BACKGROUND
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  have been described in pancreatic islets, each characterized by the expression of a particular hormone .
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  were conducted on endocrine pancreatic cells isolated from human  or rodents at adult and embryonic stages .
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 .
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  <cit> .
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 .
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  <cit> .
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  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 cdx <dig> 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.
RESULTS
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 .
acinar cells were obtained from the bac transgenic lines tg  <cit> .
the endocrine beta and delta cells were isolated, respectively, from the transgenic lines tg
ulg021tg  and tg  <cit> ; the alpha cells were obtained from the tg/tg line through selection of gfp+/mcherry cells  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  <dig> 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
ulg004tg transgenic line  <cit> , and these data were compared in the present study with endocrine and acinar cell transcriptomes.
principal component analysis  of all these pancreatic rna-seq datasets showed a tight clustering of all replicates for each pancreatic cell type , underscoring the high reproducibility of the data.
as expected, the pca also revealed a closer clustering of the three endocrine cell subtypes compared to the ductal and acinar cell types; however, when pca is performed only with the endocrine datasets, clear distinct transcriptome profiles are observed for the alpha, beta, and delta cell subtypes .
comparison of the expression levels of various known markers of each pancreatic cell type confirmed the high purity of each cell preparation.
indeed, glucagon a , insulin , and somatostatin  <dig>  were selectively detected at very high levels in alpha, beta, and delta cell libraries, respectively, representing in average 24%, 10%, and 32% of the total reads number in the corresponding libraries, while being detected at much lower levels in the other libraries .
the trypsin  and chymotrypsin-like elastase member <dig>  genes were the strongest expressed genes in acinar cells representing each about 10% of all total reads of the acinar cell libraries.
these two acinar markers were not detected at significant levels in endocrine datasets consistent with an accurate cell sorting.
among the genes expressed at highest levels in ducts, we find the cldnb, sdc <dig>  and the epcam genes coding for cell adhesion molecules, each representing less than 1% of total reads of ductal datasets.
all these results indicate an accurate and reproducible sorting of the different pancreatic cells allowing the identification of genes selectively expressed in each pancreatic cell type.
expression values for all genes in all samples are shown in additional file 2: table s <dig> and additional file 3: tables s <dig> fig.
<dig> global analysis of the zebrafish pancreatic rna-seq data.
a principal component analyses  of gene vsd  calculated by deseq package for the  <dig> zebrafish pancreatic datasets.
b pca of gene vsd for beta, alpha, and delta cells .
the pca plots show a close clustering of all replicates and distinct clusters for each pancreatic cell type.
pcas were calculated using all the  <dig>  genes annotated on zv <dig> version  <dig> ensembl
percentages of mapped reads obtained for some genes on the total number of mapped reads in each type of libraries
identification of genes enriched in endocrine, acinar, and ductal pancreatic cells
the clear distinct transcriptomic profiles observed for endocrine, acinar, and ductal cells prompt us to identify, in a first step, all genes presenting a differential expression in these three pancreatic tissues .
by using these cut-off values,  <dig>   <dig>  and  <dig> genes were found to be enriched in endocrine, ductal, and acinar cells, respectively .
as expected, the zebrafish endocrine-specific genes include several orthologs of mammalian genes known as endocrine markers such as those involved in hormone regulation and secretion, like the proprotein convertases , carboxypeptidase e , secretogranins , atp-dependent potassium channels , and the voltage-dependent type calcium channels , among others.
moreover, most of the transcription factors previously shown to be crucial for the differentiation of endocrine cells   <cit>  are detected in the endocrine signature validating our rna-seq data.
interestingly, many other transcription factors, whose function in endocrine cells is still not known, are present in this list such as egr <dig>  creb3l <dig>  lmo <dig>  cdx1b, or cdx <dig> .
gene ontology  enrichment analysis using david revealed known biological pathways in endocrine cells such as “potassium ion transport”, “regulation of secretion” and “regulation of exocytosis” as well as “response to glucose”, and “g-protein coupled receptor signaling” .
indeed, many g-couple protein receptors  and several regulators of g-protein signaling, like gpr <dig>  gpr <dig>  gpr <dig>  or gpr <dig> as well as rgs <dig>  rgs5a, rgs <dig>  or rgs <dig>  among others, were found enriched in the endocrine cells.
while some of these regulatory proteins have been previously reported to control pancreatic islet activity  <cit> , others were not yet known to have a selective expression in pancreatic endocrine cells nor to play a role in islet physiology.fig.
<dig> 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 deseq <dig> using a cut-off ratio of four-fold and adjusted p <  <dig> .
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  or new genes with endocrine-, acinar-, and ductal-enriched expression discovered by this analysis .
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  <dig> genes enriched in endocrine cells  and the  <dig> genes enriched in acinar cells  displaying the most enriched biological process
similar observations were done for the acinar and ductal cell transcriptomic signature.
indeed, as expected, many genes coding for digestive enzymes, such as trypsin , elastase , trypsin-like , amylase  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, klf <dig>  or nr0b2a .
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” .
as for the duct transcriptome, the analysis reveals novel markers such as id2a and frzb in addition to known markers like sox9b, nkx <dig> , onecut <dig>  and ctgfa, as we recently described  <cit> .
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
the evolutionarily conserved expression of a gene in a specific cell type is a strong argument for its crucial function in that cell.
this concept is widely supported by numerous studies showing conserved expression of many transcription factors in development and cell differentiation  <cit> , including pancreatic cells  <cit> .
to perform interspecies transcriptome comparison, we retrieved, from public databases, several rna-seq datasets obtained on murine and human pancreatic islets, whole pancreas , as well as a human pancreatic sample enriched in acinar tissue  <cit> .
while mouse rna-seq data are not presently available for acinar enriched preparations, the genes with either endocrine-enriched or exocrine-enriched expression  can nevertheless be identified by comparing rna-seq from purified pancreatic islets versus rna-seq from whole pancreas .
when a global comparison of the zebrafish endocrine and acinar datasets with the murine and human pancreatic data was performed using a pca plot analysis, we observed that the data clustered according to the tissue type  in the first component , while the data tended to cluster according to the species in the second component  .
thus, this global analysis suggests the existence of sets of genes displaying an evolutionary conserved endocrine and exocrine expression.
endocrine and exocrine genes were identified from human and murine datasets by selecting all genes presenting at least four-fold higher expression in each pancreatic tissue .
next, we compared the endocrine and exocrine gene lists between the three vertebrate species.
the venn diagram on fig.
3b indicates that, while a large fraction of endocrine-enriched genes are species-specific, some sets of genes display a conserved endocrine-enriched expression in two species or in the three species.
indeed, among the  <dig> zebrafish endocrine genes,  <dig> are also classified as “endocrine” in human and mice.
this endocrine signature, conserved between zebrafish, mice and human , includes many factors known to be involved in hormone maturation, secretion, and regulation.
many transcription factors crucial for endocrine cell differentiation  <cit>  are found in this zmh conserved endocrine signature , thereby validating this method to identify genes with important physiological function.
the zmh endocrine genes  also comprise components of signaling pathways reported to control pancreatic endocrine cells in mammals such as the glutamate receptor gria2a and gria2b  <cit> , regulators of g-protein signaling   <cit> , urocortin <dig>  <cit> , ion channels , and calcium dependent proteins .
this indicates that many regulatory processes controlling endocrine cell development and physiology have been maintained from fish to human.
interestingly, the zmh endocrine genes include several regulatory genes whose function in pancreatic cells is still unknown or not well defined such as the kinases map3k <dig> and mast <dig>  the signaling factor gpr <dig>  as well as the transcription factors npas4a, lmo <dig>  fev, and etv <dig>  similarly, the comparison of the exocrine-enriched genes across zebrafish, mice, and human shows that a fraction of genes displays conserved exocrine enrichment  – among the  <dig> zebrafish exocrine genes ,  <dig> show exocrine enriched expression also in human and mice.
this conserved exocrine signature  includes, as expected, many digestive enzymes as well as known exocrine transcription factors such as ptf1a, rbpjl, and nr5a <dig>  <cit> , but also regulatory genes with unknown pancreatic function, like klf <dig> fig.
<dig> conservation of the pancreatic endocrine signature among vertebrates.
a principle component analysis  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  <dig> genes displaying 1-1- <dig> orthology relationship between zebrafish, mouse, and human using a total of  <dig> rna-seq samples.
the endocrine datasets of zebrafish, mouse, and human cluster along the pc <dig> axis representing 55% of the variance, indicating a conserved endocrine signature.
the human pancreatic sample enriched in acinar tissue  <cit>  clusters with the human whole pancreatic samples  <cit>  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 .
due to gene duplications in some species and often in zebrafish, the number of corresponding murine  or human  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
transcription factors enriched in endocrine cells in zebrafish, human and/or mouse.
the level of expression in endocrine, acinar, and ductal cells is shown for each gene in the second, third, and fourth column, respectively.
the degree of expression pattern conservation is shown in the last column as zhm: endocrine-enriched in zebrafish, human, and mouse; zm: endocrine-enriched in zebrafish and mouse; zh: endocrine-enriched in zebrafish and human
the interspecies comparison of both exocrine and endocrine signature also indicates that many genes display the same enrichment in only two species .
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
while many transcription factors controlling pancreatic endocrine cell differentiation in embryos remain expressed and functional in mature cells from adults, there are nevertheless striking differences between the transcriptomes of fetal and adult cells  <cit> .
in order to determine the fraction of the endocrine-enriched genes in adult zebrafish that are already expressed in the first embryonic endocrine cells, we determined the transcriptomic profile of endocrine cells isolated from tg embryos at  <dig> hpf.
we detected  <dig> genes expressed in embryonic pancreatic cells above the threshold level of  <dig> normalized count.
by comparing these data with adult endocrine data, we found that, among the  <dig> genes enriched in adult endocrine cells,  <dig>  genes were already detected in embryonic cells .
the expression of some of these endocrine-enriched genes was further characterized by in situ hybridization  on whole zebrafish embryos .
we analyzed genes identified as endocrine enriched in the three species , in two species , or only in zebrafish .
all these genes were detected in the embryonic endocrine pancreatic cells by ish, validating the rna-seq data.
thus, these data indicate that the early embryonic endocrine cells from the dorsal pancreatic bud express a large fraction of the genes constituting the adult endocrine signature.fig.
<dig> 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 .
genes with conserved endocrine-enriched expression in zebrafish, human, and mouse , or endocrine-enriched in zebrafish and mouse , or in zebrafish and human  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
characterization of the transcriptomic signatures for the endocrine cell subtypes
in order to define the molecular signatures of alpha, beta, and delta cell subtypes, the  <dig> endocrine genes identified in adult zebrafish  were classified according to their differential expression in the distinct endocrine cell subtype ;  <dig>   <dig> and  <dig> endocrine genes were found to be enriched in alpha, beta, and delta cells, respectively .
the heatmap plot in fig.
5b presents an overview of the expression pattern for all differentially expressed genes .
pca using all  <dig> endocrine-enriched genes displays a clear discrimination of the alpha, beta, and delta cell transcriptomes .
go enrichment analysis performed on beta cell-specific genes identifies “response to organic substrate”, “ion transport”, or “response to nutrient levels” as enriched pathways among others .
the gene expressed at the highest level in the beta cells after insulin is ppdpfb.
this gene is the paralog gene of the ppdpfa gene , which was reported as specifically expressed in acinar cells and controls their differentiation  <cit> .
interestingly, the paralog ppdpfb is specifically expressed in the endocrine pancreas and mainly in beta cells.
we verified the expression of ppdpfb in  <dig> hpf zebrafish embryos by fluorescent ish  and, according to the rna-seq data, we confirmed its expression in many beta cells and in a few alpha cells but not in delta cells .
the rna-seq data also confirmed the selective expression of the zebrafish nkx <dig>  gene in beta cells, as previously reported  <cit> .
expression of the zebrafish pcsk <dig> gene is more enriched in beta cells compared to the pcsk <dig> gene , while in mouse and human, only pcsk <dig> was reported to be more enriched in beta compared to alpha cells  <cit> .
the enrichment of pcsk <dig> in beta cells was confirmed by fish on zebrafish embryos .
the zebrafish estrogen receptor  <dig>  esr <dig>  is also strongly enriched in beta cells .
finally, the beta cell-selective expression of spondin 1b , an activator of the wnt pathway, was also validated by fish on zebrafish embryos .fig.
<dig> 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 <  <dig> .
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  or new  markers identified in this analysis.
c principal component analysis performed on the nine zebrafish endocrine rnaseq datasets using the  <dig> endocrine-enriched genes.
compared to the pc plot of fig.
1b performed on all annotated zebrafish genes , this plot shows a tighter clustering of all replicates and better discrimination between the three endocrine cell subtypes
fig.
<dig> expression of ppdpfb and pcsk <dig> genes in zebrafish beta cells.
co-labeling by fluorescent in situ hybridization  of ppdpfb and pcsk <dig> with insulin, glucagon, and somatostatin.
a–c
ppdpfb is mainly expressed by beta cells  and in few alpha cells .
no expression of ppdpfb was observed in delta cells .
beta cells specifically expressed pcsk <dig>  while no expression was detected in alpha or delta cells .
as expected, the genes specifically expressed in alpha cells include those coding for the two zebrafish glucagon hormones  as well as for the arx transcription factor  but also novel markers including peptide hormones such as prepronociceptin  and neuropeptide b  or the calcium regulated factor scinderin like b .
the alpha-enriched genes also comprise the transcription factors etv <dig> and si:ch211-145o <dig>  .
the alpha-selective expression of pnoca and scinlb was confirmed by fish, validating the rna-seq data .
as for the delta cells, besides the somatostatin genes  <cit> , the rna-seq data revealed many novel markers, which include genes coding for transcription factors such as dlx3b, cdx <dig>  cdx <dig>  and tbx2b, as well as genes coding for signaling factors like bmp7b or the kinase map3k <dig>  interestingly, as demonstrated by the go enrichment analysis , many gpcr are specifically enriched in delta cells such as npy8br , glra4a , uts2r , ptger1b & 2a , adra1d , grm <dig> , oprd1b , and gpr <dig>  pinpointing these endocrine cells as targets of diverse external metabolic signals.
we confirmed the selective expression for several genes, including the cdx <dig>  cdx <dig>  lamc <dig>  and map3k <dig> genes, detected already at  <dig> hpf in delta cells .
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
the zebrafish rna-seq data led to the identification of many novel markers for each endocrine cell subtypes raising the question of whether such markers display the same cell subtype-specific expression in other species.
as the transcriptome of human and murine alpha and beta cells have been determined by a similar approach using highly purified facs cell preparations  <cit> , this allowed us to perform an interspecies comparison.
in order to identify all alpha and beta cell differentially expressed genes , we performed exactly the same procedure on the zebrafish, mouse, and human rna-seq datasets, namely a comparison of the entire alpha and beta cell transcriptomes within each species using the same software and selection criteria .
this led to the identification of  <dig>   <dig>  and  <dig> alpha-enriched genes as well as  <dig>   <dig>  and  <dig> beta-enriched genes in human, mouse, and zebrafish, respectively.
surprisingly, the interspecies comparison of these sets of genes revealed that the large majority of alpha- and beta- enriched genes are species specific .
indeed, among the  <dig> zebrafish beta-enriched genes, only three have maintained beta cell preferential expression in human and mice, namely insulin, the transcription factor pdx <dig>  and the glucagon receptor gcgra .
similarly, among the  <dig> zebrafish alpha cell-enriched genes, only  <dig> have human and murine orthologs with alpha cell-enriched expression .
as expected, glucagon and arx are part of the conserved alpha cell genes.
the adcy <dig> and adcy <dig> genes coding for adenylate cyclase are also alpha-enriched in the three vertebrate species supporting the important role of camp in alpha cells  <cit> .
the gc gene   <cit>  and fev  are among the conserved alpha cell signatures, suggesting a specific function in this endocrine cell subtype.
taken together, all these analyses indicated that, while hundreds of genes display conserved expression in endocrine and exocrine cells, there is much less conservation for the genes differentially expressed between the endocrine cell subtypes alpha and beta.
these data confirm the striking differences between species as previously reported for the human and murine alpha- and beta-enriched genes  <cit> .
importantly, many of the conserved alpha- and beta-specific genes identified in the present study were also recently highlighted as enriched in these endocrine cell types in several single cell rna-seq studies   and additional file 15: table s <dig>  for comparison).fig.
<dig> 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  and beta cells  and displaying this enrichment in zebrafish, mice, and/or human .
due to gene duplications in some species and often in zebrafish, the number of corresponding murine  or human  orthologous genes is given in brackets in each intersection.
the alpha- and beta-enriched genes were selected by deseq <dig> with fold change >  <dig> and adjusted p <  <dig>  using two murine alpha and beta cell preparations  <cit> , six human alpha and beta cell preparations  <cit> , and three zebrafish alpha and beta cell preparations .
the full list of conserved beta- and alpha-enriched genes is given in additional file 14: table s <dig> and additional file 15: tables s9
expression enrichment in alpha and beta cells is given as the log <dig> fold-change in the three species
role of the zebrafish myt1b and cdx <dig> genes in endocrine cell differentiation
to validate our cross-species approach to identify important pancreatic regulatory factors, we selected two transcriptional regulators that were expressed at high levels in endocrine cells at the early developmental stage  and performed loss of function studies.
mutations of myt1b gene  and of the cdx <dig> gene  were found to affect endocrine cell differentiation.
as cdx <dig> is selectively expressed in delta cells, we analyzed the expression of several novel delta cell markers in the cdx4
tv <dig> null zebrafish mutant  <cit> .
a previous study has shown that the zebrafish cdx <dig> mutant displays defects in the anterio-posterior patterning of the endoderm, with notably a posteriorly-shifted pancreas and an increase of pancreatic beta cell number  <cit> ; however, the selective expression of cdx <dig> in delta cells was unknown as well as its function in this endocrine cell type.
analysis of the null cdx4
tv <dig> mutant embryos revealed a complete loss of somatostatin <dig> gene expression .
similarly, expression of the new delta cell markers lamc <dig> and slc7a <dig> was almost undetectable in the mutant embryos, in contrast to the increase of insulin expression .
expression of map3k <dig> gene in delta pancreatic cells was also specifically abrogated by cdx <dig> mutation, while expression of this gene was not affected anteriorly at the level of the pronephric glomeruli .
all these data demonstrate the important role of cdx <dig> for delta cell differentiation.fig.
<dig> delta cell differentiation is disrupted in the cdx <dig> mutant embryos.
analysis of wild-type and cdx4
tv205–/– mutant embryos at  <dig> hpf by whish  and fish  using delta cell markers  as well as gcga and insulin.
cdx4
tv <dig> mutants display a loss of sst <dig> , lamc <dig> , and slc7a <dig>  pancreatic expression and an increase of beta cells.
map3k <dig> expression is strongly reduced in the pancreatic islet  while not affected in the presumptive pronephric glomeruli  .
no obvious effect is observed on gcga expression.
nuclei are stained with dapi .
our transcriptomic analyses show that embryonic pancreatic cells express both myt1a and myt1b paralogs, with myt1b being expressed at much higher levels.
this was confirmed by ish revealing expression of myt1b in the dorsal pancreatic bud of zebrafish embryos , while the paralog myt1a was barely detectable .
the adult rna-seq data indicate that the myt1a/b genes are expressed in alpha, beta, and delta cells.
simultaneous inactivation of the two zebrafish myt <dig> genes was performed by multiplex crispr/cas <dig> mutagenesis through injection of four guide rna .
injected embryos revealed a significant decrease of glucagon expression and no significant effect on insulin expression .
to confirm these data obtained in f <dig> embryos, a line harboring a null mutation in myt1b  was raised.
alpha cell mass was decreased in the myt1b
ulg029–/– embryos compared to the wild-type and heterozygous siblings .fig.
<dig> reduced glucagon expression in the myt1b zebrafish mutant embryos.
glucagon expression was analyzed by immunofluorescence in f <dig> myt1b
ulg <dig> homozygous mutant larvae and in sibling +/+  and +/– larvae at  <dig> 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
discussion
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, isl <dig>  pax <dig>  insm <dig>  ptf1a, and rbpjl, among others.
myt <dig> 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  <dig> dpf embryos.
additionally, cdx <dig>  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 gria <dig>  shown to regulate hormone synthesis and secretion in rodents  <cit> .
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 abcc <dig> and kcnj <dig> and the voltage-dependent ca++ channel cacna2d <dig>  the use of this pathway in zebrafish is further strengthened by the very high expression of the glucose transporter glut <dig>  and the glucokinase  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 sstr <dig>   <cit> , casr  <cit> , or celsr <dig>  <cit> , 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 .
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 lmo <dig> and npas4a, or the signaling factors gpr <dig>  rgs <dig>  and rgs <dig>  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 mnx <dig> and murine esr <dig>  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 .
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 mnx <dig> and esr <dig>  indicating that important genes fall in these two categories.
for proof, cdx <dig>  classified as endocrine-enriched in zebrafish and mice , was found here to be crucial for endocrine delta cell differentiation as many novel delta cell makers were drastically reduced in the cdx <dig> mutant zebrafish embryos.
our findings warrant future analyses on the murine  cdx <dig> 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 cdx <dig> mutant suggests that cdx <dig> 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 .
this is also supported by the venn diagram  showing that  the number of genes with enriched expression in both alpha and beta cells and not in delta cells  is higher than those of the “alpha-delta” enriched and “beta-delta” enriched genes , and  the number of delta cell markers is higher than the alpha and beta cell marker .
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  <cit> .
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  <cit> .
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  <dig> 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 .
differences between human and mouse alpha- and beta-enriched genes have also been previously noticed  <cit> .
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  <cit> .
similarly, pdx <dig>  one of the three conserved beta cell genes, is required to maintain beta cell identify and repress the alpha cell program  <cit> .
this first hypothesis is also supported by the ability of alpha and beta cells to transdifferentiate into each other  <cit> .
a second explanation can be the relatively stringent threshold that we used to select differentially expressed genes .
for example, the mnx <dig> gene, known to be involved in beta cell differentiation  <cit> , 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 .
these differences in enrichment between human and mouse mnx <dig> is confirmed in the recent single cell rna-seq data .
similarly, the prohormone convertase  <dig> gene pcsk <dig> required for insulin hormone maturation was only  <dig> -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, nkx <dig>  is expressed in beta cells in mice and human but not in zebrafish, where its paralog nkx <dig>  instead of nkx <dig>  is selectively expressed in mature beta cells  <cit> .
nkx <dig>  may fulfill the function of nkx <dig>  as nkx <dig>  and nkx <dig>  have equivalent biological activities  <cit> .
another example is the zn++ transporter scl30a <dig>  which is strongly expressed in beta cells in mammals but not in zebrafish, which instead strongly expresses the homologous zn++ transporter scl30a <dig>  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 neurog <dig> as pancreatic endocrine cell fate determinant is fulfilled by two other bhlh factors ascl1b and neurod <dig>  <cit> .
a functional switch is also observed for the specification of the secretory cell of the intestine, played by atoh <dig> in mice and by ascl1a in zebrafish  <cit> .
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 .
such compensations seem to occur for the murine myt <dig> gene as the myt <dig> ko mice only display very mild pancreatic defects with no decrease of endocrine cells  <cit> , while more drastic defects are observed by expression of a dominant negative myt1-eng protein  <cit> .
furthermore, adult murine endocrine pancreatic cells express the homologous myt1l and myt3/st <dig> genes  <cit>  and their expression increases in the myt <dig> ko mice  <cit> .
in zebrafish, myt1a, myt1l, and myt3/st <dig> 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  <cit>  and in zebrafish for beta cell regeneration  <cit> .
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  <cit> .
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.
CONCLUSIONS
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.

 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 have been described in pancreatic islets, each characterized by the expression of a particular hormone. 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 were conducted on endocrine pancreatic cells isolated from human or rodents at adult and embryonic stages. 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. 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. 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. 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. 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 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 cdx 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. acinar cells were obtained from the bac transgenic lines tg. the endocrine beta and delta cells were isolated, respectively, from the transgenic lines tg ulg021tg and tg; the alpha cells were obtained from the tg/tg line through selection of gfp+/mcherry cells 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 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 ulg004tg transgenic line, and these data were compared in the present study with endocrine and acinar cell transcriptomes. principal component analysis of all these pancreatic rna-seq datasets showed a tight clustering of all replicates for each pancreatic cell type, underscoring the high reproducibility of the data. as expected, the pca also revealed a closer clustering of the three endocrine cell subtypes compared to the ductal and acinar cell types; however, when pca is performed only with the endocrine datasets, clear distinct transcriptome profiles are observed for the alpha, beta, and delta cell subtypes. comparison of the expression levels of various known markers of each pancreatic cell type confirmed the high purity of each cell preparation. indeed, glucagon a, insulin, and somatostatin were selectively detected at very high levels in alpha, beta, and delta cell libraries, respectively, representing in average 24%, 10%, and 32% of the total reads number in the corresponding libraries, while being detected at much lower levels in the other libraries. the trypsin and chymotrypsin-like elastase member genes were the strongest expressed genes in acinar cells representing each about 10% of all total reads of the acinar cell libraries. these two acinar markers were not detected at significant levels in endocrine datasets consistent with an accurate cell sorting. among the genes expressed at highest levels in ducts, we find the cldnb, sdc and the epcam genes coding for cell adhesion molecules, each representing less than 1% of total reads of ductal datasets. all these results indicate an accurate and reproducible sorting of the different pancreatic cells allowing the identification of genes selectively expressed in each pancreatic cell type. expression values for all genes in all samples are shown in additional file 2: table s and additional file 3: tables s fig. global analysis of the zebrafish pancreatic rna-seq data. a principal component analyses of gene vsd calculated by deseq package for the zebrafish pancreatic datasets. b pca of gene vsd for beta, alpha, and delta cells. the pca plots show a close clustering of all replicates and distinct clusters for each pancreatic cell type. pcas were calculated using all the genes annotated on zv version ensembl percentages of mapped reads obtained for some genes on the total number of mapped reads in each type of libraries identification of genes enriched in endocrine, acinar, and ductal pancreatic cells the clear distinct transcriptomic profiles observed for endocrine, acinar, and ductal cells prompt us to identify, in a first step, all genes presenting a differential expression in these three pancreatic tissues. by using these cut-off values, and genes were found to be enriched in endocrine, ductal, and acinar cells, respectively. as expected, the zebrafish endocrine-specific genes include several orthologs of mammalian genes known as endocrine markers such as those involved in hormone regulation and secretion, like the proprotein convertases, carboxypeptidase e, secretogranins, atp-dependent potassium channels, and the voltage-dependent type calcium channels, among others. moreover, most of the transcription factors previously shown to be crucial for the differentiation of endocrine cells are detected in the endocrine signature validating our rna-seq data. interestingly, many other transcription factors, whose function in endocrine cells is still not known, are present in this list such as egr creb3l lmo cdx1b, or cdx. gene ontology enrichment analysis using david revealed known biological pathways in endocrine cells such as “potassium ion transport”, “regulation of secretion” and “regulation of exocytosis” as well as “response to glucose”, and “g-protein coupled receptor signaling”. indeed, many g-couple protein receptors and several regulators of g-protein signaling, like gpr gpr gpr or gpr as well as rgs rgs5a, rgs or rgs among others, were found enriched in the endocrine cells. while some of these regulatory proteins have been previously reported to control pancreatic islet activity, others were not yet known to have a selective expression in pancreatic endocrine cells nor to play a role in islet physiology.fig. 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 deseq using a cut-off ratio of four-fold and adjusted p < . 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 or new genes with endocrine-, acinar-, and ductal-enriched expression discovered by this analysis. 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 genes enriched in endocrine cells and the genes enriched in acinar cells displaying the most enriched biological process similar observations were done for the acinar and ductal cell transcriptomic signature. indeed, as expected, many genes coding for digestive enzymes, such as trypsin, elastase, trypsin-like, amylase 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, klf or nr0b2a. 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”. as for the duct transcriptome, the analysis reveals novel markers such as id2a and frzb in addition to known markers like sox9b, nkx, onecut 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 the evolutionarily conserved expression of a gene in a specific cell type is a strong argument for its crucial function in that cell. this concept is widely supported by numerous studies showing conserved expression of many transcription factors in development and cell differentiation, including pancreatic cells. to perform interspecies transcriptome comparison, we retrieved, from public databases, several rna-seq datasets obtained on murine and human pancreatic islets, whole pancreas, as well as a human pancreatic sample enriched in acinar tissue. while mouse rna-seq data are not presently available for acinar enriched preparations, the genes with either endocrine-enriched or exocrine-enriched expression can nevertheless be identified by comparing rna-seq from purified pancreatic islets versus rna-seq from whole pancreas. when a global comparison of the zebrafish endocrine and acinar datasets with the murine and human pancreatic data was performed using a pca plot analysis, we observed that the data clustered according to the tissue type in the first component, while the data tended to cluster according to the species in the second component. thus, this global analysis suggests the existence of sets of genes displaying an evolutionary conserved endocrine and exocrine expression. endocrine and exocrine genes were identified from human and murine datasets by selecting all genes presenting at least four-fold higher expression in each pancreatic tissue. next, we compared the endocrine and exocrine gene lists between the three vertebrate species. the venn diagram on fig. 3b indicates that, while a large fraction of endocrine-enriched genes are species-specific, some sets of genes display a conserved endocrine-enriched expression in two species or in the three species. indeed, among the zebrafish endocrine genes, are also classified as “endocrine” in human and mice. this endocrine signature, conserved between zebrafish, mice and human, includes many factors known to be involved in hormone maturation, secretion, and regulation. many transcription factors crucial for endocrine cell differentiation are found in this zmh conserved endocrine signature, thereby validating this method to identify genes with important physiological function. the zmh endocrine genes also comprise components of signaling pathways reported to control pancreatic endocrine cells in mammals such as the glutamate receptor gria2a and gria2b, regulators of g-protein signaling, urocortin, ion channels, and calcium dependent proteins. this indicates that many regulatory processes controlling endocrine cell development and physiology have been maintained from fish to human. interestingly, the zmh endocrine genes include several regulatory genes whose function in pancreatic cells is still unknown or not well defined such as the kinases map3k and mast the signaling factor gpr as well as the transcription factors npas4a, lmo fev, and etv similarly, the comparison of the exocrine-enriched genes across zebrafish, mice, and human shows that a fraction of genes displays conserved exocrine enrichment – among the zebrafish exocrine genes, show exocrine enriched expression also in human and mice. this conserved exocrine signature includes, as expected, many digestive enzymes as well as known exocrine transcription factors such as ptf1a, rbpjl, and nr5a, but also regulatory genes with unknown pancreatic function, like klf fig. conservation of the pancreatic endocrine signature among vertebrates. a principle component analysis 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 genes displaying 1-1- orthology relationship between zebrafish, mouse, and human using a total of rna-seq samples. the endocrine datasets of zebrafish, mouse, and human cluster along the pc axis representing 55% of the variance, indicating a conserved endocrine signature. the human pancreatic sample enriched in acinar tissue clusters with the human whole pancreatic samples 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. due to gene duplications in some species and often in zebrafish, the number of corresponding murine or human 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 transcription factors enriched in endocrine cells in zebrafish, human and/or mouse. the level of expression in endocrine, acinar, and ductal cells is shown for each gene in the second, third, and fourth column, respectively. the degree of expression pattern conservation is shown in the last column as zhm: endocrine-enriched in zebrafish, human, and mouse; zm: endocrine-enriched in zebrafish and mouse; zh: endocrine-enriched in zebrafish and human the interspecies comparison of both exocrine and endocrine signature also indicates that many genes display the same enrichment in only two species. 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 while many transcription factors controlling pancreatic endocrine cell differentiation in embryos remain expressed and functional in mature cells from adults, there are nevertheless striking differences between the transcriptomes of fetal and adult cells. in order to determine the fraction of the endocrine-enriched genes in adult zebrafish that are already expressed in the first embryonic endocrine cells, we determined the transcriptomic profile of endocrine cells isolated from tg embryos at hpf. we detected genes expressed in embryonic pancreatic cells above the threshold level of normalized count. by comparing these data with adult endocrine data, we found that, among the genes enriched in adult endocrine cells, genes were already detected in embryonic cells. the expression of some of these endocrine-enriched genes was further characterized by in situ hybridization on whole zebrafish embryos. we analyzed genes identified as endocrine enriched in the three species, in two species, or only in zebrafish. all these genes were detected in the embryonic endocrine pancreatic cells by ish, validating the rna-seq data. thus, these data indicate that the early embryonic endocrine cells from the dorsal pancreatic bud express a large fraction of the genes constituting the adult endocrine signature.fig. 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. genes with conserved endocrine-enriched expression in zebrafish, human, and mouse, or endocrine-enriched in zebrafish and mouse, or in zebrafish and human 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 characterization of the transcriptomic signatures for the endocrine cell subtypes in order to define the molecular signatures of alpha, beta, and delta cell subtypes, the endocrine genes identified in adult zebrafish were classified according to their differential expression in the distinct endocrine cell subtype; and endocrine genes were found to be enriched in alpha, beta, and delta cells, respectively. the heatmap plot in fig. 5b presents an overview of the expression pattern for all differentially expressed genes. pca using all endocrine-enriched genes displays a clear discrimination of the alpha, beta, and delta cell transcriptomes. go enrichment analysis performed on beta cell-specific genes identifies “response to organic substrate”, “ion transport”, or “response to nutrient levels” as enriched pathways among others. the gene expressed at the highest level in the beta cells after insulin is ppdpfb. this gene is the paralog gene of the ppdpfa gene, which was reported as specifically expressed in acinar cells and controls their differentiation. interestingly, the paralog ppdpfb is specifically expressed in the endocrine pancreas and mainly in beta cells. we verified the expression of ppdpfb in hpf zebrafish embryos by fluorescent ish and, according to the rna-seq data, we confirmed its expression in many beta cells and in a few alpha cells but not in delta cells. the rna-seq data also confirmed the selective expression of the zebrafish nkx gene in beta cells, as previously reported. expression of the zebrafish pcsk gene is more enriched in beta cells compared to the pcsk gene, while in mouse and human, only pcsk was reported to be more enriched in beta compared to alpha cells. the enrichment of pcsk in beta cells was confirmed by fish on zebrafish embryos. the zebrafish estrogen receptor esr is also strongly enriched in beta cells. finally, the beta cell-selective expression of spondin 1b, an activator of the wnt pathway, was also validated by fish on zebrafish embryos.fig. 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 < . 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 or new markers identified in this analysis. c principal component analysis performed on the nine zebrafish endocrine rnaseq datasets using the endocrine-enriched genes. compared to the pc plot of fig. 1b performed on all annotated zebrafish genes, this plot shows a tighter clustering of all replicates and better discrimination between the three endocrine cell subtypes fig. expression of ppdpfb and pcsk genes in zebrafish beta cells. co-labeling by fluorescent in situ hybridization of ppdpfb and pcsk with insulin, glucagon, and somatostatin. a–c ppdpfb is mainly expressed by beta cells and in few alpha cells. no expression of ppdpfb was observed in delta cells. beta cells specifically expressed pcsk while no expression was detected in alpha or delta cells. as expected, the genes specifically expressed in alpha cells include those coding for the two zebrafish glucagon hormones as well as for the arx transcription factor but also novel markers including peptide hormones such as prepronociceptin and neuropeptide b or the calcium regulated factor scinderin like b. the alpha-enriched genes also comprise the transcription factors etv and si:ch211-145o. the alpha-selective expression of pnoca and scinlb was confirmed by fish, validating the rna-seq data. 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, cdx cdx and tbx2b, as well as genes coding for signaling factors like bmp7b or the kinase map3k interestingly, as demonstrated by the go enrichment analysis, many gpcr are specifically enriched in delta cells such as npy8br, glra4a, uts2r, ptger1b & 2a, adra1d, grm, oprd1b, and gpr pinpointing these endocrine cells as targets of diverse external metabolic signals. we confirmed the selective expression for several genes, including the cdx cdx lamc and map3k genes, detected already at hpf in delta cells. 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 the zebrafish rna-seq data led to the identification of many novel markers for each endocrine cell subtypes raising the question of whether such markers display the same cell subtype-specific expression in other species. as the transcriptome of human and murine alpha and beta cells have been determined by a similar approach using highly purified facs cell preparations, this allowed us to perform an interspecies comparison. in order to identify all alpha and beta cell differentially expressed genes, we performed exactly the same procedure on the zebrafish, mouse, and human rna-seq datasets, namely a comparison of the entire alpha and beta cell transcriptomes within each species using the same software and selection criteria. this led to the identification of and alpha-enriched genes as well as and beta-enriched genes in human, mouse, and zebrafish, respectively. surprisingly, the interspecies comparison of these sets of genes revealed that the large majority of alpha- and beta- enriched genes are species specific. indeed, among the zebrafish beta-enriched genes, only three have maintained beta cell preferential expression in human and mice, namely insulin, the transcription factor pdx and the glucagon receptor gcgra. similarly, among the zebrafish alpha cell-enriched genes, only have human and murine orthologs with alpha cell-enriched expression. as expected, glucagon and arx are part of the conserved alpha cell genes. the adcy and adcy genes coding for adenylate cyclase are also alpha-enriched in the three vertebrate species supporting the important role of camp in alpha cells. the gc gene and fev are among the conserved alpha cell signatures, suggesting a specific function in this endocrine cell subtype. taken together, all these analyses indicated that, while hundreds of genes display conserved expression in endocrine and exocrine cells, there is much less conservation for the genes differentially expressed between the endocrine cell subtypes alpha and beta. these data confirm the striking differences between species as previously reported for the human and murine alpha- and beta-enriched genes. importantly, many of the conserved alpha- and beta-specific genes identified in the present study were also recently highlighted as enriched in these endocrine cell types in several single cell rna-seq studies and additional file 15: table s for comparison).fig. 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 and beta cells and displaying this enrichment in zebrafish, mice, and/or human. due to gene duplications in some species and often in zebrafish, the number of corresponding murine or human orthologous genes is given in brackets in each intersection. the alpha- and beta-enriched genes were selected by deseq with fold change >  and adjusted p <  using two murine alpha and beta cell preparations, six human alpha and beta cell preparations, and three zebrafish alpha and beta cell preparations. the full list of conserved beta- and alpha-enriched genes is given in additional file 14: table s and additional file 15: tables s9 expression enrichment in alpha and beta cells is given as the log fold-change in the three species role of the zebrafish myt1b and cdx genes in endocrine cell differentiation to validate our cross-species approach to identify important pancreatic regulatory factors, we selected two transcriptional regulators that were expressed at high levels in endocrine cells at the early developmental stage and performed loss of function studies. mutations of myt1b gene and of the cdx gene were found to affect endocrine cell differentiation. as cdx is selectively expressed in delta cells, we analyzed the expression of several novel delta cell markers in the cdx4 tv null zebrafish mutant. a previous study has shown that the zebrafish cdx mutant displays defects in the anterio-posterior patterning of the endoderm, with notably a posteriorly-shifted pancreas and an increase of pancreatic beta cell number; however, the selective expression of cdx in delta cells was unknown as well as its function in this endocrine cell type. analysis of the null cdx4 tv mutant embryos revealed a complete loss of somatostatin gene expression. similarly, expression of the new delta cell markers lamc and slc7a was almost undetectable in the mutant embryos, in contrast to the increase of insulin expression. expression of map3k gene in delta pancreatic cells was also specifically abrogated by cdx mutation, while expression of this gene was not affected anteriorly at the level of the pronephric glomeruli. all these data demonstrate the important role of cdx for delta cell differentiation.fig. delta cell differentiation is disrupted in the cdx mutant embryos. analysis of wild-type and cdx4 tv205–/– mutant embryos at hpf by whish and fish using delta cell markers as well as gcga and insulin. cdx4 tv mutants display a loss of sst, lamc, and slc7a pancreatic expression and an increase of beta cells. map3k expression is strongly reduced in the pancreatic islet while not affected in the presumptive pronephric glomeruli. no obvious effect is observed on gcga expression. nuclei are stained with dapi. our transcriptomic analyses show that embryonic pancreatic cells express both myt1a and myt1b paralogs, with myt1b being expressed at much higher levels. this was confirmed by ish revealing expression of myt1b in the dorsal pancreatic bud of zebrafish embryos, while the paralog myt1a was barely detectable. the adult rna-seq data indicate that the myt1a/b genes are expressed in alpha, beta, and delta cells. simultaneous inactivation of the two zebrafish myt genes was performed by multiplex crispr/cas mutagenesis through injection of four guide rna. injected embryos revealed a significant decrease of glucagon expression and no significant effect on insulin expression. to confirm these data obtained in f embryos, a line harboring a null mutation in myt1b was raised. alpha cell mass was decreased in the myt1b ulg029–/– embryos compared to the wild-type and heterozygous siblings.fig. reduced glucagon expression in the myt1b zebrafish mutant embryos. glucagon expression was analyzed by immunofluorescence in f myt1b ulg homozygous mutant larvae and in sibling +/+ and +/– larvae at 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 discussion 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, isl pax insm ptf1a, and rbpjl, among others. myt 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 dpf embryos. additionally, cdx 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 gria shown to regulate hormone synthesis and secretion in rodents. 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 abcc and kcnj and the voltage-dependent ca++ channel cacna2d the use of this pathway in zebrafish is further strengthened by the very high expression of the glucose transporter glut and the glucokinase 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 sstr, casr, or celsr, 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. 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 lmo and npas4a, or the signaling factors gpr rgs and rgs 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 mnx and murine esr 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. 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 mnx and esr indicating that important genes fall in these two categories. for proof, cdx classified as endocrine-enriched in zebrafish and mice, was found here to be crucial for endocrine delta cell differentiation as many novel delta cell makers were drastically reduced in the cdx mutant zebrafish embryos. our findings warrant future analyses on the murine cdx 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 cdx mutant suggests that cdx 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. this is also supported by the venn diagram showing that the number of genes with enriched expression in both alpha and beta cells and not in delta cells is higher than those of the “alpha-delta” enriched and “beta-delta” enriched genes, and the number of delta cell markers is higher than the alpha and beta cell marker. 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 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. differences between human and mouse alpha- and beta-enriched genes have also been previously noticed. 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, pdx 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. a second explanation can be the relatively stringent threshold that we used to select differentially expressed genes. for example, the mnx gene, known to be involved in beta cell differentiation, 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. these differences in enrichment between human and mouse mnx is confirmed in the recent single cell rna-seq data. similarly, the prohormone convertase gene pcsk required for insulin hormone maturation was only -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, nkx is expressed in beta cells in mice and human but not in zebrafish, where its paralog nkx instead of nkx is selectively expressed in mature beta cells. nkx may fulfill the function of nkx as nkx and nkx have equivalent biological activities. another example is the zn++ transporter scl30a which is strongly expressed in beta cells in mammals but not in zebrafish, which instead strongly expresses the homologous zn++ transporter scl30a 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 neurog as pancreatic endocrine cell fate determinant is fulfilled by two other bhlh factors ascl1b and neurod. a functional switch is also observed for the specification of the secretory cell of the intestine, played by atoh 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. such compensations seem to occur for the murine myt gene as the myt 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. furthermore, adult murine endocrine pancreatic cells express the homologous myt1l and myt3/st genes and their expression increases in the myt ko mice. in zebrafish, myt1a, myt1l, and myt3/st 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. 
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. comparison of these transcriptomes identified many novel markers, including transcription factors and signaling pathway components, specific for each cell type. 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 cdx 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. 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. 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. by performing interspecies comparisons, we identified hundreds of genes with conserved enriched expression in endocrine and exocrine cells among human, mouse, and zebrafish. 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. 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.
 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 cdx 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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords rna-seqcomparative transcriptomicspancreasendocrine cellsacinar cellsductal cellsfnrscdr26119285peers bernard issue-copyright-statement© the author 2017 