Imaging cytoplasmic lipid droplets in vivo with fluorescent perilipin 2 and perilipin 3 knock-in zebrafish
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Curated by eLife
Evaluation Summary:
This manuscript has generated novel and useful tools to mark cytoplasmic lipid droplets and monitor their dynamics in various tissues in live animals. It will be of interest to researchers studying lipid metabolism and related human diseases.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Cytoplasmic lipid droplets are highly dynamic storage organelles that are critical for cellular lipid homeostasis. While the molecular details of lipid droplet dynamics are a very active area of investigation, this work has been primarily performed in cultured cells. Taking advantage of the powerful transgenic and in vivo imaging opportunities available in zebrafish, we built a suite of tools to study lipid droplets in real time from the subcellular to the whole organism level. Fluorescently tagging the lipid droplet-associated proteins, perilipin 2 and perilipin 3, in the endogenous loci permits visualization of lipid droplets in the intestine, liver, and adipose tissue. Using these tools, we found that perilipin 3 is rapidly loaded on intestinal lipid droplets following a high-fat meal and later replaced by perilipin 2. These powerful new tools will facilitate studies on the role of lipid droplets in different tissues, under different genetic and physiological manipulations, and in a variety of human disease models.
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Reviewer #3 (Public Review):
In this work Farber and colleagues describe the generation of Fus(EGFP-plin2) and Fus(plin3-RFP) two knock-in zebrafish lines that alllow to study perilipins and lipid droplet biology in vivo at whole animal level. These lines could be important tools to understand how lipid droplet dynamics are affected by different genetic and physiological manipulations.
The article is well written and the work is carries out with a good methodological approach and the results support their conclusions. The weakness is the lack of originality since it does not really go behind the current knowledge in the field. Most of the data are a detailed description of zebrafish lines but I doubt that could be interested to a broad audience.
It also lacks novelty since the work does not add anything compared to what is already known …
Reviewer #3 (Public Review):
In this work Farber and colleagues describe the generation of Fus(EGFP-plin2) and Fus(plin3-RFP) two knock-in zebrafish lines that alllow to study perilipins and lipid droplet biology in vivo at whole animal level. These lines could be important tools to understand how lipid droplet dynamics are affected by different genetic and physiological manipulations.
The article is well written and the work is carries out with a good methodological approach and the results support their conclusions. The weakness is the lack of originality since it does not really go behind the current knowledge in the field. Most of the data are a detailed description of zebrafish lines but I doubt that could be interested to a broad audience.
It also lacks novelty since the work does not add anything compared to what is already known regarding peripilin 2 and 3. I think this manuscript should be submitted to a more specialized journal on lipid metabolism or to a technical "zebrafish" journal.
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Reviewer #2 (Public Review):
In this manuscript, the authors generated transgenic zebrafish reporter lines that allow observation of cytoplasmic lipid droplets in vivo. They knocked in GFP or RFP in the endogenous loci of perilipin 2 and 3, and showed that the reporter genes exhibited similar temporal and spatial expression in the intestine in response to acute high-fat feeding as the endogenous perilipin 2 and 3 transcripts. They also characterized the reporter gene expression in the liver, adipocytes, and around neuromasts. These tools open up new opportunities to study lipid droplets dynamics in live zebrafish that is not feasible in mouse models. Overall the manuscript is well written. The authors have discussed in details the strength and caveats of these reporters. The weakness is the descriptive nature of the study - many …
Reviewer #2 (Public Review):
In this manuscript, the authors generated transgenic zebrafish reporter lines that allow observation of cytoplasmic lipid droplets in vivo. They knocked in GFP or RFP in the endogenous loci of perilipin 2 and 3, and showed that the reporter genes exhibited similar temporal and spatial expression in the intestine in response to acute high-fat feeding as the endogenous perilipin 2 and 3 transcripts. They also characterized the reporter gene expression in the liver, adipocytes, and around neuromasts. These tools open up new opportunities to study lipid droplets dynamics in live zebrafish that is not feasible in mouse models. Overall the manuscript is well written. The authors have discussed in details the strength and caveats of these reporters. The weakness is the descriptive nature of the study - many interesting observations but no mechanistic study. I have the additional comments:
It is curious that in plin2 and plin3 reporter fish, the fluorescent tags were inserted at the 5' and 3' of the open reading frame, respectively. The authors did not provide any explanation. Does the location where the fluorescent tag is inserted affect the expression of the reporter genes?
GFP and TagRFP-T are not fast folding fluorescent proteins and are very stable, which may not be the best options for studying the formation and degradation of lipid droplets. How the fluorescent tags affect the stability and clearance of the protein should be carefully characterized.
Was there any indel being introduced by TALENs in these knockin fish? Is there off target effects of the TALENs?
The authors also generated transgenic fish overexpressing human PLIN2 and PLIN3 fluorescent fusion proteins. Is the subcellular localization of these fusion proteins similar to the zebrafish knockin under nofed and fed conditions? In other words, do human PLIN2 and PLIN3 proteins behave similarly as the zebrafish orthologs?
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Reviewer #1 (Public Review):
The authors find that plin2 transcript is induced in intestine of 6 dpf zebrafish larvae following a single feeding, while plin3 transcript is expressed in the fasted and fed states in the intestine. They use TALENS to knock-in EGFP and TagRFPt into the plin2 and plin3 loci, with the encoded gene products being the fusion proteins EGFP-plin2 and Plin3-TagRFPt. The EGFP-plin2 protein shows greater induction of fluorescence following a meal. The overall aim of these initial expression characterizations and development of lipid droplet reporter knock-ins is to be able to monitor the life cycle of these organelles in a living whole organism.
Higher resolution photomicrographs of lipid droplets with these knock-in lines concurrently stained with the the fluorescent lipid dyes BODIPY 558/568 C12 and BODIPY FL-C12 …
Reviewer #1 (Public Review):
The authors find that plin2 transcript is induced in intestine of 6 dpf zebrafish larvae following a single feeding, while plin3 transcript is expressed in the fasted and fed states in the intestine. They use TALENS to knock-in EGFP and TagRFPt into the plin2 and plin3 loci, with the encoded gene products being the fusion proteins EGFP-plin2 and Plin3-TagRFPt. The EGFP-plin2 protein shows greater induction of fluorescence following a meal. The overall aim of these initial expression characterizations and development of lipid droplet reporter knock-ins is to be able to monitor the life cycle of these organelles in a living whole organism.
Higher resolution photomicrographs of lipid droplets with these knock-in lines concurrently stained with the the fluorescent lipid dyes BODIPY 558/568 C12 and BODIPY FL-C12 are presented with a time series following feeding in intestine; additional cell types beyond enterocytes (i.e., hepatocytes, adipocytes, and cells surrounding lateral line structures) are presented.
The authors have provided a technical advance to the field of lipid droplet biology. With the tractable revisions set out below, their tools set the stage for chemical and genetic screens for factors and compounds that modulate the normal life cycle of these dynamic organelles.
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Evaluation Summary:
This manuscript has generated novel and useful tools to mark cytoplasmic lipid droplets and monitor their dynamics in various tissues in live animals. It will be of interest to researchers studying lipid metabolism and related human diseases.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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