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. 2017 Mar 21;15(1):21.
doi: 10.1186/s12915-017-0362-x.

Transcriptome analysis of pancreatic cells across distant species highlights novel important regulator genes

Estefania Tarifeño-Saldivia  1 , Arnaud Lavergne  1 , Alice Bernard  1 , Keerthana Padamata  1 , David Bergemann  1 , Marianne L Voz  1 , Isabelle Manfroid  1 , Bernard Peers  2
Affiliations

Transcriptome analysis of pancreatic cells across distant species highlights novel important regulator genes

Estefania Tarifeño-Saldivia et al. BMC Biol. .

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Keywords: Acinar cells; Comparative transcriptomics; Ductal cells; Endocrine cells; Pancreas; RNA-seq.

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Figures

Fig. 1
Fig. 1
Global analysis of the zebrafish pancreatic RNA-seq data. a Principal component analyses (PCA) of gene VSD (Variance stabilizing transformation) calculated by DESeq package for the 16 zebrafish pancreatic datasets. b PCA of gene VSD for beta, alpha, and delta cells (nine samples in total). The PCA plots show a close clustering of all replicates and distinct clusters for each pancreatic cell type. PCAs were calculated using all the 33,726 genes annotated on Zv9 version 75 ensembl
Fig. 2
Fig. 2
Transcriptomic signatures of zebrafish endocrine, ducta,l and acinar cells. a Venn diagram showing the number of genes with acinar-, endocrine- and ductal-enriched expression obtained with DESeq2 using a cut-off ratio of four-fold and adjusted P < 0.05. b Heatmap plot showing the expression pattern of all differentially expressed genes. Genes listed on the right side of the plot are examples of either known markers (black) or new genes with endocrine-, acinar-, and ductal-enriched expression discovered by this analysis (red). The three endocrine samples were generated in silico by combining the raw data obtained from alpha, beta, and delta cells as described in the Methods section. c and d Gene ontology enrichment analysis for the 1853 genes enriched in endocrine cells (c) and the 492 genes enriched in acinar cells (d) displaying the most enriched biological process
Fig. 3
Fig. 3
Conservation of the pancreatic endocrine signature among vertebrates. a Principle component analysis (PCA) performed on human and mouse whole pancreas and islet RNA-seq datasets and including the zebrafish endocrine and acinar datasets. The analysis was performed using the 9393 genes displaying 1-1-1 orthology relationship between zebrafish, mouse, and human using a total of 24 RNA-seq samples. The endocrine datasets of zebrafish, mouse, and human cluster along the PC1 axis representing 55% of the variance, indicating a conserved endocrine signature. The human pancreatic sample enriched in acinar tissue [9] clusters with the human whole pancreatic samples [37] due to the very high proportion of acinar cells in pancreas. b Venn diagram showing the number of endocrine-enriched genes found only in zebrafish, mouse, or human and those displaying conserved endocrine-enrichment in two species or in the three species (shown in intersections). Due to gene duplications in some species and often in zebrafish, the number of corresponding murine (M) or human (H) orthologous genes is given in brackets in each intersection. The full list of conserved endocrine-enriched genes is given in Additional file 5: Table S4
Fig. 4
Fig. 4
Expression of genes in endocrine cells of the dorsal pancreatic bud. Whole-mount in situ hybridization on zebrafish embryos showing endocrine pancreatic expression of some new zebrafish endocrine markers (n > 15). Genes with conserved endocrine-enriched expression in zebrafish, human, and mouse (ZHM), or endocrine-enriched in zebrafish and mouse (ZH), or in zebrafish and human (HM) are indicated. Z: Gene endocrine enriched only in zebrafish. N-orth: Endocrine enriched zebrafish gene with no obvious human or mouse ortholog. Arrowheads indicate the location of the dorsal pancreatic bud containing embryonic endocrine cells and insets at the top-right display higher magnification view of the pancreatic bud
Fig. 5
Fig. 5
Zebrafish genes differentially expressed in the endocrine cell subtypes. a Venn diagram displaying the number of endocrine genes with alpha-, beta- and delta-enriched expression identified with DESeq algorithm based on a cut-off ratio of four-fold and adjusted P < 0.05. b Heatmap plot showing the expression pattern of all differentially expressed genes. Genes listed at the right side of the plot are some examples of either known (black) or new (red) markers identified in this analysis. c Principal component analysis performed on the nine zebrafish endocrine RNAseq datasets using the 1853 endocrine-enriched genes. Compared to the PC plot of Fig. 1b performed on all annotated zebrafish genes (33,726 genes), this plot shows a tighter clustering of all replicates and better discrimination between the three endocrine cell subtypes
Fig. 6
Fig. 6
Expression of ppdpfb and pcsk2 genes in zebrafish beta cells. Co-labeling by fluorescent in situ hybridization (FISH) of ppdpfb and pcsk2 with insulin, glucagon, and somatostatin. a–c ppdpfb is mainly expressed by beta cells (a, arrows) and in few alpha cells (b, arrows). No expression of ppdpfb was observed in delta cells (c). Beta cells specifically expressed pcsk2 (d, arrows) while no expression was detected in alpha or delta cells (e and f). (Analyzed embryos > 10)
Fig. 7
Fig. 7
Identification of genes with conserved enriched expression in alpha and beta cells. Venn diagrams showing the number of genes presenting enriched expression in alpha cells (right panel) and beta cells (left panel) and displaying this enrichment in zebrafish, mice, and/or human (shown in intersections). Due to gene duplications in some species and often in zebrafish, the number of corresponding murine (M) or human (H) orthologous genes is given in brackets in each intersection. The alpha- and beta-enriched genes were selected by DESeq2 with fold change > 4 and adjusted P < 0.05 using two murine alpha and beta cell preparations [12], six human alpha and beta cell preparations [48], and three zebrafish alpha and beta cell preparations (this study). The full list of conserved beta- and alpha-enriched genes is given in Additional file 14: Table S8 and Additional file 15: Tables S9
Fig. 8
Fig. 8
Delta cell differentiation is disrupted in the cdx4 mutant embryos. Analysis of wild-type and cdx4 tv205–/– mutant embryos at 48 hpf by WHISH (a) and FISH (b–d) using delta cell markers (sst2, lamc2, slc7a7, and map3k15) as well as gcga and insulin. cdx4 tv205 mutants display a loss of sst2 (a), lamc2 (b), and slc7a7 (c) pancreatic expression and an increase of beta cells. Map3k15 expression is strongly reduced in the pancreatic islet (yellow arrows) while not affected in the presumptive pronephric glomeruli (white arrows) (d). No obvious effect is observed on gcga expression. Nuclei are stained with DAPI (grey staining). (Analyzed embryos > 10)
Fig. 9
Fig. 9
Reduced glucagon expression in the myt1b zebrafish mutant embryos. Glucagon expression was analyzed by immunofluorescence in F2 myt1b ulg019 homozygous mutant larvae and in sibling +/+ (WT) and +/– larvae at 2 dpf. The volume of the alpha cell mass was determined in each embryo by the imaging software Imaris. The graph shows the quantification measured for all myt1b–/– embryos and for +/– or +/+ siblings, indicating a slight by statistical significant decrease of alpha cell mass (each point is the alpha cell mass measured in one embryo)
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