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First published online 12 October 2005
doi: 10.1242/dev.02079

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A Drosophila model of the Niemann-Pick type C lysosome storage disease: dnpc1a is required for molting and sterol homeostasis

¹ Departments of Developmental Biology, Genetics, and Bioengineering, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305-5439, USA
² Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5439, USA

View larger version (57K): [in a new window]   Fig. 1. dnpc1a gene and mutant phenotypes. (A) Phylogenetic tree of yeast, worm, fly, mouse and human NPC1 proteins determined using the ClustalW analysis. According to the BDGP prediction (www.flybase.org), dnpc1b encodes a 1223 amino acid protein that lacks any signal peptide. However, another putative start codon 93 nucleotides before the BDGP-predicted ATG adds a 31 amino acid peptide that contains a predicted signal peptide and aligns well with the N termini of NPC1 and NPC1L1. (B) The gene structure of dnpc1a and deletion regions of two dnpc1a alleles. (C) Aberrant sterol accumulation in dnpc1a mutants observed using filipin staining. Left column, wild type; right column, dnpc1a mutants. Top panels show the Malpighian tubules (inset, magnified view); bottom panels show midguts. (Left) Filipin staining highlights the lumen of the Malpighian tubules (top, arrow) and the cell-cell boundaries of midgut cells (bottom, arrow) in wild-type first-instar larvae. In dnpc1a mutants (right), in addition to the normal sites of sterol, punctate accumulations of filipin staining are visible (arrowheads) inside Malpighian tubule and midgut cells.

View larger version (103K): [in a new window]   Fig. 2. Localization of dnpc1a RNA in embryos and larvae. (A-E) Anterior towards the left. (A) Ubiquitous expression of dnpc1a in a cellular blastoderm-stage embryo. (B) Higher level of expression can be seen in gastrulation furrow (arrow) of stage 7 embryo. (C) After germband extension, higher level expression is in the hindgut (arrow). (D) Aminoserosa (arrow) staining can be seen in a stage 14 embryo. (E) Putative prothoratic gland cells (arrow) in stage 16 embryos. (F) High level expression of dnpc1a mRNA in the ring gland of wandering third-instar larvae. (G) dnpc1a mRNA is also present in the garland cells (arrow). (H) In situ hybridization with dnpc1a probe detects no signals in dnpc1a1 mutant embryo.

View larger version (38K): [in a new window]   Fig. 3. dnpc1a mutants are arrested during the first instar and can be partially rescued by ecdysone feeding. (A) Comparison of different sizes of larvae of wild type (top) versus dnpc1a mutants (bottom) at different times points after egg laying (AEL) shown in B. (B) Graphic view of the body length of the wild type (n=50) and dnpc1a mutant larvae (n=50) at different times points after egg laying. (C) 20-Hydroxyecdysone feeding of developing larvae at two different time points can postpone the lethal stages of dnpc1a mutants. x-axis, stages of development.

View larger version (39K): [in a new window]   Fig. 4. High media level of cholesterol or 7-dehydrocholesterol can rescue dnpc1a. (A) Cholesterol, but not ergosterol, can partially rescue dnpc1a mutants. (B) 7-Dehydrocholesterol can rescue dnpc1a mutants to adulthood. Desmosterol and progesterone do not rescue. x-axis, stages of development.

View larger version (142K): [in a new window]   Fig. 5. No significant difference in the brain sections of wild type and dnpc1a mutants. The comparison of the brain histology between a wild-type adult animal (A) and a 7-dehydrocholesterol feeding-treated dnpc1a mutant adult (B) reveals no significant difference. (C,D) Enlarged views of the central brain regions boxed in A,B, respectively. No obvious characteristic neurodegenerative vacuoles are observed in dnpc1a mutants.

View larger version (102K): [in a new window]   Fig. 6. Sterol accumulation in dnpc1a mutants. (A,B) Filipin-stained wing imaginal discs from wild type (A) and dnpc1a mutant (B) third-instar larvae. Insets show magnified views; sterol is mainly located at cell-cell boundaries in wild type, while it accumulates in a punctate pattern in dnpc1a mutants. (C,D) Filipin-stained brains from wild-type (C) or dnpc1a mutant (D) third-instar larvae. There is no punctate accumulation present in wild type (C), but some accumulation can be seen in dnpc1a mutants (arrows in D). Insets show magnified views. (E-H) Third-instar ring glands (outlined) in different backgrounds. (E) Wild-type ring gland filipin staining. (F) Filipin staining of ring gland in ecdysone-rescued dnpc1a mutants. Punctate sterol accumulation can be seen in both ring gland and brain lobes. (G) The 2-286 driver specifically induces the expression of dnpc1a-YFP in ring gland but not brain lobes. In order to see the brain lobes, the signal was boosted. (H) dnpc1a mutant rescued by 2-286 driving dnpc1a-YFP. The ring gland shows a wild-type filipin staining pattern, while the brain lobes still have punctate sterol accumulation.

View larger version (126K): [in a new window]   Fig. 7. Ultrastructural defects in adult Malpighian tubules of dnpc1a mutants. (A,C) Wild type; (B,D), dnpc1a mutants. (A,B) Filipin staining to show the sterol distribution in wild type and dnpc1a mutants. (C,D) Electron microscopy pictures to show large multi-lamellar structures (arrow in D) present in dnpc1a mutants but not wild-type samples.

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