Figure 1.
dSeipin mutants exhibit reduced lipid storage in the fat body.
(A) Schematic of the genomic structures of wild type dSeipin and the null mutant. In the null mutant, the dSeipin locus is replaced by GFP (FP) and miniWhite (miniW+) sequences. Except for the FP and miniW+ regions, black boxes represent coding regions and grey boxes represent un-translated region. (B) Expression levels of dSeipin in different larval tissues by qRT-PCR. The error bars represent standard deviation. (C) RT-PCR analysis of wild-type and dSeipin knockout larvae. Primer positions (RT-5′ and 3′) are labeled in (A). (D) Lipid droplets labeled by Nile red (red) in larval fat bodies and young adult fat cells from wild type and dSeipin mutants. Nuclei were stained with DAPI (blue). dSeipin mutants exhibit lipid storage defects with small lipid droplets. Under starved conditions the lipid droplets in dSeipin mutant larvae are even smaller. Scale bars: 20 µm. (E) Larval fat bodies from wild type float on top of 2% sucrose solution, while fat bodies from dSeipin mutants sink to the bottom. (F) Glyceride levels in adult males of wild type, control, dSeipin mutants and transgene-rescued dSeipin mutants. dSeipin mutants have significantly lower levels of glyceride. The error bars represent standard deviation. ***: P<0.0001. (G) Average weights (adult male) from wild type, dSeipin mutants and transgene-rescued dSeipin mutants. dSeipin mutants are slightly lower in weight. The error bars represent standard deviation. **: P<0.001; *: P<0.05.
Figure 2.
dSeipin mutants may have reduced lipogenesis in the fat body.
(A) Oil Red O staining of oenocytes (arrows). Increasing lipolysis by overexpressing bmm (ppl>bmm) causes a strong Oil Red O signal in fed animals. dSeipin mutants have the same level of Oil Red O staining as wild type under fed conditions. Scale bar: 50 µm. (B) Glyceride levels in circulating hemolymph are greatly reduced in dSeipin mutants. ***: P<0.0001. (C) Genetic interaction of dSeipin with other lipid metabolism regulators. The ppl-Gal4 driver was used to overexpress DGAT or SCAP. The genotypes are as follows: wt, dSeipin, DGATqx25, dSeipin;DGATqx25, ppl>DGAT, dSeipin;ppl>DGAT, ppl>SCAP, dSeipin;ppl>SCAP, Lsd-2KG00149 and dSeipin;Lsd-2KG00149. ppl>DGAT is short for ppl-Gal4>UAS-DGAT, where DGAT is overexpressed using the ppl-Gal4 driver. Scale bar: 50 µm. (D) Quantification of the genetic interactions in (C). The lipid storage in wild type was set as 100%. The error bars represent standard deviation. NS: Non-significant; ***: P<0.0001; **: P<0.001. (E) Adult starvation test. The x-axis shows the hours of starvation and the y-axis shows the survival rate (%). dSeipin mutants are very sensitive to starvation compared to wild type and control.
Figure 3.
Ectopic lipid droplet formation in dSeipin mutants.
(A) Oil Red O staining in guts of wild type and dSeipin mutants. In wild type the proventriculus (arrow) has numerous small droplets and the anterior midgut (arrowhead) has only a few Oil Red O-positive patches. In dSeipin mutants, the droplets in the proventriculus (arrow) are larger than wild type and in the anterior midgut (arrowhead) there are more patches of Oil Red O-positive staining. The boxed regions of the proventriculus and the midgut are enlarged in the adjacent panels. Scale bar: 100 µm. (B) Nile red staining in salivary glands. In wild-type salivary glands, essentially no punctate Nile red staining was found. In dSeipin mutants, many Nile red-positive puncta are present, indicating ectopic lipid storage. Scale bar: 100 µm. (C) Ectopic lipid puncta marked by Bodipy 493/503 (green) in dSeipin mutant salivary glands are confirmed as lipid droplets by co-labeling with the lipid droplet surface marker LSD-1-mCherry (red). Nuclei were stained with DAPI (blue). Scale bar: 50 µm. The lower panels show enlarged views from a dSeipin mutant. Expression of LSD-1-mCherry in a wild-type background was used as a control.
Figure 4.
dSeipin functions tissue-autonomously in both salivary gland and fat body.
(A) Tissue-specific rescue reveals tissue-autonomous function of dSeipin. Red: Nile red staining; blue: DAPI staining. The genotypes are indicated. ppl-Gal4 drives expression in both fat body and salivary gland. lsp2-Gal4 drives expression only in fat body, while sgs3-Gal4 drives expression only in salivary gland. lsp2>dSeipin is short for lsp2-Gal4>UAS-dSeipin, where dSeipin is overexpressed using the lsp-2-Gal4 driver. Note that dSeipin;lsp2>dSeipin animals have ectopic lipid storage in the salivary gland but exhibit normal lipid storage in the fat body and dSeipin;sgs3>dSeipin animals have lipid storage defects in the fat body but no lipid storage in the salivary gland. Scale bar: 50 µm. (B) Tissue-specific RNAi confirms the tissue-autonomous function of dSeipin. Red: Nile red staining; blue: DAPI staining. UAS-dSeipin RNAi driven by salivary gland-specific 48Y-Gal4 or elav-Gal4 results in ectopic lipid storage in the salivary gland without affecting lipid storage in the fat body. Scale bar: 50 µm. (C) Quantification of (A) and (B). The error bars represent standard deviation. NS: non-significant in Student's T-test.
Figure 5.
dSeipin genetically interacts with lipogenic genes.
All images show Nile red staining of salivary glands. There are a few strongly stained fat body tissues next to the salivary glands. The genotypes are as follows: (A) Wild type, (B) bmm1, (C) Lsd-2 KG00149 (D) dSeipin, (E) 48Y>GPAT1, (F) dSeipin; 48Y>GPAT1, (G) 48Y>DGAT, and (H) dSeipin; 48Y>DGAT. dSeipin displays a strong synergistic genetic interaction with overexpression of DGAT. Scale bar: 100 µm. (I) The lipogenic pathway and the enzymes involved. (J) Quantification of (A–H). The error bars represent standard deviation. ***: P<0.0001.
Figure 6.
Seipin may affect the metabolism of PA.
All images show Nile red staining of salivary glands. There are a few strongly stained fat body tissues next to the salivary glands. The genotypes are as follows: (A) wild type, (B) dSeipin, (C) dSeipin;48Y>Lipin RNAi, (D) dSeipin;DGATqx25, (E) dSeipin;GPAT1e01407, (F) dSeipin;48Y>AGPAT1 RNAi, (G) dSeipin;48Y>APGAT2 RNAi, (H) Cct116919, (I) CdsA1, (J) dSeipin;CdsA1, (K) 48Y>CdsA and (L) dSeipin;48Y>CdsA. Ectopic lipid storage is present in B, E, F, G, I, and J. Scale bar: 100 µm. (M) Quantification of (A–L). dSeipin displays a synergistic interaction with CdsA1. The error bars represent standard deviation. NS: non-significant. ***: P<0.0001.
Figure 7.
PA levels are increased in dSeipin mutants.
(A) Heat plot showing relative levels of individual PA species and total PA species in dSeipin mutant larvae compared to wild type. The levels of total PA and most PA species are increased in dSeipin mutant larvae. *: P<0.05. (B) Heat plot showing relative levels of individual PA species and total PA species in the salivary glands of CdsA1 mutants compared to CdsA1;dSeipin double mutants. There are more PA in CdsA1;dSeipin double mutants than CdsA1 single mutants. *: P<0.05. (C) Heat plot showing relative levels of individual DAG species and total DAG species in the fat body (fb) and the salivary gland (sg) of dSeipin mutants compared to wild type. *: P<0.05; **: P<0.005. The levels of certain species of DAG are increased in the salivary gland of dSeipin mutants. (D) Heat plot showing relative levels of individual TAG species and total TAG species in dSeipin mutants compared to wild type. *: P<0.05; **: P<0.005; ***: P<0.0005. TAG levels are greatly reduced in the fat body (fb) of dSeipin mutants compared to wild type. The levels of many TAG species are increased in the salivary gland (sg) of dSeipin mutants compared to wild type.
Figure 8.
dSeipin may have distinct functions in different tissues.
(A) Sequence alignment of the C-termini of Seipin homolog proteins from yeast (FLD1p), worm (R01B10.6), fly (CG9904), mouse (Q9Z2E9.2) and human (NP116056). The arrow points to the end of the second putative transmembrane domain. The region deleted in the C-terminal truncation of dSeipin is underlined (A280 to R352). (B) Red: Nile red staining; blue: DAPI staining. Expression of the C-terminal truncation of dSeipin in dSeipin mutants rescues the reduced lipid storage phenotype in the fat body but not the ectopic lipid storage phenotype in the salivary gland. Human Seipin (hSeipin) rescues the dSeipin mutant defects in both the fat body and the salivary gland. Scale bar: 50 µm. (C) Quantification of (B). The error bars represent standard deviation. ***: P<0.0001.