In vivo CRISPR screening identifies regulators of hyperplastic and hypertrophic adipose remodelling in zebrafish
Curation statements for this article:-
Curated by eLife
eLife Assessment
This is a well-written study that presents a solid genetic screen to identify regulators of adipose morphology and remodeling in zebrafish. The authors generated a rigorous screening platform based on live, whole animal imaging and statistical methods that revealed both novel and known genes critical for adipose regulation. This work is valuable because it provides several candidate genes relevant to metabolic health and a quantitative screening pipeline that will be beneficial for future studies. A limitation of the study is that it precludes a definitive distinction between developmental and remodeling effects.
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Abstract
Abstract
Adipose tissues exhibit a remarkable capacity to expand, regress, and remodel in response to energy status. The cellular mechanisms underlying adipose remodelling are central to metabolic health. Hypertrophic remodelling - characterised by the enlargement of existing adipocytes - is associated with insulin resistance, type 2 diabetes, and cardiovascular disease. In contrast, hyperplastic remodelling – in which new adipocytes are generated - is linked to improved metabolic outcomes. Despite its clinical importance, the regulation of hypertrophic and hyperplastic adipose remodelling remains poorly understood. In this study, we first leveraged human genetic and transcriptomic data to identify candidate genes involved in adipose remodelling. We then developed a quantitative imaging pipeline to assess hyperplastic and hypertrophic morphology in zebrafish subcutaneous adipose tissue, and applied it in an F0 CRISPR mutagenesis screen targeting 25 candidate genes. This screen identified six genes that significantly altered adipose morphology; including Sushi Repeat Containing Protein (Srpx) - a gene with previously unknown roles in adipose. Among the identified genes, foxp1b mutants were notable for inducing hypertrophic morphology. To investigate further, we generated stable loss-of-function alleles for both zebrafish foxp1 genes. We found that foxp1b mutants display a developmental bias towards hypertrophic adipose growth but fail to undergo further hypertrophic remodelling in response to a high-fat diet - suggesting that early developmental patterning constrains later adaptability to diet. Together, these findings establish a scalable and tractable in vivo screening platform for identifying regulators of adipose remodelling, and reveal a potential developmental influence on the capacity for diet-induced adipose expansion.
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eLife Assessment
This is a well-written study that presents a solid genetic screen to identify regulators of adipose morphology and remodeling in zebrafish. The authors generated a rigorous screening platform based on live, whole animal imaging and statistical methods that revealed both novel and known genes critical for adipose regulation. This work is valuable because it provides several candidate genes relevant to metabolic health and a quantitative screening pipeline that will be beneficial for future studies. A limitation of the study is that it precludes a definitive distinction between developmental and remodeling effects.
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Joint Public Review:
In this manuscript, Wafer and Tandon et al. present a thoughtful and well-designed genetic screen for regulators of adipose remodeling using zebrafish as a model system. The authors cross-referenced several human adipocyte-related transcriptomic and genetic association datasets to identify candidate genes, which they then tested in zebrafish. Importantly, the authors devised an unbiased microscopy-based screening platform to document quantitative adipose phenotypes with whole animal imaging, while also employing rigorous statistical methods. From their screen, the authors identified 6 genes that resulted in robust adipose phenotypes out of a total of 25 that were tested. Overall, this work will be a useful resource for the field because of both the genes identified and the quantitative, rigorous screening pipeline. …
Joint Public Review:
In this manuscript, Wafer and Tandon et al. present a thoughtful and well-designed genetic screen for regulators of adipose remodeling using zebrafish as a model system. The authors cross-referenced several human adipocyte-related transcriptomic and genetic association datasets to identify candidate genes, which they then tested in zebrafish. Importantly, the authors devised an unbiased microscopy-based screening platform to document quantitative adipose phenotypes with whole animal imaging, while also employing rigorous statistical methods. From their screen, the authors identified 6 genes that resulted in robust adipose phenotypes out of a total of 25 that were tested. Overall, this work will be a useful resource for the field because of both the genes identified and the quantitative, rigorous screening pipeline. However, there are limitations that preclude a definitive distinction between developmental and remodeling effects that should be acknowledged and discussed, or addressed with new experiments.
Strengths:
(1) This work combines multiple omic datasets to identify candidate genes that informed a CRISPR-based screen to identify genes underlying adipose tissue development and adaptation. This approach offers a new avenue to improve our understanding and testing of new genetic mechanisms underlying the development of obesity.
(2) Using a clever screening approach, this study identifies new genes that are associated with adipose tissue lipid droplet size change. Importantly, the study provides further validation using a stable CRISPR line to show the phenotype in basal and high-fat diet conditions.
(3) The experiments are well-designed and rigorous. Sample sizes are large. Statistical analyses are highly rigorous, contributing to a high-quality study.
Weaknesses:
(1) The image quantification established in Figures 3 and 4 and used in CRISPR screening showed the relationship among zebrafish development, adipose tissue size, and lipid droplet size. Although adipose tissue development patterning is linked with adipose tissue adaptation, as shown by the evidence provided in this paper, it will be more powerful if the imaging method and pipeline were established to directly access the adipose tissue plasticity rather than just the developmental patterning. Furthermore, the authors should perform additional analysis of their existing data to more accurately determine lipid droplet size along the AP axis in response to HFD.
(2) In the absence of tissue-specific manipulations, definitively establishing the mechanisms underlying the genetic regulation of adipose tissue physiology presents limitations.
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