Abstract
Monocyte chemoattractant protein-induced protein 1 (MCPIP1), also called Regnase-1, is an RNase that has been described as a key negative modulator of inflammation. MCPIP1 also controls numerous tumor-related processes, such as proliferation, apoptosis and differentiation. In this study, we utilized a zebrafish model to investigate the role of Mcpip1 during embryogenic development. Our results demonstrated that during embryogenesis, the expression of the zc3h12a gene encoding Mcpip1 undergoes dynamic changes. Its transcript levels gradually increase from the 2-cell stage to the spherical stage and then decrease rapidly. We further found that ectopic overexpression of wild-type Mcpip1 but not the catalytically inactive mutant form resulted in an embryonic lethal phenotype in zebrafish embryos (24 hpf). At the molecular level, transcriptomic profiling revealed extensive changes in the expression of genes encoding proteins important in the endoplasmic reticulum stress response and in protein folding as well as involved in the formation of primary germ layer, mesendoderm and endoderm development, heart morphogenesis and cell migration. Altogether, our results demonstrate that the expression of zc3h12a must be tightly controlled during the first cell divisions of zebrafish embryos and that a rapid decrease in its mRNA expression is an important factor promoting proper embryo development.
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Introduction
Implantation of the blastocyst is necessary for embryonic development to occur. In mammals, uterine implantation is associated with numerous structural and molecular changes in the luminal epithelia1. Interestingly, this process is closely correlated with the inflammatory mechanism, where during the attachment of blastocysts to the uterus, modulations in the expression of different growth factors, chemokines and cytokines, such as TNF, IL-6 and prostaglandin E2 (PGE2), are observed, as well as vascularization and infiltration of immune cells from the blood to the endometrial tissue2,3,4,5. The inflammatory process is essential for implantation, but during the next stage of pregnancy, anti-inflammatory factors are induced to prevent rejection of the fetus. Thus, proper embryonic development is strictly dependent on optimal microenvironmental conditions. This microenvironment must provide optimal temperature, oxygen tension, pH, and nutrients to ensure embryo survival. Stress during the embryonic period may impair fetal development and result in embryo mortality6,7.
In human, monocyte chemoattractant protein-induced protein 1 (MCPIP1), also known as Regnase-1 and encoded by the ZC3H12A gene, possesses a PilT N-terminus domain (PIN) that exerts RNAse properties. It has been shown that MCPIP1 degrades transcripts coding for mediators of inflammation: IL-1β8, IL-69, IL-12p4010 and IL-211 and its own transcript10. Further studies have indicated that MCPIP1 also plays an important role in the suppression of miRNA activity and biogenesis via cleavage of the terminal loops of precursor miRNAs (pre-miRNAs), counteracting Dicer, a key ribonuclease in miRNA processing12. MCPIP1 transcript expression is induced by Toll-like receptor (TLR) ligands9,13 and proinflammatory cytokines, such as IL-1β and TNFα8.
In vivo studies have shown that MCPIP1 plays a critical role in preventing autoimmune conditions. Zc3h12a knockout mice display severe anemia, augmented serum immunoglobulin levels and autoantibody production. In macrophages of Zc3h12a-/- mice, elevated expression levels of IL-6, IL-12p40 and IL-1β were observed9,14. Changes in the immune response of knockout mice are not only the result of MCPIP1 involvement in mRNA decay and miRNA biogenesis but also of its influence on the activity of some transcription factors. To date, MCPIP1 has been confirmed to negatively regulate the activity of NF-ĸB and AP1, which is essential in the regulation of the synthesis of mediators controlling inflammatory and immune responses14,15,16,17.
In addition, evidence indicates that MCPIP1 is involved in cell cycle arrest18, apoptosis19 and regulation of cell differentiation20,21,22. Thus, we hypothesized that MCPIP1 might safeguard early development and regulate crucial processes in the early stages of embryonic development, such as cell differentiation, cell division, apoptosis, angiogenesis, and regulation of inflammatory processes. A zebrafish (Danio rerio) model was utilized, which is a powerful vertebrate platform widely used to investigate developmental processes. First, we investigated whether the temporal expression pattern of the zc3h12a transcript, encoding Mcpip1, is changed during embryogenesis. Subsequently, we determined how the modulation of Mcpip1 levels affects the early development of zebrafish.
Results
The zebrafish genome contains four unique members of the PIN domain-like superfamily
To begin investigating the potential function of the Mcpip1 RNase in zebrafish, we performed bioinformatic searches and alignments. According to the Ensembl and NCBI databases, the zebrafish genome contains five members of the PIN domain-like superfamily (Fig. 1a,b), also recently showed by Yang et al.23. The full-length zebrafish zc3h12a sequence (XM_021466808.1; Gene ID: 798235; NCBI) encodes a 579 amino acid Mcpip1 protein with high (82.90%) amino acid identity to common carp Mcpip1 and intermediate (~ 50%) amino acid identity to amphibian, reptilian, bird and mammalian MCPIP1. The maximum likelihood (ML) phylogenetic tree for MCPIP1 proteins (Supplementary Fig. S1) demonstrates that the fish species cluster together and form a clade separate from the nonfish species. However, a direct comparison of the zebrafish orthologs of the human PIN-domain like-superfamily members indicated that zebrafish and human MCPIP1 proteins possess a similar domain structure and 87.79% identity within the PIN domain (Fig. 1c, Supplementary Fig. S2). The most highly conserved region is the PIN domain, the catalytic center of Mcpip1 (Fig. 1d).
The zebrafish ortholog of ZC3H12A is downregulated during early embryonic development
To investigate the temporal expression pattern of the zebrafish zc3h12a gene during early embryonic development, quantitative real-time PCR (qRT‒PCR) was performed on whole embryo-derived RNA. One-cell stage fertilized zebrafish eggs were collected and incubated at a standard temperature of 28 °C as described in the methods. The embryos were monitored for cell division and collected at different developmental periods over 72 h. As expected, the first divisions were observed synchronously at ~ 20-min intervals (Fig. 2a)24. For the isolation of RNA, ~ 10 embryos were pooled at each timepoint.
We found that the zc3h12a gene follows dynamic changes in expression during early zebrafish development. The level of zc3h12a mRNA gradually increases during the first embryonic divisions, peaking at the sphere stage (4 h post fertilization [hpf]) and then significantly rapidly declining during transition into the shield stage and 1-day embryo (Fig. 2b and Supplementary Fig. S3a). In addition, we analyzed the global transcriptomic RNASeq data by White et al.25, which provides information about gene expression changes during early zebrafish embryogenesis. Both approaches showed a similar trend of zc3h12a gene expression fluctuations during zebrafish embryogenesis (Fig. 2c).
Transient thermal shock during early zebrafish development enhances the expression of zc3h12a mRNA
We next sought to investigate whether an external shock during early development affects the expression of the zc3h12a gene. The thermal stimulation approach was performed, due to the fact that the more commonly used zebrafish infection/inflammation models could not be efficiently utilized to monitor processes in early embryos (0–24 hpf)26. Synchronous populations of 2–4 cell embryos that had been cultured at 28 °C were subjected to a 30-min thermal shock at 4 °C (cold shock) or 37 °C (heat shock). Control embryos were kept at a standard temperature of 28 °C. After a transient temperature shift, the embryos were incubated at the standard 28 °C and collected at the indicated timepoints for RNA analysis (Fig. 3a).
We ensured that the developmental stages of embryos subjected to cold or heat shock followed similar timing as the control siblings (Fig. 3b) so the analysis of zc3h12a gene expression was not biased by a potential shift of developmental stage. Transient thermal shocks did not impair the overall embryonic survival rates at the 24-h stage (Fig. 3c).
Quantitative RT‒PCR analysis revealed that the expression of zc3h12a was significantly enhanced in response to the 30 min cold shock, reaching levels ~ 2.5-fold higher than those in the control embryos at the shield stage (6 hpf; Fig. 3d). The 37 °C shock suggested a similar tendency on the zc3h12a mRNA expression level at the sphere stage (4 hpf), with a ~ 1.5-fold increase (with adj. p value 0,097; Fig. 3e).
Overexpression of zc3h12a impairs early embryonic development in zebrafish
Zebrafish Mcpip1 was overexpressed by microinjection of in vitro transcribed mRNA encoding the zebrafish Mcpip1-P2A-mTurquoise protein into the yolks of one-cell stage embryos. Expression of Mcpip1 was tied to mTurquoise expression via self-cleaving P2A peptide. This strategy enabled visualization of exogenously expressed protein. For overexpression of catalytically inactive Mcpip1, one of the four conserved aspartic acid residues within the RNase domain (Fig. 1d) was substituted with asparagine (Mcpip1-D112N, herein Mcpip1 DN). It has previously been shown that a single mutation in the catalytic center of Mcpip1 is sufficient to completely abolish its RNase activity9. As a control, a construct encoding only P2A-mTurquoise was used (Fig. 4a). Prior to microinjection, agarose electrophoresis was performed, which indicated high quality of in vitro transcribed mRNAs (see Supplementary Fig. S3b).
Embryos were sampled at 4, 6 and 24 h after microinjection. Exogenous zc3h12a mRNA was clearly detected at the sphere (4 hpf) and shield (6 hpf) stages. We also found that the microinjection procedure itself did not affect the kinetics of the zc3h12a mRNA expression pattern at those two developmental stages (Fig. 4b).
We further confirmed that mTurquoise expressed from microinjected mRNA was detected in all modified embryos at the shield stage (6 hpf; Fig. 4c). We also observed that at this stage, overexpression of wild-type (WT) but not the RNase-dead mutant did not affect embryo morphology (Fig. 4d). However, embryonic lethality increased by ~ 10% (Fig. 4e). Consistent with this finding, 24 h after microinjection, most Mcpip1-overexpressing embryos showed gross morphological abnormalities; thus, the overall lethality rate was significantly higher than that of the control embryos (Fig. 4f–g).
Increased activity of Mcpip1 leads to profound transcriptomic changes in developing zebrafish embryos
Transcriptomic profiling was performed on RNA isolated from viable Mcpip1 WT and Mcpip1 DN-overexpressing embryos collected at the shield stage (n = 3 of ~ 10 pooled embryos per condition). Differentially expressed genes (DEGs) were defined with a threshold of p value < 0.05 and fold change > 1.5. Accordingly, the expressions of 247 genes were significantly upregulated and those of 303 genes were downregulated in zebrafish embryos overexpressing WT Mcpip1 compared to those overexpressing the Mcpip1 DN mutant (Fig. 5a).
Gene Ontology (GO) enrichment analyses revealed that upregulated DEGs were significantly enriched in biological processes (BP) mostly related to the response to protein folding and endoplasmic reticulum stress, examples of which are calr3a (calreticulin 3a), edem 2 (ER degradation enhancer) and hsp70 (heat shock protein 70) family genes (Fig. 5b,c).
In contrast, the downregulated DEGs were enriched in pathways related to endoderm development, left/right pattern formation, organ morphogenesis and formation of the primary germ layer and ectodermal placode development (Fig. 5d,e).
Oscillation of zc3h12a expression during early embryonic development in zebrafish inversely correlates with changes in nrarpa and rasl11b expression levels
Based on our RNASeq analysis, we next selected four significantly downregulated DEGs in zebrafish embryos overexpressing WT Mcpip1: nrarpa (Notch-regulated ankyrin repeat-containing protein A), rasl11b (RAS-like family 11 member B), rhov (ras homolog family member V) and foxh1 (forkhead box H1). These DEGs were functionally assigned to the most significantly downregulated processes (Fig. 5d,e), and their expression levels were validated by qRT‒PCR (Fig. 6a).
In the next step, the expression patterns of two transcripts, nrarpa and rasl11b, were analyzed over the course of zebrafish embryogenesis by qRT‒PCR (Fig. 2a). We found that their expression levels undergo extensive changes during the transition from 64-cell to 1-day embryos (Fig. 6b). Moreover, the relative expression levels of those genes peaked at the shield stage, which correlates with the time at which zc3h12a expression profoundly decreases (Fig. 2b).
Discussion
Zebrafish (D. rerio) is a leading model for studying developmental biology because the genome is known, fertilization occurs externally, and development is very fast. At 48 h after fertilization, zebrafish embryos form complete organ systems, including the heart, intestine and blood vessels. In addition, embryos are transparent and develop outside the uterus, making it even easier to track developmental stages24,27.
MCPIP1 expression is inducible, and mRNA levels change rapidly after stimuli associated with cell differentiation, stress induction or pathogen infection8,9,28. Consequently, an increase in MCPIP1 is correlated with a decrease in the levels of many transcripts encoding regulators of inflammation, regulators of cell differentiation and division, and regulators of apoptosis and angiogenesis. Here, we wanted to test the role of MCPIP1 in embryological development. Comparison of the amino acid sequences showed differences between human MCPIP1 and its zebrafish ortholog. However, the PIN domain has high homology with the conserved codons encoding four acidic amino acid residues that form the putative active site, which is essential for the ribonucleolytic activity of MCPIP1.
We found that the expression of zc3h12a is tightly controlled within the first cell divisions of zebrafish. Mcpip1 mRNA levels increase from the 2-cell stage up to the sphere stage (6 h after fertilization); later, its level drops dramatically. Thus, starting in the sphere stage, Mcpip1-dependent transcript level regulation is silenced. Relatively high expression of Mcpip1 during first hours post fertilization may suggest potential role of this RNase during the decay of maternal mRNA (which in zebrafish is completed by 6 hpf29), but this hypothesis requires further investigation.
MCPIP1 is encoded by an inducible gene that has the ability to rapidly respond to microenvironmental changes and responds quickly to various kinds of stress8,9,28,30,31. In this study, mild external stress was induced by a temporary change in temperature, which led to transient modulations of zc3h12a gene expression, with consequent effects on the RNA profile. To determine what consequences an increase in Mcpip1 transcript levels might have on embryonic development, we used zebrafish models overexpressing WT Mcpip1 or its mutant form containing a mutation in the active site of the PIN domain.
We found that ectopic expression of WT Mcpip1 resulted in a 10% death rate of zebrafish embryos at 6 h post-fertilization and almost 90% after 24 h, while the model expressing the inactive form of Mcpip1 resembled control models (uninjected zebrafish embryos or injected with an empty vector). These results provide evidence for a significant effect of Mcpip1 on the zebrafish embryo transcriptome. There are many studies in the literature showing that Mcpip1 regulates genes involved in apoptotic processes16,32,33,34, so high embryonic lethality may be a consequence of the dysregulation of transcripts involved in this process. In our model, activation of the endoplasmic reticulum (ER) stress response pathway observed in the Mcpip1-overexpressing embryos already at 6 hpf (Fig. 5c) could have provoked the activation of apoptotic cell death, as prolonged ER-stress is a common trigger of apoptosis35. Furthermore, Mcpip1-overexpressing embryos showed gross morphological abnormalities. Transcriptome analysis revealed expression differences in mRNA classes encoding proteins important for embryonic development. We observed that, among the genes that were activated, the largest group comprised genes encoding proteins important to the endoplasmic reticulum stress response and involved in protein folding. The activation of these genes may be an indirect consequence of the ectopic expression of Mcpip1 and the accumulation of this protein in embryonic zebrafish cells. However, among the many transcripts downregulated and thus potentially directly degraded by Mcpip1 were those involved in the formation of the primary germ layer, mesendoderm, ectoderm and endoderm development, heart morphogenesis and cell migration. Thus, the change in the expression levels of such developmentally important genes may explain why such high lethality was observed when Mcpip1 levels were elevated during early zebrafish development. For validation of RNA-Seq data by qRT-PCR, four significantly downregulated transcripts in zebrafish embryos, nrarpa (Notch-regulated ankyrin repeat-containing protein A), rasl11b (RAS-like family 11 member B), rhov (ras homolog family member V) and foxh1 (forkhead box H1) were selected. We also showed that the levels of nrarpa and rasl11b inversely correlate with fluctuations in Mcpip1 mRNA levels during early stages of development (Fig. 6b). We hypothesize that a rapid decline of Mcpip1 expression level observed at shield stage (6 hpf) contributes to a profound upregulation of nrarpa, rasl11b and possibly other developmentally important target mRNAs at this stage.
NRARP is a component of the Notch signaling pathway and participates in embryonic development in vertebrates by regulating the segmentation of the body axis. The importance of this transcript in the segmentation process has been documented in lower and higher vertebrates36,37,38. The rasl11b gene, on the other hand, encodes a small GTPase protein that is highly conserved among vertebrates; it is expressed in mesendodermal cells, and its expression is controlled by the Nodal pathway. Nodal signaling controls the expression of conserved mesendodermal transcription factors39. Interestingly, it has been shown that rasl11b knockdown induces a specific “curly tail down” phenotype in zebrafish39. Similarly, ectopic overexpression of WT Mcpip1 resulted in embryo malformations, including tail malformations (Fig. 4e). The third highly-downregulated transcript, rhov, is involved in the signal transduction of the Rho pathway, which is essential for the regulation of gastrulation and neurulation, two major developmental processes of early embryogenesis40,41. In addition, our analysis identified many other genes whose levels are directly or indirectly dependent on Mcpip1, e.g., teratocarcinoma-derived growth factor 1 (tdgf1), described as an important regulator in the development of the cardiac tube in mouse embryogenesis42, and forkhead box protein H1 (foxh1), essential during zebrafish gastrulation and head and dorsal axis formation43. Our RNAseq data also showed a decreasing trend of neurogenin 1 (neurog1) expression (p value = 0.077), a key factor directing specialization of neuroectoderm44 in 6 hpf embryos overexpressing active Mcpip1 (Supplementary Fig. S3c), which additionally proves observed lethal phenotype.
In conclusion, our studies in the zebrafish model showed that the Mcpip1 level is tightly regulated during embryonic development, while even transient stress leads to rapid induction of the zc3h12a gene. Consequently, elevated expression of the zc3h12a gene leads to abnormalities in zebrafish development as a result of altered levels of transcripts involved in processes important in embryogenesis. It can be speculated that stress leading to induction of the gene encoding Mcpip1/MCPIP1 will have similar consequences on the developing embryo in higher vertebrates, including humans.
Materials and methods
Phylogenetics and bioinformatics
To find MCPIP1 orthologs in the zebrafish genome (GRCz11), the Ensembl database was searched for genes containing a Zc3h12a-like NYN domain. Sequences were retrieved from the SwissProt, EMBL and GenBank databases using SRS and/or BLAST (Basic Local Alignment Search Tool)45. Amino acid sequence alignment was performed using the Clustal Omega program at EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/clustalo). Phylogenetic trees were constructed on the basis of amino acid differences by the maximum likelihood (ML) method with 500 bootstrap replications using Molecular Evolutionary Genetics Analysis (MEGA) version 1146. For metadata analysis of zc3h12a mRNA expression profile during zebrafish development, the RNA-seq dataset provided by the Busch-Nentwich lab and available at Expression Atlas, was used25.
Zebrafish husbandry
Zebrafish embryos/larvae were obtained by the natural spawning of adult zebrafish (line AB/TL), which were housed in a continuous recirculating closed-system aquarium with a light/dark cycle of 14/10 h at 28 °C. Larvae were incubated in E3 medium at 28 °C according to standard protocols47. The Jagiellonian University Zebrafish Core Facility (ZCF) is a licensed breeding and research facility (District Veterinary Inspectorate in Krakow registry; Ministry of Science and Higher Education record no. 022 and 0057).
Eithics statement
All experiments were conducted in accordance with the European Community Council Directive 2010/63/EU for the Care and Use of Laboratory Animals of Sept. 22, 2010 (Chapter 1, Article 1 no.3) and National Journal of Law act of Jan. 15, 2015 for Protection of animals used for scientific or educational purposes (Chapter 1, Article 2 no.1). All methods involving zebrafish embryos/larvae were in compliance with ARRIVE guidelines. The works with genetically modified microorganisms were authorized by the Polish Ministry of the Environment (No. 179/2021).
Cloning
For overexpression experiments, the full length mTurquoise cDNA was amplified by PCR from the p3E-p2a-mTurquoise plasmid (a gift from David Tobin; Addgene plasmid #135213) via the StuI and XbaI sites of the pCS2 expression vector (a gift from Amro Hamdoun48; Addgene #34931) to generate pCS2-P2A-mTurquoise (control plasmid), as presented in the schematic (Fig. 4a). Then, the cDNA coding for full length Mcpip1 (XM_021466808) was amplified by PCR to introduce EcoRI and EcoRV and cloned into the EcoRI and StuI sites of the pCS2-P2A-mTurquoise plasmid to generate the Mcpip1 WT plasmid. To clone vectors containing sequences encoding catalytically inactive Mcpip1, site-directed mutagenesis of D112 into N112 was performed. The sequences of the primers used for cloning are listed in Supplementary Table S1.
In vitro transcription
The pCS2 vectors (Control, Mcpip1 WT and Mcpip1 DN) were linearized with NotI digestion and cleaned using the PCR Mini Kit (Syngen). Then, the mRNAs were synthesized in vitro from the SP6 promoter using the mMESSAGE mMACHINE SP6 kit (Ambion AM1340) according to the manufacturer’s protocol. Transcribed mRNAs were purified using an RNA Clean & Concentrator kit (Zymo Research). A NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) was used to calculate the concentration of mRNA, which was then diluted to a concentration of 200 ng/µl. The integrity of mRNA was also confirmed by denaturing agarose electrophoresis.
Microinjection of mRNA into fertilized zebrafish eggs
For overexpression experiments, 3.5 µl (700 ng) of each mRNA was mixed with 0.5 µl phenol red (Merck, P0290) and microinjected into the yolks of approximately 100 zebrafish eggs at the one-cell stage using a WPI Picopump PV820 microinjector (2 nl). The microinjected embryos were collected at 4 and 6 hpf for further RNA analysis.
RNA isolation and quantitative PCR
For RNA isolation, ~ 10 zebrafish embryos were collected in fenozol (A&A Biotechnology), frozen and stored at -80 °C. Then, the embryos were homogenized using a homogenizer (OMNI International), and total RNA was extracted using the phenol–chloroform method. cDNA was synthesized using M-MLV reverse transcriptase (Promega), and quantitative real-time PCR was performed with SYBR Green Master Mix (A&A Biotechnology) and a QuantStudio3 thermocycler (Thermo Fisher Scientific). Rps11, eef1, acbt2 and rpl13a were used as a reference genes49,50 The primer sequences are listed in Supplementary Table S1. To validate specificity of the qRT-PCR reaction, melt curve analysis was performed at the end of each assay. Agarose gel electrophoresis was performed to ensure presence of a single product of predicted length at the end of the qRT-PCR reaction (Supplementary Fig. S3d).
RNA sequencing
The poly(A) mRNA fraction from total RNA was isolated with a Dynabeads mRNA DIRECT Micro Kit (Thermo). The sequencing library for each RNA sample was prepared according to the protocol provided by the manufacturer using the Ion Total RNA-Seq Kit v2 (Thermo). The libraries were generated from 1–15 ng of mRNA by fragmenting the mRNA with RNaseIII, purifying the fragmented RNA, and hybridizing and ligating it with Ion adaptors. Subsequently, the RNA products were reverse transcribed and amplified to double-stranded cDNA and then purified using a magnetic bead-based method. The molar concentration and size of each cDNA library were determined using the DNA HS Kit on a Bioanalyzer 2100 (Agilent). Each library was diluted to ~ 53 pM before template preparation. Up to three barcoded libraries were mixed in equal volume and used for automatic template preparation on the Ion Chef instrument (Thermo) using reagents from the Ion PI Hi-Q 200 Kit (Thermo) and Ion PI v3 Proton Chip. All samples were sequenced on the Ion Proton System (Thermo) according to the manufacturer’s instructions.
Signal processing and base calling were performed with Torrent Suite version 5.14.0. Raw reads were mapped to D. rerio Ensembl genome version GRCz11 using STAR (version 2.7.10a)51 and bowtie2 (version 2.4.4)52 for unmapped reads. Gene counts were created with htseq-count53 using the Ensembl gene model. Differential expression was analyzed with DESeq2 version (version 1.40.1). RNA sequencing data were deposited in the GEO repository (under accession no: GSE232220).
Functional annotation of DEGs (fold change > 1.5 and p value < 0.05) was performed using the R package ClusterProfiler version 4.454. Gene lists were searched using the Entrez gene annotation (ENTREZ_GENE_ID), with the D. rerio background dataset used for analyses. Volcano plots and dot plots were created using the ggplot2 libraries in R.
Imaging
Each stage of zebrafish embryo development was observed and photographed under an inverted microscope (Leica DMi1 under Flexacam C1). The fluorescence signal from mTurquoise-fused protein was observed under a fluorescence stereomicroscope (Zeiss Discovery V12 with a PentaFluar S filter slider equipped with a Zeiss Axiocam 705 mono camera). The excitation wavelength was 436 nm, and the emission wavelength was 480 nm.
Statistics
All graphs were created using CorelDRAW 2021 (Corel Corporation), and all statistical analyses, including unpaired t-test and one-way ANOVA followed by Tukey`s multiple comparisons test, were performed using GraphPad Prism 8 (GraphPad Software).
Data availability
Sequencing data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE232220. Any additional data are available from the corresponding author upon reasonable request.
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Acknowledgements
We would like to thank Dariusz Gajdzinski for his help with zebrafish maintenance and breeding. We also thank Mateusz Wilamowski for his help with cloning.
Funding
The research for this publication has been funded by MNS 5/2021 (to W.S.). T.K.P. was supported by Polish National Agency for Academic Exchange under Polish Returns 2019 project (Grant No.: PPN/PPO/2019/1/00029/U/0001). Open access publication of this article was funded by the Ministry of Education subsidy.
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A.L.-C., J.J., T.K.P., K.R. and M.C. designed the study. A.L.-C. and W.S. performed the experiments and analysed data. N.P. performed microinjection. M.K., M.D and M.M. performed RNASeq analyses. A.L.-C. and J.J. wrote the main manuscript text; W.S., K.R., M.C. and T.K.P. edited it. W.S. prepared figures. A.L.-C., W.S. and T.K.P. contributed equally to this work. All authors reviewed and approved the manuscript.
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Lichawska-Cieslar, A., Szukala, W., Prajsnar, T.K. et al. MCPIP1 functions as a safeguard of early embryonic development. Sci Rep 13, 16944 (2023). https://doi.org/10.1038/s41598-023-44294-1
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DOI: https://doi.org/10.1038/s41598-023-44294-1
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