{'fraction_non_alphanumeric': 0.036610420666195145, 'fraction_numerical': 0.02781987807753191, 'mean_word_length': 5.104440789473684, 'pattern_counts': {'":': 0, '<': 4, '<?xml version=': 0, '>': 10, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 1, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition

Summary Upon fertilization, maternal factors direct development and trigger zygotic genome activation (ZGA) at the maternal-to-zygotic transition (MZT). In zebrafish, ZGA is required for gastrulation and clearance of maternal mRNAs, which is in part regulated by the conserved microRNA miR-430. However, the factors that activate the zygotic program in vertebrates are unknown. Here, we show that Nanog, Pou5f1 and SoxB1 regulate zygotic gene activation in zebrafish. We identified several hundred genes directly activated by maternal factors, constituting the first wave of zygotic transcription. Ribosome profiling revealed that nanog, sox19b and pou5f1 are the most highly translated transcription factors pre-MZT. Combined loss of these factors resulted in developmental arrest prior to gastrulation and a failure to activate >75% of zygotic genes, including miR-430. Our results demonstrate that maternal Nanog, Pou5f1 and SoxB1 are required to initiate the zygotic developmental program and induce clearance of the maternal program by activating miR-430 expression.

methylation 7,8 . Bivalent chromatin marks are associated with zygotic genes thought to be 'poised' for activation 5 . Yet, many loci with active marks appear to be transcriptionally inactive 5 , suggesting that competent genes require induction by additional factors. ZGA is required for epiboly 9 , and the clearance of maternal mRNAs, a process regulated in part by the conserved microRNA (miRNA) miR-430 [10][11][12] . While significant advances have taken place in understanding how vertebrate embryos acquire transcriptional competency and orchestrate the clearance of the maternal program, the factors that control activation of the specific genes during ZGA remain unknown. Here we combine loss-of-function analyses, high-throughput sequencing and ribosome footprinting to identify factors that activate the first wave of zygotic transcription to initiate nuclear control of embryonic development.

Identifying the first zygotic transcripts
To define factors that mediate transcriptional activation, we first sought to identify the earliest genes transcribed from the zygotic genome. Accurate characterization of the early transcriptome faces two main challenges: (i) zygotic transcription of a gene can be masked by a large maternal contribution, and (ii) poly(A)+ selection of mRNAs can lead to apparent increases in gene expression, reflecting delayed polyadenylation of maternal mRNAs rather than transcription. We reasoned that maternal mRNAs are spliced during oogenesis, so examining introns from total RNA would allow us to quantify de novo transcription independent of polyadenylation or maternal contribution. We performed Illumina total RNA sequencing on wild type (WT) embryos after the onset of zygotic transcription (4hpf, sphere; and 6hpf, shield) (Fig. 1a) compared to embryos before MBT (2hpf, 64-cell stage) and α-amanitin treated embryos, which lack zygotic transcription. This analysis identified 608 genes with significant increases in exon or intron expression levels > 5 RPKM (reads per kilobase, per million reads) at sphere stage (P < 0.1, Benjamini-Hochberg multiple test correction) (Fig. 1b,c, Supplementary Fig. 1a-h). Intron signal identifies an additional 6602 genes with low levels of transcription by 4hpf, and 9330 transcribed genes by 6hpf, expanding the number of zygotically expressed genes previously identified 13,14 (Supplementary Fig. 1i-o). Over 74% of these are genes with maternal contributions (maternal and zygotic genes, M+Z), most of which are only identified by elevated intron signal (Fig, 1b, Supplementary Fig. 1g), reflecting the sensitivity of this method to detect de novo transcription. Next, we examined which genes are directly triggered by the maternal program in the "first wave" of transcription by 4hpf, versus those activated by zygotic factors. We reasoned that blocking zygotic gene function while leaving maternal factors unaffected would uncouple the first from subsequent waves of zygotic transcription. To this end, we inhibited splicing of zygotic mRNAs using morpholinos complementary to U1 and U2 spliceosomal RNAs (U1U2 MO) (Fig. 1d, Supplementary Fig. 1a-d) 15 . U1U2 MO embryos arrest prior to epiboly (Fig. 1a), despite remaining transcriptionally active. Illumina sequencing revealed an enrichment in intron-exon boundary reads (Fig. 1e) and activation of a subset of zygotic transcripts to levels > 5 RPKM (Methods); these genes constitute the first wave of zygotic transcription (Fig. 1f). To test that these first-wave genes are indeed independent of zygotic factors, we treated embryos with cycloheximide (CHX) prior to MBT (32-cell stage) to selectively block translation of zygotic mRNAs, while allowing translation of maternal mRNAs. CHX-treated embryos also fail to reach epiboly (Fig. 1a) and have a highly correlated transcriptome profile with U1U2 MO (Pearson's R = 0.97, Supplementary Fig. 2), confirming first-wave transcription in the absence of zygotic proteins. First-wave genes comprise both embryonic-specific and housekeeping genes ubiquitously expressed in adult tissues ( Supplementary Fig. 3a) and are enriched in pattern specification, gastrulation and chromatin modifying functions ( Supplementary Fig. 3b). We validated a subset of these genes by RT-PCR, including klf4b, nanor and isg15 ( Supplementary Fig. 3c-k). Notably, the pri-miR-430 polycistron is highly expressed as part of this first wave (>1000 RPKM) (Fig.  1c, f). Together, these results identify 269 first-wave genes expressed by sphere stage for which maternal factors are sufficient for activation.

Nanog, SoxB1 & Pou5f1 activate the first wave
Considering the specific, widespread and steep pattern of zygotic gene activation, we hypothesized that the factors that trigger the first wave may include sequence-specific transcriptional regulators highly translated prior to ZGA. We analyzed the translation levels of all maternal mRNAs using ribosome profiling data (Fig. 2a) 16 . We found that Nanog, Sox19b and Pou5f1 (Oct4) are the most highly translated sequence-specific transcription factors (TFs) in the pre-MZT transcriptome (Fig. 2b). Pou5f1, the SoxB1 family (which includes Sox2 and Sox19b) and Nanog are key TFs involved in maintaining pluripotency in embryonic stem cells (ESCs) (reviewed in 17,18 ). In zebrafish, Pou5f1 provides temporal control of gene expression 19 and together with SoxB1 regulates dorsal-ventral patterning and neuronal development 18,[20][21][22][23] , while Nanog is essential for endoderm formation through regulation of zygotic mxtx2 24 .
To examine the roles of Nanog, Sox19b and Pou5f1 in activating zygotic gene expression, we combined a maternal-zygotic loss-of-function (LOF) Pou5f1 (MZpou5f1) 21 with previously published translation blocking morpholinos for Nanog 24 and SoxB1 20 (Methods). Because Sox2, Sox3 and Sox19a have been shown to compensate for Sox19b loss, we used a combination of morpholinos targeting all four sox genes 20 ( Supplementary Fig. 4a). Simultaneous Nanog LOF in combination with SoxB1 or Pou5f1 resulted in complete developmental arrest prior to gastrulation, with >95% of the treated embryos failing to initiate epiboly (n=387 and n=52 respectively) (Fig. 2c, Supplementary Fig. 4b-e). This phenotype resembles that of α-amanitin injected embryos, suggesting that these factors play a role in activating zygotic genes. We used two different approaches to analyze the activity and specificity of these morpholinos. First, we performed ribosome profiling on WT and Nanog + SoxB1 MO injected embryos pre-MBT 16,25 . Translation efficiency for both Nanog and Sox19b was reduced >97% in the morpholino-injected embryos compared to WT (Fig.  2d, Supplementary Fig. 4f), but was largely unaffected for the rest of the transcriptome (Fig.  2e). Second, we co-injected mRNAs encoding nanog and soxB1 with the morpholinos and were able to rescue gastrulation (Fig. 2c, Supplementary Fig. 4c-e). Together, these results show that Nanog, Sox19b and Pou5f1 regulate progression through zygotic development and gastrulation.
Illumina sequencing revealed that combined loss of Nanog, SoxB1 and Pou5f1 results in widespread reduction in first-wave gene expression by 4hpf: 77% for strictly zygotic genes, 50% for M+Z genes. (Fig. 3a, b, Supplementary Fig. 5). By 6hpf, expression loss is systemic, with 86% of strictly zygotic and 79% of M+Z genes failing to be expressed to WT levels ( Fig. 3a, b, Supplementary Fig. 5), an effect that was rescued by providing back the cognate mRNAs (Fig. 3c, Supplementary Fig. 5, 6). Comparing the single and double lossof-function transcriptomes to the triple, we found that regulation is often combinatorial and redundant, with Nanog LOF having the strongest effect and SoxB1 the weakest (Fig. 3d, Supplementary Fig 7a-c). By 6hpf, affected genes include housekeeping genes, general transcription factors (e.g., gata6, otx1, irx1b, ntla) and major signaling components in gastrulation, anterior-posterior axis and dorsal-ventral axis specification (e.g., oep, fgf3, wnt11, chd, nog1, ndr2, bmp2b) ( Supplementary Fig. 7d,e). Together, these results show that Nanog, Pou5f1 and SoxB1 play a fundamental role in activating the first wave, an effect that propagates to subsequent waves resulting in a global impact on zygotic gene expression.

miR-430 is strongly activated by Nanog
Notably, among the first-wave genes co-regulated by Nanog, Pou5f1 and SoxB1 was miR-430, a miRNA that functions in the clearance of maternal mRNAs in zebrafish and Xenopus [10][11][12] . Northern analysis revealed a strong reduction of mature miR-430 levels in Nanog loss-of-function embryos (Fig. 4a). Although individual loss of SoxB1 or Pou5f1 had no detectable effect on miR-430 expression, when combined with Nanog LOF they reduced miR-430 levels even further, a phenotype that was rescued by co-injecting the respective mRNAs (Fig 4a-c). Nanog MO embryos failed to repress a GFP-reporter of endogenous miR-430 activity 26 , consistent with Nanog's role in activating miR-430 ( Supplementary Fig.  8a,b).
To determine whether Nanog specifically binds the miR-430 genomic locus, we analyzed Nanog chromatin immunoprecipitation sequencing (ChIP-Seq) data at high (3.3hpf) and dome stage (4.3hpf) 24 . Consistent with widespread Nanog regulation, 74% of first-wave genes are bound by Nanog, a significant enrichment compared to subsequent-wave genes ( Fig. 4d, Supplementary Fig. 9a). miR-430 is expressed from a 17kb genomic region on chromosome 4, which includes 55 repeated miR-430 hairpin sequences. Because this locus is repetitive, it had been excluded from previous analyses; however, the sequences are largely unique relative to the rest of the genome. Reads aligning the miR-430 locus were enriched >16-fold in the Nanog IP compared to whole cell extract (WCE), (Fig. 4e), indicating that strong Nanog binding throughout the locus correlates with strong miR-430 expression at ZGA. When the reads were aligned to the presumptive 5' end of the polycistron, we observed a strong peak of binding in a ~600 nt region between two miR-430 precursors, which contains 3 canonical Nanog binding sites (CATT[T/G][T/G]CA) 24,27 .
To determine whether Nanog induces clearance of maternal mRNAs through activation of miR-430, we analyzed the expression of an endogenous miR-430 target, cd82b 10 . cd82b mRNA is maternally deposited and cleared in WT by 6hpf (Fig. 5a). In contrast, cd82b mRNA is stabilized in MZdicer mutants or α-amanitin treated embryos, which lack miR-430 processing and expression respectively. Similar loss of regulation is observed in Nanog+SoxB1 MO, as well as triple LOF embryos, a defect that is rescued by providing the cognate mRNAs (Fig 5b, Supplementary Fig. 8c). To determine the global effect of this regulation, we examined RNA-Seq levels of maternal mRNAs containing miR-430 target sites. Loss of Nanog alone or in combination with loss of SoxB1 and MZpou5f1, resulted miR-430 target stabilization, identical to MZdicer 10,16,26 (Fig. 5c, Supplementary Fig. 8d-f) (P < 1E-51, two-sided Wilcoxon rank sum test). A significant, but weaker effect was observed in Pou5f1+SoxB1 LOF embryos (P < 1E-25) ( Supplementary Fig. 8d). These results show that Nanog together with Pou5f1 and SoxB1 activate miR-430 expression, thus revealing a genetic network that links maternal regulation of zygotic gene expression to zygotic clearance of maternal mRNAs.

Discussion
Our transcriptome analysis during the maternal-to-zygotic transition provides three major insights. First, maternal factors directly regulate hundreds of mRNAs that constitute the first wave of zygotic transcription. These targets are activated in the absence of zygotic gene function and are enriched for genes that guide early embryonic development. Transcriptional competence coincides with changes in the chromatin and DNA methylation states of the genome 4-8 . Modifications to the epigenetic landscape during the MZT may be sufficient to allow basal levels of transcription; however, we show here that maternal transcription factors play a vital role in shaping transcriptional output.
Second, we observe that Nanog, SoxB1 and Pou5f1, previously implicated in the maintenance of pluripotency, contribute to widespread activation of zygotic genes during the MZT. These maternal factors enhance transcriptional activation of more than 74% of firstwave zygotic genes, and by 6hpf influence expression of >80% genes over all. Simultaneous removal of Nanog with SoxB1 and/or Pou5f1 results in complete block of gastrulation and developmental arrest, similar to global inhibition of zygotic gene expression (Fig. 2c,  Supplementary Fig. 9c). Nanog binds 74% of first-wave genes during the early stages of ZGA (Fig. 4d). Additionally, while this manuscript was under review, Pou5f1 and Sox2 were also shown to associate with ~40% of early zygotic genes 28 . However, SoxB1+Pou5f1 LOF is insufficient to block gastrulation and zygotic development 28 (Fig. 2c). This highlights the central role of Nanog, which together with Pou5f1/SoxB1 initiates the zygotic program of development, though it is likely that additional factors cooperate with them to provide genome competency and regulate the timing of ZGA 4 . In mouse, Oct4 and Nanog have been proposed to regulate gene expression at 2-cell stage 29,30 and along with Sox2 are required for specification of the blastocyst lineages [31][32][33] . In fact, when we analyze early zygotic genes in mouse, we find that they are enriched for Nanog, Oct4 and Sox2 binding in embryonic stem cells ( Supplementary Fig. 9b). Conceptually and mechanistically, many parallels exist between the MZT and the cellular reprogramming that occurs in induced pluripotent stem cells (iPSCs) 3,12 . Indeed, reprogramming of terminally differentiated cells was first shown in the context of the early embryo through nuclear transfer 34,35 . The onset of zygotic development can be viewed as a major reprogramming event that occurs upon fusion of two terminally differentiated cells (sperm and oocyte). As shown in ES cells and iPSCs, Pou5f1, Nanog and Sox2 are central players in the induction [36][37][38][39][40] and maintenance [41][42][43] of pluripotency in vivo and in vitro 17,35 . In these contexts, part of their role is to serve as "pioneering" factors, binding to silent chromatin to facilitate de novo gene expression 44 . This pioneering activity is likely recapitulated during the MZT, where an Third, we show that Nanog together with SoxB1 and Pou5f1 directly regulates miR-430, which is responsible for clearance of maternal mRNAs during the MZT 10-12 , facilitating the transfer of developmental control to the zygotic program ( Supplementary Fig. 9c). Members of the conserved miR-430/295/302/372 family of miRNAs stabilize self-renewal fate in ES cells and enhance reprogramming efficiency 45,46 . We hypothesize that in both cases, these miRNAs are 'clearing the slate' by accelerating the removal of mRNAs from the previous program, thus facilitating the establishment of new transitional states by reprogramming factors 12 . The dramatic upregulation of miR-430 expression by Nanog, SoxB1 and Pou5f1 provides a central link between the mechanisms that drive zygotic gene activation and the clearance of the previous maternal history.

Zebrafish maintenance
MZpou5f1 hi349Tg/ hi349Tg 48 were generated as previously described by 21 . Embryos obtained from natural crosses between homozygous MZpou5f1 hi349Tg/ hi349Tg mutants were injected with 30pg of pou5f1 mRNA at the one-cell stage. MZdicer hu896/hu896 fish were generated as described in 26 . Zebrafish wild type embryos were obtained from natural crosses of TU-AB and TLF strains of mixed ages (5-17 months). Selection of mating pairs was random from a pool of 60 males and 60 females allocated for a given day of the month. Fish lines were maintained in accordance with AAALAC research guidelines, under a protocol approved by Yale University IACUC.

Treatments and mRNA injection
Embryos from all wild type crosses were pooled following collection and distributed equally between experimental conditions. Unless otherwise stated, a minimum of 30 wild type embryos were subjected to each treatment in each experimental replicate. Morpholinos were obtained from Gene Tools and resuspended in nuclease-free water. Unless otherwise stated, one nanoliter of MO solution was injected into dechorionated embryos at the one-cell stage. A combination of two MOs were used to target each gene in a 1:1 ratio as described in 49 , with one SoxB1 morpholino targeting a conserved region of both sox2 and sox3. Nanog and SoxB1 MOs were previously described in 24 49 . To rescue the loss-of-function phenotype, 50pg of Nanog and 20pg of SoxB1 mRNAs were injected either individually or together into morpholino injected embryos at one-cell stage. Triple loss-of-function embryos were additionally injected with 30pg of Pou5f1 mRNA.
Pol II inhibition: α-amanitin was obtained from Sigma Aldrich and resuspended in nucleasefree water. Dechorionated embryos were injected with 0.2ng of α-amanitin at one-cell stage 52 .
Translation inhibition: wild type embryos were collected and dechorionated at one-cell stage. To allow for translation of maternal mRNAs, at 32-cell stage, embryos were transferred to media containing Cycloheximide (50μg/mL) (Sigma Aldrich) and incubated at 28°C. Embryos were collected and frozen in liquid nitrogen at sphere and shield stage. Total RNA was extracted using Trizol (Invitrogen) and resuspended in 10μL RNase-free water.
To assay miR-430 activity, a GFP reporter was used as previously described 26 . GFP and dsRed mRNAs were in vitro transcribed using mMessage mMachine Sp6 Kit (Ambion) in accordance to the manufacturer's instructions. Embryos were injected with 150pg of GFP reporter and 100pg of dsRed loading control at the one-celled embryos.
All phenotypes were initially assayed by one experimenter and blindly confirmed and/or imaged by another. Distribution-free statistics were used to determine significance, except for calculating RNA-Seq differential expression (see below).

In situ hybridization
Template for in situ probes were amplified from shield stage cDNA and a T7-promoter sequence added for in vitro transcription. Primers are listed below. Antisense digoxigenin (DIG) RNA probes were generated by in vitro transcription in 20μL reactions consisting of 100ng purified PCR product (8μL), 2μL DIG RNA labeling mix (Roche), 2μl 10X transcription buffer (Roche), and 2μl T7 RNA polymerase (Roche) in RNAse-free water and purified using a QIAGEN RNEasy kit. In situ protocol was followed as detailed in 26 . To reduce variability, the following conditions were combined in the same tube during in situ hybridization and recognized based on their morphology: 1) wild type and α-amanitin injected embryos and 2) Nanog + SoxB1 MO with and without rescue mRNA. Prior to photo documentation, embryos were cleared using a 2:1 benzyl benzoate:benzyl alcohol solution. Images were obtained using a Zeiss stereo Discovery.V12.

Northern analysis
To detect endogenous miR-430, ten wild type and MZpou5f1 embryos injected with Nanog MO and SoxB1 MO were collected at 6hpf and flash frozen in liquid nitrogen. Total RNA was extracted using Trizol (Invitrogen) and resuspended in 5μL RNase-free water and 5 μL 2X loading buffer (8M urea, 50mM EDTA, 0.2mg/ml xylene cyanol, and 0.2mg/ml bromophenol blue). Northern protocol was followed as detailed in 16 .

Ribosome Profiling
Fifty wild type embryos injected with one nanoliter of Nanog MO (0.6mM) and SoxB1 MO (0.125mM) and fifty non-injected embryos were collected at 64-cell stage. Embryos were lysed using 800ul of a mammalian cell lysis buffer containing 100ug/ml Cycloheximide as per the manufacturers instruction (ARTseq Ribosome Profiling Kit, RPHMR12126, Epicentre). For nuclease treatment, 3ul of ARTseq Nuclease was used. Ribosome protected fragments were run and 28-29nt fragments were gel purified as previously described in 16 and cloned according to the manufacturers protocol (ARTseq Kit). Illumina libraries were constructed and sequence reads analyzed as in 16 . Subsequent to sequencing, traces of exogenous RNA corresponding to a nanog antisense probe, and ntla sense and antisense were detected outside the expected size range. Only 28 and 29nt sense sequences were used in the analysis matching the size of the ribosome footprint.

Reverse Transcription PCR (RT-PCR)
Total RNA from ten embryos was extracted using TRIzol (Invitrogen) at sphere and shield stage for each experimental condition. RNA was treated with TURBO DNase (Ambion) for 30 minutes at 37°C and extracted using phenol chloroform. cDNA was generated by reverse transcription with random hexamers using SuperscriptII (Invitrogen). RT-PCR reactions were carried out at an annealing temperature of 60°C for 35 cycles. Primers are listed below.

Illumina Sequencing
Total RNA was extracted as above, and strand-specific TruSeq Illumina RNA sequencing libraries were constructed by the Yale Center for Genome Analysis. Prior to sequencing, samples were treated with Epicentre Ribo-Zero Gold kits according to the published protocol, in order to deplete ribosomal RNA. Samples were multiplexed on Illumina HiSeq 2000 / 2500 machines to produce single-end 76 nt reads.
Hybrid gene models were constructed from the union of zebrafish Ensembl r70, RefSeq annotations (downloaded from genome.ucsc.edu on 2/8/2013), and Ensembl RNA-Seq gene models 55 . All overlapping transcript isoforms were merged in order to produce maximal exonic annotations. To quantify exonic expression levels per gene, genome-uniquely aligning reads overlapping >= 10 nts to the exonic region of a given gene were summed. To quantify intronic expression levels per gene, an annotation mask was first created consisting of repetitive sequences as annotated by RepeatMasker in addition to any region aligned by >=2 reads in the α-amanitin samples; this is to minimize false positive introns due to annotation inconsistencies, under the assumption that the transcriptionally inhibited αamanitin transcriptome should contain no intron-containing transcripts. Valid intronoverlapping reads aligned the intronic region uniquely and overlapped no more than 50% to the masked regions. For the purposes of RPKM normalization, we considered intron length to be the number of unmasked nucleotides. We additionally identified reads that mapped to at most two different genic loci (e.g., two closely related paralogs) and from these calculated "meta gene" expression values. Meta genes were treated as conventional genes for differential expression, but counted as two different genes in subsequent analyses.
The miR-430 locus is internally repetitive; therefore, reads were aligned to miR-430 in a separate step using Bowtie with switches -n 2 -k 1 on the genomic region chr4:27999472-28021845, which spans the presumed mir-430 polycistron. Reads overlapping any of the Ensembl annotated miR-430 hairpins in this region were counted as mir-430 cluster reads. Reads are counted only once, regardless of the number of times they overlap.

Differential gene expression analysis
Differential expression analysis was performed using the R package DESeq 47 with the parameters fit-type = local and sharingMode = fit-only. For exonic expression comparisons, raw exon-overlapping read counts were assembled for all genes with a raw read count of at least 10 in one or more of the samples. Genes annotated as Ensembl biotypes 'IG_C_pseudogene', 'IG_pseudogene', 'IG_V_pseudogene', 'misc_RNA', 'Mt_rRNA', 'Mt_tRNA', 'non_coding', 'nonsense_mediated_decay', 'retained_intron', 'rRNA', 'sense_intronic', 'sense_overlapping', 'snoRNA', 'snRNA' were excluded. Additionally, all Ensembl miR-430 annotations were excluded, and a meta "miR-430 hairpin" gene added in, based on the quantification described in the previous section. For intronic expression comparisons, since overall counts are lower, variance models for DESeq were calculated using both intronic counts and exonic counts as separate gene entries (i.e., at most 1 intronic count entry and 1 exonic count entry per gene). Differential expression proceeded as normal, except multiple test correction of p values was applied relative only to the intronic counts.
Zygotic transcription was determined based on significant exon and intron increases in sphere and shield stages relative to α-amanitin. 64c (pre-MBT) was used as further confirmation when no significant changes in intron level were detected or the gene was intronless (genes with <10 nts of unmasked intron sequence were considered effectively intronless). Increases in either exon signal, intron signal, or both determined positive zygotic transcription. For genes with a maternal contribution, increases in intronic signal due to zygotic transcription can be accompanied by no change or decreases in exonic signal. For genes significantly expressed, zygotic expression contribution is estimated using either intronic RPKM level; or the RPKM difference between the post-MZT condition and the maximum of 64c and α-amanitin expression levels. Expression calls are provided in Supplementary Data Table 1.
To define first-wave genes, genes that were detected as transcribed in the U1U2 MO treated embryos above an expression level of 5 RPKM were considered to be first wave, using an estimate for zygotic transcription based on intronic signal for multi-exon genes, or comparison to α-amanitin and 64c for single-exon genes as described above. Although a cut off of 5 RPKM was used for the main analyses, lower levels of transcription were observed for many genes, indicating weaker degrees of activation. Genes that were not called as transcribed in wild type sphere were removed.

Classification of loss of function expression categories
Significant changes in loss of function conditions relative to wild type were determined using either intron or exon signal, depending on the pattern of signal originally used to call the gene as zygotically expressed. For genes with no maternal contribution, decreases in either exon levels relative to wild type are considered to be loss of zygotic expression, while increases in either exon or intron levels are considered to be ectopic increases in zygotic expression. For genes with maternal contribution, we distinguish between two cases: (1) if zygotic transcription was originally detected in wild type only using intronic signal, then loss of zygotic transcription in the loss of function conditions is called only when intronic signal is lost; (2) if zygotic transcription was originally detected in wild type with both exonic and/or intronic signal, then decreases in either intronic levels or exonic levels indicate loss of zygotic expression, with intronic signal taking precedence when the directions of change disagree. For LOF embryos with the MZpou5f1 genotype, differential expression was additionally performed between uninjected and injected MZpou5f1 conditions, and expression differences between the injected conditions and wild type were required to be transitively consistent --e.g., if a gene is called significantly lower in uninjected MZpou5f1 than wild type, and a gene is significantly lower in injected MZpou5f1 than uninjected MZpou5f1, then the gene must also be considered lower in the injected compared to wild type. To ensure that expression level differences in the MZpou5f1 background are due to zygotic contributions, in addition to relying on intron signal, we filtered out any genes that were previously reported to be differentially maternally provided in MZpou5f1 19 .

ChIP-Seq analysis
Reanalysis of previously published Nanog ChIP-Seq data (GSE34683) was performed as described in 24 , except using the current version of the zebrafish genome, Zv9. For miR-430 locus alignment, reads were aligned exhaustively to the region chr4:27994413-28019085 (2kb +/-the miR-430 polycistron) using Bowtie with parameters -v 1 --best --strata --all. To estimate read depth and enrichment, reads were normalized by the number of times the read aligned the genome. To focus on the maximally non-redundant region in the locus, reads were preferentially aligned closest to the presumptive 5' boundary of the polycistron (chr4:28000732, corresponding to the 5' end of ENSDARG000000082539).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.