Imaging cytoplasmic lipid droplets in vivo with fluorescent perilipin 2 and perilipin 3 knock-in zebrafish
- Carnegie Institution for Science Department of Embryology, United States
- Department of Biochemistry and Molecular Biology, Mayo Clinic, United States
- Johns Hopkins University Department of Biology, United States
Figures
Figure 1 with 2 supplements
Generation of in-frame fluorescent reporters in the endogenous plin2 and plin3 loci.
(A,B) Representative images of whole-mount in situ hybridization (ISH) with probes against zebrafish plin2 (ENSDARG00000042332) and zgc: 77,486 (plin3/4/5) (ENSDARG00000013711) at 6 days post …
Figure 1—figure supplement 1
A deletion upstream of exon 1 in plin2 impacts gene expression following a high-fat meal.
(A) A 54 bp deletion was noted in the 5’ untranslated region upstream of exon 1 in the plin2 ENSDART00000175378.2 transcript in AB wild-type stocks. (B) RT-PCR using the primers noted in panel A …
-
Figure 1—figure supplement 1—source data 1
Source data associated with Figure 1—figure supplement 1 panel D.
- https://cdn.elifesciences.org/articles/66393/elife-66393-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 2
Whole fish images corresponding to Figure 1E.
Whole-mount images of the same fish shown in Figure 1E. Scale = 500 µm.
Figure 2
Whole-mount Plin protein expression in the intestine following a high-fat meal.
(A) Representative images of Plin3-RFP and EGFP-Plin2 fluorescence in the intestine of 6 days post fertilization (dpf) larvae at noted time-points following the onset of a 90 min high-fat meal. …
-
Figure 2—source data 1
Source data associated with Figure 2B and D.
- https://cdn.elifesciences.org/articles/66393/elife-66393-fig2-data1-v2.xlsx
Figure 3
Following a high-fat meal, Plin3-RFP and EGFP-Plin2 show an ordered recruitment to lipid droplets.
(A) Lateral views of the anterior intestine in unfed larvae and in larvae at different time-points following the start of feeding with a high-fat meal for 1 hr. Fish were heterozygous for both Fus(pl…
-
Figure 3—source data 1
Source data associated with Figure 3C.
- https://cdn.elifesciences.org/articles/66393/elife-66393-fig3-data1-v2.xlsx
Figure 4 with 5 supplements
Plin3 labels nascent lipid droplets and is replaced by a coat of Plin2, which remains with the droplets for the remainder of their lifetime.
(A) Representative confocal micrographs of Fus(plin3-RFP)/+, Fus(EGFP-plin2)/+, or Tg(fabp2:EGFP-PLIN2)/+ fish fed a high-fat meal containing either green or red BODIPY C12 fatty acid analog. Fish …
-
Figure 4—source data 1
Source data associated with Figure 4C–F.
- https://cdn.elifesciences.org/articles/66393/elife-66393-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Tg(fabp2:EGFP-PLIN2) fish over-express human PLIN2 in the yolk syncytial layer and intestine.
(A) Representative images of wild-type and transgenic Tg(fabp2:EGFP-PLIN2)/+ fish at 3 days post fertilization (dpf) highlight EGFP-PLIN2 expression in the yolk syncytial layer. Scale = 500 µm. (B) …
-
Figure 4—figure supplement 1—source data 1
Source data associated with Figure 4—figure supplement 1D and E.
- https://cdn.elifesciences.org/articles/66393/elife-66393-fig4-figsupp1-data1-v2.xlsx
Figure 4—figure supplement 2
Remaining LD area at 30 hr.
The remaining area occupied by BODIPY-positive lipid droplets at 30 hr after the onset of the meal. Mean ± SD, n = 23 WT, 12 Plin3, 17 Plin2, 9 PLIN2, Kruskal-Wallis test, p = 0.2922.
-
Figure 4—figure supplement 2—source data 1
Source data associated with Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/66393/elife-66393-fig4-figsupp2-data1-v2.xlsx
Figure 4—video 1
Plin3-RFP and BODIPY C12 time series.
Representative confocal micrographs of Fus(plin3-RFP)/+ fish fed a high-fat meal containing green BODIPY FL-C12 fatty acid analog. Fish were fed for a total of 90 min and chased at room temperature …
Figure 4—video 2
EGFP-Plin2 and BODIPY C12 time series.
Representative confocal micrographs of Fus(EGFP-plin2)/+ fish fed a high-fat meal containing red BODIPY 558/568-C12 fatty acid analog. Fish were fed for a total of 90 min and chased at room …
Figure 4—video 3
EGFP-PLIN2 and BODIPY C12 time series.
Representative confocal micrographs of Tg(fabp2:EGFP-PLIN2)/+ fish fed a high-fat meal containing red BODIPY 558/568-C12 fatty acid analog. Fish were fed for a total of 90 min and chased at room …
Figure 5
EGFP-Plin2 and Plin3-RFP are only expressed in the liver of older larvae.
(A) Lateral view of the liver in a 7 days post fertilization (dpf) zebrafish larvae heterozygous for both Fus(plin3-RFP) and Fus(EGFP-plin2). Scale = 50 µm. (B, C) Brightfield and fluorescence liver …
-
Figure 5—source data 1
Standard length data associated with Figure 5D.
- https://cdn.elifesciences.org/articles/66393/elife-66393-fig5-data1-v2.xlsx
Figure 6 with 1 supplement
EGFP-Plin2 and Plin3-RFP decorate lipid droplets in visceral adipose tissue.
(A) Cartoon of 15 days of post fertilization (dpf) larval zebrafish showing the general location (green box) of images in panels B–D. (B, C) Examples of Plin-positive adipocyte lipid droplets. Fish …
-
Figure 6—source data 1
Standard length data associated with Figure 6B–D.
- https://cdn.elifesciences.org/articles/66393/elife-66393-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Standard length measurements associated with Figure 6.
(A) Standard length measurements of siblings from two clutches of Fus(EGFP-plin2)/+ × Fus(plin3-RFP)/+ fish, associated with Figure 6F. Mean ± SD, n = 12 wild type (WT), 8 Plin3, 13 Plin2, and 18 …
-
Figure 6—figure supplement 1—source data 1
Source data associated with Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/66393/elife-66393-fig6-figsupp1-data1-v2.xlsx
Figure 7
EGFP-Plin2 decorates lipid droplets in cells surrounding neuromasts.
(A) Cartoon of 15 days of post fertilization (dpf) larval zebrafish showing the general location of lateral line neuromast images shown in panel B. The standard length of larvae imaged at this stage …
Figure 8
Whole-mount images and examples of perilipin-labeled lipid droplets corresponding to the transgenic zebrafish lines noted in Table 1.
All fish are heterozygous for the noted transgene. Heat shock transgenic lines were incubated at 37°C for 45 min prior to feeding. For whole-mount images, larvae were fed for 2 hr with a high-fat …
Tables
Table 1
Comparison of available transgenic perilipin lines.
| Transgenic line | Promoter | Coding sequence | Tissue expression |
|---|---|---|---|
| Fus(EGFP-plin2) | Integration into the endogenous plin2 locus | Zebrafish perilipin 2 ENSDART00000175378.2 | Intestine, liver, adipose, neuromasts, rare LDs in yolk syncytial layer |
| Tg(fabp2:EGFP-PLIN2) | Zebrafish intestinal fatty acid binding protein (fabp2) | Human perilipin 2 ENST00000276914.7 | Yolk syncytial layer, intestine |
| Tg(fabp10a:EGFP-PLIN2) | Zebrafish liver fatty acid binding protein (fabp10a) | Human perilipin 2 ENST00000276914.7 | Liver |
| Tg(hsp70I:EGFP-PLIN2) | Zebrafish heat shock cognate 70-kd protein, like (hsp70l) | Human perilipin 2 ENST00000276914.7 | Widespread tissue expression following heat shock; Labeled LDs observed in intestine and liver |
| Fus(plin3-RFP) | Integration into the endogenous plin3 locus | Zebrafish perilipin 3 ENSDART00000100473.5 | Intestine, liver, adipose; Cytoplasmic in addition to LDs |
| Tg(fabp2:PLIN3-EGFP) | zebrafish intestinal fatty acid binding protein (fabp2) | Human perilipin 3 ENST00000221957.9 | Yolk syncytial layer, intestine; Cytoplasmic in addition to labeled LDs in intestine |
| Tg(hsp70I:PLIN3-EGFP) | Zebrafish heat shock cognate 70-kd protein, like (hsp70l) | Human perilipin 3 ENST00000221957.9 | Widespread tissue expression following heat shock; often mosaic; Cytoplasmic in addition to labeled LDs in intestine and liver |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| gene (Danio rerio) | plin2 | Ensembl GRCz11 | ENSDARG00000042332; plin2-203 ENSDART00000175378.2 | |
| gene (Danio rerio) | zgc:77,486(plin3) | Ensembl GRCz11 | ENSDARG00000013711; zgc:77,486 ENSDART00000100473.5 | |
| Gene (Homo sapiens) | PLIN2 | Ensembl GRCh38.p13 | ENSG00000147872; PLIN2-201 ENST00000276914.7 | |
| Gene (Homo sapiens) | PLIN3 | Ensembl GRCh38.p13 | ENSG00000105355; PLIN3-201 PLIN3-201 ENST00000221957.9 | |
| Strain, strain background (Danio rerio) | AB | Zfin | ZDB-GENO-960809–7 | |
| Strain, strain background (Danio rerio) | Fus(EGFP-plin2) | This paper | Available on request from Steven Farber | |
| Strain, strain background (Danio rerio) | Fus(plin3-RFP) | This paper | Available on request from Steven Farber | |
| Strain, strain background (Danio rerio) | Tg(fabp2:EGFP-PLIN2) | This paper | Zebrafish fabp2 promoter, human PLIN2 coding sequence; Available on request from Steven Farber | |
| Strain, strain background (Danio rerio) | Tg(fabp10a: EGFP-PLIN2) | This paper | Zebrafish fabp10a promoter, human PLIN2 coding sequence; Available on request from Steven Farber | |
| Strain, strain background (Danio rerio) | Tg(hsp70l: EGFP-PLIN2) | This paper | Zebrafish hsp70l promoter, human PLIN2 coding sequence; Available on request from Steven Farber | |
| Strain, strain background (Danio rerio) | Tg(fabp2:PLIN3-EGFP) | This paper | Zebrafish fabp2 promoter, human PLIN3 coding sequence; Available on request from Steven Farber | |
| Strain, strain background (Danio rerio) | Tg(hsp70: PLIN3-EGFP) | This paper | Zebrafish hsp70l promoter, human PLIN3 coding sequence; Available on request from Steven Farber | |
| Recombinant DNA reagent | pCRII(plin3_900)(plasmid) | This paper | Zebrafish plin3 (zgc:77486) cDNA clone for in situ probe; Available on request from Steven Farber | |
| Recombinant DNA reagent | pCRII(plin2)(plasmid) | This paper | Zebrafish plin2 cDNA clone for in situ probe; Available on request from Steven Farber | |
| Recombinant DNA reagent | Pk-GoldyTal(plin2 Pair 1 LeftTAL 1)(plasmid) | This paper | TALEN targets: TTTCTGCTAACATGG; Available on request from Steven Farber | |
| Recombinant DNA reagent | Pk-GoldyTal(plin2 Pair 1 RightTAL 2)(plasmid) | This paper | TALEN target: AAATAACCAGGTTTGCC; Available on request from Steven Farber | |
| Recombinant DNA reagent | Pk-GoldyTal(plin2 Pair 2 LeftTAL 1)(plasmid) | This paper | TALEN target: TTTCTGCTAACATGGGT; Available on request from Steven Farber | |
| Recombinant DNA reagent | Pk-GoldyTal(plin2 Pair 2 RightTAL 2)(plasmid) | This paper | TALEN target: AATAACCAGGTTTGCC; Available on request from Steven Farber | |
| Recombinant DNA reagent | Pk-GoldyTal(plin3 Pair 1 LeftTAL 1) (plasmid) | This paper | TALEN target: TTGCGCCTCAGATAAC; Available on request from Steven Farber | |
| Recombinant DNA reagent | Pk-GoldyTal(plin3 Pair 1 RightTAL 2) (plasmid) | This paper | TALEN target: AATTGCCACACAACCT; Available on request from Steven Farber | |
| Recombinant DNA reagent | Pk-GoldyTal(plin3 Pair 2 LeftTAL 1) (plasmid) | This paper | TALEN target:CAGATAACAGAGAAA; Available on request from Steven Farber | |
| Recombinant DNA reagent | Pk-GoldyTal(plin3 Pair 2 RightTAL 2) (plasmid) | This paper | TALEN target:CACACAACCTAAATA; Available on request from Steven Farber | |
| Recombinant DNA reagent | pKT3Ts-goldyTALEN vector | Welker et al., 2016 | Addgene plasmid #80330; RRID:Addgene_80330 | |
| Recombinant DNA reagent | FusX TALEN assembly system | Ma et al., 2013; Ma et al., 2016 | Addgene kit #1000000063 | |
| Recombinant DNA reagent | pDestTol2Pa2(plasmid) | Kwan et al., 2007 | Plasmid #394 | http://tol2kit.genetics.utah.edu |
| Recombinant DNA reagent | P4-P1R_zPLIN2_5’UTR(plasmid) | This paper | 5’ entry plasmid for left homology arm of pDestTol2Pa2(EGFP-plin2) donor plasmid V2; Available on request from Steven Farber | |
| Recombinant DNA reagent | pME-EGFP no stop 12aa linker(plasmid) | This paper | EGFP middle entry plasmid for pDestTol2Pa2(EGFP-plin2) donor plasmid V2Includes strong zebrafish kozak sequence; Available on request from Steven Farber | |
| Recombinant DNA reagent | P3R-P3-zPLIN2_1–900 | This paper | 3’ entry plasmid for right homology arm of pDestTol2Pa2(EGFP-plin2) donor plasmid V2; Available on request from Steven Farber | |
| Recombinant DNA reagent | pDestTol2Pa2(EGFP-plin2) donor plasmid V2 | This paper | Donor plasmid for creation of Fus(EGFP-plin2) fish | |
| Recombinant DNA reagent | P4-P1R_zPLIN3_last679 | This paper | 5’ entry plasmid for left homology arm of pDestTol2Pa2(PLIN3-RFP); Available on request from Steven Farber | |
| Recombinant DNA reagent | tagRFP-t coding sequence | Horstick et al., 2015 | Modified for zebrafish from Addgene #61390 | |
| Recombinant DNA reagent | pME-tagRFP-t (plasmid) | This paper | Middle entry plasmid for pDestTol2Pa2(PLIN3-RFP); Available on request from Steven Farber | |
| Recombinant DNA reagent | P2R_P3_zPLIN3_3’UTR | This paper | 3’ entry plasmid for right homology arm of pDestTol2Pa2(PLIN3-RFP); Available on request from Steven Farber | |
| Recombinant DNA reagent | pDestTol2Pa2(plin3-RFP) donor plasmid | This paper | Donor plasmid for creation of Fus(plin3-RFP) fish; Available on request from Steven Farber | |
| Recombinant DNA reagent | pDestTol2Pa2(fabp2: EGFP-PLIN2)(plasmid) | This paper | Zebrafish fabp2 promoter, human PLIN2 coding sequence; Available on request from Steven Farber | |
| Recombinant DNA reagent | pDestTol2Pa2(fabp10a: EGFP-PLIN2)(plasmid) | This paper | Zebrafish fabp10a promoter, human PLIN2 coding sequence; Available on request from Steven Farber | |
| Recombinant DNA reagent | pDestTol2Pa2(hsp70l: EGFP-PLIN2)(plasmid) | This paper | Zebrafish hsp70l promoter, human PLIN2 coding sequence; Available on request from Steven Farber | |
| Recombinant DNA reagent | pDestTol2Pa2(fabp2: PLIN3-EGFP)(plasmid) | This paper | Zebrafish fabp2 promoter, human PLIN3 coding sequence; Available on request from Steven Farber | |
| Recombinant DNA reagent | pDestTol2Pa2(hsp70l: PLIN3-EGFP)(plasmid) | This paper | Zebrafish hsp70l promoter, human PLIN3 coding sequence; Available on request from Steven Farber | |
| Recombinant DNA reagent | pGFP-hADRP(plasmid)(plasmid) | Targett-Adams et al., 2003 | Human PLIN2 coding sequence (Alternative name ADRP) in pEGFP-C1 vector | |
| Recombinant DNA reagent | pCR8(pGFP-hADRP)(plasmid) | This paper | Human PLIN2 coding sequence (Alternative name ADRP); Available on request from Steven Farber | |
| Recombinant DNA reagent | pDNR-Dual(h Perilipin 3) (plasmid)(TIP47) | FLEXgene Repository (Harvard Medical School) | Clone ID:HsCD00004695 | Human PLIN3 coding sequence Alternative name: TIP47 |
| Recombinant DNA reagent | pCR8(Perilipin3)(TIP47)(plasmid) | This paper | Middle entry plasmid, Human PLIN3 coding sequence Alternative name: TIP47; Available on request from Steven Farber | |
| Recombinant DNA reagent | Zebrafish fabp2 promoterp5E fabp2 (–2.3 kb) fabp2 (plasmid) | Park et al., 2019 | 5’ entry plasmid, zebrafish fabp2 (ifabp) promoter sequence | |
| Recombinant DNA reagent | p5E fabp10a, (–2.8 kb) | Her et al., 2003 | zebrafish fabp10a (lfabp) promoter sequence | |
| Recombinant DNA reagent | p5E-hsp70lheat shock cognate 70 kDa protein, like | Kwan et al., 2007 | Plasmid #222 | http://tol2kit.genetics.utah.edu |
| Recombinant DNA reagent | p3E-EGFPpA | Kwan et al., 2007 | Plasmid #366 | http://tol2kit.genetics.utah.edu |
| Recombinant DNA reagent | pME-EGFP no stop | Kwan et al., 2007 | Plasmid #455 | http://tol2kit.genetics.utah.edu |
| Recombinant DNA reagent | p3E-polyA | Kwan et al., 2007 | Plasmid #302 | http://tol2kit.genetics.utah.edu |
| Sequence-based reagent | PCR primers | This paper | Please refer to Supplementary file 1 | |
| Commercial assay or kit | T3 Message Machine kit | Thermo Fisher Scientific | Catalog #AM1348 | |
| Commercial assay or kit | DIG RNA labelling kit | Roche | Catalog#11277073910 | |
| Other | T7 polymerase | Roche | Catalog #10881767001 | |
| Other | SP6 polymerase | Roche | Catalog #10810274001 | |
| Commercial assay or kit | TA Cloning Kit, Dual Promoter, with pCRII-TOPO vector | Thermo Fisher Scientific | Catalog # K207020 | |
| Commercial assay or kit | REDExtract-N-Amp Tissue PCR kit | Sigma-Aldrich | Catalog #XNAT | |
| Commercial assay or kit | RNA Clean and Concentrator Kit | Zymo Research | Catalog #R1013 | |
| Commercial assay or kit | iScript cDNA Synthesis Kit | Bio-Rad Laboratories, Inc. | Catalog #1708891 | |
| Commercial assay or kit | SsoAdvanced Universal SYBR Green Supermix | Bio-Rad Laboratories, Inc. | Catalog #1725271 | |
| Commercial assay or kit | MultiSite gateway assembly system | Invitrogen | Catalog #12537–023 | |
| Commercial assay or kit | pCR8/GW/TOPO TA cloning kit | Thermo FisherScientific | Catalog # K250020 | |
| Other | BODIPY (558/568)-C12 | Thermo Fisher Scientific | Catalog #D3835 | 4 µg/ml |
| Other | BODIPY FL-C12 | Thermo Fisher Scientific | Catalog #D3822 | 4 µg/ml |
| Other | Cholesterol | Sigma-Aldrich | Catalog#C8667 | 4% w/w |
| Other | HSC LipidTOX Green | Thermo Fisher Scientific | Catalog #H34475 | 1:5,000 |
| Other | HSC LipidTOX Red | Thermo Fisher Scientific | Catalog #H34476 | 1:5,000 |
| Other | RNAlater | Thermo Fisher Scientific | Catalog # AM7020 | |
| Other | FokI restriction enzyme | New England Biolabs | Catalog # R0109 | |
| Other | HphI restriction enzyme | New England Biolabs | Catalog # R0158 | |
| Other | Gemma Micro 75, 150 & 500 | Skretting | Zebrafish diet | |
| Software, algorithm | Mojo Hand design tool | Neff et al., 2013 | ||
| Software, algorithm | Fiji | Schindelin et al., 2012 | (Fiji, RRID:SCR_002285) | |
| Software, algorithm | ilastik | Berg et al., 2019 | Ilastik, RRID:SCR_015246 | |
| Software, algorithm | GraphPad Prism 9 | GraphPad | GraphPad Prism, RRID:SCR_002798 |
Additional files
-
Supplementary file 1
Primers used in this study.
- https://cdn.elifesciences.org/articles/66393/elife-66393-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/66393/elife-66393-transrepform1-v2.docx
