MCPIP1 functions as a safeguard of early embryonic development

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.

Full text

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MCPIP1 functions as a safeguard
of early embryonic development
Agata Lichawska‑Cieslar
1,6, Weronika Szukala
1,2,6, Tomasz K. Prajsnar
3,6,
Niedharsan Pooranachandran
3, Maria Kulecka
4,5, Michalina Dabrowska
5, Michal Mikula
5,
Krzysztof Rakus
3, Magdalena Chadzinska
3 & Jolanta Jura
1*
Monocyte chemoattractant protein‑induced protein 1 (MCPIP1), also called Regnase‑1, is an RNase
that has been described as a key negative modulator of inammation. MCPIP1 also controls numerous
tumor‑related processes, such as proliferation, apoptosis and dierentiation. In this study, we utilized
a zebrash 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
zebrash embryos (24 hpf). At the molecular level, transcriptomic proling 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 rst cell divisions of zebrash
embryos and that a rapid decrease in its mRNA expression is an important factor promoting proper
embryo development.
Implantation of the blastocyst is necessary for embryonic development to occur. In mammals, uterine implanta-
tion is associated with numerous structural and molecular changes in the luminal epithelia1. Interestingly, this
process is closely correlated with the inammatory mechanism, where during the attachment of blastocysts to
the uterus, modulations in the expression of dierent growth factors, chemokines and cytokines, such as TNF,
IL-6 and prostaglandin E2 (PGE2), are observed, as well as vascularization and inltration of immune cells from
the blood to the endometrial tissue2–5. e inammatory process is essential for implantation, but during the
next stage of pregnancy, anti-inammatory factors are induced to prevent rejection of the fetus. us, proper
embryonic development is strictly dependent on optimal microenvironmental conditions. is microenviron-
ment 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 inammation: 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 proinammatory 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
OPEN
1Department of General Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, Gronostajowa 7, 30-387 Kraków, Poland. 2Doctoral School of Exact and Natural Sciences, Jagiellonian
University, Lojasiewicza 11, 30-348 Kraków, Poland. 3Department of Evolutionary Immunology, Institute of
Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, Gronostajowa 9, 30-387 Kraków,
Poland. 4Medical Center for Postgraduate Education, Department of Gastroenterology, Hepatology and Clinical
Oncology, Marymoncka 99/103, 01-813 Warsaw, Poland. 5Maria Sklodowska-Curie National Research Institute of
Oncology, Roentgena 5, 02-781 Warsaw, Poland. 6
These authors contributed equally: Agata Lichawska-Cieslar,
Weronika Szukala and Tomasz K. Prajsnar. *email: jolanta.jura@uj.edu.pl
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decay and miRNA biogenesis but also of its inuence on the activity of some transcription factors. To date,
MCPIP1 has been conrmed to negatively regulate the activity of NF-ĸB and AP1, which is essential in the
regulation of the synthesis of mediators controlling inammatory and immune responses14–17.
In addition, evidence indicates that MCPIP1 is involved in cell cycle arrest18, apoptosis19 and regulation of cell
dierentiation20–22. us, we hypothesized that MCPIP1 might safeguard early development and regulate crucial
processes in the early stages of embryonic development, such as cell dierentiation, cell division, apoptosis,
angiogenesis, and regulation of inammatory processes. A zebrash (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 aects the early development of zebrash.
Results
The zebrash genome contains four unique members of the PIN domain‑like superfamily
To begin investigating the potential function of the Mcpip1 RNase in zebrash, we performed bioinformatic
searches and alignments. According to the Ensembl and NCBI databases, the zebrash genome contains ve
members of the PIN domain-like superfamily (Fig.1a,b), also recently showed by Yang et al.23. e full-length
zebrash 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. e maximum likelihood (ML) phylogenetic
tree for MCPIP1 proteins (Supplementary Fig.S1) demonstrates that the sh species cluster together and form
a clade separate from the nonsh species. However, a direct comparison of the zebrash orthologs of the human
PIN-domain like-superfamily members indicated that zebrash and human MCPIP1 proteins possess a similar
domain structure and 87.79% identity within the PIN domain (Fig.1c, Supplementary Fig.S2). e most highly
conserved region is the PIN domain, the catalytic center of Mcpip1 (Fig.1d).
The zebrash ortholog of ZC3H12A is downregulated during early embryonic development
To investigate the temporal expression pattern of the zebrash zc3h12a gene during early embryonic develop-
ment, quantitative real-time PCR (qRT‒PCR) was performed on whole embryo-derived RNA. One-cell stage
fertilized zebrash eggs were collected and incubated at a standard temperature of 28°C as described in the
Figure1. e zebrash ortholog of human MCPIP1 shares a similar domain structure. (a) List of genes
containing the Zc3h12a-like NYN domain in the zebrash genome based on the Ensembl database. (b) List
of genes containing the Zc3h12a-like NYN domain in the zebrash genome based on the NCBI database. (c)
Domain comparison of human MCPIP1 and its zebrash ortholog Mcpip1. UBA—ubiquitin-associated domain,
PRR—proline-rich region, PIN—PilT N-terminus nuclease domain, ZF—zinc nger motif, NDR—disordered
region, CTD—C-terminal conserved domain. (d) Alignment of the amino acid sequences of human and
zebrash PIN domains. Asterisk (*) denotes an identical residue, a colon (:) denotes conserved substitutions,
and a period (.) denotes semiconserved substitutions. e arrows indicate four aspartic acid residues, which are
critical for Mcpip1/MCPIP1 catalytic activity. Black rectangle indicates position of the D112N mutation within
the PIN domain of zebrash Mcpip1.
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methods. e embryos were monitored for cell division and collected at dierent developmental periods over
72h. As expected, the rst divisions were observed synchronously at ~ 20-min intervals (Fig.2a)24. For the isola-
tion of RNA, ~ 10 embryos were pooled at each timepoint.
We found that the zc3h12a gene follows dynamic changes in expression during early zebrash development.
e level of zc3h12a mRNA gradually increases during the rst embryonic divisions, peaking at the sphere
stage (4h post fertilization [hpf]) and then signicantly 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 zebrash
embryogenesis. Both approaches showed a similar trend of zc3h12a gene expression uctuations during zebrash
embryogenesis (Fig.2c).
Transient thermal shock during early zebrash development enhances the expression of
zc3h12a mRNA
We next sought to investigate whether an external shock during early development aects the expression of the
zc3h12a gene. e thermal stimulation approach was performed, due to the fact that the more commonly used
zebrash infection/inammation models could not be eciently 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. Aer a transient temperature shi, 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 shi 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 signicantly 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). e 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).
Figure2. e expression of zc3h12a is tightly controlled during early embryonic development in zebrash.
(a) One-cell fertilized eggs were incubated under standard conditions and collected at the indicated timepoints
for RNA analysis. Representative pictures of zebrash embryos at various developmental stages are shown.
(b) Real-time PCR analysis of the zc3h12a expression level at dierent developmental stages of zebrash. (c)
Graph represents the level of zc3h12a mRNA based on the RNA-Seq data (PMID: 29,144,233). For comparison
of both datasets, mean values of zc3h12a mRNA levels from qRT-PCR were also shown (normalized values of
data presented on Fig.2a). In both cases the level of zc3h12a mRNA was normalized to the expression level at
2-cell stage (0.75 hpf). hpf—hours post fertilization n = 3. Rps11 was used as a reference gene. Data represent
the mean ± SD, ****p < 0.0001 by one-way ANOVA (only comparisons with p < 0.0001 are shown). Scale
bar = approx. 250µm.
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Overexpression of zc3h12a impairs early embryonic development in zebrash
Zebrash Mcpip1 was overexpressed by microinjection of invitro transcribed mRNA encoding the zebrash
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. is 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sucient to com-
pletely 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 tran-
scribed mRNAs (see Supplementary Fig. S3b).
Embryos were sampled at 4, 6 and 24h aer 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
aect the kinetics of the zc3h12a mRNA expression pattern at those two developmental stages (Fig.4b).
We further conrmed that mTurquoise expressed from microinjected mRNA was detected in all modied
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 aect embryo morphology (Fig.4d). However, embryonic lethality
increased by ~ 10% (Fig.4e). Consistent with this nding, 24h aer microinjection, most Mcpip1-overexpressing
embryos showed gross morphological abnormalities; thus, the overall lethality rate was signicantly higher than
that of the control embryos (Fig.4f–g).
Increased activity of Mcpip1 leads to profound transcriptomic changes in developing zebrash
embryos
Transcriptomic proling was performed on RNA isolated from viable Mcpip1 WT and Mcpip1 DN-overexpress-
ing embryos collected at the shield stage (n = 3 of ~ 10 pooled embryos per condition). Dierentially expressed
genes (DEGs) were dened with a threshold of p value < 0.05 and fold change > 1.5. Accordingly, the expressions
of 247 genes were signicantly upregulated and those of 303 genes were downregulated in zebrash embryos
overexpressing WT Mcpip1 compared to those overexpressing the Mcpip1 DN mutant (Fig.5a).
Gene Ontology (GO) enrichment analyses revealed that upregulated DEGs were signicantly 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, le/
right pattern formation, organ morphogenesis and formation of the primary germ layer and ectodermal placode
development (Fig.5d,e).
Figure3. Eect of transient thermal shock on the expression of zebrash zc3h12a during early embryonic
development. (a) Zebrash embryos that were incubated at standard 28°C temperature for 1h were subjected
to a transient 30-min heat shock (37°C) or cold shock (4°C) as indicated on the schematic diagram. Embryos
were collected for RNA analysis at the indicated timepoint. (b) Representative pictures of zebrash embryos at
4 hpf. c. Percentages of live and dead embryos at 24 hpf. (d,e) Real-time PCR analysis of the zc3h12a expression
level in zebrash embryos subjected to each thermal shock. e arrow on the X-axis indicates the onset of the
30min thermal shock. hpf—hours post fertilization. n = 4. Rps11 was used as a reference gene. Data represent
the mean ± SD, **p < 0.01 by an unpaired t-test. Scale bar = approx. 250µm.
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Oscillation of zc3h12a expression during early embryonic development in zebrash inversely
correlates with changes in nrarpa and rasl11b expression levels
Based on our RNASeq analysis, we next selected four signicantly downregulated DEGs in zebrash 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). ese DEGs were
functionally assigned to the most signicantly 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 zebrash 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
Zebrash (D. rerio) is a leading model for studying developmental biology because the genome is known, fertiliza-
tion occurs externally, and development is very fast. At 48h aer fertilization, zebrash 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.
Figure4. Overexpression of catalytically active Mcpip1 impairs early development in zebrash. (a) Schematic
diagram of control and Mcpip1 overexpression constructs. e sequences were cloned into the pCS2 expression
vector. (b) Real-time PCR analysis of zc3h12a expression levels in uninjected and microinjected control,
Mcpip1 WT or Mcpip1 DN mRNA zebrash embryos at 4 hpf and 6 hpf. n = 3. (c) Representative uorescence
(mTurquoise) images of embryos at 6 hpf. (d) Representative bright eld images of embryos at 6 hpf. (e)
Percentages of live and dead embryos at 6 hpf. (f) Representative bright eld images of embryos at 24 hpf. (g)
Graph indicating the percentages of live and dead embryos at 24 hpf. n = 3. Rps11 was used as a reference gene.
Data represent the mean ± SD, *p < 0.05, **p < 0.01, ****p < 0.0001 by an unpaired t-test (b) or one-way ANOVA
(d,f). Scale bar = approx. 250µm.
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MCPIP1 expression is inducible, and mRNA levels change rapidly aer stimuli associated with cell dier-
entiation, 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 inammation, regulators of cell dierentia-
tion 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 dierences between human
MCPIP1 and its zebrash 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 ribonu-
cleolytic activity of MCPIP1.
We found that the expression of zc3h12a is tightly controlled within the rst cell divisions of zebrash.
Mcpip1 mRNA levels increase from the 2-cell stage up to the sphere stage (6h aer fertilization); later, its level
drops dramatically. us, starting in the sphere stage, Mcpip1-dependent transcript level regulation is silenced.
Relatively high expression of Mcpip1 during rst hours post fertilization may suggest potential role of this RNase
during the decay of maternal mRNA (which in zebrash 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
Figure5. Transcriptome analysis of zebrash embryos overexpressing Mcpip1. (a) Volcano plot for the RNA-
Seq dataset indicating dierentially expressed genes with p value < 0.05 and fold change (FC) > 1.5 between
embryos microinjected with Mcpip1 WT and Mcpip1 DN mRNA (6 hpf). n = 3. (b) Gene Ontology (GO)
enrichment analysis of upregulated biological processes in Mcpip1 WT. (c) Heatmap illustrating the expression
levels of selected upregulated DEGs. (d) Gene Ontology (GO) enrichment analysis of downregulated biological
processes in Mcpip1 WT. (e) Heatmap illustrating the expression levels of selected downregulated DEGs.
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by a temporary change in temperature, which led to transient modulations of zc3h12a gene expression, with
consequent eects on the RNA prole. To determine what consequences an increase in Mcpip1 transcript levels
might have on embryonic development, we used zebrash 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 zebrash embryos at 6h
post-fertilization and almost 90% aer 24h, while the model expressing the inactive form of Mcpip1 resembled
control models (uninjected zebrash embryos or injected with an empty vector). ese results provide evidence
for a signicant eect of Mcpip1 on the zebrash embryo transcriptome. ere are many studies in the literature
showing that Mcpip1 regulates genes involved in apoptotic processes16,32–34, 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 abnormali-
ties. Transcriptome analysis revealed expression dierences 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 fold-
ing. e activation of these genes may be an indirect consequence of the ectopic expression of Mcpip1 and the
accumulation of this protein in embryonic zebrash 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. us, 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 zebrash development. For validation of RNA-Seq data
Figure6. Expression pattern of nrarpa, rasl11b, rhov and foxh1 genes during zebrash embryogenesis. (a)
Real-time PCR analysis of nrarpa, rasl11b, rhov and foxh1 expression levels in zebrash embryos overexpressing
Mcpip1 WT or Mcpip1 DN (6 hpf). (b) Real-time PCR analysis of nrarpa and rasl11b expression levels during
early embryonic development in zebrash. n = 3. Rps11 was used as a reference gene. Data represent the
mean ± SD, *p < 0.05, **p < 0.01, ****p < 0.0001 by an unpaired t-test (a) or one-way ANOVA (b). For (b) only
selected developmental stages have been compared.
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by qRT-PCR, four signicantly downregulated transcripts in zebrash 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 uctuations 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 ver-
tebrates by regulating the segmentation of the body axis. e importance of this transcript in the segmentation
process has been documented in lower and higher vertebrates36–38. e 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 mesendo-
dermal transcription factors39. Interestingly, it has been shown that rasl11b knockdown induces a specic “curly
tail down” phenotype in zebrash39. Similarly, ectopic overexpression of WT Mcpip1 resulted in embryo malfor-
mations, including tail malformations (Fig.4e). e 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 identied 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 zebrash 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 (Supplemen-
tary Fig. S3c), which additionally proves observed lethal phenotype.
In conclusion, our studies in the zebrash 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 zebrash 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 nd MCPIP1 orthologs in the zebrash 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/ clust alo).
Phylogenetic trees were constructed on the basis of amino acid dierences 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 prole during zebrash development, the RNA-seq dataset
provided by the Busch-Nentwich lab and available at Expression Atlas, was used25.
Zebrash husbandry
Zebrash embryos/larvae were obtained by the natural spawning of adult zebrash (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. e Jagiellonian University Zebrash
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 scientic or educational purposes (Chapter1,
Article 2 no.1). All methods involving zebrash embryos/larvae were in compliance with ARRIVE guidelines.
e works with genetically modied microorganisms were authorized by the Polish Ministry of the Environ-
ment (No. 179/2021).
Cloning
For overexpression experiments, the full length mTurquoise cDNA was amplied by PCR from the p3E-p2a-
mTurquoise plasmid (a gi from David Tobin; Addgene plasmid #135213) via the StuI and XbaI sites of the pCS2
expression vector (a gi from Amro Hamdoun48; Addgene #34931) to generate pCS2-P2A-mTurquoise (control
plasmid), as presented in the schematic (Fig.4a). en, the cDNA coding for full length Mcpip1 (XM_021466808)
was amplied 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. e sequences of the
primers used for cloning are listed in Supplementary TableS1.
In vitro transcription
e pCS2 vectors (Control, Mcpip1 WT and Mcpip1 DN) were linearized with NotI digestion and cleaned
using the PCR Mini Kit (Syngen). en, the mRNAs were synthesized invitro from the SP6 promoter using the
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mMESSAGE mMACHINE SP6 kit (Ambion AM1340) according to the manufacturer’s protocol. Transcribed
mRNAs were puried using an RNA Clean & Concentrator kit (Zymo Research). A NanoDrop 2000 spectropho-
tometer (ermo Fisher Scientic) was used to calculate the concentration of mRNA, which was then diluted to
a concentration of 200ng/µl. e integrity of mRNA was also conrmed by denaturing agarose electrophoresis.
Microinjection of mRNA into fertilized zebrash 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 zebrash eggs at the one-cell stage using a WPI Picopump
PV820 microinjector (2nl). e microinjected embryos were collected at 4 and 6 hpf for further RNA analysis.
RNA isolation and quantitative PCR
For RNA isolation, ~ 10 zebrash embryos were collected in fenozol (A&A Biotechnology), frozen and stored at
-80°C. en, 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 Biotechnol-
ogy) and a QuantStudio3 thermocycler (ermo Fisher Scientic). Rps11, eef1, acbt2 and rpl13a were used as
a reference genes49,50 e primer sequences are listed in Supplementary TableS1. To validate specicity 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 (Sup-
plementary Fig. S3d).
RNA sequencing
e poly(A) mRNA fraction from total RNA was isolated with a Dynabeads mRNA DIRECT Micro Kit (ermo).
e sequencing library for each RNA sample was prepared according to the protocol provided by the manu-
facturer using the Ion Total RNA-Seq Kit v2 (ermo). e 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 amplied to double-stranded cDNA and
then puried using a magnetic bead-based method. e 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 tem-
plate preparation on the Ion Chef instrument (ermo) using reagents from the Ion PI Hi-Q 200 Kit (ermo)
and Ion PI v3 Proton Chip. All samples were sequenced on the Ion Proton System (ermo) 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. Dierential
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
ClusterProler 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 zebrash embryo development was observed and photographed under an inverted microscope
(Leica DMi1 under Flexacam C1). e uorescence signal from mTurquoise-fused protein was observed under
a uorescence stereomicroscope (Zeiss Discovery V12 with a PentaFluar S lter slider equipped with a Zeiss
Axiocam 705 mono camera). e 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 Soware).
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.
Received: 25 May 2023; Accepted: 5 October 2023
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Acknowledgements
We would like to thank Dariusz Gajdzinski for his help with zebrash maintenance and breeding. We also thank
Mateusz Wilamowski for his help with cloning.
Author contributions
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 gures. A.L.-C., W.S. and T.K.P.
contributed equally to this work. All authors reviewed and approved the manuscript.
Funding
e research for this publication has been funded by MNS 5/2021 (to W.S.). T.K.P. was supported by Pol-
ish 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.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 023- 44294-1.
Correspondence and requests for materials should be addressed to J.J.
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