Npc1aunspecified males in which lethality has been rescued by expression of Npc1aScer\UAS.T:Avic\GFP-EYFP under the control of Scer\GAL42-286 are sterile. The seminal vesicles of the mutant males are much smaller than wild type and contain essentially no mature sperm, but an accumulation of sperm debris is seen in the basal region of the testis. A mild cytokinesis defect (penetrance 5.79%) is seen, but the mutants have a similar number of spermatids per cyst as wild type at the elongated stage. The individualisation complex assembles normally at the head region of the spermatid bundle in the mutant males. However, it gradually disintegrates as it moves along the cyst and scattered actin cones are seen. Some sperm nuclei are scattered along the cyst in the mutant males (in wild-type males they are clustered at the head of the cyst) and these scattered sperm nuclei lose their needle-like shape, becoming more condensed and rounded the further away from the head region they are. 28 +/- 16% of mutant spermatids show defects in individualisation and the average number of spermatids per cyst after individualisation is reduced to 54. Mutant spermatocytes show sterol accumulation and sterol trafficking defects. Individualisation defects are more severe at higher temperatures.
The fertility of Npc1aunspecified males in which lethality has been rescued by expression of Npc1aScer\UAS.T:Avic\GFP-EYFP under the control of Scer\GAL42-286 is significantly increased by addition of 7-dehydrocholesterol in the food, but is not increased by the addition of cholesterol, ergosterol or sitosterol in the food.
Scer\GAL42-286/Npc1aUAS.EYFP partially rescues Npc1aunspecified
Scer\GAL4Feb36/Npc1aUAS.EYFP partially rescues Npc1a1
Scer\GAL42-286/Npc1aUAS.EYFP partially rescues Npc1a2
Scer\GAL4Feb36/Npc1aUAS.EYFP partially rescues Npc1a2
Scer\GAL4C587/Npc1aUAS.EYFP fails to rescue Npc1aunspecified
Scer\GAL4ptc-559.1/Npc1aUAS.EYFP fails to rescue Npc1aunspecified
Npc1aUAS.EYFP/Scer\GAL4repo fails to rescue Npc1a1
Npc1aUAS.EYFP/Scer\GAL448Y fails to rescue Npc1a1
Scer\GAL4Aug21/Npc1aUAS.EYFP fails to rescue Npc1a1
Scer\GAL4Mhc.PW/Npc1aUAS.EYFP fails to rescue Npc1a1
Npc1aUAS.EYFP/Scer\GAL448Y fails to rescue Npc1a2
Scer\GAL4Aug21/Npc1aUAS.EYFP fails to rescue Npc1a2
Scer\GAL4Mhc.PW/Npc1aUAS.EYFP fails to rescue Npc1a2
Scer\GAL4elav-C155/Npc1aUAS.EYFP fails to rescue Npc1a2
Expression of Npc1aScer\UAS.T:Avic\GFP-EYFP under the control of Scer\GAL42-286 partially rescues the Npc1aunspecified phenotype; animals are rescued to adulthood, but only 13.3% of the rescued males are fertile, and those that are fertile produce a smaller number of progeny than normal.
Expression of Npc1aScer\UAS.T:Avic\GFP-EYFP under the control of either Scer\GAL4Hsp83.PA or Scer\GAL4tub.PU rescues the lethality of Npc1aunspecified animals and results in fertile males.
Expression of Npc1aScer\UAS.T:Avic\GFP-EYFP under the control of either Scer\GAL4C587 or Scer\GAL4ptc-559.1 rescues the lethality of Npc1aunspecified animals but results in sterile males.
Neural expression of Npc1aScer\UAS.T:Avic\GFP-EYFP under the control of Scer\GAL4elav-C155 does not rescue the ecdysone-dependent larval lethality of Npc1a1 mutants. However, in cholesterol-rescued adult mutants, targeted neuronal expression strongly rescues the progressive movement defects and early lethality, extending the life span of Npc1a1 mutants to a comparable age as controls. Neuronal expression also rescues the filipin-positive cholesterol aggregates found in Npc1a1 mutant brains.
Glial expression of Npc1aScer\UAS.T:Avic\GFP-EYFP under the control of Scer\GAL4repo does not rescue the ecdysone-dependent larval lethality of Npc1a1 mutants. However, in cholesterol-rescued adult mutants, targeted glial expression strongly rescues the progressive movement defects and early lethality, extending the life span of Npc1a1 mutants. However, Scer\GAL4repo controls also exhibit a longer life span, although not as long as those expressing Npc1aScer\UAS.T:Avic\GFP-EYFP.
Expression of Npc1aScer\UAS.T:Avic\GFP-EYFP in the ring gland under the control of Scer\GAL42-286 rescues larval lethality in Npc1a1 mutants without cholesterol feeding but does not rescue the filipin-positive cholesterol aggregates found in Npc1a1 mutant brains.
Neural expression of Npc1aScer\UAS.T:Avic\GFP-EYFP under the control of Scer\GAL4elav-C155 rescues rhabdomere structure and photoreceptor vacuolization.
Neural expression of Npc1aScer\UAS.T:Avic\GFP-EYFP under the control of Scer\GAL4elav-C155 at day 45 results in a striking reduction in multi-lamellar bodies within the cytoplasm of Npc1a1 mutant neuronal cells.
Expression of NPC1Scer\UAS.T:Avic\GFP-EYFP in the ring and salivary glands, driven by Scer\GAL42-286, rescues the lethality of NPC11 and NPC12 mutants, allowing them to survive to adulthood. The subcellular accumulation of sterols in many tissues other than the ring gland is not reduced in the rescued mutants. Sterol accumulation is most severe in the Malpighian tubules, which show ultrastructural defects. Expression of NPC1Scer\UAS.T:Avic\GFP-EYFP in the ring gland, salivary glands, trachea, midgut and Malpighian tubules, driven by Scer\GAL4Feb36, also rescues NPC11 and NPC12 lethality, although some mutants still die as third instar larvae.
