Zebrafish Ddx19 deficiency causes serious apoptosis and cell proliferation

Background: DDX19 is known as for its role in mRNA transport. It is also involved in translation and innate immune responses. However, the function of Ddx19 during body development rarely reported. We know that ddx19 plays an important role in development of organisms, but we don't understand how it affects the process of cell development. Results: Here, we report ddx19-deleted mutation in zebrafish, obtained two types with base deletion of 46 bp and 7 bp using CRISPR/Cas9 gene knockout technology, show morphologic defects such as small head, small eyes, pericardial edema and trunk curvature in 24 hours post fertilization (hpf). The maximum survival time of ddx19 -/- was less than 5 day post fertilization (dpf). In comparison to the wildtype, the mutant embryos showed widespread up-regulation of cell apoptosis, and significant decrease in the number of cells. Conclusions: These data indicate loss of ddx19 is lethal, and is associated with cell apoptosis and proliferation abnormalities in the organism. Our results reveal that ddx19 is essential for the development of zebrafish embryos, which deepens the understanding of the developmental function of DEAD-box genes family.

increase in the number of cells in G1-phase, and changes in mRNAs transcripts in the cytoplasm [18].
In 2013, Jao, L.E. et al.. obtained the ddx19 -/using CRISPR/Cas9 gene editing technology that mediated multiple allele knockouts in zebrafish genome, and found that ddx19 gene knockout leads to phenotypic defects and homozygous death [19]. However, which cell processes are involved in ddx19 and how ddx19 affects cell development in vivo remain to be explored.
Previous studies have linked DEAD-box proteins to cell apoptosis and cell proliferation [8,10,[20][21][22], two cellular processes that manifest growth and development. Apoptosis, or programmed cell death, is a physiological processes closely related to cancer, autoimmunity and degenerative diseases. This process is initiated and regulated by the caspase family, which is composed of seven proteins in zebrafish [23] and activated through a variety of independent pathways. The internal mitochondrial pathway, death receptor-mediated external pathway, and endoplasmic reticulum pathway are all known to mediate apoptosis [24][25][26]. Cell proliferation is often regulated by the cell cycle. As a conserved mechanism of eukaryotic cell self-replication, cell cycle regulation is crucial for tissue homeostasis and cell replacement during organism development. Multiple regulatory factors are involved in the cell cycle regulation process [27]. Any wrong expression of these factors likely will lead to malignant proliferation and differentiation, which are associated with a variety of diseases and tumors. Such as, cell proliferation and tumorigenesis of esophageal cancer are inhibited in ddx5 -/- [7]; Overexpression DDX20 promotes the proliferation of prostate cancer cells through the NF-κB pathway [8].
Here, we hypothesized that the mRNA transport factor Ddx19 regulates the process of cell proliferation and apoptosis, and consequently affects body development in zebrafish. We tested this hypothesis by deleting ddx19 in zebrafish, using CRISPR/Cas9 gene editing technology. We obtained two types of deletion mutated phenotypes with apparent development defects. Both types survived up to 4-5 dpf and showed up-regulation in apoptosis levels and decreased numbers of cells in the Sphase.

Ddx19was persistently expressed in zebrafish embryos
The amino acid residues of Ddx19 were 89% identical in human, mouse and zebrafish (Fig.1a). Only one ddx19 gene was found in zebrafish, although two homologous genes (DDX19A/Ddx19a) and (DDX19B/Ddx19b) have been identified in human and mouse (Fig.1b). To confirm the expression of ddx19 in early development of zebrafish, we measured ddx19 mRNA transcripts in the embryos by q-RT PCR firstly. The ddx19 mRNA was present from the 1-cell stage to 125 hpf ( Fig.1c-d). The expression level of ddx19 decreased from the 1-cell stage to the 6-somite stage (Fig.1c). In comparison to 24 hpf, the expression level was higher during 24-125 hpf, and subsequently became less variable (Fig. 1d). We speculated that ddx19 may play a role in early embryonic development of zebrafish.
The ddx19 gene was knocked out using CRISPR/Cas9 The total length of ddx19 gene was 13424bp, with 12 exons, of which the first exon was targeted using CRISPR/Cas9 (Fig.2a). The non-injected WT group showed one band using T7E1 enzyme digestion, and the sequencing peak was single, while T7E1 enzyme digestion showed three bands, which were 472bp, 196bp and 176bp, respectively (Fig.2b) and the sequencing peak was chaotic near the target in positive F 0 (Fig.2c).
The F 0 positive was mated with the wildtype to produce F 1 offspring. Electrophoresis analyses of fin clips DNA showed two bands in heterozygotes (Fig.3a). Sequence analyses of the cDNA showed two types of mutants, with 7 bp and 46 bp deletion, respectively (Fig.3b). Furthermore, we analyzed the protein structure of Ddx19 in wildtype and mutants. Ddx19 had 487 amino acids, contained DEXDc and HELICc two domain, with five conserved motifs. Both types of deletion caused frame-shift mutation and premature termination (Fig.3c). The ddx19 in the mutation of 7 bp deletion coded 17 amino acids while the ddx19 in the 46 bp deletion mutants was missing the starting codon.
Ddx19 was essential for embryonic development of zebrafish Compared with the control group, the 10-tailed zebrafish in the experimental group showed morphologic defect (Fig.4a). We further verified the corresponding relationship between genotype and phenotype, and found that all morphological defects were corresponding to ddx19 -/- (Fig.4b).
Compared with the control groups, the ddx19 -/mutants showed smaller heads, smaller eyes, pericardial edema and trunk curvature. When the mutants were injected with 100-250 ng/μL ddx19 mRNA in the 1-cell stage, the morphologic defects observed in the negative control was rescued completely (Fig.5a), Compared to the control group, the experimental group rescued with ddx19 mRNA injection showed a decreased proportion of phenotypic defects (p = 0.029, 2 -test; Fig. 5b). We quantified ddx19 transcripts by q-PCR in the wild type and ddx19 -/-, and detected that its transcription level was significantly decreased in ddx19 -/-(p = 0.007, Student t-test; Fig.5c).
We counted defect phenotypes in the offspring from three pairs of parents and calculated the proportion in the total. The proportion of phenotypic defects was about 20%. Our data supports that the phenotypes consistent with Mendelian's law in the ddx19 -/with 46 bp deletion (p = 0.970, 2 -test; Apoptotic signals were up-regulated and cell proliferation was decreased in ddx19 -/-Compared with the wildtype (Fig.7a), apoptotic signals were up-regulated in ddx19 -/missing 46 bp at 24 hpf (Fig.7b), and ddx19 -/missing 7 bp showed the same performance (Fig.7c). Apoptosis was widespread throughout the body in ddx19 -/at 24 hpf (Fig.S1a), while the apoptosis signal decreased in ddx19 -/at 48 hpf than that at 24 hpf (Fig.S1b). At 48hpf, confocal imaging showed that unlike cell proliferation in wildtype groups (Fig.8a), partial embryos of ddx19 -/sibling were detected a decrease in the number of cells in S-phase, suggesting that cell proliferation may be blocked in ddx19 +/- (Fig.8b), and the number of cells in S-phase was significantly decrease in ddx19 -/-, indicating that cell proliferation was significantly blocked (Fig.8c). Our results were confirmed by repeated experiments ( Fig.S2a-b). The detection results of 60hpf were also consistent with 48hpf ( Fig.S2c).

Discussion
Here we have shown that the deletion of ddx19 causes abnormal apoptosis and proliferation in zebrafish embryos, and subsequently death. The phenotypes from our ddx19 -/are similar to that of homozygous ddx19 mutants (ddx19 hi1464/hi1464 ), resulting in phenotypic defects and homozygous death [28]. In addition to these findings, we found that the absence of DDX19 caused in the arrest of cells in the S-phase. The ddx19 -/appeared to have more serious defects in body development, and this could be a reason of early death in embryos. It is also possible that abnormality of apoptosis and cell proliferation contributed to deformity of embryonic development and death in ddx19 -/-.
The mechanism for causing changes in apoptosis and cell proliferation is not known. Here, we suggest a possible mechanism which DNA damage results in apoptosis and abnormal cell proliferation. Some studies have shown that R-loop may be a major source of replication stress and genomic instability, leading to DNA damage responses [29][30][31]. The regulation of R-loop levels is complex and varied.
Depletion of RNase H activity impairs the removal of R-loop in saccharides, causing DNA damage [32].
Helicase DHX9 interacts with PARP1 to prevent R-loop dependent DNA damage. The integrity of DNA is the key to the survival and reproduction of organisms [33]. Therefore, the repair of DNA damage is very important. Many researchers have found that DNA damage response is often accompanied by apoptosis and abnormal cell proliferation [34][35][36][37][38]. The DNA damage checkpoint kinases Chk1 and Chk2 are closely related to apoptosis and cell cycle [39]. Overexpression of E2F3a induces DNA damage, leading to ATM-dependent apoptosis [40]. Here, we focused on the functionality of Ddx19.
According to the report that Ddx19 cleared the R-loop caused by DNA damage to stabilized genomic DNA in HeLa cells [31]. Ddx19 acts as a bridge between mRNA nuclear export and transcription and replication, inhibiting genomic instability after DNA damage in proliferating cells [41]. Combined with our findings on its role in the process of apoptosis and proliferation, we speculate that ddx19 deletion induces DNA damage due to the R-loop accumulation. This leads to widespread cell apoptosis and proliferation abnormalities, leading to death of zebrafish embryos and larvae. To test this hypothesis, the next work can be perform by construct a fusion protein of the DNA-RNA hybrid binding domain to further examine whether R-loop accumulated in ddx19 -/-, and to detect and compare the expression of DNA damage checkpoint protein kinases in wildtype and ddx19 -/-.
Many members of the DEAD-box family are known to regulate development [42][43][44][45][46]. For example, Ddx27 deletion blocked proliferation and regeneration ability of skeletal muscle in zebrafish [42]. Loss of Ddx18 results in G1 cell-cycle arrest, and specifically regulates the amount of primitive myeloid and erythroid cells [45]. Unlike Ddx19, Ddx27 and Ddx18 are mainly involved in the ribosomal biogenesis process. Ddx46 is related to the splicing of pre-mRNA, and is required for development of digestive organs and brain as well as differentiation of hematopoietic stem cells in zebrafish [43,44].
Ddx39ab, also known as a nucleoplasm transport factor, specifically participates in myocyte and lens development in zebrafish, and its deletion blocked mRNA splicing of members of the kmt2 gene family [46]. Similar to ddx19 -/-, the homozygous death of ddx39ab occurs at an early developmental stage. But the defect phenotypes caused by ddx19 -/are more extensive and severe than those caused by of ddx39ab -/-. Nucleoplasm transport factor DDX3 maintains cell cycle and genome stability in HCT116 [28], and our observation of the roles of Ddx19 during development in vivo was similar to it. Therefore, we speculated that the nucleoplasm transport factor in the DEAD-box family may be involved in the regulation of apoptosis and proliferation, which provided a theoretical reference for our further understanding and classify of the family.

Conclusion
We obtained ddx19 heritable effective mutants by CRISPR/Cas9 genome editing technology in zebrafish. The ddx19 -/were lethal. ddx19 knockout caused severe deformity, up-regulation in apoptosis, a significant decrease in the number of cells in the S-phase, and largely blocking of cell proliferation. We conclude that Ddx19 is involved in the regulation of cell apoptosis and proliferation, thus affecting the normal development process in zebrafish embryos and larvae.

Zebrafish source and maintenance
The experimental wildtype zebrafish was the AB strain, purchased from Shanghai Institute of primer was universal primer, which sequences was 5'-AAAAAAAGCACCGACTCGGTGCCAC-3'. We designed the targeting sequence consistent with previous studies [19]. gRNA was synthesized by in vitro transcription, and co-injected with Cas9 protein into the animal pole at the one-cell stage. The final concentration of gRNA for injection was 100 ng/μL and Cas9 protein was 800 ng/μL, and the injection volume was 1nL. We washed away the TUNEL reaction solution using 1x hoechst ® 33342 solution incubation 2 h at 37°C . Finally, samples were fixed with 1%-1.5% low melting glue and photographed under a confocal microscope.

Edu labeling
Edu labeling was carried out as described [42]. We analyzed the proliferating cells in zebrafish embryos using Click-iT ® Plus Edu Imaging Kits (Invitrogen, C10639). Embryos of 24 dpf were incubated for 3 h at a concentration of 400 μm Edu. We removed the Edu label solution, collected embryos at 48 hpf and 60 hpf, fixed with 4% PFA-PBS overnight at 4 °C. Then, the samples were incubated with protease k for 15-30 min at room temperature. The samples were fixed again for 20 min at room temperature. The detection reaction solution is proportionally configured according to the requirements of the instruction, and fully mixed. Samples were incubated overnight at 37 °C. We removed the reaction solution, and incubated the samples in 1x hoechst ® 33342 solution (final concentration 5 g/mL) for 2 h at 37 °C. Samples were fixed with 1%-1.5% low melting glue. Samples were examined with confocal photography. Imaging Images were obtained using Zeiss Axio Imager2. A Leica TCS SP8 laser scanning confocal microscope was used for fluorescence imaging.

Statistical analysis
All experimental results were from three or more biological replicates. Phenotypic analysis was performed using the 2 -test. Student t-test was used for the pairwise difference analysis. Results were considered significant when p<0.05. Declaration plasmid. We thank Jing Xie for maintenance of the laboratory and the fish care and members of our laboratory for comment and modify on the writing.

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