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Mycn regulates intestinal development through ribosomal biogenesis in a zebrafish model of Feingold syndrome 1 [1]

['Yun-Fei Li', 'Institute Of Genetics', 'Department Of Human Genetics', 'Zhejiang University School Of Medicine', 'Hangzhou', 'Tao Cheng', 'Women S Hospital', 'Ying-Jie Zhang', 'Xin-Xin Fu', 'Jing Mo']

Date: 2022-11 

Feingold syndrome type 1, caused by loss-of-function of MYCN, is characterized by varied phenotypes including esophageal and duodenal atresia. However, no adequate model exists for studying the syndrome’s pathological or molecular mechanisms, nor is there a treatment strategy. Here, we developed a zebrafish Feingold syndrome type 1 model with nonfunctional mycn, which had severe intestinal atresia. Single-cell RNA-seq identified a subcluster of intestinal cells that were highly sensitive to Mycn, and impaired cell proliferation decreased the overall number of intestinal cells in the mycn mutant fish. Bulk RNA-seq and metabolomic analysis showed that expression of ribosomal genes was down-regulated and that amino acid metabolism was abnormal. Northern blot and ribosomal profiling analysis showed abnormal rRNA processing and decreases in free 40S, 60S, and 80S ribosome particles, which led to impaired translation in the mutant. Besides, both Ribo-seq and western blot analysis showed that mTOR pathway was impaired in mycn mutant, and blocking mTOR pathway by rapamycin treatment can mimic the intestinal defect, and both L-leucine and Rheb, which can elevate translation via activating TOR pathway, could rescue the intestinal phenotype of mycn mutant. In summary, by this zebrafish Feingold syndrome type 1 model, we found that disturbance of ribosomal biogenesis and blockage of protein synthesis during development are primary causes of the intestinal defect in Feingold syndrome type 1. Importantly, our work suggests that leucine supplementation may be a feasible and easy treatment option for this disease.

In this study, we generated a large deletion in mycn in zebrafish using the CRISPR/Cas9 system. Homozygous mycn mutant fish are viable and fertile, and most importantly, the mutants carry a series of developmental defects similar to those of Feingold syndrome type 1, such as an abnormal pharyngeal arch (cartilage defects) and intestinal deficiency. Using this model, we studied the mechanism of the Mycn deficiency leading to intestinal developmental defects and discovered a potential therapeutic strategy for alleviating the intestinal defects in patients with Feingold syndrome type 1.

The vertebrate alimentary canal is derived from the primitive gut tube, which originates from the endodermal layer [ 9 ] and gives rise to the digestive system organs, including the pancreas, liver, gall bladder, and intestines. Developmental defects in this process can lead to serious human diseases, such as intestinal atresia, malrotation, hypoplasia, and epithelial defects, which cause malabsorptive or secretory diarrheal syndromes [ 10 ]. Although the zebrafish digestive system differs morphologically from that of mammals, a high degree of homology exists between zebrafish intestines and mammalian intestines in terms of their cellular composition and molecular pathways regulating intestinal development [ 11 ]. Many zebrafish models for studying congenital diseases affecting the intestines have been reported because of experimental tractability; these models have provided novel insights into the developmental mechanisms, pathogenesis, and therapeutics of intestinal congenital diseases [ 12 , 13 ].

The MYC proto-oncogene family is a class of transcription factors with a basic helix–loop–helix domain and includes MYC, MYCL, and MYCN [ 3 ]. MYCN amplification or overexpression has been described in many cancers, including neuroblastoma, retinoblastoma, rhabdomyosarcoma, and lung cancer, which are frequently of embryonic or neuroendocrine origin [ 4 ]. Like other members of the MYC family, MYCN controls the expression of its target genes and regulates many fundamental cellular processes such as proliferation, differentiation, apoptosis, protein synthesis, and metabolism [ 5 ]. Research on chicken embryos has shown that overexpression of MYCN drives the neural crest toward a neural stem cell fate [ 6 ]. MYCN homozygous mutant mice die at embryonic day E11.5, and multiple organs, including the nervous system, mesonephros, lungs, and gut, are affected. Besides, this study show that the homozygous mutant embryos bleed easily upon manipulation, exhibit distended aortas, and are severely anemic when examined at E12. These observations suggest a failure in the developing cardiovascular system, leading to spontaneous bleeding that would also result in embryonic death [ 7 ]. Conditional disruption of MYCN in mouse neural progenitor cells has shown that MYCN is essential for neural progenitor cell expansion and inhibits its differentiation [ 8 ]. However, the function of MYCN in organogenesis remains uncertain, and the mechanism through which MYCN regulates intestinal development remains unknown.

Feingold syndrome is a skeletal dysplasia caused by loss-of-function mutations of either MYCN (type 1) or MIR17HG (type 2), which encodes miR-17-92 microRNAs [ 1 ]. The syndrome is characterized by autosomal dominant inheritance of microcephaly and limb malformations, notably hypoplastic thumbs, and clinodactyly of the second and fifth fingers. Feingold syndrome type 1 is always accompanied by gastrointestinal atresia (primarily esophageal and/or duodenal atresia) [ 2 ]. However, whether the digestive system deficiency in Feingold syndrome 1 is the direct result of MYCN loss-of-function or a sequence effect of other developmental defects caused by MYCN mutation remains unknown. Further, the mechanism of the intestinal defects in patients with Feingold syndrome type 1 is also unclear. This unclear pathogenesis of Feingold syndrome type 1 is a major obstacle to developing treatments for the disease.

Raw sequencing reads were analyzed using the 10X Cellranger pipeline, version 3.0.2, with the default parameters. The expression matrix was obtained after running Cellranger. The R package Seurat (version 4.0.2) [ 24 ] was used to perform downstream analysis. We created a Seurat object with the CreateSeuratObject() function with min.cells = 3, min.genes = 100. Next, we performed a standard analysis procedure with the functions FilterCells(), NormalizeData(), FindVariableGenes(), FindClusters(), and FindAllMarkers(), with appropriate parameters. Finally, all clusters were visualized in 2 dimensions using t-SNE or UMAP. These clusters were annotated based on differentially expressed markers in each cluster or by comparison with published single-cell datasets. The WT and mycn mutant datasets were integrated using the Seurat integration procedure. First, variable features for each dataset were normalized and identified independently with nfeatures = 2,000. The FindIntegrationAnchors function was used to identify anchors; the anchors were used as input for the IntegrateData function to integrate the 2 datasets. Finally, the same procedures were performed on the integrated datasets as done for the single dataset with appropriate parameters. The Seurat subset function was used to create an intestinal cell Seurat object for downstream analysis.

Samples were prepared for the single-cell RNA-seq as previously described [ 23 ]. Approximately 30 mycn mutant or WT zebrafish embryos at 3 dpf were transferred to 1.5 mL low binding microcentrifuge tubes (Eppendorf 022431021). Trypsin-EDTA solution (Beyotime, C0201) was added, then the solution was pipetted up and down several times through a P200 tip every 5 min for 30 min. After all embryos were dissociated into single cell, the cells were centrifuged into a pellet and resuspended by adding 200 μL 0.05% bovine serum albumin/Ringer’s solution. Cell density was quantified manually using hemacytometers (QIUJING), and cell viability was analyzed using propidium iodide staining. Libraries were prepared using the Chromium Controller and Chromium Single Cell 3′ Library (10× Genomics, PN-1000074) per the manufacturer’s protocol for 10,000-cell recovery. Final libraries were sequenced on the Illumina Novaseq6000 (Genergy Bio-technology, Shanghai).

WT and mycn mutant zebrafish embryos were collected for RNA sequencing at 2 and 3 dpf. Library construction and sequencing were completed by Novogene (Novogene Bioinformatics Technology, Beijing, China). Paired-end sequencing (Novaseq6000, 150-bp reads) was performed. The sequencing reads were aligned to the zebrafish GRCz11 genome using STAR ( https://github.com/alexdobin/STAR ) [ 19 ], and reads mapped to multiple genomic locations were removed. Gene expression counts for each sample were calculated using featureCounts [ 20 ]. Differential expression analysis was performed using the DESeq2 package ( https://github.com/mikelove/DESeq2 ) [ 21 ]. Differentially expressed genes were obtained by comparing the mycn mutant to the WT samples with padj ≤ 0.1 and |log2foldchange| < 0. Finally, overlapped down-regulated genes of the 2 and 3 dpf samples were selected. GO biological process analysis was performed with the clusterProfiler package ( https://bioconductor.org/packages/clusterProfiler/ ) [ 22 ].

For gene set enrichment analysis (GSEA), the mappings linking zebrafish gene to gene ontology (GO) terms were achieved through org.Dr.eg ( https://bioconductor.org/packages/org.Dr.eg.db/ ). A ranked list was formed for GSEA using sign (log2FC) *(−log10PValue) as the ranking statistic. GSEA was performed by clusterProfiler package ( https://bioconductor.org/packages/clusterProfiler/ ), using fgsea ( https://github.com/ctlab/fgsea/ ) with 10e5 interactions, and the results were visualized by enrichplot package ( https://bioconductor.org/packages/enrichplot/ ).

FastQC ( https://github.com/s-andrews/FastQC ) was used to perform quality issues inspection for raw fastq files. For quality trimming, cutadapt ( http://cutadapt.readthedocs.org/en/stable/ ) was used to remove adapter sequences and filter out reads that became shorter than 20 nt (-m parameter). Trimmed clean reads were aligned to the zebrafish GRCz11 genome using STAR ( https://github.com/alexdobin/STAR ), and reads mapped to multiple genomic location were removed. featureCounts [ 20 ] was used to calculated gene expression counts of each sample. The expression counts of all samples were transformed to fragments per kilobase of exon per million mapped fragments (FPKM). Translation efficiency was defined by the ratio of Ribo-seq FPKM and RNA-seq FPKM. Differential translation efficiency analysis was performed by R package limma ( https://bioconductor.org/packages/limma/ ). More than 3,000 genes were identified as down-regulated genes in mycn mutant samples (p-value < 0.05 and log2 fold change < −0.5). Those genes were uploaded to KOBAS ( http://bioinfo.org/kobas/ ) web tool to perform KEGG pathway enrichment analysis (p-value < 0.01, Edge weight = 0.2, Top cluster = 3).

WT and mycn mutant embryos were collected at 3 dpf, digested with cold 0.5% trypsin to a single-cell suspension, and homogenized in lysis buffer for 20 to 50 times. The homogenate centrifuged at 12,000g for 15 min at 4°C, and the supernatants were digested with RNase for 30 min and aborted by 1 M EGTA followed by layering on top of a 10% to 50% sucrose-gradient solution. Ultracentrifugation was performed at 36,000 rpm for 2 h at 4°C. After centrifugation, the fractions were collected according to the absorbance at optical density OD260 by a TRAIX detector. Then the RNA was purified and used to construct the library for sequencing.

Total RNA was extracted from fish samples using TRIpure Reagent (Aidlab, RN0102) according to the manufacturer’s instructions. Digoxigenin (DIG)-labeled 5′ETS-1, ITS1, and ITS2 probes were obtained by PCR with specific primers ( S1 Table ) and the corresponding plasmid DNA as the template, together with the DIG DNA Labeling Mix (Roche Diagnostics,11277065910). Northern blot hybridization was performed as previously described [ 18 ]. DIG-labeled probes were detected with CDP-Star Chemiluminescent Substrate (Roche, Cat#12041677001), according to the manufacturer’s instructions.

WT and mycn mutant embryos with ET33J1: EGFP transgenic backgrounds were collected at 3 dpf. For proliferation analysis, the embryos were immersed in 3 mg/mL BrdU in 0.3× Danieau buffer at 60 hpf overnight in the dark, then washed 3 times with PBST and digested with cold 1% trypsin to a single-cell suspension. The cells were incubated and labeled with BrdU primary antibody and Alexa Fluor 594–labeled secondary antibody. For apoptosis analysis, the cells were stained with the Annexin V-APC/7-AAD apoptosis kit (Multisciences) per the manufacturer’s instructions, and the signal was detected by a DxFLEX flow cytometer.

2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) (5 mg/mL, Sigma) was used to label the zebrafish intestines at 7 dpf. Embryos were treated with DCFH-DA in 0.3× Danieau buffer for 2 h. PED6 (D23739, Thermo Fisher) and EnzChek (E6639, Thermo Fisher) were used to test the digestive ability of the intestinal proteins and lipids. Embryos at 7 dpf were treated with 3 μg/mL PED6 or 20 μg/mL EnzChek in a 0.3× Danieau buffer for 3 h. Rapamycin was used to inhibit the mTORC1 pathway. Embryos were exposed to 400 and 800 nM rapamycin (Sangon Biotech, China) in a 0.3× Danieau buffer from 10 hours postfertilization (hpf) to 3 dpf. L-Leu and rheb mRNA were used to elevate the mTORC1 pathway. Embryos were injected with L-Leu (500 nM, Sigma) at 30 hpf or rheb mRNA (100 pg and 150 pg) at 1-cell stage, then harvested at 3 dpf.

The sucrose-gradient centrifugations were performed as previously described [ 17 ]. WT and mycn mutant embryos were collected at 3 dpf, digested with cold 0.5% trypsin to a single-cell suspension, and homogenized in lysis buffer for 20 to 50 times. The homogenate was centrifuged at 12,000g for 15 min at 4°C, and the supernatants were layered on top of a 10% to 50% sucrose-gradient solution. Ultracentrifugation was performed at 36,000 rpm for 2 h (hours) at 4°C. After centrifugation, the absorbance at optical density OD260 of the fractions collected from the top of the tube was detected using a TRAIX detector (Thermo Scientific, USA).

The 7 dpf embryos were fixed in 4% PFA with 1% 5N NaOH at 4°C overnight. The embryos were washed several times with PBS, then stained with Alizarin red solution (0.4 ml saturated Alizarin red S in ethanol/10 ml 0.5% KOH) overnight at room temperature. After wash in 0.5% aqueous KOH for several times, the embryos were transferred in graded series of glycerol (15%, 30%, 60%, and 80% in 0.5% KOH) and stored in 100% glycerol.

The 5 days postfertilization (dpf) embryos were fixed in 4% PFA at 4°C overnight. The embryos were washed several times with PBS, then stained with 0.1% Alcian blue 8GX in acid alcohol (70% ethanol and 30% glacial acetic acid) overnight at room temperature. After rehydration in PBS, the embryos were digested with 1% trypsin for 1 h at 37°C. Embryos were then washed with PBS several times and stored in glycerol.

Embryos were fixed in 4% PFA overnight at 4°C for immunofluorescence or HE staining. After washing in PBST (0.1% Tween20 in PBS), the embryos were dehydrated, transparentized with xylene, and mounted in paraffin overnight at 4°C. The sections were cut serially to 3-μm thick and collected on poly-L-lysine-coated glass slides (CITOGLAS, 188105). HE staining was performed using Dyeing and sealing machine (GEMINI AS). Immunofluorescence staining was performed as described [ 16 ]. PCNA antibody was purchased from Sigma (P8825, 1:1,000); puromycin antibody was purchased from Merck (MABE343, 1:1,000). Alexa Fluor 488–labeled secondary antibody (Invitrogen A21206) was used for visualization. The TUNEL assay was performed with the In Situ Cell Death Detection Kit (Roche) per the manufacturer’s instructions.

Embryos were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight at 4°C. The probes were labeled with digoxigenin (Roche Diagnostics). The ifabp, lfabp, prss1, insulin, foxa3, foxa1, gata6, slc15a1b, pyyb, rpls, rps, lmo7a, baiap2l1a, fabp6, id2a, MT-ND2, tcnl, tm4sf4, and apoc2 probes were generated and used for whole mount in situ hybridization (WISH) as previously described [ 15 ]. DNA fragments of above genes were cloned into the pEASY-blunt-zero vector (Transgen), then sequenced. Primers used were listed in S1 Table .

The zebrafish AB strain was used in all experiments to generate knock-in or mutant lines. To generate the mycn mutants, we synthesized 3 gRNAs against the second exon of the zebrafish mycn gene as previously described [ 14 ]. The Cas9 protein and mycn-targeting gRNAs were coinjected into the wild-type (WT) embryos at 1-cell stage. The mycn mutant lines were identified in the F1 generation by analyzing the PCR product using the primer pair listed in S1 Table . To construct mycn:EGFP knock-in zebrafish, we generated a gRNA targeting the second intron, and a donor DNA with EGFP reporter just before the stop codon of mycn flanked with 2 homologous arms. We coinjected Cas9, gRNA, and the donor into zebrafish embryos at the 1-cell stage, then screen embryos with correct EGFP expression.

All animal procedures were performed per the requirements of the “Regulation for the Use of Experimental Animals in Zhejiang Province.” The Animal Ethics Committee of the School of Medicine, Zhejiang University, approved this study. The protocol number is NO. 24278.

Results

mycn was highly enriched in the developing digestive system of the zebrafish embryos To use zebrafish as a model for studying Feingold syndrome type 1 and the function of Mycn during organogenesis, we first explored the spatiotemporal expression patterns of mycn during embryonic development in zebrafish. mycn transcription can be detected by RNA in situ hybridization (ISH) at the onset of gastrulation and is enriched in the neural ectoderm. At the end of gastrulation, mycn was specifically expressed in both the anterior and posterior neural plate, consistent with previous reports on the role of MYCN in neural development and oncogenesis [25]. Beginning at 18 hpf, mycn expression could be detected in the epiphysis, eye, optic tectum, spinal cord, and endoderm (S1A Fig). After 24 hpf, mycn expression was progressively restricted to the central nervous system, pharyngeal arch, and digestive system (Fig 1A). PPT PowerPoint slide

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TIFF original image Download: Fig 1. mycn expression patterns in zebrafish during early development. (A) Expression patterns of mycn in zebrafish at 2 and 3 dpf by whole-mount WISH. Lateral view (left), dorsal view (right). (B) mycn expressed along the whole intestines of the embryos at 3 dpf shown by section via ISH. Arrows indicate the embryo intestines. Sections were cut along the sagittal plane. (C) Fluorescence images show the mycn expression patterns by EGFP-knock-in fish at 2 and 3 dpf (lateral view). (D) mycn expressed along the whole intestines of the zebrafish at 4 dpf shown by section of mycn:EGFP fish. (E) UMAP plot shows unsupervised clustering of the cells in WT embryos of 3 dpf; cells are colored by their cell type annotations. (F) Violin plots show the mycn expression levels of different cell types of WT embryonic scRNA-seq data at 3 dpf. Scale bars: 200 μm (A and C), 50 μm (B and D). dpf, days postfertilization; ISH, in situ hybridization; scRNA-seq, singe-cell RNA-seq; UMAP, uniform manifold approximation and projection; WISH, whole mount in situ hybridization; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001856.g001 To validate the ISH results and investigate the mycn expression dynamics during zebrafish development, we generated an mycn:EGFP knock-in fish by inserting an EGFP sequence just before the stop codon of mycn with a P2A linker between the 2 proteins (S1C Fig) to avoid possibly distorting the Mycn protein structure. The mycn:EGFP line confirmed that mycn was mainly expressed in the central nervous and digestive systems during organogenesis (Fig 1C). Interestingly, mycn was also expressed in the migrating neuromast cells of the lateral line, indicating that Mycn might also function in the sensory organs. High-magnification imaging of both the mycn ISH and mycn:EGFP showed that mycn was expressed in the intestinal epithelial cells, suggesting that Mycn functions directly in intestinal development (Fig 1B and 1D). To further characterize the mycn expression pattern, we performed single-cell RNA-seq for WT embryos at 3 dpf. A total of 23 clusters were identified and annotated after strict quality control (Fig 1E). We then explored the mycn expression level across these clusters and found that mycn was highly expressed in the central nervous system, neural crest cells, and endoderm-derived tissues such as the intestines, liver, and pancreas (Fig 1F). Additionally, we investigated mycn expression in published single-cell RNA-seq datasets [26–28]. During the gastrulation and somitogenesis stages, the brain and optic cells showed high mycn expression (S1D and S1E Fig). High mycn expression levels were detected in intestinal cells at 2 dpf, and mycn expression decreased in the intestines at 5 dpf (S1B and S1F–S1I Fig), which was consistent with the ISH experiment on mycn.

Single-cell transcriptomics showed that Mycn loss-of-function reduced the specific intestinal cell types during embryonic development To systematically investigate the phenotypes resulting from Mycn loss-of-function at a higher resolution, we performed single-cell RNA-seq in the mycn mutant fish at 3 dpf. We integrated mycn mutant scRNA-seq datasets with WT datasets (see Material and methods). A total of 27 cell types were identified and annotated based on their expression markers (Figs 3A and S5A). Calculating the cell ratio of each cell type revealed that the cranial neural crest, blood vessels, and digestive organs, including the intestines, liver, and pancreas, were dramatically decreased in the mycn mutants (Fig 3B). To more deeply characterize the intestinal cell clusters, we selected and reclustered the intestinal cells and identified 9 subclusters (Fig 3C). We noticed that cluster 8 highly expressed mycn, and this cluster showed stemness characteristics based on the pseudotime trajectory analysis (S5C and S5D Fig); this cluster was completely disappeared in the mycn mutants. Besides, clusters 4 and 6 were significantly reduced in the mycn mutant. To characterize these subclusters, we performed differential expression analysis, selected the most significant markers (Fig 3D), and verified their expressions via ISH in both WT and mycn mutant embryos. Expressions of all those selected cluster markers were decreased in the mycn mutant intestines at 3 dpf, especially for those of clusters 4, 6, 7, and 8, which could hardly be detected in the mutants (Fig 3E). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Single-cell RNA-seq analysis of WT and mycn mutant embryos at 3 dpf. (A) Unsupervised clustering of cells in the mycn mutants and WT embryos at 3 dpf. Cells are colored according to their cell type annotations inferred from expressed marker genes and published datasets. (B) Bar plot shows the percentages of each cell type in mycn mutants (blue) or WT embryos (red). (C) UMAP plot shows the subclusters of intestinal cells selected from A. Cells are colored by cell type clusters. A total of 9 clusters were identified by unsupervised clustering. (D) Dot plot shows the expressions of marker genes in each subcluster of intestinal cells. Heatmap represents average expression level and dot size represents percentage of cell expression across mycn mutant and WT embryos. (E) WISH results show the expression of marker genes of each cluster: id2a (cluster 0), tm4sf4 (cluster 2), apoc2 (clusters 1, 3, and 5), lmo7a (cluster 4), tcnl (cluster 6), baiap2l1a (cluster 7), fabp6 (clusters 4 and 7), and MT-ND2 (cluster 8) in WT and mycn mutant embryos. Arrow heads indicate the intestines. Scale bar: 100 μm. dpf, days postfertilization; UMAP, uniform manifold approximation and projection; WISH, whole mount in situ hybridization; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001856.g003

Proliferation arrest, but not apoptosis, led to intestinal defects in the mycn mutants The morphological, molecular, and single-cell RNA-seq results indicated that the intestinal defects in the mycn mutants may be due to reduced cell numbers rather than to a differentiation blockade. Thus, we analyzed the levels of proliferation and apoptosis in the intestines of the mycn mutant and WT embryos at 3 dpf. Immunofluorescence showed that the PCNA signals were significantly reduced in the intestinal bulbs of the mycn mutants (Fig 4A). However, TUNEL assay revealed no obvious apoptotic signals in the intestinal bulb region of either the mycn mutant or WT embryos (Fig 4B). To further confirm the intestinal proliferation defects of the mycn mutants, we dissociated the 3 dpf embryos of the mycn−/−; ET33J1: EGFP line and performed fluorescence-activated cell sorting to sort out the intestinal cells. We then used BrdU and Annexin V-APC analysis to mark the cells that were proliferating or undergoing apoptosis, respectively. Consistent with the immunofluorescence results, the flow cytometry results also showed a significantly lower proliferation rate in the mycn mutants, but apoptosis could hardly be detected in the intestines of either the WT or mutant embryos at 3 dpf (Fig 4C–4E). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Detection of cell proliferation and apoptosis in intestines of WT and mycn mutant. (A) Cell proliferation in the intestines was detected by PCNA immunofluorescence staining (green signal) of the tissue sections from the WT and mycn mutant embryos. The statistical analysis of the proliferating cells of the sections of intestine in the right. (B) Apoptosis in the intestines was detected via TUNEL assay (red signal) for the tissue sections from the WT, mycn mutant, and camptothecin-treated (as positive control) embryos. Sections were cut along the transverse plane. Dotted lines indicate the intestine positions. (C) Schematic representation of the experimental workflow of the flow cytometry analysis. (D) Cell proliferation in the intestines was detected by BrdU incorporation assay. (E) Apoptosis in the intestines was detected by APC-Annexin V staining. Embryos used in the flow cytometry analysis were descendants of ET33J1: EGFP reporter line crossed with WT or mycn mutants (D and E). Scale bar: 50 μm. PCNA, proliferating cell nuclear antigen; WT, wild-type. https://doi.org/10.1371/journal.pbio.3001856.g004

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