Transcriptome analysis of pancreatic cells across distant species highlights novel important regulator genes
© Peers et al. 2017
Received: 3 November 2016
Accepted: 1 March 2017
Published: 21 March 2017
Defining the transcriptome and the genetic pathways of pancreatic cells is of great interest for elucidating the molecular attributes of pancreas disorders such as diabetes and cancer. As the function of the different pancreatic cell types has been maintained during vertebrate evolution, the comparison of their transcriptomes across distant vertebrate species is a means to pinpoint genes under strong evolutionary constraints due to their crucial function, which have therefore preserved their selective expression in these pancreatic cell types.
In this study, RNA-sequencing was performed on pancreatic alpha, beta, and delta endocrine cells as well as the acinar and ductal exocrine cells isolated from adult zebrafish transgenic lines. Comparison of these transcriptomes identified many novel markers, including transcription factors and signaling pathway components, specific for each cell type. By performing interspecies comparisons, we identified hundreds of genes with conserved enriched expression in endocrine and exocrine cells among human, mouse, and zebrafish. This list includes many genes known as crucial for pancreatic cell formation or function, but also pinpoints many factors whose pancreatic function is still unknown. A large set of endocrine-enriched genes can already be detected at early developmental stages as revealed by the transcriptomic profiling of embryonic endocrine cells, indicating a potential role in cell differentiation. The actual involvement of conserved endocrine genes in pancreatic cell differentiation was demonstrated in zebrafish for myt1b, whose invalidation leads to a reduction of alpha cells, and for cdx4, selectively expressed in endocrine delta cells and crucial for their specification. Intriguingly, comparison of the endocrine alpha and beta cell subtypes from human, mouse, and zebrafish reveals a much lower conservation of the transcriptomic signatures for these two endocrine cell subtypes compared to the signatures of pan-endocrine and exocrine cells. These data suggest that the identity of the alpha and beta cells relies on a few key factors, corroborating numerous examples of inter-conversion between these two endocrine cell subtypes.
This study highlights both evolutionary conserved and species-specific features that will help to unveil universal and fundamental regulatory pathways as well as pathways specific to human and laboratory animal models such as mouse and zebrafish.
KeywordsRNA-seq Comparative transcriptomics Pancreas Endocrine cells Acinar cells Ductal cells
Pancreas is a vital organ playing crucial function in the metabolism of all vertebrates. Acinar cells, the most abundant cell type of the pancreas, produce the digestive enzymes that are conveyed to the gut by the pancreatic ducts. The pancreatic endocrine cells are grouped in the Langerhans islets and secrete diverse hormones controlling metabolism and glucose homeostasis. Five endocrine cell subtypes (alpha, beta, delta, PP, and epsilon cells) have been described in pancreatic islets, each characterized by the expression of a particular hormone (glucagon, insulin, somatostatin, pancreatic polypeptide, and ghrelin, respectively). Many transcriptomic studies have been focused on pancreatic endocrine cells, and notably on beta cells, due to their implication in the development of diabetes. Microarrays and RNA sequencing (RNA-seq) were conducted on endocrine pancreatic cells isolated from human (healthy or diabetic persons) or rodents at adult and embryonic stages [1–9]. More recently, RNA-seq performed on endocrine cell types isolated by FACS or at single-cell level allowed to define the genes enriched in each endocrine cell type in human and mice [7, 10–21]. However, no comprehensive interspecies comparison has been performed so far to define the conserved signatures for each pancreatic cell type, except for two studies comparing human and murine beta cells and reporting some notable differences between these two mammalian species [12, 21]. Comparison of transcriptomes between species is a straightforward approach to identify tissue-specific or cell type-specific genes playing crucial functions in the physiology of the studied tissue or cell. Indeed, if a gene is essential in a differentiated cell, strong constraints will maintain its expression throughout evolution and its tissue-specific expression will be detected in most species. In accordance to this view, several studies have reported conservation of organ-specific expression for a large set of genes among species [22–24]. Nevertheless, there are also striking divergences in gene expression patterns even between close vertebrate species such as human and mice, which may contribute to physiological adaptations [25, 26]. Comparative studies between evolutionary distant species are useful to identify the set of genes displaying highly conserved cell type-specific expression and likely playing a fundamental function in the studied cells. Such analyses have not been performed yet on pancreatic cells due to the lack of transcriptomic data from pancreatic cells isolated from lower vertebrates such as zebrafish. To tackle this lack of knowledge, we first determined the transcriptomic landscape of the three major endocrine cell types (alpha, beta, and delta cells) as well as of the acinar and ductal cells from zebrafish. Analysis of these zebrafish datasets allowed us to define the signature of each cell type. Then, comparison with published human and murine pancreas data led to a definition of the conserved signatures. Furthermore, by determining the transcriptome of endocrine cells from early stage embryos, we identified genes expressed during endocrine cell differentiation and putatively involved in this process; among them, myt1b and cdx4 are shown to be essential for endocrine cell differentiation in zebrafish. Thus, our list of pancreatic conserved genes represents a useful resource for studies related to pancreatic development and disease such as diabetes and pancreatic cancer.
Transcriptomic profiles of the different pancreatic cell types isolated from adult zebrafish
We purified the different pancreatic cell types from adult zebrafish using a series of transgenic reporter lines allowing the selection of these distinct cells by fluorescence-activated cell sorting (FACS). Acinar cells were obtained from the BAC transgenic lines Tg(ptf1a:GFP) . The endocrine beta and delta cells were isolated, respectively, from the transgenic lines Tg(ins:GFP) ulg021Tg (see Methods section) and Tg(sst2:GFP) ; the alpha cells were obtained from the Tg(gcga:GFP)/Tg(ins:NTR-mCherry) line through selection of GFP+/mCherry cells (as many beta cells were found to express Tg(gcga:GFP) transgene at a lower level, Additional file 1: Figure S1). RNA-seq was performed on three independent preparations for each cell type, except for acinar cells, for which four replicates were prepared. About 60 million of paired-end reads were obtained from each Illumina library, 80% of which mapped to the zebrafish genome. We previously reported the transcriptome of pancreatic ductal cells by using the same procedure on the Tg(nkx6.1:GFP) ulg004Tg transgenic line , and these data were compared in the present study with endocrine and acinar cell transcriptomes.
Percentage of the reads obtained for highest expressed markers in each type of library
Identification of genes enriched in endocrine, acinar, and ductal pancreatic cells
Similar observations were done for the acinar and ductal cell transcriptomic signature. Indeed, as expected, many genes coding for digestive enzymes, such as trypsin (try), elastase (i.e., ela and cela), trypsin-like (tryl/zgc:66382), amylase (amy2a) as well as the known acinar transcription factors ptf1a and rbpjl, were found enriched in acinar cells. The acinar gene list also includes novel markers such as genes encoding for the transcription factors esr2a, klf15, or nr0b2a (Fig. 2b and Additional file 4: Table S3). As expected, GO enrichment analysis revealed, among the biological processes active in acinar cells, “Gland development” and “Digestive system development”, “Cellular amino acid metabolic process”, “Proteolysis” as well as “ATP synthesis coupled electron transport” (Fig. 2d). As for the duct transcriptome, the analysis reveals novel markers such as id2a and frzb in addition to known markers like sox9b, nkx6.1, onecut1, and ctgfa, as we recently described . All together, these analyses confirm that many known markers and biological pathways display the same pancreatic enrichment in zebrafish as in mammals, and also highlight many novel cell type-specific genes not previously reported to display such selective expression in mammals. This led us to compare comprehensively the endocrine- and exocrine-enriched genes across zebrafish and mammalian species, thereby defining the conserved specific signatures among vertebrates.
Comparison of the pancreatic endocrine and exocrine transcriptomic signatures across zebrafish, mouse, and human
List of conserved endocrine-enriched transcription factors
The interspecies comparison of both exocrine and endocrine signature also indicates that many genes display the same enrichment in only two species (e.g., “HM”: between human and mice; “ZM”: zebrafish and mouse). As expected, the number of conserved HM endocrine and exocrine genes is higher than the number of conserved ZH and ZM genes. Overall, this global analysis indicates that, while significant divergence is observed between species at the level of endocrine and exocrine signatures, hundreds of genes have nevertheless maintained a common expression pattern, among which are most of the known pancreatic regulatory genes.
Identification of genes expressed in embryonic endocrine cells
Characterization of the transcriptomic signatures for the endocrine cell subtypes
As expected, the genes specifically expressed in alpha cells include those coding for the two zebrafish glucagon hormones (gcga and gcgb) as well as for the Arx transcription factor (arxa) but also novel markers including peptide hormones such as prepronociceptin (pnoca) and neuropeptide B (npb) or the calcium regulated factor scinderin like b (scinlb). The alpha-enriched genes also comprise the transcription factors etv1 and si:ch211-145o7.3 (a forkhead domain factor). The alpha-selective expression of pnoca and scinlb was confirmed by FISH, validating the RNA-seq data (Additional file 12: Figure S5).
As for the delta cells, besides the somatostatin genes , the RNA-seq data revealed many novel markers, which include genes coding for transcription factors such as dlx3b, cdx1, cdx4, and tbx2b, as well as genes coding for signaling factors like bmp7b or the kinase Map3k15. Interestingly, as demonstrated by the GO enrichment analysis (Additional file 10: Figure S3), many GPCR are specifically enriched in delta cells such as npy8br (neuropeptide Y receptor), glra4a (glycine receptor), uts2r (urotensin receptor), ptger1b & 2a (prostaglandin receptor), adra1d (adrenoreceptor), grm3 (glutamate receptor), oprd1b (opioid receptor), and gpr123, pinpointing these endocrine cells as targets of diverse external metabolic signals. We confirmed the selective expression for several genes, including the cdx4, cdx1, lamc2, and map3k15 genes, detected already at 24 hpf in delta cells (Additional file 13: Figure S6). All these results validate the RNA-seq data and show that many endocrine cell subtype markers acquire their selective expression in the first embryonic endocrine cells.
Comparison of transcriptomic signatures of alpha and beta cells across species
List of genes with evolutionary conserved alpha and beta cell-enriched expression
Conserved beta cell-specific genes
Conserved alpha cell-enriched genes
Role of the zebrafish myt1b and cdx4 genes in endocrine cell differentiation
In this study, we have explored the transcriptomic landscape of the major pancreatic cell types in zebrafish, thereby defining the transcriptomic signature of acinar and endocrine cells, as well as of the three major endocrine cell subtypes alpha, beta, and delta. By this analysis, we identified many novel cell type-specific markers with still unknown pancreatic function. By comparing the endocrine and exocrine transcriptomic signatures from zebrafish, human, and mouse, we could define an evolutionarily conserved signature for these two pancreatic tissues, pinpointing genes and pathways that likely represent key players in the pancreas of vertebrates. Consistent with this notion, more than half of all transcription factors in these endocrine or exocrine conserved signatures are known regulators of pancreatic cell differentiation, such as the transcription factors neuroD, isl1, pax6, insm1, ptf1a, and rbpjl, among others. Myt1 is part of the endocrine conserved signature and we show here that inactivation of the myt1b gene in zebrafish leads to a decrease of alpha cell mass in 2 dpf embryos. Additionally, cdx4, which is selectively expressed in delta cells, is also necessary for their differentiation.
The endocrine signature conserved among the three vertebrate species reveals that several signaling pathways regulating hormone secretion and cellular homeostasis are commonly used by pancreatic cells from fish to mammals. For example, the evolutionarily conserved signature comprises the RNA binding protein Elavl4/Hud and the ionotropic glutamate receptor Gria2, shown to regulate hormone synthesis and secretion in rodents [40, 52, 53]. Similarly, the glucose response system regulating insulin and glucagon secretion in mammals seems to be also used in zebrafish as various components of this pathway are found in the conserved endocrine genes such as the ATP sensitive K+ channels abcc8 and kcnj11 and the voltage-dependent Ca++ channel cacna2d2. The use of this pathway in zebrafish is further strengthened by the very high expression of the glucose transporter glut2 (slc2a2) and the glucokinase (gck) in zebrafish beta-endocrine cells. Additionally, some GPCR receptors which have been reported to control the activity of pancreatic islet cells in mice, such as the Sstr3 (somatostatin receptor) , CasR , or Celsr3 , are included in the conserved endocrine signature. Thus, this list of conserved endocrine-enriched genes strongly suggests that the function of several signaling pathways controlling the formation and activity of pancreatic cells has been maintained from fish to humans. This is consistent with many studies using zebrafish as a model for pancreas development and diabetes that revealed conserved physiological regulations (reviewed in [34, 35]). The conserved endocrine and exocrine pancreatic signatures also pinpoint to many novel candidate regulatory genes that deserve special attention for future studies. For instance, the transcription factors lmo1 and npas4a, or the signaling factors gpr158, rgs7, and rgs17, all selectively expressed in pancreatic endocrine cells in zebrafish, mice, and human, more than likely play an important role in pancreatic cells.
In the present study, we identified the human and murine endocrine- and exocrine-enriched genes by comparing transcriptomic data from purified islets with data obtained from whole pancreas as well as from one human pancreatic sample enriched in acinar tissue. While this approach identified almost all expected endocrine and exocrine markers, some markers may have been missed due to the low purity of tissue preparations. For example, the human MNX1 and murine Esr1 (Estrogen receptor alpha) genes were not enriched in the human or murine endocrine samples, respectively, while these two genes were reported to be important for endocrine beta cells in both species [57–60]. These misclassifications may be due to the use of RNA-seq data obtained from whole pancreas instead of highly purified exocrine cells; such potential errors will be corrected when RNAseq data from purified ductal and acinar cells will be available. The recent single cell transcriptomic data reported for human and murine pancreas bypass the need for purified cells and will probably constitute a useful resource for doing such interspecies comparison. However, murine acinar cells have not been captured in these single-cell studies preventing the identification of acinar-enriched and endocrine-enriched genes. When such data is available, it will be interesting to perform a global analysis of all pancreatic single cell transcriptomic studies and compare it with the zebrafish pancreatic data. In the meantime, genes classified in our list of conserved “ZM” or “ZH” endocrine genes should be also considered as they indeed include mnx1 and esr1, indicating that important genes fall in these two categories. For proof, cdx4, classified as endocrine-enriched in zebrafish and mice (i.e., “ZM”), was found here to be crucial for endocrine delta cell differentiation as many novel delta cell makers were drastically reduced in the cdx4 mutant zebrafish embryos. Our findings warrant future analyses on the murine (and human) Cdx4 gene to decipher its expression and function during pancreas development in mammals. Interestingly, the loss of delta cells with the concomitant increase of beta cells observed in the zebrafish cdx4 mutant suggests that Cdx4 could act on the balance of delta versus beta cells by determining the fate of endocrine precursors.
The present study also provides a comprehensive list of new markers of the zebrafish endocrine alpha, beta, and delta cell subtypes. An overall view indicates that the alpha and beta cells are slightly more similar to each other compared to delta cells, as shown by the PC analysis (Figs. 1b and 5c). This is also supported by the Venn diagram (Fig. 5a) showing that (1) the number of genes with enriched expression in both alpha and beta cells and not in delta cells (“alpha-beta” enriched genes) is higher than those of the “alpha-delta” enriched and “beta-delta” enriched genes (123 genes vs. 28 and 68 genes, respectively), and (2) the number of delta cell markers is higher than the alpha and beta cell marker (192 vs. 70 and 73 genes, respectively). These observations are consistent with a recent study in zebrafish larva showing the higher capacity of alpha cells to transdifferentiate to beta cells compared to delta cells . Whether this is also true in adult zebrafish must be verified as it has been demonstrated in mice that the transdifferentiation competence of alpha or delta cells toward beta cells is age dependent .
An unexpected observation of our study is the low conservation of the alpha and beta cell transcriptomic signatures between zebrafish, mice, and human. Indeed, only 20 and three genes defined the conserved alpha and beta cell signatures, respectively. Such low conservation cannot be attributed to a low quality of cell samples or RNA-seq data because, for the three species datasets, there is a very high enrichment of alpha and beta cell markers in the corresponding libraries confirming the good purity of alpha and beta cell preparations. Further, it is noteworthy that this low conservation is not only observed between zebrafish and the two mammalian species but also between human and mouse (see Venn diagram in Fig. 8). Differences between human and mouse alpha- and beta-enriched genes have also been previously noticed [12, 21]. Several non-exclusive explanations can be proposed for this apparent low conservation of the alpha- and beta-signatures in both distant and closer species. First, as the alpha and beta cells have relatively similar transcriptomes, as shown on Fig. 1a, the identity of these two cell subtypes could rely on the action of only few regulatory genes. This hypothesis is supported by the presence of Arx among the few conserved alpha cell genes, which is the key determinant of alpha cell identity and its inactivation in mice is sufficient to transdifferentiate alpha to beta cells . Similarly, Pdx1, one of the three conserved beta cell genes, is required to maintain beta cell identify and repress the alpha cell program . This first hypothesis is also supported by the ability of alpha and beta cells to transdifferentiate into each other [65, 66]. A second explanation can be the relatively stringent threshold that we used to select differentially expressed genes (four-fold enrichment, adjusted P < 0.05). For example, the mnx1 gene, known to be involved in beta cell differentiation [67, 68], is enriched in beta cells in the three vertebrate species but was not classified as beta-enriched in human and zebrafish due to a rather low beta versus alpha cell enrichment (three-fold in human, 1.9-fold in zebrafish versus 70-fold in mice). These differences in enrichment between human and mouse Mnx1 is confirmed in the recent single cell RNA-seq data [18–21]. Similarly, the prohormone convertase 1 gene pcsk1 required for insulin hormone maturation was only 2.7-fold enriched in zebrafish beta cells versus alpha cells. It is possible that small differences in expression levels may be sufficient for some genes to give a different physiological response. A third explanation of the low cell subtype conservation could stem from functional switches occurring between homologous genes able to perform the same function. For example, Nkx6.1 is expressed in beta cells in mice and human but not in zebrafish, where its paralog nkx6.2 instead of nkx6.1 is selectively expressed in mature beta cells . nkx6.2 may fulfill the function of Nkx6.1 as Nkx6.1 and Nkx6.2 have equivalent biological activities . Another example is the Zn++ transporter scl30a8, which is strongly expressed in beta cells in mammals but not in zebrafish, which instead strongly expresses the homologous Zn++ transporter scl30a2. Such functional switches may be difficult to identify as they can occur between genes of the same superfamily but belonging to different subclasses. For example, we have shown that, in zebrafish, the role of the bHLH Neurog3 as pancreatic endocrine cell fate determinant is fulfilled by two other bHLH factors Ascl1b and Neurod1 . A functional switch is also observed for the specification of the secretory cell of the intestine, played by Atoh1 in mice and by Ascl1a in zebrafish . The capacity of homologous factors to fulfill the same function is also supported by the observations that many null mutations can be compensated by a homologous gene [72–74]. Such compensations seem to occur for the murine Myt1 gene as the Myt1 KO mice only display very mild pancreatic defects with no decrease of endocrine cells , while more drastic defects are observed by expression of a dominant negative Myt1-Eng protein [1, 76]. Furthermore, adult murine endocrine pancreatic cells express the homologous Myt1l and Myt3/St18 genes  and their expression increases in the Myt1 KO mice . In zebrafish, myt1a, myt1l, and myt3/st18 are expressed at much lower level compared to myt1b; this probably explains the decrease in alpha cell mass observed in the single myt1b mutant. Further experiments will be required to determine whether some compensation occurs through myt1a and if the defects are more drastic in the double myt1a/b mutant.
The stronger expression of glucagon receptor in the beta cells of the three vertebrate species highlights the importance of paracrine regulations between alpha and beta cells and indicates a role of glucagon on beta cell physiology, as previously reported in rodents  and in zebrafish for beta cell regeneration . Interestingly, our transcriptomic analyses highlight fev as an endocrine conserved gene as well as an alpha cell conserved gene, suggesting a role of this transcription factor in alpha cells. Glucose tolerance test in Fev knockout mice previously revealed a slower response apparently due to a reduction in insulin production while the level of most crucial transcription factors of beta cells were unchanged . As our transcriptomic analysis indicates that fev is expressed more than 20-fold higher in alpha cells versus beta cells in zebrafish, mice, and human, this argues for an important function in alpha cells and warrants further phenotypic analyses.
The present transcriptomic analysis of the distinct zebrafish pancreatic cell types identifies novel pancreatic regulatory genes and thereby constitutes a valuable resource for future studies of the pancreas not only in zebrafish but also in mammals giving interesting clues on genes and signaling pathways active in these cells. The comparison of the present zebrafish pancreatic RNA-seq data with the recent single cell pancreatic transcriptomic data or with future data of pure pancreatic cells obtained not only from human and mouse but also from other species will help to better define gene regulatory networks controlling pancreas ontogeny and physiology in vertebrates. This will be useful for studies aimed at understanding dysfunction of pancreatic endocrine cells in human diseases like diabetes and to design novel drugs or therapies.
Generation of the Tg(Insulin:GFP) ulg021 Tg line
The (Insulin:GFP) transgene was generated by first cloning a 897 pb PCR fragment, amplified with O97 and O98 primers (Additional file 17: Table S10), that includes 745 bp of the insulin promoter, the exon 1 and intron 1 and 7 bp of exon 2 just upstream of the ATG of the insulin ORF, into the gateway vector pCR8/GW/TOPO to produce pMV90-G2a plasmid. Second, a triple LR recombination using p5E-MCS, pMV90-G2a, and p3E-EGFP-PA inserted into pDestTol2pA2, provided by the tol2kit , generated the transgene Tg(insulin:GFP) that has been introduced into AB embryos by coinjection with the Tol2 transposase to generate the Tg(insulin:GFP) ulg021 Tg line.
Preparation of zebrafish pancreatic cells by FACS
The endocrine cells were prepared using the zebrafish transgenic lines Tg(Insulin:GFP) ulg021Tg , Tg(gcga:GFP/ins:mCherry)ia1, and Tg(st22:GFP) to isolate beta, alpha, and delta cells, respectively [28, 81]. Acinar and ductal cells were isolated by using Tg(ptf1a:GFP)  and Tg(nkx6.1:GFP) ulg004Tg , respectively. The endocrine tissue was dissected under an epifluorescence stereomicroscope before dissociation by enzymatic treatment for 30 min at 28 °C with 1× Tryple Select (Life Techologies), 40 μg/mL proteinase K (Roche), and 10 μg/mL collagenase IV (Life Technologies), combined with mechanical disruption by pipetting every 5 minutes. For acinar cell preparations, pancreata were digested with a mix of collagenase IV (500 μg/mL), collagenase P (350 μg/mL), and dispase II (1 mg/mL) for 15 min at 28° with mechanical disruption every 5 minutes. After dissociation, cells were washed twice with 1× PBS containing 1% BSA and pelleted at 4 °C for 5 min at 300 g and immediately sorted by FACS. Cells were selected based on GFP expression using FACS Aria II by two consecutive sorting steps: the first sorting was done in the “yield” mode and the second in the “purity” mode. Purity was estimated by FACS Aria II after cell sorting (more than 99% purity of GPF+ cells) and by fluorescence microscopy (from 95–99% purity). Each replicate sample was prepared from four adult zebrafish. Approximately 10,000–20,000 endocrine cells, 20,000–40,000 acinar cells, and 2000–10,000 ductal cells were obtained after FACS and used for library preparations.
RNA extraction, cDNA amplification, library preparation, and sequencing
Total RNA was extracted from FACS sorted cells using the RNeasy plus micro kit (Qiagene). RNA from endocrine and acinar cells was eluted in 10 μL with a concentration of 100–400 pg/μL. RNA integrity was assessed by a capillary electrophoresis using Agilent RNA 6000 pico chip (Agilent technologies), the RIN value for each sample was from 8 to 10. The Smarter Ultra low RNA input kit (clontech)  was used to for the synthesis and amplification of cDNA synthesis using up to 10 ng of total RNA following the manufacturer’s instructions and performing no more than 12 cycles of PCR in order to minimize amplification biases. The quality of cDNA was verified by 2100 High Sensitivity DNA assay (Agilent technologies). Truseq DNA Illumina libraries were prepared and sequenced to obtain approximately 90 million reads (100 bp paired-end reads) per library using the Hiseq 2000 Illumina sequencer.
RNA-seq data analysis
Sequences were trimmed in order to remove adaptors and low quality bases. Trimmed reads were mapped in to the genome (Zv9, Ensembl genome version 75) using Tophat v.2.0.9 . Tophat’s options were set according to the library features (-r 220 --mate-std-dev 82 --segment-length 18) and the option --min-intron-length was set up to 30 nucleotides according to the intron length described by Moss et al. . For the mouse and human datasets, raw data were downloaded from the public databases: human pancreas (four samples from Fagerberg et al.  and one acinar cell-enriched sample from Morán et al. : the E-MTAB-1294 dataset), human islets (three samples from Nica et al.  and four samples from Morán et al. ), murine pancreas (two samples from Holmstrom et al. ), and murine islets (three samples from Morán et al. ). The alpha and beta cell RNA-seq datasets were obtained from Benner et al.  for mouse (two samples of each) and from Blodgett et al.  for human (six samples each) . Reads were mapped into the mouse GRCm38 and the human GRCh37 genomes (Ensembl genome version 75) using default options. Gene expression was measured from the mapped reads by using HT-seq-count . PCA, using princomp function of R, was calculated for the whole dataset using the variance stabilization transformation values obtained by DESeq R package from the gene expression values . For differential expression analysis, we used the R package DESeq2 . DESeq2 employs shrinkage estimation for dispersions and fold change; it uses Wald test for significance with posterior adjustment of P values using the procedure of Benjamini and Hochberg (giving adjusted P values). Genes differentially expressed were selected with an adjusted P < 0.05 and a fold change > 4. For the comparison of the acinar, ductal and endocrine cell data, in-silico endocrine datasets were simulated by combining alpha, beta and delta RNA-seq data. This approach was taken to decrease the number of pairwise comparisons and could be used since the sequencing deepness of each library was in a similar range (below of a factor 2). Endocrine dataset #1 was obtained by combining the mapped reads of alpha1 (47 × 106 reads), beta1 (48 × 106 reads), and delta1 (31 × 106 reads) datasets; endocrine dataset #2 is a mix of alpha2 (46 × 106 reads), beta2 (29 × 106 reads), and delta2 (30 106 reads); endocrine dataset #3 is the combination of alpha3 (59 × 106 reads), beta3 (62 × 106 reads), and delta3 (59 × 106 reads) datasets. The RNA-seq raw data have been deposited on ENA under the accession number PRJEB10140.
Comparison of the human, murine, and zebrafish pancreatic transcriptomes
The predicted orthologs among zebrafish, mouse and human were obtained from Ensembl . The information from the three species was retrieved using Biomart tool . Two different orthology tables were generated (tables available upon request). The first table contains all zebrafish, murine and human genes with 1-1-1 orthology relationship. Orthology table two comprises all 1-1-1 orthologs as well as the genes presenting 1-many-many orthology relationships and thus includes all duplicated genes (paralogs), which are notably found in the zebrafish genome. The interspecies PC analysis was performed using only the genes with a 1-1-1 orthology relationship. The genes presenting an endocrine-enriched or an exocrine-enriched expression were identified in mouse and in human by using DESeq2 software selecting genes with at least four-fold higher expression in islet dataset or in whole pancreas dataset (adjusted P < 0.05). The interspecies comparison of endocrine- and exocrine-enriched genes was performed using the orthology table two.
GO enrichment analysis
Tissue- and cell type-enriched genes were converted and uploaded to DAVID bioinformatics resource . Using the Functional annotation tool, we run a GO enrichment analysis for endocrine and acinar enriched genes as well as for the alpha-, beta- and delta-enriched genes. Enriched processes were identified by GOTerm_BP_FAT with a P < 0.1.
In situ hybridization
Antisense RNA probe for the different genes were prepared as described by Thisse et al. , except for fev . Briefly, primers were designed to amplify a part of the transcript that is used as a template to synthesize the probe. The reverse primer at the 5’ end contains the minimal promoter sequence for T3 RNA polymerase (5’-AATTAACCCTCACTAAAGGGAG-3’), templates were amplified by RT-PCR using the set of primers shown in Additional file 17: Table S10. Whole mount in situ hybridization and fluorescent in situ hybridization (WISH and FISH) were performed as described by Mavropoulos et al. , applying some modification to this protocol. Briefly, larvae of 3 dpf or older were incubated during 20 minutes in methanol and 3% H2O2 at room temperature, prior to dehydration. Antisense probe hybridization was performed using 10–50 ng of DIG- and DNP-probes in hybridization buffer containing 5% dextran sulfate (MW: 500,000) at 65 °C overnight. Antibodies were pre-absorbed on homogenized larvae (mix of different developmental stages) for 2 h at room temperature and then diluted to 1/3000 DIG-AP, 1/1500 DIG-HRP, and 1/800 DNP-HRP (PerkinElmer).
Inactivation of myt1a and myt1b genes by multiplex CRISPR/cas9 mutagenesis
Mutations in the myt1a and myt1b genes were generated by multiplex CRISPR/Cas9 technology essentially as described previously [94, 95]. The nls-zCas9-nls mRNA was synthesized by transcription of the plasmid pT3TS-nCas9n (Addgene). CRISPR guide RNAs were selected using CRISPR design and chopchop software to target the beginning of Myt1a and Myt1b coding regions (guides 1) and the regions coding for the first zinc finger domain (guides 2). The following target sites were used: GCCAAGACGCAGATGATAAGCGG and GATGGTTTAGGCCATGTCAGTGG for myt1a, and GTCTGAGGGAGGGCCGGCAGCGG and TGCCATTGCATCCTGGAGTGGGG for myt1b (PAM motifs are underlined). The DNA templates were prepared by annealing and filling two oligonucleotides containing the T7 promoter sequence and the target sequences as previously described . After synthesis and purification of gRNA, fertilized zebrafish eggs were injected with approximately 1 nL of a solution containing 50 ng of the four gRNA and 300 ng of nls-zCas9-nls mRNA. The efficiency of mutagenesis was verified by genotyping using Heteroduplex Migration Assays after amplification of targeted genomic sequences. A few injected embryos were fixed in PFA at 48 hpf for phenotypic analysis and the others were raised until adulthood. Founder fish transmitting a germline mutation in myt1b were outcrossed with wild type fish; F1 fish harboring a 5 bp insertion in myt1b (myt1b ulg039 allele) causing a frameshift in the coding sequences were incrossed to generate myt1b –/– embryos and +/–, +/+ siblings, which were analyzed by immunohistochemistry.
Expression of insulin and glucagon was analyzed by immunofluorescence on whole-mount zebrafish embryos. After overnight fixation in 2% PFA at 4 °C, 1 hour incubation in PBS 1% Triton X-100 for 2 hours at room temperature in blocking solution (4% BSA, 10% DMSO, 0.3% Triton X-100 in PBS), embryos were incubated with the primary antibodies anti-Mouse Glucagon 1/200 (Sigma, G2654) and Anti-Guinea Pig Insulin 1/300 (MP, 64714). After washing, embryos were incubated with the secondary antibodies Anti-Mouse Alexa 568 or 633 (Invitrogen, A-11004 and A-21052) and Anti-Guinea Pig Alexa 568 or 633 (Invitrogen, A-11075 and A-21105) diluted 1/300. After washing and mounting, embryos were scanned with a Leica SP5 confocal microscope and images were analyzed using Imaris 7.2.3 software. Alpha and beta cell mass was measured using Imaris based on 3D reconstitution of Fluorescence signal obtained by immunofluorescence; the same confocal and Imaris parameter settings were used for wild-type and mutant embryos. To compare the number of alpha cells and beta cells in wild-type and mutants, glucagon+ and insulin+ cells were counted in every 6-μm optical section throughout the whole principal islet. Expression of GFP and mCherry in the pancreas of Tg(gcga:GFP);(ins:NTR-mCherry) adult transgenic fish as well as of Tg(sst2:GFP) adult fish was analyzed by immunofluorescence on cryosections (Additional file 1: Figure S1). The antibodies used are the same than described above and also include the anti-Rabbit Somatostatin 1/300 (Dako, a0564) and Anti-Rabbit Alexa 568 or 633 1/300 (Invitrogen, A-11011 and A-21070). Expression of GFP in beta cells was also verified for the Tg(insulin:GFP) larvae (Additional file 1: Figure S1).
Days post fertilization
Fluorescent in situ hybridization
G-couple protein receptors
Hours post fertilization
Principal component analysis
Whole-mount in situ hybridization
Genes with conserved expression pattern in zebrafish and human
Genes with conserved expression pattern in zebrafish and mice
Genes with conserved expression pattern in zebrafish, mice and human
We are very grateful to Francesco Argenton for the transgenic line Tg(gcga:GFP), to Zhen Li and Zhiyuan Gong for the transgenic line Tg(st22:GFP), to Steven Leach for the transgenic line Tg(ptf1a:GFP), and to Leonard Zon for the kgg/cdx4 mutant line. We thank the following GIGA technical platforms: GIGA-Zebrafish (H. Pendeville), GIGA-Cell Imaging and Flow Cytometry platform (S. Ormenese and S. Raafat), GIGA-Genotranscriptomic (B. Hennuy, W. Coppieters and L. Karim), and GIGA-Immunohistochemistry (C. Humblet and E. Dortu). ET-S was supported by WBI, Becas Chile and Leon Fredericq fund, AL by FRIA, KP by WBI, MLV, IM, and BP are Chercheur qualifié FNRS. This work was funded by the FNRS-FRS, the Belgian State's “Interuniversity Attraction Poles” Program (SSTC, PAI) and the “Fonds Speciaux” from ULg.
Availability of data and materials
The RNA-seq raw data have been deposited on ENA (http://www.ebi.ac.uk/ena) under the accession number PRJEB10140. The gene expression values are given in Additional file 2: Tables S1 and Additional file 3: Table S2.
ET-S, MLV, IM, and BP designed the experiments and ET-S, AL, AB, KP, DB, and IM performed the experiments. ET-S, MLV, IM, and BP wrote the manuscript. All authors read and approved the article.
The authors declare that they have no competing interests.
All animal work has been conducted according to national guidelines and all animal experiments described herein were approved by the ethical committee of the University of Liège (protocol numbers 1328).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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