Use of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells

Xin, Yurong; Kim, Jinrang; Ni, Min; Wei, Yi; Okamoto, Haruka; Lee, Joseph; Adler, Christina; Cavino, Katie; Murphy, Andrew J.; Yancopouloš, George D.; Lin, Hong‐Cheu; Gromada, Jesper

doi:10.1073/pnas.1602306113

Cited by 148 publications

(145 citation statements)

References 23 publications

(22 reference statements)

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“…S1). The expression of ATF5 in β cells of the murine pancreatic islet is consistent with several single-cell transcriptomic studies that identified Atf5 transcript in murine and human β cells (27)(28)(29).…”

Section: Resultssupporting

confidence: 87%

ATF5 regulates β-cell survival during stress

Juliana

Yang

Rozo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The stress response and cell survival are necessary for normal pancreatic β-cell function, glucose homeostasis, and prevention of diabetes. The homeodomain transcription factor and human diabetes gene pancreas/duodenum homeobox protein 1 (Pdx1) regulates β-cell survival and endoplasmic reticulum stress susceptibility, in part through direct regulation of activating transcription factor 4 (Atf4). Here we show that Atf5, a close but less-studied relative of Atf4, is also a target of Pdx1 and is critical for β-cell survival under stress conditions. Pdx1 deficiency led to decreased Atf5 transcript, and primary islet ChIP-sequencing localized PDX1 to the Atf5 promoter, implicating Atf5 as a PDX1 target. Atf5 expression was stress inducible and enriched in β cells. Importantly, Atf5 deficiency decreased survival under stress conditions. Loss-of-function and chromatin occupancy experiments positioned Atf5 downstream of and parallel to Atf4 in the regulation of eIF4E-binding protein 1 (4ebp1), a mammalian target of rapamycin (mTOR) pathway component that inhibits protein translation. Accordingly, Atf5 deficiency attenuated stress suppression of global translation, likely enhancing the susceptibility of β cells to stress-induced apoptosis. Thus, we identify ATF5 as a member of the transcriptional network governing pancreatic β-cell survival during stress.educed pancreatic β-cell number and function characterize all forms of diabetes. Insulin-secreting β cells are notoriously susceptible to stress, including endoplasmic reticulum (ER), cytokine, and oxidative stress (1-4). Thus, understanding apoptotic cell-fate decisions during stress could provide new targets that could be exploited for the prevention or amelioration of diabetes.In secretory cells such as the β cell, the unfolded protein response (UPR) and regulation of translation, particularly in response to stress, are key factors in maintaining cellular homeostasis, as clearly demonstrated in mouse models with deficiencies of critical regulators such as protein kinase R-like ER kinase (PERK) and EIF2α (5-7). In humans, PERK mutation causes Wolcott-Rallison syndrome, a rare autosomal recessive disorder characterized by permanent neonatal diabetes (8, 9). Downstream of PERK, the basic leucine zipper (bZIP) transcription factor activating transcription factor 4 (ATF4) regulates the expression of 4ebp1, a member of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein family (4EBPs). 4EBP1 is the most abundant mammalian isoform in the pancreas (10) and functions as an inhibitor of translation initiation by binding the capbinding protein eIF4E, thereby preventing formation of the eIF4F translational initiation complex (11,12). Expression of 4EBP1 is induced by stress, and 4ebp1 deficiency results in deregulated translational control and increased susceptibility to ER stressmediated apoptosis in β cells (13).We previously demonstrated that the homeodomain transcription factor and human diabetes gene pancreas/duodenum homeobox protein 1 (Pdx1) regulates p...

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Section: Resultssupporting

confidence: 87%

ATF5 regulates β-cell survival during stress

Juliana

Yang

Rozo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…1C). Cells expressing less than 3500 genes (n = 72) also exhibited high mitochondrial alignment rates and other reported transcriptional metrics of cell death and/or poor quality (Ilicic et al 2016;Xin et al 2016) and were removed from subsequent analyses (Fig. 1C).…”

Section: Resultsmentioning

confidence: 99%

Single-cell transcriptomes identify human islet cell signatures and reveal cell-type–specific expression changes in type 2 diabetes

et al. 2016

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Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type-specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis.[Supplemental material is available for this article.]Pancreatic islets of Langerhans are clusters of at least four different hormone-secreting endocrine cell types that elicit coordinatedbut distinct-responses to maintain glucose homeostasis. As such, they are central to diabetes pathophysiology. On average, human islets consist mostly of beta (54%), alpha (35%), and delta (11%) cells; up to a few percent gamma/pancreatic polypeptide (PP) cells; and very few epsilon cells (Brissova et al. 2005;Cabrera et al. 2006;Blodgett et al. 2015). Human islet composition is neither uniform nor static but varies between individuals and across regions of the pancreas (Brissova et al. 2005;Cabrera et al. 2006;Blodgett et al. 2015). Cellular heterogeneity complicates molecular studies of whole human islets and may mask important role(s) for less common cells in the population (Dorrell et al. 2011b;Bramswig et al. 2013;Nica et al. 2013;Blodgett et al. 2015;Liu and Trapnell 2016). Moreover, it complicates attempts to identify epigenetic and transcriptional signatures distinguishing diabetic from nondiabetic (ND) islets, leading to inconsistent reports of genes and pathways affected (Gunton et al. 2005;Marselli et al. 2010;Taneera et al. 2012;Dayeh et al. 2014). Conventional sorting and enrichment techniques are unable to specifically purify each human islet cell type (Dorrell et al. 2008;Nica et al. 2013;Bramswig et al. 2013;Hrvatin et al. 2014;Blodgett et al. 2015), thus a precise understanding of the transcriptiona...

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“…Single cells were captured and processed on a Fluidigm C1 instrument in collaboration with Regeneron Pharmaceuticals as described in ref. 45. For analysis of cells isolated from grafted arches from CD68-GFP reporter mice, samples were processed as described above, and flow cytometry was conducted on a BD LSRII UV.…”

Section: Methodsmentioning

confidence: 99%