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First published online 31 July 2007
doi: 10.1242/jcs.006551

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The fatty acid elongase NOA is necessary for viability and has a somatic role in Drosophila sperm development

Anita Jung^*,, Martin Hollmann^* and Mireille A. Schäfer

FB18 Zoologie/Entwicklungsbiologie, Universität Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany and III. Zool. Institut-Entwicklungsbiologie, Humboldtallee 34A, 37073 Göttingen, Germany

View larger version (46K): [in this window] [in a new window]   Fig. 1. Enhancer trap pattern and gene structure. (A,B) -Galactosidase activity is demonstrated in adult testis tissue (A) and in larval testis anlagen (B). The terminal epithelium is indicated by an arrowhead in A. An arrow points to the apical expression seen at both stages (A,B). The relationship between germ cells and cyst cells is shown in the diagram with the cyst cells in red and the interconnected germ cells in grey. The situation is depicted for the premeiotic phase where the two cyst cells with their unstained nuclei are equivalent (as an example surrounding four germ cells) as well as for the postmeiotic phase (with four instead of the 64 elongated spermatids) in which the head and tail cyst cells with -galactosidase-positive nuclei surround the germ cells in a directed manner. (C) Separated spermatid bundles show two stained nuclei (arrow and arrowhead) proving that both head and tail cyst cell are active. (D) The position of the spermatid nuclei is shown by Hoechst 33258 staining (double arrow). (E) The genomic organisation of the noa gene is shown (top). Exons 1-5 are shown as white boxes. The integration sites for the three P-element lines are indicated by arrows. Restriction sites within the gene are indicated for EcoRI (E), HindIII (H), SacI (S) and XbaI (X). In head RNA an alternative form of the first exon (1') was found which extends the first exon by 60 nt in 3' direction thus leading to an alternative splice donor site. The two bars (A,A') in the 3' UTR indicate poly(A) rich regions mentioned in the RNaseH experiment (see Fig. 2B). The two transcripts are outlined below the diagram. The exons show the transcribed sequences in grey and the coding region in black. The bracketed line corresponds to a PCR fragment that was used as probe to prove the existence of the long RNA species. The sequences contained within the rescue construct are given at the bottom (see Materials and Methods).

View larger version (62K): [in this window] [in a new window]   Fig. 2. Transcript characterization. (A) Northern blot showing the two sizes of noa mRNA of female (F) and male (M) OreR flies after hybridization with a noa cDNA next to a northern blot that was hybridized with the PCR fragment indicated in Fig. 1B. Transcript sizes are given in kb on the left. (B) RNaseH analysis. OreR mRNA was hybridized with oligo(dT), RNaseH treated to varying degrees (+, ++) and analyzed with a noa cDNA probe in parallel with untreated samples (–). The size of the different cleavage products is indicated in kb at the side of the blots.

View larger version (62K): [in this window] [in a new window]   Fig. 3. Amino acid alignment of NOA and its closest vertebrate relative ELOVL6. (Hs, Homo sapiens; Mm, Mus musculus; Dr, Danio rerio). Identical amino acids are shown on a black background, similar amino acids/conservative changes on grey background. The position of the transmembrane domains (Hofmann and Stoffel, 1993), which are common to all ELO proteins, is indicated by dashed lines above the sequences for the mammalian homologues and below the sequences for NOA. The conserved histidine motif HXXHH in the centre of the protein (aa 147-151) is indicated. It is a Fe-chelating ligand used for electron transfer in O2-dependent redox reactions (Shanklin et al., 1994). A putative ER localization signal is found at the C terminus (di-lysine motif) (Gaynor et al., 1994; Schröder et al., 1995). Two potential N-glycosylation signals (NYS and NWT) at the N terminus suggest, that the N terminus resides in the ER and the C terminus on the cytoplasmic side as proposed for other ELO proteins (Tvrdik et al., 2000).

View larger version (63K): [in this window] [in a new window]   Fig. 4. Transcript levels in mutants and gonads. (A) RNA was isolated from wild-type (wt) third instar larvae and from larvae homozygous for the alleles l(3)01895 (1895) or l(3)04106 (4106). Noa transcripts were detected using a cDNA probe. (B) RNA was isolated from OreR ovaries (O) and testes (T) and separated next to RNA from female (FC) and male (MC) carcasses. RNA amounts in the samples of A and B were compared by hybridization to RpL9 mRNA (Schmidt et al., 1996).

View larger version (93K): [in this window] [in a new window]   Fig. 5. Expression analysis. (A) Expression pattern in the germarium at the beginning of oogenesis is shown via the enhancer trap function. Germ cells show -galactosidase activity in all regions of the germarium (1-3) and in all follicle stages (e.g. S2). (B-H) In situ hybridizations to noa sequences are shown for a stage 10 follicle (B), an embryo at the syncytial blastoderm stage expressing maternal noa RNA (C), an embryo at the cellular blastoderm stage (D,E) with apical localization of the transcripts (black arrow in E) and pole cells without noa transcripts (white arrow, E), and an embryo around stage 8-9 with expression in the central nervous system (F) and in the PNS (G). Two imaginal discs (wing and leg) also contain noa transcripts (H). (I) Expression in a third instar larval brain is detected by X-Gal staining after expression of a noa-lacZ reporter gene.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Diagram of noa-lacZ reporter constructs. Grey lines represent flanking sequences of the noa gene. Their extent is indicated by the start nucleotide of the sequence. Grey boxes represent transcribed regions of the noa gene (for orientation pA signals are indicated in the 3' UTR). The lacZ coding region is shown in black. The top two constructs contain only a portion of the 5' UTR and a synthetic ATG in front of the lacZ coding region. Termination of transcription occurs in the hsp70 3' UTR (white box). The constructs in the middle contain the entire 5' UTR with the noa translation start codon together with varying sizes of the promoter region. The constructs at the bottom contain varying lengths of the noa 3' UTR. The relative intensities of resulting -galactosidase activity are indicated on the right and refer to expression in all tissues.

View larger version (90K): [in this window] [in a new window]   Fig. 7. Noa expression in the adult testis. (A) A group of cells in the testis tip of the enhancer trap line exhibits -galactosidase activity. (B) Confocal microscopy after staining with anti-Fasciclin III antibody (to locate the hub cells, red) and anti--galactosidase antibody (green) proves close proximity of the two cell types. (C) Both head (arrowhead) and tail (arrow) cyst cell express -galactosidase in an isolated cyst with elongating spermatids. (D) Nuclei are visualized using Hoechst 33258 staining. (E) In situ hybridizations on spermatid bundles are shown. The noa transcripts are found along the spermatid bundles, i.e. in the cyst cells (top). For comparison, the germ cell-specific transcripts of Mst87F are detected within the spermatid bundles (bottom). (F) NOA-lacZ fusion proteins are also detected around the spermatid bundles and not within the bundles.

View larger version (80K): [in this window] [in a new window]   Fig. 8. Noa expression in cyst cells is dependent of germ cells. Expression patterns are compared for postmeiotic cyst cell expression in noal(3)01895/+ testes (A,C,E,G) and premeiotic expression in l(3)04708/+ testes (B,D,F,H).The normal enhancer trap pattern is shown in adult (A,B) and in larval testes (C,D). Apical expression is indicated by a double arrow in A,C and E. The larval testis anlagen demonstrate the postmeiotic expression in noal(3)01895/+, since they contain only premeiotic stages of spermatogenesis and show no staining in cyst cells. In addition, expression in the nuclei of the fat body cells is visible (C). Premeiotic expression, starting with the progenitor cells, is clearly visible in larval l(3)04708/+ testes (D). The enhancer trap pattern is altered but -galactosidase activity can still be observed in bgcn mutant testes (E,F) and in germ cell free tudor testes (G,H). The patterns are distinctly different and in the case of l(3)04708/+ correlate well with premeiotic expression (F). The modification in the pattern is due to the altered testis shape resulting from lack of elongated stages. In the case of noa, expression at the testis base is apparently due to activity in the cells of the terminal epithelium (E,G), as can best be seen in G, where the various parts of the testis base are well extended. The beginning of the terminal epithelium is indicated by an arrowhead, the beginning of the seminal vesicle by an arrow in A,E,G,H. Note that enhancer trap activity is in the terminal epithelium in noal(3)01895/+ testes, but clearly above that region in l(3)04708/+ testes (G,H).

View larger version (84K): [in this window] [in a new window]   Fig. 9. Noa expression in cyst cells is necessary for sperm development. Wild-type testes (A,C,E) and testes from transgenic animals carrying a PPY-Gal4-driven UAS-noa-RNAi construct (B,D,F) were analyzed by microscopy. Testis shape is altered (testis tip is indicated by a white arrowhead) and the small seminal vesicle (sv) remains empty (compare A with B). The testis shown in B represents a severe case of the variable RNAi phenotypes. (C-F) Squash preparations of testes with Hoechst 33258 (green) and TRITC-coupled phalloidin (red) staining. (D) Testes with a weak phenotype showing displaced nuclei that do not show an individualization cone even though the rest of the cyst does (arrows in D). In the wild-type sometimes nuclei are also displaced but these then also contain an individualization cone (arrowhead in C). Individualization cones of the 64 spermatids in a cyst are found dispersed after staining with TRITC-coupled phalloidin (region between the white arrowheads in F) in contrast to the synchronized individualization complexes observed in wild-type cysts (E). The testis in F represents a weak phenotype after RNAi. The expression characteristics of the PPY-driver line are demonstrated by a UAS-GFP transgene (G,H). Cysts containing early stages of spermatogenesis (e.g. spermatocytes, arrows in G,H) are stained all around proving premeiotic expression in both cyst cells by the PPY driver.

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