SRSF3 is a key regulator of epicardial formation

  1. Irina-Elena Lupu
  2. Susann Bruche
  3. Anob M. Chakrabarti
  4. Ian R. McCracken
  5. Tamara Carsana
  6. Andia N. Redpath
  7. Nicola Smart

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Abstract

The epicardium is a fundamental regulator of cardiac development and regeneration, functioning to secrete essential growth factors and to produce epicardium-derived cells (EPDCs) that contribute most coronary mural cells and cardiac fibroblasts. The molecular mechanisms controlling epicardial formation have not been fully elucidated. In this study, we found that the RNA-binding protein SRSF3 is highly expressed in the embryonic proepicardium and epicardial layer. Deletion of Srsf3 from the murine proepicardium led to proliferative arrest, which prevented proper epicardial formation. Induction of Srsf3 deletion after the proepicardial stage resulted in impaired epicardial proliferation and EPDC formation by E13.5. Single-cell RNA-sequencing showed SRSF3-depleted epicardial cells were eliminated, however, the surviving non-recombined cells became hyperproliferative and, remarkably, compensated for the early deficit, via a mechanism that involved Srsf3 up-regulation This unexpected finding attests the importance of SRSF3 in controlling epicardial proliferation, and highlights the significant confounding effect of mosaic recombination on embryonic phenotyping. Mapping the SRSF3–RNA interaction network by endogenous irCLIP identified binding to major cell cycle regulators, such as Ccnd1 and Map4k4 , with both splicing and non-splicing roles. This research defines SRSF3 as a key regulator of epicardial cell proliferation.

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  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Lupu et al have identified a role for the RNA binding protein SRSF3 in epicardial development. In a well written manuscript containing many experiments the authors show that this protein is required at different time points in epicardial development to control a range of processes, in particular cell proliferation. This advances understanding of the complex roles of this RNA binding protein in the heart - and raises an important message about how incomplete Cre recombination needs to be considered in interpreting conditional mutant phenotypes. The following points should be addressed.

    1. SRSF3 is known to play essential developmental roles in the myocardium where it regulates capping of transcripts involved in contraction. This point should be mentioned in addition to roles in proliferation. To facilitate understanding, the authors should say more about the subset of cardiomyocytes labelled by Gata5-Cre. For example, is this the result of stochastic activation of the transgene or is a specific subset of cells labelled? How much of the myocardium is targeted?
    2. The authors show failure of ventricular compaction at E13.5 using Wt1-CreERT2 and go on to assess proliferation in epicardial cells. As epicardial-derived signals are known to promote compact myocardial growth, they should also show whether there are indirect defects in proliferation in compact layer myocardium that might explain the non-compaction phenotype. The authors should also indicate if any of the large number of genes bound by SRSF3 encode known or potential pro-proliferative signals from the epicardium or EPDCs to the myocardium and potentially validate their altered expression in mutant hearts.
    3. The rescue by expansion of non-recombined cells is a most interesting aspect of this study. Can the authors see any such outcompeting in the explant experiments (for example in Figure 2)? Do the authors consider this to be an exclusively in vivo competition phenomenon? Given the known roles of Myc in cell competition can the authors use their single cell transcriptomic data to score Myc expression levels in cells from Srsf3 iKO hearts or determine if Myc transcripts are bound by SRSF3?
    4. The authors suggest that this rescue occur by upregulation of Srsf3 in non-recombined cells. It would be helpful to provide additional lines of evidence supporting the hypothesis that SRSF3 expression is upregulated due to hypoxia. Do the CLIP experiments reveal whether SRSF3 binds to it's own transcript?
    5. The authors imply that SRSF3 may regulate Ccnd1 mRNA stability. Can the authors directly evaluate this point? Please clarify if this gene is also affected in the knock-down experiments in MEC1 cells.
    6. Please brighten the immunofluorescence panels in Figure 1 to more clearly show nuclear labelling and tissue structure.
    7. Given the broad roles of SRSF3 is the adjective key necessary in the title?

    Significance

    This ms advances understanding of the complex roles of this RNA binding protein in the heart - and raises an important message about how incomplete Cre recombination needs to be considered in interpreting conditional mutant phenotypes. This study would be of interest to reseachers in the fields of heart development and RNA-protein interactions. Although there are a number of major points to be addressed, these could be potentially dealt with rapidly.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    In this study, Lupu et al. analyzed the role of the RNA-binding protein SRSF3 for epicardial development. The authors found that Srsf3 is highly expressed in the proepicardial organ and during early stages of epicardial layer formation. Conditional inactivation of SRSF3 in the proepicardial organ stage using a Gata5-Cre driver line resulted in defective formation of the epicardium, accompanied by a proliferation arrest of the proepicardium, resulting in embryonic lethality at E12.5. In contrast, epicardial-specific Srsf3 deletion at later stages using the inducible Wt1CreERT2 line caused a less severe phenotype indicated by impaired coronary vasculature formation, reduced cardiac compaction, and myocardial hypoxia. Mosaic recombination yielded a small population of epicardial cells that upregulate Srsf3, hyperproliferate and compensate for the depleted Srsf3 negative lineage. Single-cell RNA sequencing of control and epicardial Srsf3 knock out hearts, combined with infrared CLIP to map SRSF3 binding sites in the transcriptome identified a number of putative SRSF3 targets involved in mitotic cell cycle control. Among others, SRSF3 binds directly to transcripts encoding key regulators of proliferation, such as Cyclin D1, and senescence, including MAP4K4. The authors conclude that SRSF3 exerts different functions in processing of RNAs, including splicing.

    Overall, this is a well-written and well-organized manuscript, describing interesting findings in the field of epicardial development. However, the mechanistic part is not overly strong. The authors detected some moderate changes in the distribution of different Map4k4 splicing isoforms after knockdown of Srsf3 in an immortalized epicardial cell line but did not go any deeper. The cause for the reduced presence of transcripts for the SRSF3-target Ccnd1 after knockdown of Srsf3 remains enigmatic.

    Significance

    The authors raise a number of speculations why remaining Srsf3-expressing cell start to hyperproliferate after inactivation of Srsf3 but it does not become clear which mechanism is critical. How do non-targeted epicardial cells in the mosaic recombination model sense the loss of SRSF3 knock out cells, resulting in hyperproliferation and enhanced Srsf3 expression? Is a loss of lateral inhibition, e.g. by activated YAP/TAZ, causative for enhanced proliferation of the remaining epicardial cells and an elevated expression level of WT1 and SRSF3? Immunofluorescence staining and/or qRT-PCR of YAP/TAZ and TEADs might provide an answer.

    Is the elevated expression of Srsf3 in non-targeted epicardial cells due to enhanced transcription and/or by altered post-transcriptional processes? How does this observation fit to previous reports indicating that Srsf3 overexpression promotes inclusion of an autoregulatory cassette exon (exon 4) containing a premature (in-frame) stop codon in Srsf3, thereby confining this SRSF3 isoform to nonsense mediated decay (NMD) (doi: 10.1093/emboj/16.16.5077, doi: 10.1186/gb-2012-13-3-r17, doi: 10.1038/srep14548, doi: 10.1161/CIRCRESAHA.118.31451)? In contrast, Srsf1 as well as PTBP1/2 have been previously reported to regulate Srsf3 expression by promoting exon 4 skipping. The authors should perform RNA seq and/or qRT-PCRs validation to check the inclusion of Srsf4 Exon4 as well as Srsf1 and PTBP1/2 expression levels in control and knock out epicardial cells.

    It remains unclear by which mechanisms (alternative splicing, alternative polyadenylation, miRNA processing, or others) SRFS3 mainly exerts its function in the embryonic epicardial lineage. The selection and validation of Map4k4 as a splicing target is not based on an unbiased splicing analysis. In my opinion it is mandatory to provide a full assessment of splicing changes in Srsf3-deficient cells, either by long-range sequencing or by analysis of exon-junction reads.

    Likewise, it is completely enigmatic what SRFS3 does to Ccnd1 transcripts. Does SRFS3 increase the half-life of Ccnd1, does it impact trafficking? At least, the authors have to determine changes in the half-life of Ccnd1 after depletion of SRFS3.

    An unbiased bioinformatics analysis addressing alternative splicing, alternative polyadenylation, and mRNA processing is necessary. Ideally, primary epicardial cells should be used and not an immortalized epicardial cell lines. It is well known, that splicing in cell lines differs substantially from splicing in primary cells.

    I am not convinced that the moderate changes of different Map4k4 splicing isoforms after knockdown of Srsf3 are really responsible for the rather drastic phenotype. Additional experiments are needed to prove a decisive function of a shift in Map4k4 splicing isoforms for hyperproliferation of epicardial cells.

    The authors claim that inactivation of Srsf3 inhibits cell proliferation and causes a senescence-like phenotype. The claim for acquisition of senescence is solely based on transcriptional changes. No attempts were made to visualize an increase of senescent cells in Srsf3-mutant embryos. The authors need to perform SA-bGAL assays or use other techniques to analyse the appearance of senescent cells in the mutants.

    Fig. 2E indicates that WT1 positive / tdTom negative epicardial cell population is enriched in a specific region of the pre-epicardial organ from Srsf3KOs. However it is not clear whether these cells proliferate. The authors should quantify Ki67positive cells in both the WT1-positive / tdTom-positive and the WT1positive / tdTom-negative epicardial population.

    In the headline on page 6, the authors stated that "SRSF3 depletion in the PEO results in impaired ... migration of epicardial progenitor cells", which they deduced from the reduced outgrowth of ventricular epicardial explants. However, the reduced outgrowth from the PEO could be caused by both, reduced proliferation and/or reduced migration. Therefore, the authors should provide additional data clearly indicating reduced migration, e.g. by blocking transcription. Scratch assays of SRFS3 knockout/knockdown vs. control epicardial cells would strengthen the analysis, Is there a change in the GO term "regulation of migration"?

    To prove the reduced proliferation ratio in Figure 4B, quantification of Cyclin D1 positive cells in both SRSF3 positive and negative cells is required.

    Minor issues

    Abstract line 12: a full stop is missing at the end of the sentence.

    Figure 1A: E11.5, figure label 'DAPI WT1' is missing.

    Page 8: Bracket in front of Fig. 4B is missing

    Page 8: G2M phase change uniformly to G2/M phase

    Page 9: 'Srsf3-depleted hearts also demonstrated an increased abundance of epicardial cells with upregulated expression of genes associated with quiescence, such as Clu48, and senescence, for example Map4k4, Tmem30a and Pofut249 (Fig. 4F)'. The sentence is misleading, implying Srfs3 inactivation in all cardiac cell types ('Srsf3-depleted hearts').

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The study by Irina Lupu and colleagues highlights SRSF3 as a key regulator of epicardial development by regulating epicardial cell proliferation. This was demonstrated via two murine knockout models; the first to assimilate the role SRSF3 plays in epicardial formation as a whole, and the second to address its importance post a pivotal maturation point. Through scRNA sequencing and irCLIP, several SRSF3 targets were ascertained and identified as cell cycle regulators. Those epicardial cells that did not lose SRSF3 compensated the loss of some of their mates by increasing SRSF3 expression and over-proliferating. Overall, the paper is interesting and the conclusions are largely supported by the provided data.

    Major comments:

    1. Authors claim that a "reduction in SRSF3 expression levels coincided with the downregulation of WT1 in the epicardium". This was evidenced by immunofluorescence imaging (figure 1A). I suggest conducting a qRT-PCR to quantify Wt1 expression over time, similar to the experiment they performed in figure 1B.
    2. A western blot depicting SRSF3 protein production in controls compared to the knockout model may provide stronger evidence of its depletion (figure 1E).
    3. Authors state that they were unable to directly identify the absence of exons 2 and 3 in individual cells. Please provide evidence that exons 2 and 3 have been knocked out, at least by performing a qRT-PCR.
    4. To prove the functional implication in the observed phenotype of the identified SRSF3 targets, please interfere with Map4k4 activity or expression and check whether the defective epicardial cell proliferation is reverted. This should be done at least in vitro, ideally in vivo.

    Minor comments:

    1. Several minor typos and spacing issues were observed. Please correct.
    2. It would be good for the reader if the authors would simplify their rationale for the use of the two mouse models. It is slightly convoluted and not easy to follow.
    3. In figure 4, it is recommended to add a stacked bar plot to represent the percentage of each cell cluster/population after 4A. This would help the reader
    4. Figure 4B. It is confusing for the reader to understand the fact that the majority of tdTomato+ sorted cells in Srsf3 iKO keep expressing Srsf3. Including the quantification of the image could help.

    Significance

    The paper will be of interest to readers in the field of cardiology, embryology and molecular biology. It will advance the field especially in the study of the development of the epicardium. The models are sophisticated and the experiments carefully performed.

    My field is molecular cardiology, with interest in RNA-binding proteins.

  5. Version published to 10.1101/2021.11.25.470003v2 on bioRxiv
  6. Version published to 10.1101/2021.11.25.470003v1 on bioRxiv