m6A epitranscriptomic modification regulates neural progenitor-to-glial cell transition in the retina
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Evaluation Summary:
This work describes the function of N6-methyladenosine (m6A) modification in developing retina. Enriched scRNA-seq and MeRIP-seq data will be an excellent resource for neurodevelopmental community.
(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. The reviewers remained anonymous to the authors.)
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Abstract
N 6 -methyladenosine (m 6 A) is the most prevalent mRNA internal modification and has been shown to regulate the development, physiology, and pathology of various tissues. However, the functions of the m 6 A epitranscriptome in the visual system remain unclear. In this study, using a retina-specific conditional knockout mouse model, we show that retinas deficient in Mettl3 , the core component of the m 6 A methyltransferase complex, exhibit structural and functional abnormalities beginning at the end of retinogenesis. Immunohistological and single-cell RNA sequencing (scRNA-seq) analyses of retinogenesis processes reveal that retinal progenitor cells (RPCs) and Müller glial cells are the two cell types primarily affected by Mettl3 deficiency. Integrative analyses of scRNA-seq and MeRIP-seq data suggest that m 6 A fine-tunes the transcriptomic transition from RPCs to Müller cells by promoting the degradation of RPC transcripts, the disruption of which leads to abnormalities in late retinogenesis and likely compromises the glial functions of Müller cells. Overexpression of m 6 A-regulated RPC transcripts in late RPCs partially recapitulates the Mettl3 -deficient retinal phenotype. Collectively, our study reveals an epitranscriptomic mechanism governing progenitor-to-glial cell transition during late retinogenesis, which is essential for the homeostasis of the mature retina. The mechanism revealed in this study might also apply to other nervous systems.
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Evaluation Summary:
This work describes the function of N6-methyladenosine (m6A) modification in developing retina. Enriched scRNA-seq and MeRIP-seq data will be an excellent resource for neurodevelopmental community.
(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. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The manuscript by Xin et al. investigated the function of N6-methyladenosine (m6A) modification in the retina. They conditionally deleted Mettl3, which is the key enzyme depositing m6A on mRNAs, in the mouse retina from the beginning of retinogenesis, and studied the consequences of Mettl3 deletion-mediated m6A depletion. They found that Mettl3 deletion led to disorganization of the retina structure at postnatal day 14 (P14), and claimed that Muller glial dysfunction was underlying these morphological abnormalities. They then focused on investigating how Mettl3 deletion affected late retinogenesis after birth and claimed that Mettl3cko retinal progenitor cells (RPCs) withdrew from the cell cycle slower than control RPCs. As a result, there was an increased number of Muller glial cells at P7. They then …
Reviewer #1 (Public Review):
The manuscript by Xin et al. investigated the function of N6-methyladenosine (m6A) modification in the retina. They conditionally deleted Mettl3, which is the key enzyme depositing m6A on mRNAs, in the mouse retina from the beginning of retinogenesis, and studied the consequences of Mettl3 deletion-mediated m6A depletion. They found that Mettl3 deletion led to disorganization of the retina structure at postnatal day 14 (P14), and claimed that Muller glial dysfunction was underlying these morphological abnormalities. They then focused on investigating how Mettl3 deletion affected late retinogenesis after birth and claimed that Mettl3cko retinal progenitor cells (RPCs) withdrew from the cell cycle slower than control RPCs. As a result, there was an increased number of Muller glial cells at P7. They then performed single cell RNA seq and MeRIP-seq to uncover the underlying molecular mechanisms. They claimed that Mettl3-mediated m6A modification promotes the degradation of RPC-related mRNAs, facilitates termination of retinogenesis and fine-tuning the transcriptomic transition from RPCs to Muller glial cells.
The authors performed a significant amount of analyses. But they didn't integrate and explain their data clearly enough for readers to follow. The logic behind the study is not strong. The lack of rigor, data inconsistency and confusing explanations further reduced the weight for this work.
Major concerns:
1. The authors showed disrupted retina structure in Mettl3cko and claimed that dysfunction of Muller glial cells was the underlying cause. However, based on the data presented in the manuscript, it is not clear whether retinal structure abnormalities are direct effects of Muller glial defects or secondary effects induced by defects in RPCs or other cell types.
2. The authors claimed that m6A is important for RPC to Muller glial transition but didn't clearly state whether m6A regulates gliogenesis, which can be easily assessed by quantifying the number of Muller glial cells. The authors showed that the number of Muller glial cells was increased in mettl3cko at P7 based on Sox9 staining, but the number of Muller glial cells in single cell RNA-seq data stayed unchanged between Mettl3cko and controls. In addition, the number of Muller glial cells in adult retinas (for example, at P14) was not examined. Based on images in Fig 1E, the number of Muller glial cells seemed to be slightly decreased compared to controls. Moreover, in line 130, the authors mentioned that Mettl3cko retinas resembled those with loss of Muller glia in the literature. These discrepancies need to be addressed.
3. The authors showed that there was an increased number of RPCs in Mettl3cko at late stages of retinal development compared to controls. However, some of the data do not seem to be consistent with published literature and their own supplementary data. Specifically, at P7, based on their single cell RNA-seq data, 6% of retinal cells were RPCs in control retinas, and 11% of retinal cells were RPCs in Mettl3cko (Fig 3-figure supplement 2). However, the number seems to be very high for control P7 retinas. In published single cell datasets (Clark et al. 2019), the percentage of retinal cells identified as RPCs was close to 0 at around P7. In addition, in Figure 3-Figure supplement 3, the authors stained P7 Mettl3cko and control retinas with Ki67. There was a very limited number of Ki67+ cells in Mettl3cko, which doesn't quite match the 11% found in single cell RNA-seq data.
4. The stage of the retina was not consistent across experiments. The single cell RNA-seq experiment was performed using P7 retinas, whereas MeRIP-seq used P6 retinas. Twenty-four hours could make a big difference in terms of transcription at this stage.
5. Many claims in the manuscript are not fully supported by the data. For example, the authors over-expressed candidate m6A-regulated genes in RPCs and showed that they prevented the RPCs from exiting the cell cycle. However, the data presented in Fig 6 cannot fully support this conclusion. PCNA is not a pan cell cycle marker. Reduction of GFP+PCNA- cells in Mettl3cko doesn't necessarily mean that mutant cells failed to exit the cell cycle.
6. Individual data points and N numbers were not shown in the bar graphs.
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Reviewer #2 (Public Review):
In this study, Xin et al. investigated the role of m6A epitranscriptomic modification in the developing mouse retina. They found the structural disorganization and functional deficits of P14 retinae in Mttl3CKO mice. Combined immunohistological and single-cell transcriptome analyses showed that RPCs and Müller glia are subject to Mettl3 deficiency. Further integrative analyses of scRNA-seq and MeRIP-seq suggested the RPC-specific transcript degradation at the late stages of retina development. Finally, misexpression of specific m6A-regulated RPC-enriched transcripts at P1 could partially recapitulate the phenotype as Mettl3-deficient retinae. The finding is potentially interesting, but a direct mechanistic link remains inadequate.
Strength: The study clearly demonstrated the defects of Mettl3CKO retinae …
Reviewer #2 (Public Review):
In this study, Xin et al. investigated the role of m6A epitranscriptomic modification in the developing mouse retina. They found the structural disorganization and functional deficits of P14 retinae in Mttl3CKO mice. Combined immunohistological and single-cell transcriptome analyses showed that RPCs and Müller glia are subject to Mettl3 deficiency. Further integrative analyses of scRNA-seq and MeRIP-seq suggested the RPC-specific transcript degradation at the late stages of retina development. Finally, misexpression of specific m6A-regulated RPC-enriched transcripts at P1 could partially recapitulate the phenotype as Mettl3-deficient retinae. The finding is potentially interesting, but a direct mechanistic link remains inadequate.
Strength: The study clearly demonstrated the defects of Mettl3CKO retinae mice, including cellular disorganization and abnormal physiological responses. Enriched scRNA-seq and MeRIP-seq data presented here will be an excellent resource to study the function of m6A modification in retinogenesis.
Weakness: While identifying the essential role of m6A modification in gliogenesis by late RPCs is potentially interesting, the direct evidence remains missing mainly. Overall mechanistic exploration was conducted mainly at the populational level such that many results lose cellular specificity, which is critical to uncover the direct link between late RPC-specific m6A-modified transcripts and gliogenesis. Besides, some results seem inconsonant, which needs further clarification.
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