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Institut Jacques Monod, Department of Developmental Biology, CNRS, Université Paris 7 Denis-Diderot and Université Paris 6 Pierre et Marie Curie, Paris, France
Institut Jacques Monod, Department of Developmental Biology, UMR 7592, CNRS, Université Paris 7 Denis-Diderot and Université Paris 6 Pierre et Marie Curie, Tour 43 2, Place Jussieu, F-75251 Paris Cedex 05, France
Intracellular lipid droplets store neutral lipids such as triacylglycerols (TAG) or sterol esters in eukaryotes. These organelles are composed of a monolayer of amphipathic phospholipids that encircles a core of neutral lipids (see Murphy, 2001 for review). In mammals, four proteins have been identified to be physically linked to the surface of lipid droplets: adipose differentiation related protein (ADRP) also called Adipophilin, Perilipins (Peri), S3-12, and tail-interacting 47-kDa protein (TIP47; Blanchette-Mackie et al., 1995; Brasaemle et al., 1997; Lu et al., 2001; Miura et al., 2002; Wolins et al., 2003). The N-terminal regions of Perilipin, ADRP, and TIP47 are conserved between species and define the PAT domain protein family (Miura et al., 2002). ADRP is ubiquitously expressed in all mammalian cells and is associated with small lipid droplets (Jiang and Serrero, 1992; Brasaemle et al., 1997; Heid et al., 1998). In contrast, Perilipin is only expressed in differentiated adipocytes and in steroidogenic cells and is associated with large lipid droplets (Greenberg et al., 1991, 1993; Brasaemle et al., 1997). Perilipin is likely to be involved in the regulation of lipid storage (Brasaemle et al., 2000; Clifford et al., 2000). Indeed, targeted disruption of the perilipin gene in mice leads to a reduction of adipose tissue mass, elevated basal lipolysis, and resistance to diet-induced obesity (Martinez-Botas et al., 2000; Tansey et al., 2001).
Several nonmammalian PAT domain proteins have been identified in organisms such as Xenopus, Drosophila, or Dictyostelium (Lu et al., 2001; Miura et al., 2002). In the Drosophila genome, Lipid storage droplet 1 and 2 (Lsd-1 and Lsd-2) encode PAT domain proteins (Lu et al., 2001; Miura et al., 2002). Green fluorescent protein (GFP) -tagged LSD-1 and LSD-2 are located at the surface of lipid droplets in Drosophila and mammalian cultured cells (Miura et al., 2002). Lsd-2 is expressed during all stages of Drosophila development (Gronke et al., 2003; Teixeira et al., 2003). During oogenesis, Lsd-2 is expressed in nurse cells and maternal transcripts accumulate in the oocytes (Gronke et al., 2003; Teixeira et al., 2003). In subsequent embryonic stages, Lsd-2 transcript is detected in specific structures such as germline precursor cells, amniosera, fat body, or midgut (Gronke et al., 2003; Teixeira et al., 2003). Later, at the third instar, Lsd-2 is expressed in the fat body (Gronke et al., 2003; Teixeira et al., 2003). Lsd-2 is involved in lipid metabolism in Drosophila. Mutation of Lsd-2 in Drosophila is associated with a reduction of TAG content in embryos and in adults and abnormalities in lipid deposition in germ line cells and in oocytes (Gronke et al., 2003; Teixeira et al., 2003). Moreover, overexpression of the Lsd-2 gene is correlated with an increase in TAG level in adult flies (Gronke et al., 2003).
During wing formation, Vestigial (VG) interacts physically with the Scalloped (SD) gene product (Paumard-Rigal et al., 1998; Simmonds et al., 1998). Several studies have shown that the SD/VG dimer functions as an essential regulator of the wing transcriptional program that controls wing differentiation and cell proliferation and is sufficient to reprogram cells to adopt a wing cell fate (Kim et al., 1996; Halder et al., 1998; Delanoue et al., 2004). We identified the Lsd-2 gene during a screen designed to find genes whose expression pattern is closely related to that of the pro-wing vestigial (vg) gene. We isolated a GAL4 enhancer trap strain (X20) in which a P[GAL4] element is inserted into Lsd-2 (Brand and Perrimon, 1993). GAL4 expression, monitored with a UAS-GFP construct, was detected in the wing pouch of the wing imaginal disc in this strain. We demonstrated by in situ hybridization and immunochemistry experiments that Lsd-2 is expressed in the wing pouch and in the notum of the wing imaginal disc. This expression of Lsd-2 is correlated with the accumulation of lipid droplets in the wing pouch of wing imaginal discs. Moreover, we see a strong decrease of lipid droplet staining in an Lsd-2 strong hypomorphic mutant and an increase of staining in an Lsd-2 overexpressor. These results suggest an active role of Lsd-2 in lipid storage in the wing imaginal disc.
The similarities in the gene expression pattern of Lsd-2 and vg prompted us to analyze the effect of vg on Lsd-2 expression. Results indicate that vg induces Lsd-2 expression and LSD-2 accumulation. These results suggest that Lsd-2 mediates vg activity that requires neutral lipid accumulation.
RESULTS AND DISCUSSION
Lsd-2 Is Expressed in Wing Imaginal Discs
In Drosophila, enhancer trapping has been used to identify genes that are expressed in specific spatial and developmental patterns (O'Kane and Gehring, 1987). We analyzed different enhancer trap lines to identify genes that potentially interact with VG. We screened several P[GAL4] enhancer trap strains in Drosophila for expression in the wing imaginal disc by mating them with a UAS-GFP strain (Brand and Perrimon, 1993). We isolated the X20 strain that shows strong GAL4 expression in the wing pouch and at a lower level in the presumptive notum of third-instar wing imaginal discs (Fig. 1A). The wing pouch of the wing imaginal disc corresponds to the wing primordium. The presumptive notum corresponds to the presumptive thoracic structures. We used plasmid rescue to recover genomic DNA flanking the P[GAL4] element. Sequencing indicates that the P[GAL4] element is inserted two nucleotides downstream of the start of the 5′UTR region of the Lsd-2 gene (Fig. 1B). We performed in situ hybridization experiments using an Lsd-2 antisense RNA probe to confirm that GAL4 expression in the X20 strain reflects Lsd-2 expression. Results shown in Figure 1C demonstrate strong Lsd-2 expression in the wing pouch and lower expression in the notum of the wing disc. No staining was observed using an Lsd-2 sense RNA probe in wild-type larvae (data not shown), confirming the specificity of the staining. Strong staining in the wing pouch and less staining in the notum was also observed when we performed immunocytochemical staining of wing imaginal discs with LSD-2 antibodies (Fig. 1E). To determine whether staining in the hinge region of the wing disc is unspecific or represents low endogenous expression, we performed LSD-2 immunocytochemistry staining of wing discs homozygous for an amorphic Lds-2 allele (Lsd-21; Teixeira et al., 2003). As shown in Figure 1F,H, the same staining was observed in the hinge region of wild-type discs and of homozygous Lsd-21 discs, indicating that this signal is unspecific. These results are in good agreement with a recent study using high-density DNA microarrays to determine genes whose expression differs between the wing primordium and body wall primordium of the wing imaginal discs (Butler et al., 2003). This study showed that the Lsd-2 gene is highly expressed in the wing pouch relative to the notum (Butler et al., 2003).
We analyzed Lsd-2 expression in the X20 strain (Fig. 1B) to determine whether this was a hypomorphic Lsd-2 allele. We observed a very strong reduction of Lsd-2 transcript and LSD-2 protein (Fig. 1D,G), indicating that the X20 strain is a strong hypomorphic allele of Lsd-2.
Lsd-2 transcript and LSD-2 protein are not uniformly expressed in the wing pouch of the wing disc. We observed a strong reduction of Lsd-2 transcript and LSD-2 protein at the dorsoventral (D/V) boundary by in situ hybridization, GFP staining, and immunohistochemistry (Fig. 1A,C,E, arrowheads). We also observed a low expression region juxtaposed to a high expression region of Lsd-2 transcripts along the anteroposterior (A/P) boundary (Fig. 1A,C, arrow and crossed arrow, respectively). Of interest, the D/V boundary is the presumptive zone of the adult wing margin, and the rest of the wing pouch corresponds to the adult wing blade. These two structures strongly differ, Lsd-2 appears to be necessary only in the formation of the wing blade.
The Lsd-2 transcript and LSD-2 protein are also present in the notum of the wing imaginal disc (Fig. 1C,E). The same pattern is observed with GFP in the X20:UAS-GFP strain. Two layers of cells constitute this region of the wing disc: epithelial cells that will give rise to the body wall cuticle of dorsal (notum) and ventral (pleura) thoracic structures and adepithelial cells that correspond to muscle progenitor cells giving rise to direct and indirect flight musculature (Bate et al., 1991). The adepithelial cells express a high level of twist and correspond to myoblasts (Bate et al., 1991). To determine which layer expresses Lsd-2, we acquired a z-section of the notum of wing imaginal discs using confocal microscopy (Fig. 1I). The twist-expressing cells were not labeled by LSD-2 antibodies, indicating that Lsd-2 expression was restricted to epithelial cells. Together, these data indicate that Lsd-2 expression is associated with cuticular structures.
Expression of Lsd-2 in Wing Imaginal Disc Is Correlated With Lipid Storage
Recent studies in Drosophila have shown that the LSD-2 protein localizes at the surface of lipid droplets deposited in fat bodies and in the germ line of females (Miura et al., 2002; Gronke et al., 2003; Teixeira et al., 2003). To determine whether LSD-2 in the wing pouch is associated with a high concentration of lipids, we stained wing discs with Nile red, which labels neutral lipids (Brown et al., 1992). As shown in Figure 2A, we observed a higher concentration of lipid deposition in the wing pouch cells than in notum cells. This pattern is very similar to that of Lsd-2 (Fig. 1) and is consistent with an effect of LSD-2 on neutral lipid accumulation. To determine the subcellular localization of LSD-2 in wing imaginal disc cells, we stained imaginal discs with Nile red and with LSD-2 antibodies. We observed a localization of LSD-2 at the lipid droplet surface (Fig. 2D), suggesting that the function of LSD-2 in wing disc cells may be mediated through its association with lipid droplets. However, LSD-2 expression seems not to be restricted to the surface of lipid droplet. To analyze more precisely LSD-2 subcellular localization, we stained nuclei with 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI; Fig. 2E) and endoplasmic reticulum (ER; Fig. 2F) by using a transgenic strain expressing a fusion protein between GFP and Pdi (protein disulfide isomerase) that marks ER (Bobinnec et al., 2003). We observed that LSD-2 was excluded from nuclei (Fig. 2E). However, LSD-2 appears to colocalize with the ER (Fig. 2F), although we do not observe perfect overlapping. A connection between lipid droplets and tubular structures labeled with Pdi has been observed in the oocyte (Teixeira et al., 2003). However, in the oocyte, LSD-2 is not detected in ER. In addition, a similar distribution of lipid droplets and ER has been observed in the nurse cells (Teixeira et al., 2003). We observed a similar distribution of LSD-2 and ER, suggesting that, in the wing discs, LSD-2 subcellular localization might be different than during oogenesis and could be involved in lipid droplet biogenesis associated with ER (Murphy, 2001).
To further investigate the relationship between Lsd-2 expression and lipid droplet accumulation in the wing pouch, we analyzed lipid droplet deposition in an Lsd-2 mutant (X20) and in wing discs overexpressing Lsd-2. Lsd-2 mutant discs display a decrease in lipid droplet staining in the wing pouch compared with wild-type, indicating that Lsd-2 expression is required for lipid accumulation in this structure (compare Fig. 2C and B). In these experiments, wing discs were dissected from ptc-LacZ larvae (Fig. 2B) and X20/X20:ptc-LacZ larvae (Fig. 2C) and counterstained with anti–β-galactosidase antibodies to provide a signal reference. Similar results were observed with the Lsd-21 amorphic allele (Teixeira et al., 2003; data not shown). Overexpression of Lsd-2 with the ptc-GAL4 driver expressing GAL4 along the A/P boundary of the wing disc (Fig. 3A) promotes an accumulation of neutral lipids according to the ptc domain (Fig. 3B). High magnification shows an increase in staining in the wing pouch (Fig. 3C, left panels) and in the hinge and in notum regions of the wing disc (Fig. 3C, right panels, arrows and arrowheads, respectively). Neutral lipid accumulation was increased exactly where Lsd-2 was overexpressed (as visualized by GFP), suggesting that this phenomenon is cell-autonomous. Taken together, these results indicate that Lsd-2 expression is required for regulation of neutral lipid storage in wing imaginal discs in a cell-autonomous manner.
The vestigial Pro-Wing Gene Product Induces Lsd-2 Expression
Wing pouch-specific expression of Lsd-2 led us to hypothesize that a correlation links Lsd-2 to the wing cell fate. Wing cell identity is controlled by the vestigial gene, which encodes a nuclear protein expressed in the wing imaginal disc. VG is both necessary and sufficient for wing formation (Williams et al., 1994; Kim et al., 1996). vg null mutants are associated with a loss of the wing blade and ectopic vg expression induces wing outgrowths (Kim et al., 1996). This ability of vg to form ectopic wing tissue led to the proposal that vg is a master control gene. We therefore tested the hypothesis that the vestigial pro-wing gene might regulate Lsd-2 expression.
Because vg mutant wing discs show a loss of the wing pouch and vg mutant cells fail to proliferate, it was not possible to analyze Lsd-2 expression in vg mutants. We chose to analyze the effect of vg on Lsd-2 with the UAS/GAL4 system (Brand and Perrimon, 1993; Fig. 4A,B) or in cell clones overexpressing vg (Fig. 4C–F). In en-GAL4, UAS-GFP; UAS-vg wing discs, the Lsd-2 transcript (Fig. 4A) and the LSD-2 protein (Fig. 4B) were significantly increased in the posterior compartment. Moreover, we observed an enlargement of the Lsd-2 expression domain consistent with the wing pouch enlargement observed in vg overexpression. We observed a similar effect in the posterior region of the notal part of the wing disc (Fig. 4A,B, arrows). Flip-out clones overexpressing vg were generated in the wing disc using a flip-out GAL4 transposon driving expression of UAS-vg (Fig. 4C–E). Clones overexpressing vg in the hinge part of the disc show strong LSD-2 immunostaining (Fig. 4C–E, arrows). vg overexpression in other regions of the disc does not result LSD-2 overexpression, probably because of the strong endogenous LSD-2 staining. Of interest, expression of other vg target genes such as blistered (bs) or Distal-less (Dll) were induced in vg overexpression clones in the wing hinge but wild-type expression of these genes was not modified in the wing pouch (Baena-Lopez and Garcia-Bellido, 2003). In the leg disc, neither vg expression nor Lsd-2 transcripts can be detected, although a low level of staining is detected with anti–LSD-2 (not shown). We generated flip-out clones in the leg disc and observed that cell clones overexpressing vg in all parts of the leg disc display an up-regulation of LSD-2 protein expression consistent with a positive regulation of Lsd-2 by vg (Fig. 4F, arrows). However, LSD-2 expression was not induced in all clones, suggesting that additional factors are required for LSD-2 expression. Similar results have been observed for other vg target genes such as nubbin (nub), serum responsive factor (SRF) enhancer, and vg enhancers (VgQE and Vg BE). In these experiments, gene expression was not induced in all cells of the clones or in all cell clones (Halder et al., 1998; Baena-Lopez and Garcia-Bellido, 2003). In addition, vg seems to act in a cell-autonomous manner. Indeed, LSD-2 overexpression is exclusively restricted to vg overexpressing cells visualized by GFP (Fig. 4D,E).
Surprisingly, vg overexpression driven by en-GAL4 seems unable to induce Lsd-2 expression at the D/V boundary of the wing disc (Fig. 4A,B, arrowheads). These data are in agreement with the hypothesis of a transcriptional repression of Lsd-2 at the D/V boundary.
It has been shown that Lsd-2 is expressed in fat bodies and that an Lsd-2 null mutation is associated with a decrease in neutral lipid content (Gronke et al., 2003; Teixeira et al., 2003). Results presented in this study show that Lsd-2 expression and lipid droplet deposition is not restricted to tissues directly involved in lipid metabolism such as fat bodies, as already shown in ovary (Teixeira et al., 2003). Furthermore, lipid droplets were not uniformly distributed through the wing disc but preferentially localized in the wing pouch where Lsd-2 is highly expressed. Neutral lipid accumulation was severely reduced in an Lsd-2 mutant background. We do not observe any obvious wing phenotype associated with the Lsd-2 amorphic alleles (Lsd-21 allele [Teixeira et al., 2003], Lsd-251allele [Gronke et al., 2003]) and with the Lsd-2 hypomorphic allele (X20). However, another Lsd gene has been identified in Drosophila (Lsd-1), and we cannot exclude the possibility that Lsd-1 and Lsd-2 genes have overlapping roles in the wing disc, therefore masking the effect of an Lsd-2 mutation in wing formation. According to this hypothesis, we were able to detect expression of Lsd-1 by real-time polymerase chain reaction experiments performed on wing imaginal discs (data not shown). Of interest, Lsd-1 expression was increased up to fourfold in the Lsd-2 mutants (X20, Lsd-21, and Lsd-251), suggesting a feed-back regulation between both genes (data not shown).
During the larval state, extensive cell growth and proliferation take place in wing imaginal discs. This finding requires intensive membrane lipid synthesis and energy expenditure at the cellular level. One attractive possibility is that Lsd-2 expression and its regulation of lipid metabolism is correlated with cell proliferation.
We have observed that Lsd-2 expression is strongly reduced at the D/V boundary of the wing disc. This structure corresponds to the domain that will give rise to chemosensory and mechanosensory neurons located at the wing margin. This finding suggests that Lsd-2 expression is specifically associated with cuticular differentiation of the wing blade and is repressed in margin cells. Further studies will be required to clarify the relationship between wing cell proliferation/differentiation and the role of Lsd-2 in lipid metabolism.
The wild-type control stock used throughout this work is w1118. The X20 strain comes from a pGaw P element mutagenesis performed by T Préat. The Lsd-21 strain was provided by L. Teixeira (Teixeira et al., 2003). The Lsd-251 strain was provided by R. Kühnlein. The UAS-vg and UAS-Lsd-2 strains were generated in our laboratory (Paumard-Rigal et al., 1998). The en-GAL4, ptc-GAL4, ptc-LacZ, and UAS-GFP strains came from the Bloomington Drosophila center. The en-GAL4, UAS-GFP, X20, and UAS-GFP strains were established for experimental needs. The hsp70-flp;Tub-FRT>cd2>FRT-Gal4, UAS-GFP /TM3 was generated from Tub-FRT>cd2>FRT-Gal4 (Pignoni and Zipursky, 1997).
Clones expressing the VG protein were generated using a flip-out Gal4 transposon and UAS-vg. Females with the genotype hsp70-flp;Tub-FRT>cd2>FRT-Gal4, UAS-GFP/TM3 were mated to UAS-vg males (Pignoni and Zipursky, 1997). Progeny were grown at 25°C, heat-shocked for 1 hr at 38°C, 30 to 54 hr after egg-laying, then allowed to grow at 25°C until the late third-instar stage.
Dissections were performed on wandering third-instar larvae. In situ hybridization followed the protocol of Tautz and Pfeifle (1989). Digoxigenin (DIG) -labeled antisense or sense RNA probes of Lsd-2 were generated with T3 or T7 RNA polymerase (Promega) and DIG-UTP (Roche) from expressed sequence tag LD32616 digested either by EcoRI or ScaI. These probes were used for whole-mount in situ hybridization of fixed larval imaginal discs. The DIG-labeled RNA probes were detected by an anti-DIG antibody coupled to alkaline phosphatase (Roche) and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) as the substrate.
Stainings were performed according to protocols described in Brower et al. (1984). Anti–LSD-2 (gift from N. Vanzo) was reacted with either CY3-conjugated goat anti-rat IgG (Jackson ImmunoResearch) or CY5-conjugated goat anti-mouse (Beckman–Coulter) at a dilution of 1:200. Anti-Twist (gift from S. Roth [Roth et al., 1989]) was reacted with fluorescein isothiocyanate–conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch). Anti–β-galactosidase (Jackson Immuno Lab) was reacted with CY3-conjugated goat anti-rat IgG (Jackson ImmunoResearch). Nile red was from SIGMA. One microliter of saturated solution of Nile red in acetone was added to 2 ml of PBT (phophate buffered saline 0.3% Triton). Nile red staining was performed on discs either after immunostaining or after a 30-min 4% paraformaldehyde fixation step. PBT plus Nile red was applied for 30 min, and discs were washed three times for 10 min. Images were acquired by using a Leica DMR microscope equipped with a Nikon DXM 1200 camera (×10, ×20, and ×63 objectives) and a Leica TSC SP2 laser scanning confocal microscope. Images were processed with Adobe Photoshop and ImageJ software.
The authors thank N. Vanzo, L. Teixeira, and R. Kühnlein for reagents and flies. The authors acknowledge the technical assistance of B. Legois and A. Dutriaux. This work was funded by an Action thématique Concertée (ATC) viellissement grant from the Institut National pour la Santé et la Recherche Médicale (INSERM). J.D.F. is a fellow from the Association pour la Recherche sur le Cancer (ARC).