mushroom body neuroblast clones have a significant reduction in cell number compared to control clones.
shot3 germline clones do not produce viable eggs. shot3 clones of cells are often double-layered in egg chambers from stage 6-7 onwards, with actin accumulation at the contacts between the two layers. Double-layered clones are only detected in cells overlying the oocyte, and at stage 10 of oogenesis are generally found in the posterior half of the follicle cells covering the oocyte. Microtubule levels and organisation, integrin localisation and oocyte elongation are normal in shot3 mutant cells.
Mutant embryos show defects in motor axon extension; the ISN stalls at the approximate position where it would normally make the second dorsalmost muscle contact, the SNa stalls where it would normally bifurcate and the ISNb stalls in the ventral muscle field and fails to reach muscle 12. Mutant embryos also show defects in sensory axon extension.
The tracheal dorsal trunk lumen is discontinuous at 75% of anastomosis sites in stage 14 or older mutant embryos. Lateral trunk connections and all anastomoses at the dorsal midline are affected. The overall branching pattern of trachea within hemisegments appears normal. 6% of tracheal dorsal trunk connections form apparently normally and 19% are constricted relative to wild type. Fusion cells appear to be present at the dorsal trunk anastomosis sites. Microtubules are not apically located at anastomosis sites of the tracheal dorsal trunk in mutant embryos, in contrast to wild type, though they remain largely at the cell periphery. F-actin accumulates apically in the tracheal cells but the lumen does not extend through the fusion cells at the anastomosis site.
shot3 mutants show strong reduction of output synapses at the NMJ, while this phenotype is not seen in the CNS.
The lateral chordotonal sensory axons arrest at variable stages before reaching the central nervous system (CNS); 93% extend towards the intersegmental nerve (ISN) and stall or fail to reach the ISN, 7% travel a significant distance within the ISN, but none of the axons reach the CNS border. The morphology of the LCH growth cones is normal, with the growth cone adopting the general morphology of wild-type growth cones at the place where they stall. Defects in LCH dendrite morphology are seen in shot3 and shot2/shot3 mutant embryos. 90-100% of the motor axons of all three major pathways (ISN, SNa and ISNb) stall prematurely in late stage 16/stage 17 homozygous embryos. 3% of ISN axons lack the most dorsal terminal arborisation, 68% lack the 2 most dorsal terminal arborisations and 24% lack all three terminal arborisations. 1% of ISN axons stall before entering the dorsal muscle field. The SNa and ISNb motor axons stall before reaching their muscle targets. 75% of ISNb motor axons stall in the ventral muscle field (somewhere between muscle 6 and muscle 13) and 14% stall at the muscle field border. 33% of SNa axons have one branch missing (wild-type axons form two branches), 38% lack both branches and 17% stall over the ventral muscles or are absent. Some SNa and ISNb motor axon bundles fail to exit the CNS successfully or stall earlier than the muscle field entry points in shot3/Df(2R)CX1 embryos. 61% of ISNb motor axons stall in the ventral muscle field (somewhere between muscle 6 and muscle 13) and 20% stall at the muscle field border. 40% of ISN axons lack the 2 most dorsal terminal arborisations and 42% lack all three terminal arborisations. 17% of ISN axons stall before entering the dorsal muscle field. 24% of SNa axons have one branch missing (wild-type axons form two branches), 60% lack both branches and 8% stall over the ventral muscles or are absent. 6% of ISN axons lack the most dorsal terminal arborisation, 48% lack the 2 most dorsal terminal arborisations and 43% lack all three terminal arborisations in shot2/shot3 embryos. 57% of ISNb motor axons stall in the ventral muscle field (somewhere between muscle 6 and muscle 13) and 30% stall at the muscle field border. 46% of SNa axons have one branch missing (wild-type axons form two branches) and 28% lack both branches. Muscle morphology and number is normal in late stage 16 homozygous embryos.
Mushroom body neuroblast clones induced using the MARCM system exhibit drastic axon projection defects compared with wild type controls. The origin of the pathfinding errors appears to be near the dendritic projection area. In the majority of cases the abnormal axon bundles do project in a generally wild type direction, towards the midline region. Some even eventually fuse with the medial lobe, as they do in the wild type.
Mutant phenotype as assayed by Ecol\lacZrp staining: longitudinal connectives missing. Mutant phenotype of lateral chordotonal axons includes: missing axons.