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Cited by 310Pancreatic function and diabetesDiabetes and associated disordersSingle-cell and spatial transcriptomics

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The transcriptional landscape of mouse beta cells compared to human beta cells reveals notable species differences in long non-coding RNA and protein-coding gene expression

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Benner, Christopher, van der Meulen, Talitha, Cacéres, Elena, Tigyi, Kristof, Donaldson, Cynthia J, Huising, Mark O·2014·BMC Genomics
DOI 10.1186/1471-2164-15-620PMC PMC4124169
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Abstract

BACKGROUND: Insulin producing beta cell and glucagon producing alpha cells are colocalized in pancreatic islets in an arrangement that facilitates the coordinated release of the two principal hormones that regulate glucose homeostasis and prevent both hypoglycemia and diabetes. However, this intricate organization has also complicated the determination of the cellular source(s) of the expression of genes that are detected in the islet. This reflects a significant gap in our understanding of mouse islet physiology, which reduces the effectiveness by which mice model human islet disease. RESULTS: To overcome this challenge, we generated a bitransgenic reporter mouse that faithfully labels all beta and alpha cells in mouse islets to enable FACS-based purification and the generation of comprehensive transcriptomes of both populations. This facilitates systematic comparison across thousands of genes between the two major endocrine cell types of the islets of Langerhans whose principal hormones are of cardinal importance for glucose homeostasis. Our data leveraged against similar data for human beta cells reveal a core common beta cell transcriptome of 9900+ genes. Against the backdrop of overall similar beta cell transcriptomes, we describe marked differences in the repertoire of receptors and long non-coding RNAs between mouse and human beta cells. CONCLUSIONS: The comprehensive mouse alpha and beta cell transcriptomes complemented by the comparison of the global (dis)similarities between mouse and human beta cells represent invaluable resources to boost the accuracy by which rodent models offer guidance in finding cures for human diabetes.

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Figures

Figure 1 Generation of a beta cell reporter mouse that faithfully and selectively marks all beta cells. A fusion of histone-2b (H2b) and monomeric cherry (mCherry) was inserted downstream of the long Ins1 promoter fragment (A) to generate a mIns1-H2b-mCherry beta cell reporter mouse that features nuclear expression of mCherry in all beta cells (B). Male (C) and female (D) mIns1-H2b-mCherry mice demonstrate normal glucose control as demonstrated by glucose tolerance test compared to wild type littermates.
Figure 1 Generation of a beta cell reporter mouse that faithfully and selectively marks all beta cells. A fusion of histone-2b (H2b) and monomeric cherry (mCherry) was inserted downstream of the long Ins1 promoter fragment (A) to generate a mIns1-H2b-mCherry beta cell reporter mouse that features nuclear expression of mCherry in all beta cells (B). Male (C) and female (D) mIns1-H2b-mCherry mice demonstrate normal glucose control as demonstrated by glucose tolerance test compared to wild type littermates.CC BY · PubMed Central
Figure 2 Validation of comprehensive transcriptomes of mouse beta and alpha cells. Bitransgenic offspring of mIns1-H2b-mCherry x S100b-eGFP bitransgenic reporter mice (A) enable the FACS purification of pure populations of beta and alpha cells (B). Expression of eGFP is negligible in mCherry + beta cells (C), and mCherry expression in eGFP + alpha cells is not detected (D). A volcano plot highlights key alpha and beta cell-enriched genes (E). The most abundantly expressed genes in beta (F) and alpha (G) cells are expressed by the fraction of total reads that maps to each gene and compared to expression in the opposite cell type. Expression of the beta cell markers Ins1 (H), Ins2 (I), Ucn3 (J) and Mafa (K) in alpha cells measures on average less than 2.39% of expression in beta cells. Conversely, expression of the alpha cell markers Gcg (L), Arx (M), Irx1 (N) and Mafb (O) in mouse beta cells is on average even lower. See also Additional files 1, 2 and 3.
Figure 2 Validation of comprehensive transcriptomes of mouse beta and alpha cells. Bitransgenic offspring of mIns1-H2b-mCherry x S100b-eGFP bitransgenic reporter mice (A) enable the FACS purification of pure populations of beta and alpha cells (B). Expression of eGFP is negligible in mCherry + beta cells (C), and mCherry expression in eGFP + alpha cells is not detected (D). A volcano plot highlights key alpha and beta cell-enriched genes (E). The most abundantly expressed genes in beta (F) and alpha (G) cells are expressed by the fraction of total reads that maps to each gene and compared to expression in the opposite cell type. Expression of the beta cell markers Ins1 (H), Ins2 (I), Ucn3 (J) and Mafa (K) in alpha cells measures on average less than 2.39% of expression in beta cells. Conversely, expression of the alpha cell markers Gcg (L), Arx (M), Irx1 (N) and Mafb (O) in mouse beta cells is on average even lower. See also Additional files 1, 2 and 3.CC BY · PubMed Central
Figure 3 Comparison of mouse beta and alpha cell transcriptomes based on functional annotation. Establishment and maintenance of alpha and beta cell identity is regulated by a complex interplay of transcription factors (A), whose expression pattern is accurately reflected by our mouse alpha and beta cell transcriptomes. Actual transcription factor expression in beta and alpha cells is represented by a dot plot where each dot represents an individual gene (B). Genes that are significantly enriched (p < 1 × 10-7) in beta or alpha cells are highlighted in red and green, respectively, with genes enriched with p < 1 × 10-50 displayed in bold for emphasis. Ins1, Ins2, and Gcg are marked for reference. See also Additional files 5 and 6.
Figure 3 Comparison of mouse beta and alpha cell transcriptomes based on functional annotation. Establishment and maintenance of alpha and beta cell identity is regulated by a complex interplay of transcription factors (A), whose expression pattern is accurately reflected by our mouse alpha and beta cell transcriptomes. Actual transcription factor expression in beta and alpha cells is represented by a dot plot where each dot represents an individual gene (B). Genes that are significantly enriched (p < 1 × 10-7) in beta or alpha cells are highlighted in red and green, respectively, with genes enriched with p < 1 × 10-50 displayed in bold for emphasis. Ins1, Ins2, and Gcg are marked for reference. See also Additional files 5 and 6.CC BY · PubMed Central
Figure 4 Validation of known differences in gene expression between mouse and human alpha and beta cells. We assessed the expression of two sets of genes, Mafa/Mafb and Ucn3/Crh, that markedly differ in distribution between alpha and beta cells in mouse (A) and human (B) islets. Mafa is restricted to beta cells of both species, while Mafb is selectively expressed in mouse alpha cells (A), but expressed in human beta cells as well (B). Ucn3 is highly selective for mouse beta cells while Crh is detected in neither mouse islet population (A, C, D). By contrast, UCN3 expression is a common feature of human alpha and beta cells, while CRH is enriched in human alpha cells (B, E, F). Human RNA-seq data are from [16]. See also Additional file 7 for single channel images.
Figure 4 Validation of known differences in gene expression between mouse and human alpha and beta cells. We assessed the expression of two sets of genes, Mafa/Mafb and Ucn3/Crh, that markedly differ in distribution between alpha and beta cells in mouse (A) and human (B) islets. Mafa is restricted to beta cells of both species, while Mafb is selectively expressed in mouse alpha cells (A), but expressed in human beta cells as well (B). Ucn3 is highly selective for mouse beta cells while Crh is detected in neither mouse islet population (A, C, D). By contrast, UCN3 expression is a common feature of human alpha and beta cells, while CRH is enriched in human alpha cells (B, E, F). Human RNA-seq data are from [16]. See also Additional file 7 for single channel images.CC BY · PubMed Central
Figure 5 Comparison of the mouse and human beta cell transcriptomes. The unbiased comparison of mouse and human transcriptomes reveals a common core of over 9900 genes that are expressed at an RPKM value > 1 in beta cells of both species (A). A total of 725 genes (569 unique and 156 enriched) was robustly (>10-fold) and significantly (p < 1 × 10-7) higher expressed in mouse beta cells (B, red) while 815 genes (666 unique and 149 enriched) were by the same criteria significantly higher expressed in human beta cells (B, blue). Genes enriched in mouse beta cells were co-enriched for mouse-specific PDX1 binding and vice versa (C). Moreover, enrichment of PDX1 ChIP-Seq peaks in either species is associated with species-specific PDX1 binding motifs (D), as illustrated for Sytl4 (E). Iapp transcript levels in purified beta cells (F) and islet IAPP peptide content (G) are markedly reduced in human compared to mouse. Additional browser plots demonstrate notable examples of significantly enriched or selectively expressed genes in either species including Gad2 (H), Prlr (I), Ghr (J) and Cntfr (K). Human RNA-seq data are from [16], ChIP-Seq data are from [38, 49]. See also Additional files 8, 9 and 10.
Figure 5 Comparison of the mouse and human beta cell transcriptomes. The unbiased comparison of mouse and human transcriptomes reveals a common core of over 9900 genes that are expressed at an RPKM value > 1 in beta cells of both species (A). A total of 725 genes (569 unique and 156 enriched) was robustly (>10-fold) and significantly (p < 1 × 10-7) higher expressed in mouse beta cells (B, red) while 815 genes (666 unique and 149 enriched) were by the same criteria significantly higher expressed in human beta cells (B, blue). Genes enriched in mouse beta cells were co-enriched for mouse-specific PDX1 binding and vice versa (C). Moreover, enrichment of PDX1 ChIP-Seq peaks in either species is associated with species-specific PDX1 binding motifs (D), as illustrated for Sytl4 (E). Iapp transcript levels in purified beta cells (F) and islet IAPP peptide content (G) are markedly reduced in human compared to mouse. Additional browser plots demonstrate notable examples of significantly enriched or selectively expressed genes in either species including Gad2 (H), Prlr (I), Ghr (J) and Cntfr (K). Human RNA-seq data are from [16], ChIP-Seq data are from [38, 49]. See also Additional files 8, 9 and 10.CC BY · PubMed Central
Figure 6 The mouse alpha and beta cell transcriptomes reveal unique beta cell-specific lncRNAs. Mouse islet lncRNAs demonstrate significant enrichment for binding by the beta cell-specific transcription factors PDX1 (A) and NKX6.1 (B), are enriched for H3K4me1 and H3K4me3 markers of transcriptionally active loci (C) and are co-regulated with their nearest protein-coding neighbor (D). This is illustrated by lncRNAs adjacent to Nkx6.1 (E) and Pparg (F), which are co-enriched in mouse beta cells with these protein-coding genes and strongly associate with binding of beta cell-specific transcription factors. Overnight stimulation of mouse islets with 16.8 mM glucose strongly regulates a subset of lncRNAs in comparison to protein-coding transcripts (G) as illustrated by lncRNAs that are significantly up regulated by glucose at chromosome X (H), 8 (I) and 12 (J) or down regulated by glucose at chromosome 16 (K). ChIP-Seq and H3K4 data are from [38, 45, 48, 69]. Numbers inside the grey boxes refer to lncRNA coordinates and correspond to Additional file 11.
Figure 6 The mouse alpha and beta cell transcriptomes reveal unique beta cell-specific lncRNAs. Mouse islet lncRNAs demonstrate significant enrichment for binding by the beta cell-specific transcription factors PDX1 (A) and NKX6.1 (B), are enriched for H3K4me1 and H3K4me3 markers of transcriptionally active loci (C) and are co-regulated with their nearest protein-coding neighbor (D). This is illustrated by lncRNAs adjacent to Nkx6.1 (E) and Pparg (F), which are co-enriched in mouse beta cells with these protein-coding genes and strongly associate with binding of beta cell-specific transcription factors. Overnight stimulation of mouse islets with 16.8 mM glucose strongly regulates a subset of lncRNAs in comparison to protein-coding transcripts (G) as illustrated by lncRNAs that are significantly up regulated by glucose at chromosome X (H), 8 (I) and 12 (J) or down regulated by glucose at chromosome 16 (K). ChIP-Seq and H3K4 data are from [38, 45, 48, 69]. Numbers inside the grey boxes refer to lncRNA coordinates and correspond to Additional file 11.CC BY · PubMed Central
Figure 7 The Il1r1 locus is uniquely regulated in mouse beta cells. Il1r1 is highly enriched in mouse beta cells when compared to both mouse alpha cells (A) and human beta cells (B). This expression pattern is mimicked by a beta cell-specific lncRNA upstream of the Il1r1 transcription start in the mouse but not human locus. Both the Il1r1 transcription start site and the upstream lncRNA in mouse are associated with strong transcription factor binding for PDX1, NKX6.1, MAFA and NEUROD1, while there is only limited NKX6.1 binding at the human locus (B). Cell surface expression of IL1R1/CD121a on mIns1-H2b-mCherry + beta cells is confirmed by FACS (C) and immunofluorescence (D, E). Both Il1r1 and its associated lncRNA are significantly up regulated by overnight stimulation of mouse islets with 16.8 mM glucose (F). The Il1r1 lncRNA features 13 repeats of the ETS1 consensus binding site within a 300 bp stretch of DNA (MCAST p value = 0.0009), which may offer an explanation for the strong glucose regulation of Il1r1 and its associated lncRNA, as Ets1 expression is significantly increased by elevated glucose (G). Human RNA-seq data are from [16], ChIP-Seq data are from [38, 48, 69]. Numbers inside the grey box refer to lncRNA coordinates and correspond to Additional file 11.
Figure 7 The Il1r1 locus is uniquely regulated in mouse beta cells. Il1r1 is highly enriched in mouse beta cells when compared to both mouse alpha cells (A) and human beta cells (B). This expression pattern is mimicked by a beta cell-specific lncRNA upstream of the Il1r1 transcription start in the mouse but not human locus. Both the Il1r1 transcription start site and the upstream lncRNA in mouse are associated with strong transcription factor binding for PDX1, NKX6.1, MAFA and NEUROD1, while there is only limited NKX6.1 binding at the human locus (B). Cell surface expression of IL1R1/CD121a on mIns1-H2b-mCherry + beta cells is confirmed by FACS (C) and immunofluorescence (D, E). Both Il1r1 and its associated lncRNA are significantly up regulated by overnight stimulation of mouse islets with 16.8 mM glucose (F). The Il1r1 lncRNA features 13 repeats of the ETS1 consensus binding site within a 300 bp stretch of DNA (MCAST p value = 0.0009), which may offer an explanation for the strong glucose regulation of Il1r1 and its associated lncRNA, as Ets1 expression is significantly increased by elevated glucose (G). Human RNA-seq data are from [16], ChIP-Seq data are from [38, 48, 69]. Numbers inside the grey box refer to lncRNA coordinates and correspond to Additional file 11.CC BY · PubMed Central

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Metadata

Authors
Benner, C., van der Meulen, T., Cacéres, E., Tigyi, K., Donaldson, C. J., & Huising, M. O.
Affiliations
Salk Institute for Biological Studies (US)
Corresponding author
Huising, Mark O
ORCID
Venue
BMC Genomics
Publisher
Springer Science and Business Media LLC
Published
2014-07-22
Volume
15
Issue
1
Pages
620-620
ISSN
1471-2164 (ISSN-L)
DOI
10.1186/1471-2164-15-620
PMC
PMC4124169
OpenAlex
W2139458690
Cited by
302
References
76
Citations by year
2026: 9 · 2025: 18 · 2024: 11 · 2023: 44 · 2022: 24 · 2021: 30 · 2020: 33 · 2019: 20 · 2018: 27 · 2017: 41 · 2016: 24 · 2015: 18 · 2014: 1
FWCI
27.78
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • BMC Genomics (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • Vanderbilt University
  • National Institutes of Health (DK026741)
  • National Institute of Diabetes and Digestive and Kidney Diseases (P01DK026741)
Topics
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Pancreatic function and diabetes
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Diabetes and associated disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Keywords
pancreatic beta cells, alpha cells, islet transcriptome, long non-coding RNA, species differences, human-mouse comparison, receptor repertoire, diabetes model, glucose homeostasis, insulin, glucagon
MeSH terms
Red Fluorescent Protein, Animals, Flow Cytometry, Glucagon, Humans, Luminescent Proteins, Receptors, Cell Surface, RNA, Transcription Factors, Gene Library, Sequence Analysis, RNA, Glucagon-Secreting Cells, Insulin-Secreting Cells, Mice, Transcriptome, RNA, Long Noncoding

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