SUPPLEMENTARY INFORMATION

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1 Supplementary Discussion Rationale for using maternal ythdf2 -/- mutants as study subject To study the genetic basis of the embryonic developmental delay that we observed, we crossed fish with different ythdf2 genotypes and counted embryos with either wild-type or developmentally delayed phenotypes (Extended Data Fig. 1c). The results showed that maternal genotype is the sole determinant for developmental delay phenotypes in the offspring: wild-type females produce no delayed embryos while ythdf2 -/- females produce ~95% developmentally delayed embryos, regardless of paternal genotype. In addition, we have also observed marked paternally-associated embryonic lethality and/or sterility from ythdf2 homozygous mutant males. As shown in Extended Data Fig. 1d, ythdf2 -/- males produce on average 70% progeny whose development never progresses past the 1-cell stage. This phenotype occurs regardless of whether ythdf2 -/- males are mated to wild type, ythdf2 +/-, or ythdf2 -/- females. Further, the percentage of embryos suffering from cell cycle 12 delay during the maternal-zygotic transition is identical between offspring from m(ythdf2 -/- ) p(ythdf2 +/+ ) or m(ythdf2 -/- ) p(ythdf2 -/- ) matings (Extended Data Fig. 1c). Thus, we infer that Ythdf2 must be pleiotropic, acting both to degrade maternal transcripts and also to produce normal sperm or some other paternal effect, which we intend to study further. Importantly, the surviving embryos from matings in which the paternal genotype is ythdf2 -/- (regardless of the maternal genotype) appear normal, survive to adulthood with no obviously disabilities, and harbor a transcriptome that is essentially indistinguishable from wild-type after the maternal phase of Ythdf2 action has ended (Extended Fig. 9). We, therefore, consider the maternal effect and the paternal effect of Ythdf2 separate gene functions, ones that arise in different animals (fathers versus offspring) in different 1

2 biological processes (perhaps spermatogenesis or fertilization versus embryonic development) and can segregate independently of one another. Taken together, in order to more easily and specifically investigate the function of maternal ythdf2, apart from the paternally-associated lethality we observed for ythdf2 homozygous mutant males, we chose to cross female ythdf2 -/- fish to male wild-type fish in order to produce embryos without only maternal ythdf2 as our primary study subject. Characterization of transcript superclusters A total of 11,631 genes that are detected by high-throughput sequencing during the studied period (0 8 h.p.f.) were included in a clustering analysis (see Online Methods). The clustering analysis generated three superclusters we describe as maternal, semi-stable, and zygotic based on their expression dynamics. Each supercluster comprises two subclusters with distinct trends. The maternal supercluster (containing 5,211 genes) exhibited high expression levels at early time points and were degraded to very low levels afterwards (Fig. 2a). These genes formed two subclusters: the first started to decrease early on from 1 h.p.f. (maternal-early; 2,167 genes), while the second declined rapidly from 4 h.p.f. onward (maternal-late; 3,044 genes). The methylation ratio of maternal-late genes is 43.6%, higher than that of maternal-early ones at 25.9%. The expression of genes in the zygotic supercluster (containing 2,472 genes) was non-existent initially but started to increase at later stages. The first subcluster was activated at approximately 3 h.p.f. (zygotic-early; 831 genes), and the second started expression after 4 h.p.f. (zygotic-late; 1,641 genes). The methylation ratios of both zygotic subclusters are similar at around 31%. The semi-stable supercluster (containing 3,948 genes), unlike the other two superclusters, 2

3 featured relatively stable expression patterns. One of the subclusters exhibited medium expression levels before further increasing after 4 h.p.f. (semi-stable-1; 1,527 genes), while the other subcluster showed high expression levels initially and slowly decreased in abundance starting from 4 h.p.f. (semi-stable-2; 2,421 genes). Both semi-stable subclusters show high levels of methylation, with semi-stable-2 (51.5%) slightly higher than semi-stable-1 (45.8%). Collectively, the above clustering analysis highlights the dynamics of gene expression patterns and RNA methylation levels during zebrafish early development. Determination of Ythdf2 direct targets Considering that over 60% of upregulated, methylated transcripts are maternal genes at 4 h.p.f. in maternal ythdf2 -/- embryos, (Extended Data Fig. 5b), ythdf2 LOF is likely to cause a shift in m 6 A abundance when compared to wild-type embryos. If Ythdf2 primarily functions to degrade maternal mrna, then in maternal ythdf2 -/- mutants, the abundance of m 6 A should increase with the retention of maternal mrna; if Ythdf2 primarily functions to promote the stabilization of zygotic transcripts, m 6 A abundance should otherwise decrease with the delayed activation of zygotic gene expression (Fig. 2d). Mass spectrometry results showed over 50% increase of m 6 A abundance in ythdf2 -/- embryos compared to wild-type ones at 4 h.p.f. (Extended Data Fig. 5a), thus confirming that maternal mrnas are the major direct targets of Ythdf2. Ythdf2 primarily promote the decay of maternal but not zygotic mrnas during MZT In order to investigate the specificity of Ythdf2, we use mrna-seq to study the transcriptome change of embryos devoid of both maternal and zygotic ythdf2 (mz-ko) and maternal ythdf2 -/- 3

4 (m-ko) mutants. At 6 h.p.f, mz-ko mutants showed an extended delay of maternal clearance compared to m-ko mutants (Extended Data Fig. 8a, c) and a continued delay of zygotic gene activation up to 8 h.p.f. (Extended Data Fig. 8a-d), indicating that zygotic Ythdf2 might also participate in maternal RNA clearance. However, at later time points (12 and 24 h.p.f.), when maternal RNA degradation is largely finished, ythdf2 KO has no global effects on the zygotic transcriptome (Extended Data Fig. 9a, b). This is also true when considering m-ko vs. mz-ko embryos that are stage-matched instead of time-matched and m-ko embryos injected with ythdf2-mo (to achieve maternal-zygotic LOF without male-associated lethality) (Extended Data Fig. 9c-f). Therefore, Ythdf2 appears to primarily promote the decay of maternal but not zygotic mrnas during MZT. Crosstalk between maternal and zygotic modes of maternal RNA clearance The existence of both maternally-driven and zygotically-driven modes of maternal mrna clearance within animal species is a fascinating but cryptic phenomenon. For instance, the mir-430 pathway is known to facilitate maternal transcripts degradation and may overlap with the Ythdf2-based regulation. mir-430 is responsible for the translational inhibition and clearance of a few hundred zebrafish maternal mrnas 4,35 but is itself transcribed zygotically and dependent on the maternally provided pluripotency-inducing factors, Nanog, Pou5f3, and Sox19b for its transcription 36. We investigated potential overlap between the maternal Ythdf2- and the zygotic mir-430-based pathways. We found that: i) The targets of mir and Ythdf2 exhibit a significant overlap (37%, p = , Fisher's Exact Test, Extended Data Fig. 10a), and the common targets are mainly clustered in functions related to cell cycle regulation and intracellular transport (Extended Data Fig. 4

5 10b). ii) The specific targets of mir-430 decayed later than both the specific targets of Ythdf2 and targets in common between Ythdf2 and mir-430 in wild-type zebrafish embryos, and these specific targets of mir-430 were also less affected upon ythdf2 knockout (Extended Fig. 10c-f). The decay of common targets occurs the earliest, suggesting the necessity of timely removal of these critical developmental factors with parallel and/or redundant pathways. Further, the expression of these common targets doesn t rebound as much at 8 h.p.f. compared to 6 h.p.f. as the other two groups (Extended Fig. 10c-f), which may indicate they are not pleiotropic and do not need to be transcribed appreciably at future times. iii) Maternal ythdf2 -/- embryos showed similar delays in zygotic genome activation to Nanog+Sox19b+Pou5f3 LOF embryos at 4 h.p.f. (Extended Data Fig. 10g) and the disruption of the two pathways exhibits similar developmental arrest phenotypes 3. According to our aforementioned definition (the most stringent Ythdf2 targets are the top 20% of upregulated genes differentially expressed in both maternal ythdf2 -/- mutants and MO-injected samples and also present in both m 6 A-seq and m 6 A-CLIP-seq at 4 h.p.f.), nanog, pou5f3, and sox19b transcripts are not stringent Ythdf2 RNA targets (Supplementary Data 1). By checking the correlation of mrna-seq data upon ythdf2 LOF and the published mrna-seq data with combined loss of all three factors 3, we found that zygotic gene expression is significantly decreased upon the removal of the respective regulators in both datasets (Extended Data Fig. 10g). In addition, we observed that nanog and pou5f3 transcripts were under-represented in ythdf2 -/- samples at 4 h.p.f. (Supplementary Data 1). These data suggest that both Ythdf2- and mir430-mediated pathways regulate the clearance of critical maternal transcripts and may function to facilitate activation of the zygotic genome through promoting the maternal factors required for mir-430 expression. Such co-existing mechanisms for the clearance of maternal transcripts have also been indicated in other species such as pathways mediated by 5

6 pumilio 23 and brain tumor 24 in Drosophila and poly(c)-binding proteins in C. elegans

7 Supplementary Figure Supplementary Figure 1: Uncropped gel image related to ED Figure 4b. Supplementary Data Supplementary Data 1: Excel file showing a, mir430 vs Ythdf2 targets with 1.5-fold change threshold. b, Methylation of nanog, pou5f3, and sox19b transcripts detected by m 6 A-seq and m 6 A-CLIP-seq. c, Expression level change of nanog, pou5f3, and sox19b transcripts upon ythdf2 LOF (mutant/wild-type). Supplementary Data 2: Excel file showing a, a list of high-throughput sequencing samples generated in this study. b, a summary of processed sequencing data which directly led to the main conclusion in this study. 7