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Origin of new genes after zygotic genome activation in vertebrate

Origin of new genes after zygotic genome activation in vertebrate doi:10.1093/jmcb/mjx057 Journal of Molecular Cell Biology (2018), 10(2), 139–146 j 139 Published online February 14, 2018 Article Origin of new genes after zygotic genome activation in vertebrate 1,2,† 1,3,† 4 1,3 1,3,4 1,3, *, Hai-Bo Xu , Yong-Xin Li , Yan Li , Newton O. Otecko , Ya-Ping Zhang , Bingyu Mao 1,3, and Dong-Dong Wu * State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China College of Life Science, Anhui University, Hefei, China Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, China State Key Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China These authors contributed equally to the work. * Correspondence to: Dong-Dong Wu, E-mail: wudongdong@mail.kiz.ac.cn; Bingyu Mao, E-mail: mao@mail.kiz.ac.cn Edited by Luonan Chen New genes are drivers of evolutionary innovation and phenotypic evolution. Expression of new genes in early development raises the possibility that new genes could originate and be recruited for functions in embryonic development, but this remains undocu- mented. Here, based on temporal gene expression at different developmental stages in Xenopus tropicalis, we found that young protein-coding genes were significantly enriched for expression in developmental stages occurring after the midblastula trans- ition (MBT), and displayed a decreasing trend in abundance in the subsequent stages after MBT. To complement the finding, we demonstrate essential functional attributes of a young orphan gene, named as Fog2, in morphological development. Our data indicate that new genes could originate after MBT and be recruited for functions in embryonic development, and thus provide insights for better understanding of the origin, evolution, and function of new genes. Keywords: young gene evolution, zygotic genome activation, new gene origin Introduction New genes, as fundamental materials for evolutionary innov- in Drosophila lead to either lethality at diverse development stages, ation, have been investigated for many years (Long et al., 2003, or tissue-specific morphological defects (Chen et al., 2010). In human, 2013). Many new genes rapidly acquire important and even new genes display a substantially high expression levels in fetal brain essential functions driven by positive selection (Chen et al., compared with adult brain (Zhang et al., 2011; Wu et al., 2015). The 2010, 2013), with most pronounced roles being in reproduction, investigations raise the possibility that new genes could originate dur- development, and brain (Kaessmann, 2010; Tautz and Domazet- ing the process of embryonic development. Particularly, during early Loso, 2011; Chen et al., 2013). For instance, a testis-bias development after the midblastula transition (MBT), zygotic genome expression of new genes is recapitulated in different animals activation is initiated, where remarkable epigenetic modifications (Kaessmann, 2010). As seen in human, many new genes exhibit a occur to induce the transcription of the zygotic genome to gradually brain, particularly a neocortex-biased expression, suggesting that take control of development. These dynamic changes in epigenetic they may be recruited for the accelerated evolution of the human modifications enhance transcriptional activity and promiscuous tran- brain and may be involved in the acquisition of high cognitive scription (Ostrup et al., 2013). Theseeventsare mirrored in testis, ability (Li et al., 2010; Wu et al., 2011; Zhang et al., 2011). where widespread demethylation of CpG dinucleotide-enriched pro- New genes also play integral roles in development (Chen et al., moter sequences occurs, resulting in a transcriptionally active chro- 2010, 2012). For instance, in a pioneering study by Chen et al. matin state that facilitates the access of transcriptional machinery (2010), knockdown of many new genes by RNA interference (RNAi) and promiscuous transcription of genes (Kleene, 2001; Kaessmann, 2010). Studies have observed that new genes are frequently recruited for new function in the testis due to the promiscuous tran- Received July 16, 2017. Revised November 20, 2017. Accepted December 19, scription (Kaessmann, 2010; Tautz and Domazet-Loso, 2011; Chen et al., 2013). Therefore, we hypothesized that new genes might be © The Author (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. recruited during early development, particularly after MBT. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 140 j Xu et al. To test the hypothesis that new gene could frequently be recruited modules of coexpressed genes with shared functionality. A total for key roles in early animal development, we examined the temporal of 30 distinct modules, representing clusters of genes with cor- expression of new genes during the early development of a frog, related expression, were identified (Figure 2A). Many of the and found that expression of new genes was significantly enriched modules exhibit expression patterns significantly correlated in developmental stages after embryonic genome activation. with specific developmental stages (Figure 2A, Supplementary Furthermore, we examined the roles of two young orphan genes, Figure S3). For example, the M2 module is a stage 9-dominant named Fog1 and Fog2, and demonstrated that they have important module, with the significance of its association having a P-value −8 functions in frog development. After MBT, zygotic genome activation of 1.0 × 10 (Figure 2B). (ZGA) is initiated, where remarkable epigenetic modifications occur We then examined the enrichment of new genes within these that induce the transcription of the zygotic genome (Ostrup et al., modules. Young duplicated genes of X. tropicalis were significantly 2013), with a similar pattern observed in the testis (Kleene, 2001; over-represented in 13 of the 30 modules (Figure 2C, P < 0.01), Kaessmann, 2010). We propose that the induced transcription of the with 12 modules harboring significant correlation with a develop- zygotic genome might facilitate the origination of new genes. mental stage after MBT (Figure 2A, P < 0.01 after Bonferroni cor- rection by χ test). The 12 modules cover the gastrulation Results processes (stages 9, 10,and 11.12,with 119 young duplicated Temporal expression profiling reveals high expression of young genes involved), neurulation (stages 16.18, 19,and 20.21,with 76 protein-coding genes after MBT young duplicated genes involved), and part of the organogenesis In the present study, we categorized young protein-coding period (stages 24.26, 38.39,and 44.45,with 66 young duplicated genes in the frog (Xenopus tropicalis), i.e. duplicate genes and genes involved). This enrichment indicates that these new genes orphan genes, according to their mechanisms of origin have evolved important functions in critical gene–gene inter- (Supplementary Tables S1 and S2). Duplicate genes are gener- action networks. Interestingly, the level of enrichment for new ated by gene duplication from existing old genes, while orphan genes showed a decreasing trend with developmental stage genes do not have homology with genes in other species, and after MBT (P = 0.028, Figure 2D; P = 0.048, Supplementary are likely originate de novo or from rapidly evolved genes, Figure S4), corroborating the expression trajectory of new genes thereby losing similarity with their ancestral sequences (Tautz (Figure 1). The decreasing pattern after MBT could not be attrib- and Domazet-Loso, 2011). Genes that were specifically dupli- uted to changes in global gene expression levels or changes in cated in X. tropicalis were retrieved by BioMart (Smedley et al., the proportions of expressed genes among developmental 2009) from Ensembl (http://www.ensembl.org/, version 72). stages, as correlation was not observed between enrichment The ages of X. tropicalis genes were obtained from levels of new genes and transcriptional levels of developmental ProteinHistorian (Capra et al., 2012). Genes with an age of zero stages (Supplementary Figure S5). were taken as new genes that newly originated during the evo- Young orphan genes were significantly enriched within two mod- lution of frogs. Recently duplicated genes were defined as ules (P < 0.01 after Bonferroni correction by χ test), with modules X. tropicalis-specific duplicated genes with an age of 0. Orphan M5 (29 genes) corresponding to developmental stage 10,and M24 genes, which are X. tropicalis specific, were identified from hom- (15 genes) (Supplementary Figure S6). It is notable that another 47 ologous searches of protein databases of other animal species young duplicated genes are over-represented within module M5. using BLASTP with an E-value of 1e−10 (Altschul et al., 1997). Gene enrichment analysis performed by the DAVID program (Huang Profiling of temporal gene expression from the 2-cell stage to et al., 2008) found that genes in M5 were significantly enriched in stage 44–45 (Tan et al., 2013) revealed that the expression of categories associated with development such as ‘pattern specifica- young protein-coding genes peak after MBT, particularly from gas- tion process’ (GO:0007389, P = 0.01, 10 genes: T, HHEX, NOG, trulation to neurulation (Figure 1, Supplementary Figure S1A). GSC, LHX1, DYNC2LI1, GATA4, ZIC1, TCF7L1,and ZIC3)and ‘region- Expression levels of the new genes displayed a decreasing trend alization’ (GO:0003002, P = 0.04) (Supplementary Table S3). with developmental stages after MBT. When gene expression These results indicate that the young genes might have evolved levels were normalized to the whole genome level, this pattern developmental roles through interactions with other genes. still held for new genes (Figure 1, Supplementary Figure S1B). Although expression quantification may not be accurate for young Expression patterns of two young orphan genes reveal potential duplicate genes using RNA-sequencing data, we reasoned that it functions in development would not change our conclusion as orphan genes also exhibited High-expression levels of young protein-coding genes after the signature peak expression after MBT (Figure 1). MBT, particularly from gastrulation to neurulation, raise the possi- bility that some of these new genes might have acquired import- Weighted gene co-expression network analysis ant functions in these developmental stages. To complement this Genes function by interacting with other genes, generating finding, we chose some young protein-coding genes for further gene–gene interaction networks. To gain insight into involve- functional assessments. Two genes (ENSXETG00000027093,i.e. ment of new genes in networks, we performed weighted gene LOC100170590, which we named Fog1, frog orphan gene 1,and co-expression network analysis (WGCNA), an unsupervised and ENSXETG00000030468,i.e.LOC100158459,which we named unbiased analysis (Langfelder and Horvath, 2008) to identify Fog2,frogorphangene 2) stand out, as they exhibit high expression Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Origin of new genes after zygotic genome activation in vertebrate j 141 2012). The above expression correlation analyses suggest that Fog1 and Fog2 genes might be coupled to embryonic development. We further investigated the potential functions of the two genes in another model frog, Xenopus laevis. Real-time PCR confirmed their increased expression levels at the gastrula and the neurula stages, but lower levels in the tailbud stage of development (Supplementary Figure S7). After that, we used in situ hybridiza- tion to examine the temporal expression patterns during embry- onic development, and found high expression of Fog1 and Fog2 in somites (muscle) (Figure 3). This is consistent with the above expression correlation analysis, where many genes involved in somite development displayed correlated expression with Fog1 Figure 1 Expression level of young protein-coding genes at different and Fog2. In addition, Fog1 is also expressed in the branchial developmental stages in X. tropicalis. log (FPKM+1) was used to 2 arches, optic vesicle, and tail end, while Fog2 shows expression in calculate the expression value of each gene. Two groups of orphan the nervous system and optic vesicle (Figure 3). genes were identified by BLASTP with cutoff E = 1e−10 and 1e−4, respectively. Functional study of Fog1 and Fog2 The above expression analysis suggested that Fog1 and Fog2 levels after MBT, particularly from gastrulation to neurulation might have functions in development. To further interrogate the (Supplementary Figure S7). To interrogate the epigenetic aspects functions of Fog1 and Fog2 in development, we knocked down associated with expression, we examined the levels of H3K4me3 mRNA expression of these two genes by injecting gene-specific modification, which is associated with active transcription of MO (morpholino) into X. laevis embryos. By visual inspection, nearby genes (Guenther et al., 2007), across thetwo genesat dif- no noticeable phenotypic change was observed in development ferent developmental stages. As expected, the changes of after injecting Fog1 MO (Supplementary Figure S9). While this H3K4me3 modification levels displayed a pattern paralleling the does not annul the potential functional importance of this gene, changes in the expression of these two genes (Supplementary it highlights the need for more systematic phenotyping assays Figure S8). Among the gene–gene expression networks, the two to capture the phenotypic changes after knocking down Fog1. genes are located within module M7, which exhibited high In stark contrast, a serious change in body axis curvature was expression level at the stages 10−14 after MBT (Figure 2A). Gene observed in embryos after knocking down Fog2 compared to enrichment analysis showed that genes in M7 are significantly wild-type embryos (Figure 4A−D, Supplementary Figure S11). enriched in many categories associated with development such as: This malformation could be partially rescued when Fog2 mRNA ‘somite development’, ‘pattern specification process’, ‘regionaliza- was co-injected (Figure 4E and F, Supplementary Figure S11). In tion’, ‘segmentation’ and ‘anterior/posterior pattern specification’ consideration of expression of Fog2 in somites (muscle) and (Supplementary Table S4). These findings suggest that Fog1/Fog2 mesoderm (Figure 3, Supplementary Figure S12), and expres- might be functionally linked with development. sion correlation with other genes involved in mesoderm devel- To explore potential functions of these two genes, we firstly per- opment, we further examined the expression of MyoD,a crucial formed an expression correlation analysis to identify genes whose gene in somites formation as an early response to mesoderm expression correlate significantly with these two genes. A total of induction in Xenopus embryos. Fog2 MO injection caused downre- 190 genes showed correlation with Fog1 (R > 0.9, by Pearson cor- gulation of MyoD (Figure 5). This phenotype could be partially res- relation), and gene enrichment analysis revealed that these genes cued by co-injection with Fog2 mRNA (Figure 5). Since the rescue were enriched in development categories. For example, 31 genes experiment was performed by co-injecting MO and Fog2 mRNA, it are enriched in the category ‘anatomical structure development’ is important to note that MO can block not only the endogenous (GO:0048856, P = 0.029), five genes in ‘mesenchyme development’ mRNA but also the injected mRNA. Hence, the rescued phenotype (GO:0060485, P = 0.02), four genes in ‘neural crest cell differenti- might be due an overall increase in Fog2 mRNA. Altogether, these ation’ (GO:0014033, P = 0.016), and four genes in ‘somite devel- results suggest that Fog2 is tightly coupled to the morphological opment’ (GO:0061053, P = 0.0379) (Supplementary Table S5). development of X. laevis. BLAST analysis did not find the homolo- On the other hand, 12 genes are correlated with Fog2 (R > gous gene of Fog2 in thegenomeofTibetan frog (Nanorana par- 0.9, by Pearson correlation), with 7 of the genes involved in keri)(Sun et al., 2015), supporting Fog2 as a newly evolved gene. ‘anatomical structure development’ (GO:0048856, P = 0.0002) The experiment reiterates that some new genes could be recruited (Supplementary Table S6). Among these genes, Msgn1 (meso- in embryonic development. genin 1) is a master regulator of paraxial presomitic mesoderm differentiation (Chalamalasetty et al., 2014), and Szl, encodes Discussion the secreted frizzled related protein Sizzled that negatively reg- In the present study, we show that young protein-coding genes ulates Tolloid-like activity to control deposition of a fibronectin are significantly enriched in embryonic developmental stages after (FN) matrix between the mesoderm and endoderm (Kenny et al., MBT. A decreasing trend in the level of enrichment and expression Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 142 j Xu et al. AB 1 0.5 0 –0.5 –1 Z score –4 –2 0 2 4 M15 M24 M23 M28 M25 M26 M27 M17 M16 M18 M29 M22 M21 M19 M30 M20 M4 M1 M7 M5 M2 M3 M6 M8 M14 M13 M10 M11 M9 M12 CD 12 y = –0.28x + 6.462 R = 0.282, P = 0.028 * 6 * * * * 2 * Figure 2 WGCNA of young protein-coding genes. (A) Heatmaps displaying the correlations (and corresponding P-values) between modules and developmental stages or adult tissues. Color legend at the top indicates the level of correlation. eld0−4 represent five adult tissues: brain, liver, kidney, heart, and skeletal muscle, respectively. (B) Heatmap of gene expression in module M2.(C) Enrichment level of young duplicated genes for the different modules. Values are the proportion of young duplicated genes in each module divided by the proportion of other genes in the module. Asterisks (*) represent significant enrichments (P < 0.01 after Bonferroni correction by χ test). (D) Level of enrichment of young duplicated genes at each developmental stage shows a decreasing trend after MBT. of new genes was seen in the developmental stages after MBT, a site (Ka), to the number of synonymous substitutions per syn- pattern that had not been previously reported. For example, onymous site (Ks)) of Fog1 and Fog2 is 0.283 and 0.744,respect- knockdown of new genes in Drosophila (Chen et al., 2010) gener- ively (Supplementary Figure S13). It seems that Fog1 evolves ally lead to pupal rather than embryonic lethality. A repetitive ana- under purifying selection, suggesting that it still may harbor an lysis of time-course developmental data in zebrafish showed no important function, while Fog2 evolves more rapidly which occurs peak expression in the early developmental stages (Domazet-Loso commonly for recently evolved new genes. and Tautz, 2010; Zhong et al., 2016). Numerous possibilities such After MBT, zygotic genome activation is initiated, where as different bioinformatics practices, different data profiling strat- remarkable epigenetic modifications occur to induce the tran- egies, as well as specie-specific variations might explain the con- scription of the zygotic genome to gradually take over the con- flicts in these observations. In particular, we studied functions of trol of development (Ostrup et al., 2013). An example of the two genes Fog1 and Fog2 exhibiting high expression levels after epigenetic changes that occur with this transition is the genomic MBT, and found that Fog2 might have an important role in devel- enrichment of H3K4me3, an indicator of active promoters, which opment. When comparing orthologous gene coding sequences is observed at the time of ZGA in zebrafish (Vastenhouw et al., between X. laevis and X. tropicalis,wefound that Ka/Ks(theratio 2010) and Xenopus (Akkers et al., 2009). Another example is of the number of nonsynonymous substitutions per nonsynonymous DNA methylation, which is considered to be a negative regulator Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Enrichemtn level 2cell 4cell M1 8cell M2 16cell M3 M4 stage6 M5 stage8 M6 stage9 M7 stage10 M8 stage11–12 M9 M10 stage13–14 M11 stage15 M12 stage16–18 M13 stage19 M14 M15 stage20–21 M16 stage22–23 M17 stage24–26 M18 stage28 M19 M20 stage31–32 M21 stage33–34 M22 stage38–39 M23 stage40 M24 M25 stage41–42 M26 stage44–45 M27 eld0 M28 eld1 M29 eld2 M30 eld3 eld4 Enrichment Level 2cell 4cell stage9 8cell stage10 16ell stage11.12 stage6 stage13.14 stage8 stage9 stage15 stage10 stage16.18 stage11–12 stage19 stage13–14 stage15 stage20.21 stage16–18 stage22.23 stage19 stage24.26 stage20–21 stage28 stage22–23 stage24–26 stage31.32 stage28 stage33.34 stage31–32 stage38.39 stage33–34 stage38–39 stage40 stage40 stage41.42 stage41–42 stage44.45 stage44–45 Origin of new genes after zygotic genome activation in vertebrate j 143 Figure 3 Expression patterns of Fog1 and Fog2 in X. laevis embryos. (A−H) Expression patterns of Fog1 mRNA. (A and B) Fog1 is weakly expressed in the animal pole at the blastula stage. (C−E) Fog1 is weekly expressed in the neural system at the neurula stage. (F−H) Fog1 is mainly expressed in the muscle, brain, optic vesicle, branchial arches, and tail end. (A’−H’) Expression patterns of Fog2 mRNA. (A’−C’) Fog2 is weakly expressed in the animal pole at the blastula stage. (D’ and E’) Fog2 is expressed in the neural system at the neurula stage. (F’−H’) Fog2 is mainly expressed in the brain, optic vesicle, and muscle. n, neural system; ov, optic vesicle; mu, muscle; ba, branchial arches; te, tail end. Figure 4 Knockdown of Fog2 causes defects or malformation in embryonic development of Xenopus at late stages of development. Embryos were injected on the left side. (A) Wild-type (WT) embryos. (C) Embryos injected with Fog2-MO. (E) Embryos injected with both Fog2-MO and mRNA. (B, D, F) The same embryo for A, C, E, respectively. Numbers at the bottom corners represent the number of embryos with the corre- sponding effect and the number of embryos used in this experiment. The side of the injection is indicated by lacZ (red dots). Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 144 j Xu et al. Figure 5 Knockdown of Fog2 causes downregulation of MyoD. (A) Wild-type embryos. (C) Embryos injected with Fog2-MO. (E) Embryos injected with both Fog2-MO and mRNA. (B, D, F) The opposite side for A, C, E, respectively. Numbers at the bottom corners represent the number of embryos with the corresponding effect and the number of embryos used in this experiment. The side of the injection is indicated by lacZ (red dots). of gene expression. Methylated DNA is efficiently transcriptionally Analysis of transcriptome data repressed in Xenopus oocytes, but DNA methylation-dependent Transcriptome data from 23 developmental stages from a pre- transcriptional repression is greatly reduced after ZGA, and the vious study (Tan et al., 2013) were downloaded from the NCBI repressive effect of DNA methylation is only restored during SRA (http://www.ncbi.nlm.nih.gov/sra/). Detailed information on organogenesis and terminal differentiation (Bogdanovic et al., each developmental stage is available elsewhere (Tan et al., 2011; Ostrup et al., 2013). Thesedynamic changesinepigenetic 2013). All reads were processed using Btrim to remove low- modifications enhance transcriptional activity after ZGA. The pro- quality reads with parameters –l 30 –q 20 (Kong, 2011), with the miscuous transcription then enables proto-genes to be selected paired and unpaired short reads generated then aligned to the due to beneficial functions that might accidentally be gained, and X. tropicalis genome (JGI_4.2) (Ensembl release 72) using the thus allow them to evolve into bona fide genes (Kaessmann, 2010). read gapped alignment program Tophat 2.0.9 (Trapnell et al., This epigenetic aspect is synonymous with that observed in testis, 2009; Kim et al., 2011). After merging paired and unpaired align- where widespread demethylation of CpG dinucleotide-enriched pro- ments from the same sample, the program Cufflinks 2.0.2 was moter sequences occurs, resulting in a transcriptionally active chro- used to assemble the transcriptomes (Trapnell et al., 2010) matin state that facilitates the access of transcriptional machinery (Supplementary Figure S14). All isoforms assembled by Cufflinks and promiscuous transcription of genes (Kleene, 2001; Kaessmann, from the 45 samples were sent to the Cuffcompare utility, along 2010). Previous studies have evidenced the recruitment of new with the Ensembl annotation file, to generate an integrated com- genes for new functions in the testis (Kaessmann, 2010; Tautz and bined gtf annotation file. With the purpose of minimizing annota- Domazet-Loso, 2011; Chen et al., 2013). In conclusion, our study tion artifacts, we processed the Cuffcompare output file through identifies a genomic hotbed for the origination of new genes, and the following steps. First, we excluded all new single exon tran- illustrates likely developmental functions of new genes besides the scripts and sequences with lengths < 200 bp. Transcript with traditional roles such as in the testis and brain. FPKM values of at least 1 in any one sample or could be assembled in at least two samples were considered credible. Credible tran- Materials and methods scripts were added to the original Ensembl annotation gtf file as Animal ethics new isoforms of known X. tropicalis genes, and labeled with the The handling of animals used in this study followed the guidelines class code ‘j’. We used this new annotation as a reference and and regulations of Kunming Institute of Zoology on animal experi- re-run Cuffcompare with transcripts labeled with the class codes mentation and was approved by the Institutional Animal Care and ‘i’, ‘x’, and ‘u’. We selected these loci as the transcripts labeled Use Committee of the Kunming Institute of Zoology. with class codes ‘i’, ‘x’, or ‘u’ to represent sense intronic locus, Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Origin of new genes after zygotic genome activation in vertebrate j 145 antisence exonic locus, or intergenic locus transcripts, respect- CAAAT-3′ and reverse: 5′-GACGAGGAAGATGAGGAGATGGAA-3′. Fog2, ively. Finally, we combined the known annotated Ensembl genes forward: 5′-TTTGTGCGTCCTACCCTATGC-3′ and reverse: 5′-CCAG and their new assembled isoforms together with genes from the TTCAGCTAACCAGTCCCT-3′. Similarly, full-length ORF of Fog1 and new loci identified to obtain a new set of annotations. This final Fog2 were cloned into the pCS2 -C-FLAG vector for mRNA tran- annotation was processed by Cuffdiff together with the original scription. Primer sequences were as follows: Fog1,forward: alignment file to calculate the FPKM values of each gene in each 5′-CCGCTCGAGATGGGCCCTGTCCCCCCAACC-3′ and reverse: 5′-CG sample. CTCCGGAATGAAGCTTATTCAACCCTTTTTG-3′. Fog2, forward: 5′-CCG Based on our robust transcript reconstruction, we analyzed the CTCGAGATGGAAGCTCCACCTGGAATATAC-3′ and reverse: 5′-CGCAG coding potential of the transcripts encoded by the newly identified ATCTGGTAACCCCAGTAACAAGTGGAC-3′. The cloned plasmids were loci using Coding Potential Calculator (CPC) (Kong et al., 2007). used as templates for the in vitro transcription of RNA probes and Transcript sequences were extracted by gffread, a utility of the mRNA using the SP6 mMessage Machine kit (Ambion). Cufflinks package (Trapnell et al., 2010). Transcripts with a score ≥0 were deemed as coding, those with scores <−1 as noncoding, Real-time PCR assay and all others as weak noncoding. If all transcripts within a new Embryos of different stages (including 0, 3, 8, 10, 11, 14, 16, locus were classified as noncoding, we defined it as a lncRNA 18, 24, 25, 28, 32,and 39) were collected, with total RNA acquired locus, with those in intergenic regions having no overlap with any from each sample separately. RNA was then reverse transcribed known gene locus considered as lincRNA loci. using the Fermentas RevertAid First Strand cDNA Synthesis Kit to prepare templates for real-time PCR on a LightCycler (Roche Weighted gene co-expression network analysis Diagnostics). Expression of the two genes was examined using Weighted gene networks were constructed using the WGCNA SYBR green qPCR using the following primers: Fog1,forward: package implemented in R (Langfelder and Horvath, 2008). The 5′-CCAAGCCCAGGACATTCACC-3′; reverse: 5′-CCGTCTCAGGGATTA power of 7, for which the scale-free topology fit index curves GTTCAGC-3′. Fog2,forward: 5′-GCTACAACACCTTTGTGGGTGA-3′; flatten out at roughly 0.9, was interpreted as a soft-threshold reverse: 5′-GCTAACCAGTCCCTCCTTTCCT-3′. Primers for GAPDH for the adjacency matrix. In total, 30 modules were identified. were used as described in Nichane et al. (2008). The following cyc- Module membership is defined by the calculated Pearson correl- ling conditions were used: denaturation at 95°C(10 sec), anneal- ation between the level of expression of a given gene and a ing at 60°C(10 sec), and extension at 72°C(10 sec). given module eigengene. The module eigengene, which is defined as the first principal component of a given module, is Western blot of embryos considered as representative of the gene expression profiles for Embryoswerelysed in lysisbuffer(50 mM Tris-HCl, pH 7.4, this module. We then correlated eigengenes with binary 150 mM NaCl, 5 mM EDTA, pH 8.0,and 1%TritonX-100) and pro- phenotype to each time point/sample. The statistical signifi- tease inhibitors (Roche) mixture for 30 minonice.The lysates cance of the correlations was estimated using the Student were cleared of debris by centrifugation at 4°Cfor 10 min at t-test. Simply, a module that has the highest association with 14000 rpm. SDS loading buffer was added to the supernatant, the status of the time point/sample was inferred to probably which was then heated at 95°Cfor 10 min. Total lysates were then have a biological function that underlies the specific traits of subjected to SDS-PAGE and western blot analyses. Antibodies used this time point/sample. in this experiment were as follows: anti-FLAG (Sigma, 1:1000)and anti-β-actin (Abcam, 1:5000), with HRP-conjugated anti-rabbit or Identification of X. tropicalis-specific new genes anti-mouse IgG (Pierce, 1:5000) used as secondary antibodies. Theagesof X. tropicalis geneswereobtainedfromProteinHis- torian (Capra et al., 2012). Genes with an age of zero were taken as new genes that newly originated during the evolution of frogs. Embryo culture, microinjection, whole-mount in situ Genes that were specifically duplicated in X. tropicalis were hybridization retrieved by BioMart (Smedley et al., 2009) from Ensembl (http:// Embryos were staged as previously outlined (Nieuwkoop and www.ensembl.org/). Recently, duplicated genes were defined as Faber, 1967). At the 4-cell stage, 12 ng morpholino (MO) and/or X. tropicalis specific duplicated genes with an age of 0. Orphan 0.6 ng mRNA for the genes were injected into the embryos. In all genes, which are X. tropicalis specific, were identified from hom- experiments, embryos were co-injected with mRNA for LacZ to ologous searches of protein databases of other animal species identify the manipulated side of the embryo. Sequences of the using BLASTP with an E-value of 1e−10 (Altschul et al., 1997). MO used were: Fog1 MO: CCATCGGAACACTAATTCTGAACCT; Fog2 MO: TCCAGGTGGAGCTTCCATGATGCAG. RNA probes for Fog1 and Gene cloning and in vitro transcription Fog2 were used to examine levels of mRNA expression. Embryos Approximately 570 bp of the coding sequences of the Fog1 and at the appropriate stages were fixed in MEMFA (Harland, 1991). Fog2 genes from X. laevis were amplified by PCR and cloned into Whole-mount in situ hybridization was performed as described pGEM-T vector to generate a probe to detect RNA transcription. previously (Harland, 1991). The injected areas were identified by RNA from embryos of 16 stages was used as a template. The PCR staining for LacZ using red-gal. Some stained embryos were primers were as follows: Fog1,forward: 5′-CCTGACTGGACTGGAGG embedded in paraffin and sectioned at 15 μm. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 146 j Xu et al. Kleene, K.C. (2001). 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TopHat2: accurate alignment of Zhong, Z., Yang, L., Zhang, Y.E., et al. (2016). Correlated expression of retro- transcriptomes in the presence of insertions, deletions and gene fusions. copies and parental genes in zebrafish. Mol. Genet. Genomics 291, Genome Biol. 14,R36. 723–737. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Cell Biology Oxford University Press

Origin of new genes after zygotic genome activation in vertebrate

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doi:10.1093/jmcb/mjx057 Journal of Molecular Cell Biology (2018), 10(2), 139–146 j 139 Published online February 14, 2018 Article Origin of new genes after zygotic genome activation in vertebrate 1,2,† 1,3,† 4 1,3 1,3,4 1,3, *, Hai-Bo Xu , Yong-Xin Li , Yan Li , Newton O. Otecko , Ya-Ping Zhang , Bingyu Mao 1,3, and Dong-Dong Wu * State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China College of Life Science, Anhui University, Hefei, China Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, China State Key Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming, China These authors contributed equally to the work. * Correspondence to: Dong-Dong Wu, E-mail: wudongdong@mail.kiz.ac.cn; Bingyu Mao, E-mail: mao@mail.kiz.ac.cn Edited by Luonan Chen New genes are drivers of evolutionary innovation and phenotypic evolution. Expression of new genes in early development raises the possibility that new genes could originate and be recruited for functions in embryonic development, but this remains undocu- mented. Here, based on temporal gene expression at different developmental stages in Xenopus tropicalis, we found that young protein-coding genes were significantly enriched for expression in developmental stages occurring after the midblastula trans- ition (MBT), and displayed a decreasing trend in abundance in the subsequent stages after MBT. To complement the finding, we demonstrate essential functional attributes of a young orphan gene, named as Fog2, in morphological development. Our data indicate that new genes could originate after MBT and be recruited for functions in embryonic development, and thus provide insights for better understanding of the origin, evolution, and function of new genes. Keywords: young gene evolution, zygotic genome activation, new gene origin Introduction New genes, as fundamental materials for evolutionary innov- in Drosophila lead to either lethality at diverse development stages, ation, have been investigated for many years (Long et al., 2003, or tissue-specific morphological defects (Chen et al., 2010). In human, 2013). Many new genes rapidly acquire important and even new genes display a substantially high expression levels in fetal brain essential functions driven by positive selection (Chen et al., compared with adult brain (Zhang et al., 2011; Wu et al., 2015). The 2010, 2013), with most pronounced roles being in reproduction, investigations raise the possibility that new genes could originate dur- development, and brain (Kaessmann, 2010; Tautz and Domazet- ing the process of embryonic development. Particularly, during early Loso, 2011; Chen et al., 2013). For instance, a testis-bias development after the midblastula transition (MBT), zygotic genome expression of new genes is recapitulated in different animals activation is initiated, where remarkable epigenetic modifications (Kaessmann, 2010). As seen in human, many new genes exhibit a occur to induce the transcription of the zygotic genome to gradually brain, particularly a neocortex-biased expression, suggesting that take control of development. These dynamic changes in epigenetic they may be recruited for the accelerated evolution of the human modifications enhance transcriptional activity and promiscuous tran- brain and may be involved in the acquisition of high cognitive scription (Ostrup et al., 2013). Theseeventsare mirrored in testis, ability (Li et al., 2010; Wu et al., 2011; Zhang et al., 2011). where widespread demethylation of CpG dinucleotide-enriched pro- New genes also play integral roles in development (Chen et al., moter sequences occurs, resulting in a transcriptionally active chro- 2010, 2012). For instance, in a pioneering study by Chen et al. matin state that facilitates the access of transcriptional machinery (2010), knockdown of many new genes by RNA interference (RNAi) and promiscuous transcription of genes (Kleene, 2001; Kaessmann, 2010). Studies have observed that new genes are frequently recruited for new function in the testis due to the promiscuous tran- Received July 16, 2017. Revised November 20, 2017. Accepted December 19, scription (Kaessmann, 2010; Tautz and Domazet-Loso, 2011; Chen et al., 2013). Therefore, we hypothesized that new genes might be © The Author (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. recruited during early development, particularly after MBT. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 140 j Xu et al. To test the hypothesis that new gene could frequently be recruited modules of coexpressed genes with shared functionality. A total for key roles in early animal development, we examined the temporal of 30 distinct modules, representing clusters of genes with cor- expression of new genes during the early development of a frog, related expression, were identified (Figure 2A). Many of the and found that expression of new genes was significantly enriched modules exhibit expression patterns significantly correlated in developmental stages after embryonic genome activation. with specific developmental stages (Figure 2A, Supplementary Furthermore, we examined the roles of two young orphan genes, Figure S3). For example, the M2 module is a stage 9-dominant named Fog1 and Fog2, and demonstrated that they have important module, with the significance of its association having a P-value −8 functions in frog development. After MBT, zygotic genome activation of 1.0 × 10 (Figure 2B). (ZGA) is initiated, where remarkable epigenetic modifications occur We then examined the enrichment of new genes within these that induce the transcription of the zygotic genome (Ostrup et al., modules. Young duplicated genes of X. tropicalis were significantly 2013), with a similar pattern observed in the testis (Kleene, 2001; over-represented in 13 of the 30 modules (Figure 2C, P < 0.01), Kaessmann, 2010). We propose that the induced transcription of the with 12 modules harboring significant correlation with a develop- zygotic genome might facilitate the origination of new genes. mental stage after MBT (Figure 2A, P < 0.01 after Bonferroni cor- rection by χ test). The 12 modules cover the gastrulation Results processes (stages 9, 10,and 11.12,with 119 young duplicated Temporal expression profiling reveals high expression of young genes involved), neurulation (stages 16.18, 19,and 20.21,with 76 protein-coding genes after MBT young duplicated genes involved), and part of the organogenesis In the present study, we categorized young protein-coding period (stages 24.26, 38.39,and 44.45,with 66 young duplicated genes in the frog (Xenopus tropicalis), i.e. duplicate genes and genes involved). This enrichment indicates that these new genes orphan genes, according to their mechanisms of origin have evolved important functions in critical gene–gene inter- (Supplementary Tables S1 and S2). Duplicate genes are gener- action networks. Interestingly, the level of enrichment for new ated by gene duplication from existing old genes, while orphan genes showed a decreasing trend with developmental stage genes do not have homology with genes in other species, and after MBT (P = 0.028, Figure 2D; P = 0.048, Supplementary are likely originate de novo or from rapidly evolved genes, Figure S4), corroborating the expression trajectory of new genes thereby losing similarity with their ancestral sequences (Tautz (Figure 1). The decreasing pattern after MBT could not be attrib- and Domazet-Loso, 2011). Genes that were specifically dupli- uted to changes in global gene expression levels or changes in cated in X. tropicalis were retrieved by BioMart (Smedley et al., the proportions of expressed genes among developmental 2009) from Ensembl (http://www.ensembl.org/, version 72). stages, as correlation was not observed between enrichment The ages of X. tropicalis genes were obtained from levels of new genes and transcriptional levels of developmental ProteinHistorian (Capra et al., 2012). Genes with an age of zero stages (Supplementary Figure S5). were taken as new genes that newly originated during the evo- Young orphan genes were significantly enriched within two mod- lution of frogs. Recently duplicated genes were defined as ules (P < 0.01 after Bonferroni correction by χ test), with modules X. tropicalis-specific duplicated genes with an age of 0. Orphan M5 (29 genes) corresponding to developmental stage 10,and M24 genes, which are X. tropicalis specific, were identified from hom- (15 genes) (Supplementary Figure S6). It is notable that another 47 ologous searches of protein databases of other animal species young duplicated genes are over-represented within module M5. using BLASTP with an E-value of 1e−10 (Altschul et al., 1997). Gene enrichment analysis performed by the DAVID program (Huang Profiling of temporal gene expression from the 2-cell stage to et al., 2008) found that genes in M5 were significantly enriched in stage 44–45 (Tan et al., 2013) revealed that the expression of categories associated with development such as ‘pattern specifica- young protein-coding genes peak after MBT, particularly from gas- tion process’ (GO:0007389, P = 0.01, 10 genes: T, HHEX, NOG, trulation to neurulation (Figure 1, Supplementary Figure S1A). GSC, LHX1, DYNC2LI1, GATA4, ZIC1, TCF7L1,and ZIC3)and ‘region- Expression levels of the new genes displayed a decreasing trend alization’ (GO:0003002, P = 0.04) (Supplementary Table S3). with developmental stages after MBT. When gene expression These results indicate that the young genes might have evolved levels were normalized to the whole genome level, this pattern developmental roles through interactions with other genes. still held for new genes (Figure 1, Supplementary Figure S1B). Although expression quantification may not be accurate for young Expression patterns of two young orphan genes reveal potential duplicate genes using RNA-sequencing data, we reasoned that it functions in development would not change our conclusion as orphan genes also exhibited High-expression levels of young protein-coding genes after the signature peak expression after MBT (Figure 1). MBT, particularly from gastrulation to neurulation, raise the possi- bility that some of these new genes might have acquired import- Weighted gene co-expression network analysis ant functions in these developmental stages. To complement this Genes function by interacting with other genes, generating finding, we chose some young protein-coding genes for further gene–gene interaction networks. To gain insight into involve- functional assessments. Two genes (ENSXETG00000027093,i.e. ment of new genes in networks, we performed weighted gene LOC100170590, which we named Fog1, frog orphan gene 1,and co-expression network analysis (WGCNA), an unsupervised and ENSXETG00000030468,i.e.LOC100158459,which we named unbiased analysis (Langfelder and Horvath, 2008) to identify Fog2,frogorphangene 2) stand out, as they exhibit high expression Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Origin of new genes after zygotic genome activation in vertebrate j 141 2012). The above expression correlation analyses suggest that Fog1 and Fog2 genes might be coupled to embryonic development. We further investigated the potential functions of the two genes in another model frog, Xenopus laevis. Real-time PCR confirmed their increased expression levels at the gastrula and the neurula stages, but lower levels in the tailbud stage of development (Supplementary Figure S7). After that, we used in situ hybridiza- tion to examine the temporal expression patterns during embry- onic development, and found high expression of Fog1 and Fog2 in somites (muscle) (Figure 3). This is consistent with the above expression correlation analysis, where many genes involved in somite development displayed correlated expression with Fog1 Figure 1 Expression level of young protein-coding genes at different and Fog2. In addition, Fog1 is also expressed in the branchial developmental stages in X. tropicalis. log (FPKM+1) was used to 2 arches, optic vesicle, and tail end, while Fog2 shows expression in calculate the expression value of each gene. Two groups of orphan the nervous system and optic vesicle (Figure 3). genes were identified by BLASTP with cutoff E = 1e−10 and 1e−4, respectively. Functional study of Fog1 and Fog2 The above expression analysis suggested that Fog1 and Fog2 levels after MBT, particularly from gastrulation to neurulation might have functions in development. To further interrogate the (Supplementary Figure S7). To interrogate the epigenetic aspects functions of Fog1 and Fog2 in development, we knocked down associated with expression, we examined the levels of H3K4me3 mRNA expression of these two genes by injecting gene-specific modification, which is associated with active transcription of MO (morpholino) into X. laevis embryos. By visual inspection, nearby genes (Guenther et al., 2007), across thetwo genesat dif- no noticeable phenotypic change was observed in development ferent developmental stages. As expected, the changes of after injecting Fog1 MO (Supplementary Figure S9). While this H3K4me3 modification levels displayed a pattern paralleling the does not annul the potential functional importance of this gene, changes in the expression of these two genes (Supplementary it highlights the need for more systematic phenotyping assays Figure S8). Among the gene–gene expression networks, the two to capture the phenotypic changes after knocking down Fog1. genes are located within module M7, which exhibited high In stark contrast, a serious change in body axis curvature was expression level at the stages 10−14 after MBT (Figure 2A). Gene observed in embryos after knocking down Fog2 compared to enrichment analysis showed that genes in M7 are significantly wild-type embryos (Figure 4A−D, Supplementary Figure S11). enriched in many categories associated with development such as: This malformation could be partially rescued when Fog2 mRNA ‘somite development’, ‘pattern specification process’, ‘regionaliza- was co-injected (Figure 4E and F, Supplementary Figure S11). In tion’, ‘segmentation’ and ‘anterior/posterior pattern specification’ consideration of expression of Fog2 in somites (muscle) and (Supplementary Table S4). These findings suggest that Fog1/Fog2 mesoderm (Figure 3, Supplementary Figure S12), and expres- might be functionally linked with development. sion correlation with other genes involved in mesoderm devel- To explore potential functions of these two genes, we firstly per- opment, we further examined the expression of MyoD,a crucial formed an expression correlation analysis to identify genes whose gene in somites formation as an early response to mesoderm expression correlate significantly with these two genes. A total of induction in Xenopus embryos. Fog2 MO injection caused downre- 190 genes showed correlation with Fog1 (R > 0.9, by Pearson cor- gulation of MyoD (Figure 5). This phenotype could be partially res- relation), and gene enrichment analysis revealed that these genes cued by co-injection with Fog2 mRNA (Figure 5). Since the rescue were enriched in development categories. For example, 31 genes experiment was performed by co-injecting MO and Fog2 mRNA, it are enriched in the category ‘anatomical structure development’ is important to note that MO can block not only the endogenous (GO:0048856, P = 0.029), five genes in ‘mesenchyme development’ mRNA but also the injected mRNA. Hence, the rescued phenotype (GO:0060485, P = 0.02), four genes in ‘neural crest cell differenti- might be due an overall increase in Fog2 mRNA. Altogether, these ation’ (GO:0014033, P = 0.016), and four genes in ‘somite devel- results suggest that Fog2 is tightly coupled to the morphological opment’ (GO:0061053, P = 0.0379) (Supplementary Table S5). development of X. laevis. BLAST analysis did not find the homolo- On the other hand, 12 genes are correlated with Fog2 (R > gous gene of Fog2 in thegenomeofTibetan frog (Nanorana par- 0.9, by Pearson correlation), with 7 of the genes involved in keri)(Sun et al., 2015), supporting Fog2 as a newly evolved gene. ‘anatomical structure development’ (GO:0048856, P = 0.0002) The experiment reiterates that some new genes could be recruited (Supplementary Table S6). Among these genes, Msgn1 (meso- in embryonic development. genin 1) is a master regulator of paraxial presomitic mesoderm differentiation (Chalamalasetty et al., 2014), and Szl, encodes Discussion the secreted frizzled related protein Sizzled that negatively reg- In the present study, we show that young protein-coding genes ulates Tolloid-like activity to control deposition of a fibronectin are significantly enriched in embryonic developmental stages after (FN) matrix between the mesoderm and endoderm (Kenny et al., MBT. A decreasing trend in the level of enrichment and expression Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 142 j Xu et al. AB 1 0.5 0 –0.5 –1 Z score –4 –2 0 2 4 M15 M24 M23 M28 M25 M26 M27 M17 M16 M18 M29 M22 M21 M19 M30 M20 M4 M1 M7 M5 M2 M3 M6 M8 M14 M13 M10 M11 M9 M12 CD 12 y = –0.28x + 6.462 R = 0.282, P = 0.028 * 6 * * * * 2 * Figure 2 WGCNA of young protein-coding genes. (A) Heatmaps displaying the correlations (and corresponding P-values) between modules and developmental stages or adult tissues. Color legend at the top indicates the level of correlation. eld0−4 represent five adult tissues: brain, liver, kidney, heart, and skeletal muscle, respectively. (B) Heatmap of gene expression in module M2.(C) Enrichment level of young duplicated genes for the different modules. Values are the proportion of young duplicated genes in each module divided by the proportion of other genes in the module. Asterisks (*) represent significant enrichments (P < 0.01 after Bonferroni correction by χ test). (D) Level of enrichment of young duplicated genes at each developmental stage shows a decreasing trend after MBT. of new genes was seen in the developmental stages after MBT, a site (Ka), to the number of synonymous substitutions per syn- pattern that had not been previously reported. For example, onymous site (Ks)) of Fog1 and Fog2 is 0.283 and 0.744,respect- knockdown of new genes in Drosophila (Chen et al., 2010) gener- ively (Supplementary Figure S13). It seems that Fog1 evolves ally lead to pupal rather than embryonic lethality. A repetitive ana- under purifying selection, suggesting that it still may harbor an lysis of time-course developmental data in zebrafish showed no important function, while Fog2 evolves more rapidly which occurs peak expression in the early developmental stages (Domazet-Loso commonly for recently evolved new genes. and Tautz, 2010; Zhong et al., 2016). Numerous possibilities such After MBT, zygotic genome activation is initiated, where as different bioinformatics practices, different data profiling strat- remarkable epigenetic modifications occur to induce the tran- egies, as well as specie-specific variations might explain the con- scription of the zygotic genome to gradually take over the con- flicts in these observations. In particular, we studied functions of trol of development (Ostrup et al., 2013). An example of the two genes Fog1 and Fog2 exhibiting high expression levels after epigenetic changes that occur with this transition is the genomic MBT, and found that Fog2 might have an important role in devel- enrichment of H3K4me3, an indicator of active promoters, which opment. When comparing orthologous gene coding sequences is observed at the time of ZGA in zebrafish (Vastenhouw et al., between X. laevis and X. tropicalis,wefound that Ka/Ks(theratio 2010) and Xenopus (Akkers et al., 2009). Another example is of the number of nonsynonymous substitutions per nonsynonymous DNA methylation, which is considered to be a negative regulator Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Enrichemtn level 2cell 4cell M1 8cell M2 16cell M3 M4 stage6 M5 stage8 M6 stage9 M7 stage10 M8 stage11–12 M9 M10 stage13–14 M11 stage15 M12 stage16–18 M13 stage19 M14 M15 stage20–21 M16 stage22–23 M17 stage24–26 M18 stage28 M19 M20 stage31–32 M21 stage33–34 M22 stage38–39 M23 stage40 M24 M25 stage41–42 M26 stage44–45 M27 eld0 M28 eld1 M29 eld2 M30 eld3 eld4 Enrichment Level 2cell 4cell stage9 8cell stage10 16ell stage11.12 stage6 stage13.14 stage8 stage9 stage15 stage10 stage16.18 stage11–12 stage19 stage13–14 stage15 stage20.21 stage16–18 stage22.23 stage19 stage24.26 stage20–21 stage28 stage22–23 stage24–26 stage31.32 stage28 stage33.34 stage31–32 stage38.39 stage33–34 stage38–39 stage40 stage40 stage41.42 stage41–42 stage44.45 stage44–45 Origin of new genes after zygotic genome activation in vertebrate j 143 Figure 3 Expression patterns of Fog1 and Fog2 in X. laevis embryos. (A−H) Expression patterns of Fog1 mRNA. (A and B) Fog1 is weakly expressed in the animal pole at the blastula stage. (C−E) Fog1 is weekly expressed in the neural system at the neurula stage. (F−H) Fog1 is mainly expressed in the muscle, brain, optic vesicle, branchial arches, and tail end. (A’−H’) Expression patterns of Fog2 mRNA. (A’−C’) Fog2 is weakly expressed in the animal pole at the blastula stage. (D’ and E’) Fog2 is expressed in the neural system at the neurula stage. (F’−H’) Fog2 is mainly expressed in the brain, optic vesicle, and muscle. n, neural system; ov, optic vesicle; mu, muscle; ba, branchial arches; te, tail end. Figure 4 Knockdown of Fog2 causes defects or malformation in embryonic development of Xenopus at late stages of development. Embryos were injected on the left side. (A) Wild-type (WT) embryos. (C) Embryos injected with Fog2-MO. (E) Embryos injected with both Fog2-MO and mRNA. (B, D, F) The same embryo for A, C, E, respectively. Numbers at the bottom corners represent the number of embryos with the corre- sponding effect and the number of embryos used in this experiment. The side of the injection is indicated by lacZ (red dots). Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 144 j Xu et al. Figure 5 Knockdown of Fog2 causes downregulation of MyoD. (A) Wild-type embryos. (C) Embryos injected with Fog2-MO. (E) Embryos injected with both Fog2-MO and mRNA. (B, D, F) The opposite side for A, C, E, respectively. Numbers at the bottom corners represent the number of embryos with the corresponding effect and the number of embryos used in this experiment. The side of the injection is indicated by lacZ (red dots). of gene expression. Methylated DNA is efficiently transcriptionally Analysis of transcriptome data repressed in Xenopus oocytes, but DNA methylation-dependent Transcriptome data from 23 developmental stages from a pre- transcriptional repression is greatly reduced after ZGA, and the vious study (Tan et al., 2013) were downloaded from the NCBI repressive effect of DNA methylation is only restored during SRA (http://www.ncbi.nlm.nih.gov/sra/). Detailed information on organogenesis and terminal differentiation (Bogdanovic et al., each developmental stage is available elsewhere (Tan et al., 2011; Ostrup et al., 2013). Thesedynamic changesinepigenetic 2013). All reads were processed using Btrim to remove low- modifications enhance transcriptional activity after ZGA. The pro- quality reads with parameters –l 30 –q 20 (Kong, 2011), with the miscuous transcription then enables proto-genes to be selected paired and unpaired short reads generated then aligned to the due to beneficial functions that might accidentally be gained, and X. tropicalis genome (JGI_4.2) (Ensembl release 72) using the thus allow them to evolve into bona fide genes (Kaessmann, 2010). read gapped alignment program Tophat 2.0.9 (Trapnell et al., This epigenetic aspect is synonymous with that observed in testis, 2009; Kim et al., 2011). After merging paired and unpaired align- where widespread demethylation of CpG dinucleotide-enriched pro- ments from the same sample, the program Cufflinks 2.0.2 was moter sequences occurs, resulting in a transcriptionally active chro- used to assemble the transcriptomes (Trapnell et al., 2010) matin state that facilitates the access of transcriptional machinery (Supplementary Figure S14). All isoforms assembled by Cufflinks and promiscuous transcription of genes (Kleene, 2001; Kaessmann, from the 45 samples were sent to the Cuffcompare utility, along 2010). Previous studies have evidenced the recruitment of new with the Ensembl annotation file, to generate an integrated com- genes for new functions in the testis (Kaessmann, 2010; Tautz and bined gtf annotation file. With the purpose of minimizing annota- Domazet-Loso, 2011; Chen et al., 2013). In conclusion, our study tion artifacts, we processed the Cuffcompare output file through identifies a genomic hotbed for the origination of new genes, and the following steps. First, we excluded all new single exon tran- illustrates likely developmental functions of new genes besides the scripts and sequences with lengths < 200 bp. Transcript with traditional roles such as in the testis and brain. FPKM values of at least 1 in any one sample or could be assembled in at least two samples were considered credible. Credible tran- Materials and methods scripts were added to the original Ensembl annotation gtf file as Animal ethics new isoforms of known X. tropicalis genes, and labeled with the The handling of animals used in this study followed the guidelines class code ‘j’. We used this new annotation as a reference and and regulations of Kunming Institute of Zoology on animal experi- re-run Cuffcompare with transcripts labeled with the class codes mentation and was approved by the Institutional Animal Care and ‘i’, ‘x’, and ‘u’. We selected these loci as the transcripts labeled Use Committee of the Kunming Institute of Zoology. with class codes ‘i’, ‘x’, or ‘u’ to represent sense intronic locus, Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 Origin of new genes after zygotic genome activation in vertebrate j 145 antisence exonic locus, or intergenic locus transcripts, respect- CAAAT-3′ and reverse: 5′-GACGAGGAAGATGAGGAGATGGAA-3′. Fog2, ively. Finally, we combined the known annotated Ensembl genes forward: 5′-TTTGTGCGTCCTACCCTATGC-3′ and reverse: 5′-CCAG and their new assembled isoforms together with genes from the TTCAGCTAACCAGTCCCT-3′. Similarly, full-length ORF of Fog1 and new loci identified to obtain a new set of annotations. This final Fog2 were cloned into the pCS2 -C-FLAG vector for mRNA tran- annotation was processed by Cuffdiff together with the original scription. Primer sequences were as follows: Fog1,forward: alignment file to calculate the FPKM values of each gene in each 5′-CCGCTCGAGATGGGCCCTGTCCCCCCAACC-3′ and reverse: 5′-CG sample. CTCCGGAATGAAGCTTATTCAACCCTTTTTG-3′. Fog2, forward: 5′-CCG Based on our robust transcript reconstruction, we analyzed the CTCGAGATGGAAGCTCCACCTGGAATATAC-3′ and reverse: 5′-CGCAG coding potential of the transcripts encoded by the newly identified ATCTGGTAACCCCAGTAACAAGTGGAC-3′. The cloned plasmids were loci using Coding Potential Calculator (CPC) (Kong et al., 2007). used as templates for the in vitro transcription of RNA probes and Transcript sequences were extracted by gffread, a utility of the mRNA using the SP6 mMessage Machine kit (Ambion). Cufflinks package (Trapnell et al., 2010). Transcripts with a score ≥0 were deemed as coding, those with scores <−1 as noncoding, Real-time PCR assay and all others as weak noncoding. If all transcripts within a new Embryos of different stages (including 0, 3, 8, 10, 11, 14, 16, locus were classified as noncoding, we defined it as a lncRNA 18, 24, 25, 28, 32,and 39) were collected, with total RNA acquired locus, with those in intergenic regions having no overlap with any from each sample separately. RNA was then reverse transcribed known gene locus considered as lincRNA loci. using the Fermentas RevertAid First Strand cDNA Synthesis Kit to prepare templates for real-time PCR on a LightCycler (Roche Weighted gene co-expression network analysis Diagnostics). Expression of the two genes was examined using Weighted gene networks were constructed using the WGCNA SYBR green qPCR using the following primers: Fog1,forward: package implemented in R (Langfelder and Horvath, 2008). The 5′-CCAAGCCCAGGACATTCACC-3′; reverse: 5′-CCGTCTCAGGGATTA power of 7, for which the scale-free topology fit index curves GTTCAGC-3′. Fog2,forward: 5′-GCTACAACACCTTTGTGGGTGA-3′; flatten out at roughly 0.9, was interpreted as a soft-threshold reverse: 5′-GCTAACCAGTCCCTCCTTTCCT-3′. Primers for GAPDH for the adjacency matrix. In total, 30 modules were identified. were used as described in Nichane et al. (2008). The following cyc- Module membership is defined by the calculated Pearson correl- ling conditions were used: denaturation at 95°C(10 sec), anneal- ation between the level of expression of a given gene and a ing at 60°C(10 sec), and extension at 72°C(10 sec). given module eigengene. The module eigengene, which is defined as the first principal component of a given module, is Western blot of embryos considered as representative of the gene expression profiles for Embryoswerelysed in lysisbuffer(50 mM Tris-HCl, pH 7.4, this module. We then correlated eigengenes with binary 150 mM NaCl, 5 mM EDTA, pH 8.0,and 1%TritonX-100) and pro- phenotype to each time point/sample. The statistical signifi- tease inhibitors (Roche) mixture for 30 minonice.The lysates cance of the correlations was estimated using the Student were cleared of debris by centrifugation at 4°Cfor 10 min at t-test. Simply, a module that has the highest association with 14000 rpm. SDS loading buffer was added to the supernatant, the status of the time point/sample was inferred to probably which was then heated at 95°Cfor 10 min. Total lysates were then have a biological function that underlies the specific traits of subjected to SDS-PAGE and western blot analyses. Antibodies used this time point/sample. in this experiment were as follows: anti-FLAG (Sigma, 1:1000)and anti-β-actin (Abcam, 1:5000), with HRP-conjugated anti-rabbit or Identification of X. tropicalis-specific new genes anti-mouse IgG (Pierce, 1:5000) used as secondary antibodies. Theagesof X. tropicalis geneswereobtainedfromProteinHis- torian (Capra et al., 2012). Genes with an age of zero were taken as new genes that newly originated during the evolution of frogs. Embryo culture, microinjection, whole-mount in situ Genes that were specifically duplicated in X. tropicalis were hybridization retrieved by BioMart (Smedley et al., 2009) from Ensembl (http:// Embryos were staged as previously outlined (Nieuwkoop and www.ensembl.org/). Recently, duplicated genes were defined as Faber, 1967). At the 4-cell stage, 12 ng morpholino (MO) and/or X. tropicalis specific duplicated genes with an age of 0. Orphan 0.6 ng mRNA for the genes were injected into the embryos. In all genes, which are X. tropicalis specific, were identified from hom- experiments, embryos were co-injected with mRNA for LacZ to ologous searches of protein databases of other animal species identify the manipulated side of the embryo. Sequences of the using BLASTP with an E-value of 1e−10 (Altschul et al., 1997). MO used were: Fog1 MO: CCATCGGAACACTAATTCTGAACCT; Fog2 MO: TCCAGGTGGAGCTTCCATGATGCAG. RNA probes for Fog1 and Gene cloning and in vitro transcription Fog2 were used to examine levels of mRNA expression. Embryos Approximately 570 bp of the coding sequences of the Fog1 and at the appropriate stages were fixed in MEMFA (Harland, 1991). Fog2 genes from X. laevis were amplified by PCR and cloned into Whole-mount in situ hybridization was performed as described pGEM-T vector to generate a probe to detect RNA transcription. previously (Harland, 1991). The injected areas were identified by RNA from embryos of 16 stages was used as a template. The PCR staining for LacZ using red-gal. Some stained embryos were primers were as follows: Fog1,forward: 5′-CCTGACTGGACTGGAGG embedded in paraffin and sectioned at 15 μm. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/2/139/4769427 by Ed 'DeepDyve' Gillespie user on 20 June 2018 146 j Xu et al. Kleene, K.C. (2001). 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Journal of Molecular Cell BiologyOxford University Press

Published: Feb 14, 2018

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