Tissue-specific modifier alleles determine Mertk loss-of-function traits
Abstract
Knockout (KO) mouse models play critical roles in elucidating biological processes behind disease-associated or disease-resistant traits. As a consequence of gene KO, mice display certain phenotypes. Based on insight into the molecular role of said gene in a biological process, it is inferred that the particular biological process causally underlies the trait. This approach has been crucial towards understanding the basis of pathological and/or advantageous traits associated with Mertk KO. MERTK is a receptor tyrosine kinase with a critical role in phagocytosis of apoptotic cells or cellular debris. Therefore, early-onset, severe retinal degeneration was described to be a direct consequence of failed phagocytosis of photoreceptor outer segments by retinal pigment epithelia. Similarly, enhanced anti-tumor immunity was inferred to result from the failure of macrophages to dispose cancer cell corpses, resulting in a pro-inflammatory tumor microenvironment. Here we report that the loss of Mertk alone is not sufficient for retinal degeneration. This trait only manifests when the function of the paralog Tyro3 is concomitantly lost. Additionally, the dramatic resistance against two syngeneic mouse tumor models observed in Mertk KO cannot, at least entirely, be ascribed to the loss of Mertk . The widely used Mertk KO carries multiple coincidental changes in its genome that affect the expression of a number of genes, including Tyro3 . Nonetheless, neither Tyro3 , nor macrophage phagocytosis by alternate genetic redundancy, accounts for the absence of anti-tumor immunity in two independent Mertk KOs. Collectively, our results indicate that context-dependent epistasis of independent modifier alleles determine Mertk KO traits.
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Evaluation Summary:
The authors show that a widely used knock-out mouse for Mertk carries multiple additional changes in its genome, affecting the expression of a number of genes besides Mertk. They show that, although the line was back-crossed to the C57 background, these changes are due to the original 129P2 genome of the embryonic stem cells in which the knock-out was originally created. Through the generation of two new knock-out mouse strains, in C57 embryonic stem cells, the authors here show only part of the phenotype of the original Mertk knock-out mouse can be reproduced. Overall, this study raises awareness as to the limitations of the Mertk-/- v1 model and limits direct inference of Mertk-/-v1 observed phenotypes to Mertk deficiency alone.
(This preprint has been reviewed by eLife. We include the public reviews from the …
Evaluation Summary:
The authors show that a widely used knock-out mouse for Mertk carries multiple additional changes in its genome, affecting the expression of a number of genes besides Mertk. They show that, although the line was back-crossed to the C57 background, these changes are due to the original 129P2 genome of the embryonic stem cells in which the knock-out was originally created. Through the generation of two new knock-out mouse strains, in C57 embryonic stem cells, the authors here show only part of the phenotype of the original Mertk knock-out mouse can be reproduced. Overall, this study raises awareness as to the limitations of the Mertk-/- v1 model and limits direct inference of Mertk-/-v1 observed phenotypes to Mertk deficiency alone.
(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 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript by Akalu et al. is well written and contains thoroughly executed experiments, that challenge previous conclusions obtained using the Mertk-/-V1 mouse model, in different tissue contexts.
Using several new mouse models, the authors functionally show that Mertk loss alone is not sufficient to trigger the retinal degeneration phenotype characteristic of Mertk-/-v1. Rather, they demonstrate that retinal degeneration requires the combined loss of Mertk and Tyro3, a second TAM receptor exhibiting hypomorphic expression in the Mertk-/-v1 model, due to its expression from a DNA portion carried over from the 129 ES cell background used to generate the Mertk-/-v1 line. The study further provides compelling functional data by demonstrating that Tyro3 ablation in the newly generated Mertk-/- v2 BL6 model, …
Reviewer #1 (Public Review):
The manuscript by Akalu et al. is well written and contains thoroughly executed experiments, that challenge previous conclusions obtained using the Mertk-/-V1 mouse model, in different tissue contexts.
Using several new mouse models, the authors functionally show that Mertk loss alone is not sufficient to trigger the retinal degeneration phenotype characteristic of Mertk-/-v1. Rather, they demonstrate that retinal degeneration requires the combined loss of Mertk and Tyro3, a second TAM receptor exhibiting hypomorphic expression in the Mertk-/-v1 model, due to its expression from a DNA portion carried over from the 129 ES cell background used to generate the Mertk-/-v1 line. The study further provides compelling functional data by demonstrating that Tyro3 ablation in the newly generated Mertk-/- v2 BL6 model, is required to recapitulate the retinal degeneration phenotype.
Interestingly, the work presented here also reports different outcomes in cancer contexts, where the authors show that the Mertk-/- v1 exhibits remarkable anti-tumor resistance in two independent "cold" cancer models (YUMM1.7 sc model and GL261 intracranial GBM model), which were not recapitulated in the newly generated Mertk-/- mouse lines on a BL6 background, namely the Mertk-/- v2 and Mertk-/- v3 as well as Mertk-/- v2 Tyro3-/-v2 that lack both Mertk and Tyro3, pointing to additional gene modifiers being at play. These findings are highly relevant for the cancer field and would certainly benefit from future experiments suggested by the authors to identify the modifier genes to bolster their significance.
Overall, this well-executed study demonstrates that the Mertk-/-v1 model carries additional changes in its genome that affect the expression of several genes, including Tyro3, that act as confounding events and limit the direct inference of observed phenotypes to Mertk deficiency alone.
A potential weakness in this aspect resides in the fact that the newly generated mouse models Mertk-/- v2 and Mertk-/- v3 appear to display a compensatory increase in the levels of TYRO3 protein, which needs to be discussed by the authors.
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Reviewer #2 (Public Review):
Through a thorough review of the genome of this specific (Mertk) knock-out mouse, the authors found multiple additional (unwanted) changes in the genome. These genetic changes were due to the way the knock-out mouse was generated. The authors created two novel mouse knock-out lines, without the additional changes, in which only part of the findings observed in the original knock-out mouse could be reproduced. This article leads to the generation of two novel knock-out lines relevant to researchers studying MERTK and rectification of part of the previous findings in the original Mertk knock-out mouse. Besides, being relevant to a broader audience, this paper clearly demonstrates that the original background and methods used for generating knock-out mice should be evaluated and taken into account when planning …
Reviewer #2 (Public Review):
Through a thorough review of the genome of this specific (Mertk) knock-out mouse, the authors found multiple additional (unwanted) changes in the genome. These genetic changes were due to the way the knock-out mouse was generated. The authors created two novel mouse knock-out lines, without the additional changes, in which only part of the findings observed in the original knock-out mouse could be reproduced. This article leads to the generation of two novel knock-out lines relevant to researchers studying MERTK and rectification of part of the previous findings in the original Mertk knock-out mouse. Besides, being relevant to a broader audience, this paper clearly demonstrates that the original background and methods used for generating knock-out mice should be evaluated and taken into account when planning mouse in vitro and in vivo studies.
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