Zebrafish Rif1 impacts zygotic genome activation, replication timing, and sex determination

Reviewer #1 (Public Review):

In this manuscript authors examined the effect of rif1 knockout on replication timing and transcription in early embryos of zebrafish. Contrary to the expectation, genome-wide replication timing domains did not significantly change upon Rif1 knockout, although the replication timing became less dynamic in the mutant, meaning the entire genomes are replicated toward the mid S. In contrast, transcriptional profiles change by rif1 mutation throughout the embryo stage. These effects were more predominantly observed after gastrulation at the early stages of zebrafish development.

The results presented in this manuscript provide new information on the effects of rif1 mutation on early zebrafish development, although the underlying mechanism has not been explored. The information is useful for researchers in the field of early development, with specific focus on replication and transcription regulation.

The genome wide analyses of replication timing has been conducted and analyzed properly. The transcriptional analyses are conducted by RNA-seq and SLAM-seq (determining the nascent mRNA), and the results convincingly show the overall transcriptional patterns at different developmental stages.

This work shows that Rif1 regulates replication timing and transcription in zebrafish embryos, while the extents of the effects vary during the developmental process. Although the data convincingly illustrate the whole picture of Rif1 KO on replication and transcription during zebrafish development, the mechanistic insight is missing. Especially, how Rif1 may or may not coordinately regulate replication and transcription during the zebrafish development has not been addressed.

Reviewer #2 (Public Review):

This study by Masser et al. analyzes global replication timing and gene expression in rif-1 null zebrafish. This work is an extension of their previous report on the normal replication timing pattern during wild-type zebrafish development. The major valuable finding here is that Rif1 is not essential for viability in zebrafish, and - counter to expectation from studies in cultured cells and other species - late replication does not strongly depend on Rif1. Instead, the data suggest that Rif1 subtly sharpens replication timing pattern during normal development rather than function generally to delay replication timing. In the absence of Rif1, the normal pattern establishment is somewhat delayed. The authors also document some changes in expression during development with more genes being repressed by Rif1 than activated at some early stages.

The study and analysis are generally rigorous, and the conclusions are supported by convincing data. The manuscript is well written, though there are aspects of the presentation that could be improved for a broader scientific audience. Given the strong link between replication timing and cell type/development, studying timing in a whole developing organism is important. The experimental approach is technically challenging, particularly the bioinformatic analysis. The scientific advance here is largely confined to documenting the timing of Rif1-affected transcription, the unanticipated effect of the rif1 deletion on replication timing and on sex determination, though the latter is not explored. The work is descriptive and feels like two relatively unconnected studies, transcription and replication plus a small bit of development, and the difference in timing of the transcription phenotypes and replication phenotypes suggests they may be very distinct Rif1 roles. There isn't a lot of new insight into the mechanism of how Rif1 affects either replication timing or gene expression. As such, the overall study is an useful set of findings and detailed data for future work, but it doesn't make a big step forward in understanding the role of Rif1 or the biological processes it affects.

Weaknesses worth addressing include the following:

1. Loss of Rif1 did not affect viability, but it did strongly influence sex determination, resulting in a lower population of females. This effect is the strongest organismal phenotype, but the study provides no explanation for the loss of females from the data gathered here.
2. The approach to distinguish nascent zygotically expressed mRNAs from maternal mRNAs is a strength. Are the differentially expressed genes related at all to regions of the genome whose replication timing is most affected? Are any of them related to the sex determination or developmental phenotypes?

Reviewer #3 (Public Review):

Using the zebrafish model system, this manuscript assessed the roles of Rif1 protein in replication timing control and transcription during early development, and successfully demonstrated the differential impact of Rif1 protein in replication timing control and transcription. Moreover, the comprehensive assessments of the impacts of mutating Rif1 on animal development (including animal survival and sexual development) were assessed. Although there are works that examined Rif1's implications in replication timing and transcription separately, this work is unique in assessing all these points at once.

The strength of this manuscript is the genomic analyses of replication timing and transcription being combined in a single model system. Consequently, this manuscript clearly demonstrates the differential impact of Rif1 in these processes during zebrafish development.

The weakness of this manuscript is, as the authors comment in the Discussion, analyses of replication timing and transcription were performed using bulk embryos. There is a possibility that tissue-specific changes could have been masked. Tissue-specific or single-cell analysis in the future will fill the gap in the knowledge.

Some of the findings presented in this manuscript are consistent with previous findings using different models such as Drosophila and mice, whereas other findings do not necessarily agree. I hope further studies will reveal more clearly what is common in these systems, and what is different.

Also, the suggestion that the Rif1 protein may be implicated in a function similar to Fanconi-Anemia genes/proteins is very intriguing.

Overall, the data presented in this manuscript sufficiently justify the authors' claims. Moreover, this manuscript provides interesting insights into Rif1's function, as well as how development could be controlled.