1. Author Response:

    Reviewer #1 (Public Review):

    This work from Park et al represents a large, ambitious study utilizing a variety of mouse models (several novel) to establish mechanisms underlying cardiac pathologies observed upon loss of imprinting at the H19/IGF2 locus. The studies indicate 1) that mice recapitulate key cardiovascular features observed in humans with BWS, 2) that developmental cardiomegaly and progressive cardiomyopathy are distinct, non-correlated phenotypes driven by disparate mechanisms (upregulation of IGF2 and reduction of H19, respectively), and that H19 associated pathologies are driven by reduced interaction with let7 microRNAs. There is considerable novelty and potential impact of the work, as it presents substantial mechanistic insight into the consequences of LOI and the development of BWS. The authors use a variety of appropriate approaches, including mouse echocardiography, tissue IHC, pressure myography, and in vitro studies of cell size regulation to support these primary conclusions. In its current form, however, some primary conclusions are insufficiently supported, and additional quantification and controls would be needed.

    Major

    1. The conclusion of transient neonatal cardiomegaly that resolves by 2 months is insufficiently supported. Increased cell surface area is useful, but can be driven by cell spreading, not necessarily hypertrophy, and no data is shown at the 2-month time point to suggest reversion of cardiomegaly.

    Data for heart weight/tibia length at 2 months is described on lines 116-117.

    1. It seems an important validation is needed for the Let7 binding site deletion model - I do not see any data confirming that the gene editing was indeed successful nor that Let7 binding to H19 was effectively disrupted.

    As described above, we performed new experiments to validate the Let-7 binding site deletion model. See Lines 329-33 and Figure 6-figure supplement 1. Briefly, we purify H19 lncRNA from wild type and from H19let7/H19let7 extracts using biotinylated oligonucleotides. We show that this method is equally efficient in purifying wild type H19 and H19let7 lncRNAs. However, let-7 miRNAs copurify only with wild type H19.

    Further, it is unclear at what age the Let7 binding site deletion mice were assayed for cardiomegaly/hypertrophy.

    Mice were analyzed at one year of age. This information is now provided in the main text as well as in Figure 6 legend.

    The HW/TL values (WT = 7.5) are different from most others reported throughout the manuscript.

    The reviewer is correct that there are some differences in heart weight/tibia length values across the various mouse models.

    Based on the differences between genetic backgrounds, it would not be appropriate to compare between different data sets. Our study was designed so that each model is compared always to wild type littermates. Essentially, our paper describes 4 independent studies: WT vs LOI, LOI+BAC vs LOI, WT vs H19DEx1/+, and WT vs H19Dlet7/H19Dlet7. In each case we see a 15-30% increase in heart size associated with loss of H19 function. We consider it a strength of our study that we consistently account for potential strain effects and that we demonstrate H19 function in 4 completely independent comparisons.

    However, we also understand the reviewer’s confusion and modified the main text to make clear that each model is being compared to its wild type littermates only.

    Finally, WT/TL ratios for WT vs LOI and WT vs H19DEx1/H19+ models were measured on independent sets of mice by two different labs. We consistently measured a 20-30% increase in mice lacking H19 lncRNA. Also, echocardiography results showed a 25% increase in average heart mass.

    1. Many observations or conclusions are not sufficiently supported by quantification. For example, there is a lack of quantification of any western blots.

    As described above, all data are now quantitated. Especially, see Figure 2-figure supplement 1, Figure 3-figure supplement 1, Figure 4-figure supplement 1, and Figure 6-figure supplement 2 for quantification of Western blots.

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  2. Evaluation Summary:

    This manuscript models a particular class of genetic lesions observed in the imprinting disorder and overgrowth syndrome, Beckwith-Wiedemann syndrome (BWS), using a highly tractable mouse model. Because more than one gene is abnormally expressed in BWS that is caused by loss of imprinting of H19 and IGF2, the authors vary the expression of both genes to investigate the source of the cardiovascular phenotypes and are able to ascribe independent heart phenotypes resulting from IGF2 overexpression and H19 loss of expression.

    (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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  3. Reviewer #1 (Public Review):

    This work from Park et al represents a large, ambitious study utilizing a variety of mouse models (several novel) to establish mechanisms underlying cardiac pathologies observed upon loss of imprinting at the H19/IGF2 locus. The studies indicate 1) that mice recapitulate key cardiovascular features observed in humans with BWS, 2) that developmental cardiomegaly and progressive cardiomyopathy are distinct, non-correlated phenotypes driven by disparate mechanisms (upregulation of IGF2 and reduction of H19, respectively), and that H19 associated pathologies are driven by reduced interaction with let7 microRNAs. There is considerable novelty and potential impact of the work, as it presents substantial mechanistic insight into the consequences of LOI and the development of BWS. The authors use a variety of appropriate approaches, including mouse echocardiography, tissue IHC, pressure myography, and in vitro studies of cell size regulation to support these primary conclusions. In its current form, however, some primary conclusions are insufficiently supported, and additional quantification and controls would be needed.

    Major

    1. The conclusion of transient neonatal cardiomegaly that resolves by 2 months is insufficiently supported. Increased cell surface area is useful, but can be driven by cell spreading, not necessarily hypertrophy, and no data is shown at the 2-month time point to suggest reversion of cardiomegaly.

    2. It seems an important validation is needed for the Let7 binding site deletion model - I do not see any data confirming that the gene editing was indeed successful nor that Let7 binding to H19 was effectively disrupted. Further, it is unclear at what age the Let7 binding site deletion mice were assayed for cardiomegaly/hypertrophy. The HW/TL values (WT = 7.5) are different from most others reported throughout the manuscript.

    3. Many observations or conclusions are not sufficiently supported by quantification. For example, there is a lack of quantification of any western blots.

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  4. Reviewer #2 (Public Review):

    BWS caused by loss of imprinting of H19 and IGF2 results in overexpression of the growth factor IGF2 and reduced expression of H19. Because overgrowth and various other phenotypes associated with this disorder can result from misexpression of either of these genes, the investigators have used a mouse model that recapitulates the human phenotypes. Specifically, they use a series of endogenous mouse mutations and transgenes to test the role of Igf2 overexpression and H19 loss in neonatal cardiomegaly and adult onset progressive heart pathology, including fibrosis and reduced ventricular function. The use of H19/Igf2 ICR mutations to increase Igf2 expression shows that the neonatal cardiomegaly phenotype is largest caused by increased expression of IGF2. This phenotype ultimately resolves when IGF2 decreases. In contrast the adult phenotype is consistent with loss of H19, which is rescued using an H19 BAC. Finally, because H19 encodes a 2.3 kb lncRNA and a microRNA, the researchers used two additional mutants that test the function of each, demonstrating that reduced H19 expression can cause progressive heart pathologies and that interaction of H19 lncRNA with let7 microRNA is essential for normal cardiac physiology.

    This is an interesting and elegant manuscript that shows the importance of the H19/IGF2 axis for normal heart physiology. Moreover, this work brings together some of the disparate functions that have been ascribed to H19. The work is convincing and the only remaining issues concern the level of expression of H19 and Igf2 in the various mutant backgrounds and target cells of IGF2 expression. Also, it is unclear if BWS individuals with H19/IGF2 lesions have heart disease later in life, and it would have been helpful for the readers if the authors had commented on this.

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  5. Reviewer #3 (Public Review):

    Park et al were seeking to understand the effects of loss of imprinting at the H19/Igf2 locus on the physiology of the developing mouse heart. With the extensive molecular characterisation of the mechanisms of gene regulation at this locus in the public domain, they used this knowledge to move to the next step of making a detailed phenotypic study using a suite of mouse mutants.

    Park et al utilize a suite of different approaches including choosing a set of mouse mutants that together have the capacity to ratchet up and down the expression of the two imprinted genes Ig2f and H19, and because of their known coordinate regulation, they can also distinguish their individual effects. This results in a predicted gene expression ratio and the resultant phenotypes in these mutants are characterized thoroughly. Heart phenotypes were examined in WT, LOI, LOI+BAC Tg and H19 deficient neonates which had different levels of each Igf2 & H19 and represent genetic rescues. Dysmorphology of the heart was observed and characterised. In aged animals, cardiac hypertrophy was evident in mice with LOI. Echocardiography revealed mild CV phenotypes in LOI mice.

    The morphology of the heart overall and in detail (eg aorta strength/size) are investigated. Cardiac hypertrophy is assessed using markers for cell proliferation and cell assays are employed and revealed that exogenous IGF2 peptide induces cellular hypertrophy in WT cardiomyocytes through mTOR pathways.

    However the authors noticed that the phenotypes under study elicited some odd parent of origin behaviours, namely, LOI female adult mice showed weak phenotypes compared to male mice but they were equivalent in neonatal mice. The authors therefore thought that neonatal hypertrophy and adult disease phenotypes were complicated by something unknown. This called for a genetic rescue experiment to carefully check for H19 and Igf2 expression independently, something feasible given the molecular detail in which these two genes have been studied mechanistically and the strains of mice available. It was revealed that H19 does not contribute to neonatal hypergrowth, instead, hypertrophy results from biallelic Igf2. This they justify by published work on TOR signalling.

    The study undertook transcriptomic analysis of WT vs H19 deficient hearts followed by gene enrichment studies to pin point EMT deficiencies which they went on to validate with assays in isolated cells in culture. They study determined that endothelial cell transition was increased in the absence of H19. Prior studies pointed to let7 miRNAs binding H19 and to find out whether without this, cardiomyopathy results, CRISPR/Cas9 genome editing to delete let-7 binding sites in the H19 gene indeed supported this.

    This work overall shows show that neonatal cardiomegaly is dependent on increased Igf2 compared to WT. Deletion of H19 is sufficient to induce cardiomyopathy in adult mice. A role in the regulation of cardiac endothelial cell growth is attributed to H19 through let7. A huge body of work.

    Overall, the authors achieved their aims and their results support their conclusions.

    This work moves ahead from mechanism to functional characterisation of these imprinted genes and focuses on mammalian development and physiology in the heart. It does this through a thorough and exhaustive examination of morphology, cell biology and quantitative methods in what are an informed and well characterised set of mutant strains. There is a lot of work in this study and it will impact researcher's views of the importance of imprinted genes in normal development. The importance of tight gene expression levels in development and differentiation and how this can be achieved using co-ordinately regulated gene pairs. The H19/Igf2 locus and its control elements are of interest because in humans there is an association with BWS. It will therefore provide an excellent model in which to study cardiomyopathy as it related to the human disease BWS.

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