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Centroacinar cells are progenitors that contribute to endocrine

regeneration.

Fabien Delaspre*1, Rebecca L. Beer*1, Meritxell Rovira2, Wei Huang1, Guangliang

Wang1, Stephen Gee1, Maria del Carmen Vitery1, Sarah J. Wheelan1, 3,

and Michael J. Parsons1, 4†

1 McKusickNathans Institute for Genetic Medicine, Johns Hopkins University,

Baltimore, MD

2 Genomic Programming of BetaCells Laboratory, Institut d’Investigacions

Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain

3 Department of Oncology, Johns Hopkins University, Baltimore, MD

4 Department of Surgery, Johns Hopkins University, Baltimore, MD

* Authors contributed equally to this work

† Corresponding author [email protected]

Short title: CACs are βcell progenitors

Correspondence: Michael J. Parsons, Johns Hopkins University School of Medicine, 733 N. Broadway, MRB 467, Baltimore, MD 21205; [email protected]

3,994 words, 7 Figures

1

Diabetes Publish Ahead of Print, published online July 7, 2015 Diabetes Page 2 of 50

Abstract

Diabetes is associated with a paucity of insulinproducing β cells. With the goal of finding therapeutic routes to treat diabetes, we aim to find molecular and cellular mechanisms involved in βcell neogenesis and regeneration. To facilitate discovery of such mechanisms we utilize a vertebrate organism where pancreatic cells readily regenerate. The larval zebrafish pancreas contains Notchresponsive progenitors that during development give rise to adult ductal, endocrine, and centroacinar cells

(CACs). Adult CACs are also Notchresponsive and are morphologically similar to their larval predecessors. To test our hypothesis that adult CACs are also progenitors we took two complementary approaches: 1) we established the transcriptome for adult

CACs. Using gene ontology, transgenic lines, and in situ hybridization we found that the CAC transcriptome is enriched for progenitor markers. 2) Using lineage tracing we demonstrated that CACs do form new endocrine cells following βcell ablation or partial . We concluded that CACs and their larval predecessors are the same cell type and represent an opportune model by which to study both βcell neogenesis and βcell regeneration. Furthermore, we show that in cftr lossoffunction mutants there is a deficiency of larval CACs, providing a possible explanation for pancreatic complications associated with cystic fibrosis.

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The zebrafish has a remarkable regenerative capacity, including an ability to

regenerate pancreatic β cells (1; 2). The regenerative capacity of the fish has already

revealed new avenues by which therapies may be developed to replace tissues in

patients that otherwise would not normally heal (3). Humans and other mammals can

also undergo somewhat limited βcell regeneration (46), yet whether such

regeneration includes βcell neogenesis is still under debate (79). Investigation into

whether βcell neogenesis occurs in mammals has likely been hindered by the more

limited regenerative capacity of those models. Knowledge of the mechanisms utilized

in the adult zebrafish pancreas to regenerate endocrine tissue has the potential to

reveal routes by which missing β cells could be replaced in diabetic patients.

Pancreata from zebrafish and mammals share considerable similarity in terms of

morphology and gene expression. The larval zebrafish pancreas contains a single

principal islet and, starting at 5 days post fertilization (dfp), secondary islets that form

throughout the pancreas parenchyma (10). The adult zebrafish pancreas consists of 4

lobes: spleen, middle, left, and ventral. β cells are arranged as isolated

single cells and in islets along with other endocrine cells. The principal islet is

normally located in the gallbladderspleen lobe (11).

During zebrafish development, pancreatic Notchresponsive cells (PNCs) give rise to

endocrine, ductal and centroacinar cells (CACs) (10; 12). CACs are defined as

specialized ductal epithelial cells located at the ends of ducts within the acinar lumen.

Both PNCs and CACs are ductal cells that share the following characteristics: 1)

possession of longcytoplasmic extensions (13); 2) distinctive ultrastructure (14); and,

3) they are responsive to Notch signaling (10; 15; 16). CACs have been proposed to

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be adult multipotent progenitors in rodents (1519). Although the progenitor status of mammalian CACs remains controversial (2024), the idea is supported by the observation that mammalian CACs proliferate in response to different injuries, including streptozotocin induced destruction of β cells (25), partial pancreatectomy

(26), and acute or chronic administration of caerulein (27).

To characterize CACs in more detail we used RNAseq to define their transcriptome.

Gene ontology analysis revealed CACs are enriched for genes important in progenitor biology. To explore whether CACs are involved in βcell regeneration we have utilized two injury models in zebrafish: 1) a transgenic method to specifically ablate β cells; and 2) partial pancreatectomy (PPx), a surgical procedure that removes the left lobe of the pancreas. Using inducible genetic lineage tracing, we find that CACs do indeed contribute to βcell regeneration. Our discovery that CACs act as endocrine progenitors in a rapidly regenerating, genetically tractable model can reveal mechanisms important in βcell neogenesis that have been difficult to study in mammals, and that may ultimately be applied to improve regenerative capacity in diabetic patients to restore βcell mass.

Research Design and Methods

Transgenic lines. Tg(HS4sst2:CFP;ins:PhiYFPmdest12TAnfsB)lmc009

(abbreviated ins:NTR) carries a transgene containing: 1) insulin promoter driving a zebrafish codon optimized NTRT2AYFP open reading frame; and 2) somatostatin2 promoter/enhancer driving CFP transcription (Fig. 4A). Other lines listed in

Supplemental Table 2.

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CAC isolation. 3month post fertilization (mpf) adult Tp1:GFP (Method 1) and

Tp1:gfp; fli1:DsRed fish (Method 2) were sacrificed and pancreata dissected in 1X

Hanks Balanced Salt Solution without Ca2+ or Mg2+ (HBSS). Pancreata were

dissociated in 0.7 mg/mL Collagenase P (Roche) (37 °C, 20 minutes), followed by

addition of FBS (Gibco) to 5% final concentration on ice. Cells were collected by 3 X

5 minute centrifugation (700 X g, 4 ºC) with resuspension in 1X HBSS with 5%

FBS. Cells were then resuspended in 1X HBSS, 0.01% Trypsin (Gibco) (37 °C, 5

minutes), followed by 3 X 5 minute centrifugation (700 X g, 4 ºC) with resuspension

in 1X HBSS, 5% FBS, and filtration through 70 µM and 40 µM nylon filters (BD

Falcon). For Method 1, APCconjugated antiCD105 (STEMCELL Technologies,

60039AD) was used to label endothelial cells. Cells were sorted on a BD Biosciences

FACS Aria IIu cell sorter.

RNA Sequencing. Total RNA was isolated (RNeasy Mini, Qiagen) and RNAseq

libraries were prepared (Illumina TruSeq RNA Sample Preparation Kit v2). 50cycle

singleend reads were collected (Illumina Genome Analyzer II). Reads were

processed and mapped to Zv9/danRer7 using RSEM (28). EBseq (29) was used to

determine differential expression and significance values.

Quantitative PCR. cDNAs were made (SuperScript® III FirstStrand Synthesis Kit,

Invitrogen). qPCR was performed on a BioRad C1000 BioRad thermal cycler, and

analyzed using the CFX Manager software (BioRad). qPCR primers are listed in

Supplemental Table 3.

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Drug treatments. To ablate βcells, 30 mM metronidazole (MTZ, M3761, Sigma) in

PBS was intracoelomically (ic) injected into ins:NTR fish at a dose of 0.25 g/kg of body weight. Control fish were injected with PBS. To activate creERT2, 20 l of 2

M 4Hydroxytamoxifen (4OHT, T176, Sigma) (12) was ic injected daily for 3 days.

To induce 2° islets, 50 M DAPT in E3 medium (30) was applied to larvae from 35 days post fertilization (dpf) (10).

Partial Pancreatectomy. Fish were anesthetized in 0.168 mg/ml tricane (Sigma)

(30). An incision was made on the left flank and most of the left pancreatic lobe was removed. Sham surgeries were performed as controls. Fish were kept in still water

(28ºC) that was changed after daily feeding. For the first three days following surgery,

EdU was added to the water (2.5 M). After PPx +3 days, EdU was delivered by ic injection of 20 L of 25 M EdU.

Immunohistochemistry, in situ hybridization, and microscopy. 5 dpf larvae were fixed in 4% PFA (4 °C overnight). Antibody staining and in situ hybridization was performed as described (12; 31). Primers for making riboprobes are listed in

Supplemental Table 3. For adult pancreata, 3 mpf zebrafish were fixed overnight in

10% formalin at 4 °C. Viscera were then dissected, embedded in paraffin blocks, and processed using standard procedures (Abcam). Antibodies are listed in Supplemental

Table 4. Images were collected using a Nikon AZ100 microscope or a Nikon A1si

Laser Scanning Confocal microscope. For adult quantification, at least 5 sections of each individual islet >1000 m were counted per fish.

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Glucose assays. Adult fish were fasted (24 hours), sacrificed, and blood glucose

measured using an OneTouch Ultra (LifeScan) glucose meter (32). Larval glucose

levels were determined in 5 dpf larvae using a glucose assay kit (BioVision). The

cftrpd1049 mutant larvae were genotyped by observing Kupffer’s vesicle in 10somite

stage embryos (33). As a positive control, ins:nfsbmCherryjh4 larvae were treated

with 10mM metronidazole (Sigma) or vehicle from 3 to 5 dpf (2).

Results

Transcriptome analysis of CACs

During development, endocrine cells, ductal cells, and CACs originate from ductal

PNCs, which are the only Notchresponsive cells in the larval pancreas (Fig. 1A) (10;

12). CACs are the only PNCderived cells that remain Notchresponsive in adults (12)

(Fig. 1B); hence, we hypothesized that CACs may function as adult progenitors. To

explore this possibility, we sought to characterize the CAC transcriptome using RNA

seq. To flow sort adult CACs, we dissociated pancreata from Tp1:GFP adult fish and

used two methods to remove contaminating Notchresponsive endothelial cells:

Method 1) a CD105 APCconjugated antibody to label endothelial cells red, and

Method 2) a second transgene, fli:dsRed, which drives RFP expression in endothelial

cells (Supplementary Fig. 1A). Using either red fluorescent blood vessel marker (bv),

we collected four populations from dissociated adult pancreata: GFP+/bv,

GFP+/bv+, GFP/bv+, and GFP/bv (Supplementary Fig. 1B). As expected, qPCR

revealed high expression of the CAC marker sox9b in GFP+ populations and of the

vascular marker cdh5 in bv+ populations (Supplementary Fig. 1C).

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Using each method, we sequenced RNA from GFP+/bv (CAC) populations and

GFP/bv (nonCAC) populations. Considering the intersection of both sequencing experiments, we identified 353 genes that were differentially expressed with >1.5 fold change difference in CACs vs. nonCACs, including 236 upregulated genes (Fig. 1C

D and Supplementary Table 1). As expected for an epithelial cell type, these genes included a number of ion channels (clcn1b, cftr) but also transcription factors important in regulating pancreas progenitors such as the Notchdownstream targets her15.1/her15.2 (34). Using Method 2 we generated another independent preparation of RNA and quantified expression of cftr, clcn1b, her15 by qPCR. These genes were significantly upregulated in CACs vs nonCACs (Fig. 1E), confirming that our catalog is enriched for CAC markers.

Using the functional annotationclustering algorithm in DAVID (35), we investigated whether our 236 CAC markers were enriched for particular gene ontology terms. This analysis identified four biological clusters with a statistically significant enrichment score >1.3 (Fig. 1F): 1) developmental programming, 2) epithelial cell biology, 3) epithelial development, and 4) cell motility/cytoskeletal organization (Supplementary

Fig. 2). This analysis confirms that CACs express a genetic program consistent with an epithelial progenitor cell population, supporting our hypothesis that CACs function as an adult progenitor cell pool.

We next investigated the expression pattern of three CAC markers in adult pancreas:

Nkx6.1, Cftr, and Nkx2.2a. Nkx6.1 is important for endocrine differentiation and is a known marker of PNCs (36). Using the Tp1:hmgb1mCherry reporter line, in which all Notchresponsive cells express nuclear mCherry, we observed Nkx6.1 expression

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in all CACs (Fig. 2A). Using TgBAC(cftrgfp) fish, we observed the endogenous

expression pattern of a CftrGFP fusion protein (33), which was localized to the thin

cellular extensions of Nkx6.1expressing cells (Fig. 2B). Without the advantage of

using CAC or cell polarity markers, others have concluded that Cftr marks the apical

surface of ductal cells (37). In fact, the protein is restricted to the thin extensions of

CACs that cover the inner lumen of the pancreatic (see also Fig. 3D). Lastly, we

used TgBAC(nkx2.2a:meGFP) fish to examine the endogenous expression pattern of

nkx2.2a, a transcription factor important in endocrine differentiation (38). In these

fish, Nkx6.1expressing CACs expressed meGFP. (Fig. 2C). Overall, our analysis

establishes a new set of markers that are consistent with the predicted role of CACs as

progenitor cells.

Larval Notchresponsive progenitors express CAC markers

Our transcriptome analysis suggested that CACs are adult progenitors, so we next

hypothesized that CACs and PNCs may represent a single cell type. Thus, we used in

situ hybridization and the abovementioned transgenic lines to examine the expression

pattern of several of our new CAC markers in larval pancreata. To visualize PNCs we

used our Notchresponsive reporter lines, Tp1:GFP or Tp1:hmgb1mCherry. In 5 dpf

fish, we observed clcn1b, her15, Nkx6.1, CftrGFP, and nkx2.2a:meGFP expression

in PNCs (5/5 genes examined, Fig. 3AE). As PNCs are progenitors for adult CACs

(12) and share marker gene expression, cell morphology, and ultrastructure with

them (10), we conclude that PNCs are in fact an early population of CACs.

Henceforth, we will refer to PNCs as larval CACs.

CACs proliferate and are more proximal to islets during βββcell regeneration

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Larval CACs contribute to endocrine and ductal cell populations during development

(12). Thus, we next set out to discover whether adult CACs play a role in βcell regeneration. To ablate β cells, we used HS4sst2:CFP;ins:PhiYFPmdest12TA nfsBlmc009 zebrafish (abbreviated ins:NTR). In these animals, β cells express destabilized YFP and nitroreductase, which converts metronidazole (MTZ) to a cytotoxin (Fig. 4A). Fish were initially injected with MTZ (MTZ +0 days) or vehicle and then were sacrificed at MTZ +3, +5, +7, +10 and +17 days. We then analyzed blood glucose levels and pancreas morphology. At MTZ +3 days, we observed near total βcell ablation and an unaffected α cell population (99.7%, Fig. 4BD,

Supplementary Fig. 3AB). These fish were severely hyperglycemic but returned to euglycemia after two weeks of recovery (Fig. 4E), confirming previous studies (1).

By MTZ +5 to +7 days, the number of single β cells and βcell clusters (small groups of β cells not containing α cells) initially increased and then resolved between MTZ

+10 and +17 days (Supplementary Fig. 3CD). This observation is consistent with a model of βcell neogenesis, where single cells differentiate, proliferate and form islets

(25).

To visualize CACs following βcell ablation we used the Notch reporter, Tp1:GFP.

Single β cells were often seen proximal to or in contact with CACs in controls (Fig.

5A). By measuring the distance between single βcell nuclei and the nuclei of the two nearest GFPpositive cells, we determined that at MTZ +5 and +7 days CACs were significantly closer to single β cells (14.35 +/ 0.6 µm and 14.74 +/ 0.7 µm, respectively) than in controls (22.81 +/ 1.56 µm, p < 0.0001, Fig. 5AD).

Additionally, adjacent to CACs we occasionally saw single Insulinexpressing cells that were GFPpositive and lacked extensions (6/19 fish at MTZ +5 and +7 days, Fig.

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5C). These results are consistent with a cellfate transition from CAC to single β cell

via a doublepositive stage.

Large islets were associated with CACs in regenerating and control pancreata (Fig.

5E). During regeneration the distance between large islets and CACs significantly

decreased (10.84 +/ 0.6 m in controls vs 6.27 +/ 0.4 m at MTZ +17, p < 0.0001)

(Fig. 5H). GFPpositive cells, many of which coexpressed Insulin (59.1% at MTZ

+10 days, 7/7 fish), could even be found inside regenerating islets (Fig. 5FG, I).

Together, these results are consistent with CAC migration during regeneration – a

hypothesis supported by similar observations in rodents (39).

We also observed cell proliferation during βcell regeneration using EdU

incorporation (40). Fish were coinjected with EdU on MTZ +0 days and every other

day thereafter until sacrifice. In contrast to controls, by MTZ +17 days many β cells

were labeled with EdU (4% vs. 50%, n=4 and 6 fish, respectively), and CACs

peripheral to islets were also EdU labeled (6% vs. 30%, respectively, Fig. 5JK).

Thus, proliferation of both β cells and CACs had occurred during regeneration

following cellspecific ablation. Altogether, our observations led us to hypothesize

that CACs play an active role in βcell regeneration.

CACs directly contribute to βcell regeneration

To directly test whether CACs are a bone fide adult progenitor population, we used

our crebased strategy to lineage trace Notchresponsive cells (12; 41). This system

uses two transgenes: 1) a Notchresponsive cre driver, Tp1:CreERT2; and 2) the cre

responder, βactin:loxstoploxhmgb1mCherry. Zebrafish carrying both transgenes

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are called lineagetracing fish (LT). Addition of 4OHT to LT fish indelibly labels

Notchresponsive cells with nuclearmCherry (nucmCherry). To quantify the efficiency of 4OHTdependent labeling in adults, we used LT fish that also carried the

Notchresponsive reporter, Tp1:GFP. After injecting adult LT; Tp1:GFP fish with

4OHT daily for 3 days, 75% of GFPpositive cells were labeled with nucmCherry

(n=5, Fig. 6AC).

LT; ins:NTR tripletransgenic zebrafish were injected daily for three days with 4OHT to label CACs and on the next day were injected with MTZ to induce βcell ablation.

Control fish received identical 4OHT injections followed by vehicle injection. In control pancreata, nucmCherry label was seen in CACs but never in Insulin expressing cells (n=1191 cells from 5 fish). During early regeneration at MTZ +3 days, β cells were ablated and only CACs were nucmCherry labeled, as in controls.

However, by MTZ +7, days, in addition to CACs, cells within recovering islets were also labeled. At MTZ +10 days, 43% of Insulinexpressing cells were nucmCherry labeled (n=320 cells from 4 fish, Fig. 6DF). Hence, we concluded that CACs are progenitors that contribute to βcell neogenesis.

CACs contribute to ductal cells and endocrine cells after partial pancreatectomy

We predicted that adult CACs represent a progenitor source for other pancreatic cell types. To test this capacity of CACs during regeneration, we next used PPx in LT;

Ptf1a:eGFP doubletransgenic fish, which express eGFP in all acinar cells. We injected fish with 4OHT and EdU as previously, before surgically removing the left pancreatic lobe and treating with EdU for the following 7 days. By PPx +7 days the operated pancreata appeared indistinguishable from sham controls, and 27.6% of the

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cells in the regenerating lobe had incorporated EdU (vs 1% in sham controls, n=5 and

5 respectively), demonstrating proliferation had occurred (Supplementary Fig. 4). We

detected nucmCherrylabeled endocrine cells, ductal cells, and CACs in the

regenerated lobe, but did not observe nucmCherry label in eGFPexpressing acinar

cells (Fig. 6GI). Accordingly, we concluded that CACs represent a progenitor pool

for both endocrine and ductal cells during regeneration, as do larval CACs during

development (12).

cftr is necessary for pancreas development

Having established that CACs are endocrine and ductal progenitors during

development and regeneration, we next became interested in whether any of the CAC

markers we identified were important for pancreas development. The expression of

cftr in CACs is of particular interest, as loss of function of this gene in humans leads

to cystic fibrosis (CF) – a debilitating disorder associated in 90% of cases with

pancreatic insufficiency, pancreatic ductal blockage, and CFrelated diabetes (CFRD)

(42). Analysis of the pancreata from cftr mutant larvae showed that by 5 dpf there was

a small but significant deficit in pancreas size and fewer Nkx6.1expressing larval

CACs (Fig. 7AC, GH). Consequently, the potential of cftr/ larvae to produce

secondary islets was also significantly reduced (Fig. 7DF, I), although this reduction

did not affect glycemia (Supplementary Fig. 5). Taken together, our results suggest

that Cftr plays a role in zebrafish CAC development and function. Ultimately, cftr

mutant fish may serve as a critically important CFRD disease model.

Discussion

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Previously, we described a pancreatic Notchresponsive population that resides in the larval ducts. These progenitor cells differentiate and give rise to at least three kinds of cells in the adult: endocrine cells, ductal cells and CACs. Two pieces of data suggested that CACs may also be progenitors: 1) as shown previously by us and others, CACs closely resemble their larval precursors in terms of morphology and ultrastructure (10; 13; 39); and, 2) as reported here, our newly discovered markers for adult CACs are also expressed in the larval progenitors. As we are interested in the cellular origins of βcell regeneration, we decided that CACs were a promising candidate and set out to see if these cells were involved in pancreas recovery following tissue damage.

Using a Notchresponsive reporter, we observed CACs physically responding to the regeneration process, even appearing to infiltrate damaged islets. However, the formal possibility existed that nonCAC cells of unknown origin became Notchresponsive during the regenerative response. Therefore, we employed genetic lineage tracing of

Notchresponsive cells in our LT fish to label CACs and their progeny before tissue damage. The presence of labeled cells near the periphery of islets suggests that lineagetraced CACs migrate from their centroacinar position towards damaged islets.

Interestingly, our functional annotation analysis revealed that the CAC transcriptome is significantly enriched for genes important for cell migration in other contexts (e.g., cxcl12, npr2a). Investigation into the role these genes play during βcell regeneration may reveal the mechanism behind this remarkable movement.

Clearly, β cells can regenerate via neogenesis from CACs in the adult zebrafish pancreas in a manner analogous to endocrine formation during development.

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However, the precise extent to which CACs contribute to βcell neogenesis cannot be

reliably quantified using our methods. For reasons discussed below, our data (and

those of other lineage tracing experiments) should not be over interpreted.

Additionally, we appreciate that other cell types also contribute to pancreas

regeneration, such as α and δ cells that have been shown to transdifferentiate in

murine and zebrafish models of βcell loss (4345).

Hepatocytes in the of zebrafish can regenerate from biliary epithelial cells

(BECs) in larvae and adults (41). Like CACs, BECs are Notchresponsive ductal

cells. regeneration is dependent on the function of the transcription factor

Sox9b, which is upregulated as biliary epithelial cells dedifferentiate (41). Sox9b

function is also required for the differentiation of pancreas progenitors toward an

endocrine fate (31). In contrast to BECs, we observe that CACs maintain

developmental programs, as evidenced by their continued expression of sox9b and

nkx6.1. Indeed, the master regulators of βcell differentiation nxk6.1 and nkx2.2a are

expressed in fish CACs but not in mammalian CACs. (34; 38). This difference may

underlie the enhanced ability of the zebrafish pancreas to regenerate following β cell

loss, as maintenance of developmental programs in fish CACs may allow them to be

continually available for differentiation once the need for new β cells arises.

The notion that CACs and/or ductal cell types contribute to adult βcell neogenesis in

mammals has been the focus of intense scrutiny. A number of studies have utilized

various loci to lineage trace CACs/ductal cells during murine pancreas development

and regeneration including: CAII (20), Hnf1β (21), Muc1 (22), Hes1 (23), and Sox9

(24). While all of the labeled cell populations contributed to the endocrine

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compartment during embryogenesis, most of these studies were unable to demonstrate that adult pancreatic ductal cells contribute to βcell neogenesis following either pancreatic ductal ligation (PDL) or βcellspecific ablation in adults (2124). However the results of these studies come with several caveats. If genes important in maintaining progenitor status (thus blocking differentiation) are used to drive lineage tracing, the highest creER activity will likely occur in cells that are the least expected to differentiate. As such, unless 100% of a particular cell population can be reliably labeled, one cannot definitively rule out the contribution of ductal cells to βcell regeneration in mammals. Therefore, the contribution of zebrafish CACs to βcell regeneration presents an intriguing and potentially critical model of βcell neogenesis from endogenous progenitors.

Despite conflicting evidence for CAC contribution to mammalian pancreas regeneration, injury does seem to reactivate developmental pathways in the adult pancreas. Following PDL, Sox9expressing ductal cells give rise to a few cells expressing Ngn3 (24), a gene that encodes a master regulator of embryonic endocrine differentiation. In another study, lineage tracing of Ngn3expressing cells demonstrated that some of these cells do indeed complete differentiation and become

β cells (46). Although this latter result clearly demonstrates that neogenesis can occur in the mammalian pancreas, this form of regeneration is very limited and dependent on the severity of the injury. Interestingly, isolated ALDH1+ murine CACs/terminal ductal cells can produce endocrine cells in both pancreatosphere and dorsal bud explant cultures (47). However, whether these ALDH1+ cells are in fact CACs is unclear, as they do not highly express Hes1, a downstream target of canonical Notch signaling and a hallmark of CACs (10; 15; 16). In the fish, Aldh1expressing cells

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have recently been described in association with, but separate from, larval and

juvenile CACs (48). Whether CACs represent a genetically uniform or heterogeneous

population in either fish or mammals remains to be determined. Regardless, all

together these observations suggest that it may be possible to manipulate

developmental pathways in the adult in order to improve the regenerative capacity of

the mammalian pancreas.

Knowing that CACs are adult progenitors in the pancreas will facilitate future studies

into other human diseases. As an example, we decided to study the cystic fibrosis

transmembrane conductance regulator (cftr), transcripts for which are highly

enriched in zebrafish CACs. Abnormal CFTR channel function causes Cystic Fibrosis

(CF), a debilitating disease often associated with CFRelated Diabetes (CFRD).

CFRD is characterized by diminished βcell mass and insulin dependence (42). Our

examination of cftr mutant larvae (5 dpf) revealed these fish had a small but

significant reduction in pancreas size. Such early pancreatic phenotypic consequences

of Cftr loss of function were not observed by Navis et al (37). There are numerous

reasons for this apparent discrepancy between studies: 1) the welldocumented

occurrence of phenotypic variation in CF due to modifiers in the genetic background

(49); 2) confocal microscopy on microdissected pancreata provides better resolution;

and 3) potential differences in animal husbandry, which is known to affect phenotype

in other CF animal models (50). Using our Notchresponsive reporters and micro

dissection to facilitate careful observation, we detect fewer CAC progenitors in cftr

mutant larvae. Possibly due to having fewer progenitors, these fish develop fewer

secondary islets. As patients with CFRD have a diminished βcell mass, these

observations in mutant fish are intriguing and warrant further investigation.

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In summary, regeneration occurs readily in the adult zebrafish pancreas and we believe that studies with this model organism will continue to shed light onto mechanisms that can be exploited to improve regeneration in diabetic patients.

Furthermore, our new insights into the importance of cftr in pancreas development suggest the role of CFTR in the human pancreas should be reevaluated.

Contributions

F. D. researched data, wrote manuscript. R. L. B. researched data, wrote manuscript.

M. R. researched data, reviewed manuscript. W. H. researched data. G. W. researched data. S. G. researched data. M. C. V. researched data. S. J. W. analyzed data, contributed discussion. M. J. P. wrote/edited manuscript, contributed discussion.

Acknowledgements

The authors would like to thank Dr. Garry Cutting and Dr. David Hackam of Johns

Hopkins University for critical reading of this manuscript and Dr. Sarah Kucenas of

Vanderbilt University and Dr. Michel Bagnat of Duke University for fish. We would also like to thank Frazer Matthews for his invaluable fish facility management and laboratory support. Dr. Michael Parsons is the guarantor of this work, and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. This work was supported by JDRF 17

2012408, NIH R01DK080730, 5P30DK079637, 1F32DK101289, and the MSCRF

2013 Postdoctoral Fellowship. The authors have no conflicts of interest to report.

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17. Gasslander T, Ihse I, Smeds S: The importance of the centroacinar region in cerulein-induced mouse pancreatic growth. Scand J Gastroenterol 1992;27:564- 570 18. Hayashi K, Takahashi T, Kakita A, Yamashina S: Regional differences in the cellular proliferation activity of the regenerating rat pancreas after partial pancreatectomy. Arch Histol Cytol 1999;62:337-346 19. Seymour PA, Freude KK, Tran MN, Mayes EE, Jensen J, Kist R, Scherer G, Sander M: SOX9 is required for maintenance of the pancreatic progenitor cell pool. Proc Natl Acad Sci U S A 2007;104:1865-1870 20. Inada A, Nienaber C, Katsuta H, Fujitani Y, Levine J, Morita R, Sharma A, Bonner-Weir S: Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth. Proc Natl Acad Sci U S A 2008;105:19915-19919 21. Solar M, Cardalda C, Houbracken I, Martin M, Maestro MA, De Medts N, Xu X, Grau V, Heimberg H, Bouwens L, Ferrer J: Pancreatic exocrine duct cells give rise to insulin-producing beta cells during embryogenesis but not after birth. Dev Cell 2009;17:849-860 22. Kopinke D, Murtaugh LC: Exocrine-to-endocrine differentiation is detectable only prior to birth in the uninjured mouse pancreas. BMC Dev Biol 2010;10:38 23. Kopinke D, Brailsford M, Shea JE, Leavitt R, Scaife CL, Murtaugh LC: Lineage tracing reveals the dynamic contribution of Hes1+ cells to the developing and adult pancreas. Development 2011;138:431-441 24. Kopp JL, Dubois CL, Schaffer AE, Hao E, Shih HP, Seymour PA, Ma J, Sander M: Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas. Development 2011;138:653-665 25. Nagasao J, Yoshioka K, Amasaki H, Tsujio M, Ogawa M, Taniguchi K, Mutoh K: Morphological changes in the rat endocrine pancreas within 12 h of intravenous streptozotocin administration. Anat Histol Embryol 2005;34:42-47 26. Hayashi KY, Tamaki H, Handa K, Takahashi T, Kakita A, Yamashina S: Differentiation and proliferation of endocrine cells in the regenerating rat pancreas after 90% pancreatectomy. Arch Histol Cytol 2003;66:163-174 27. Gasslander T, Smeds S, Blomqvist L, Ihse I: Proliferative response of different exocrine pancreatic cell types to hormonal stimuli. I. Effects of long-term cerulein administration. Scand J Gastroenterol 1990;25:1103-1110 28. Li B, Dewey CN: RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC bioinformatics 2011;12:323 29. Leng N, Dawson JA, Thomson JA, Ruotti V, Rissman AI, Smits BM, Haag JD, Gould MN, Stewart RM, Kendziorski C: EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments. Bioinformatics 2013;29:1035-1043 30. Westerfield M: The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). University of Oregon Press, Eugene, 2000 31. Manfroid I, Ghaye A, Naye F, Detry N, Palm S, Pan L, Ma TP, Huang W, Rovira M, Martial JA, Parsons MJ, Moens CB, Voz ML, Peers B: Zebrafish sox9b is crucial for hepatopancreatic duct development and pancreatic endocrine cell regeneration. Dev Biol 2012;366:268-278 32. Eames SC, Philipson LH, Prince VE, Kinkel MD: Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis. Zebrafish 2010;7:205-213

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33. Navis A, Marjoram L, Bagnat M: Cftr controls lumen expansion and function of Kupffer's vesicle in zebrafish. Development 2013;140:1703-1712 34. Schaffer AE, Taylor BL, Benthuysen JR, Liu J, Thorel F, Yuan W, Jiao Y, Kaestner KH, Herrera PL, Magnuson MA, May CL, Sander M: Nkx6.1 controls a gene regulatory network required for establishing and maintaining pancreatic Beta cell identity. PLoS genetics 2013;9:e1003274 35. Huang da W, Sherman BT, Tan Q, Collins JR, Alvord WG, Roayaei J, Stephens R, Baseler MW, Lane HC, Lempicki RA: The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists. Genome Biol 2007;8:R183 36. Huang W, Wang G, Delaspre F, Vitery Mdel C, Beer RL, Parsons MJ: Retinoic acid plays an evolutionarily conserved and biphasic role in pancreas development. Dev Biol 2014;394:83-93 37. Navis A, Bagnat M: Loss of cftr function leads to pancreatic destruction in larval zebrafish. Dev Biol 2015; 38. Sussel L, Kalamaras J, Hartigan-O'Connor DJ, Meneses JJ, Pedersen RA, Rubenstein JL, German MS: Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells. Development 1998;125:2213-2221 39. Pour PM: Pancreatic centroacinar cells. The regulator of both exocrine and endocrine function. Int J Pancreatol 1994;15:51-64 40. Chehrehasa F, Meedeniya AC, Dwyer P, Abrahamsen G, Mackay-Sim A: EdU, a new thymidine analogue for labelling proliferating cells in the nervous system. Journal of neuroscience methods 2009;177:122-130 41. He J, Lu H, Zou Q, Luo L: Regeneration of liver after extreme hepatocyte loss occurs mainly via biliary transdifferentiation in zebrafish. Gastroenterology 2014;146:789-800 e788 42. Hameed S, Jaffe A, Verge CF: Cystic fibrosis related diabetes (CFRD)--the end stage of progressive insulin deficiency. Pediatric pulmonology 2011;46:747-760 43. Thorel F, Nepote V, Avril I, Kohno K, Desgraz R, Chera S, Herrera PL: Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss. Nature 2010;464:1149-1154 44. Chera S, Baronnier D, Ghila L, Cigliola V, Jensen JN, Gu G, Furuyama K, Thorel F, Gribble FM, Reimann F, Herrera PL: Diabetes recovery by age-dependent conversion of pancreatic delta-cells into insulin producers. Nature 2014;514:503-507 45. Ye L, Robertson MA, Hesselson D, Stainier DY, Anderson RM: glucagon is essential for alpha cell transdifferentiation and beta cell neogenesis. Development 2015;142:1407-1417 46. Van de Casteele M, Leuckx G, Cai Y, Yuchi Y, Coppens V, De Groef S, Van Gassen N, Baeyens L, Heremans Y, Wright CV, Heimberg H: Partial duct ligation: beta-cell proliferation and beyond. Diabetes 2014;63:2567-2577 47. Rovira M, Scott SG, Liss AS, Jensen J, Thayer SP, Leach SD: Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas. Proc Natl Acad Sci U S A 2010;107:75-80 48. Matsuda H, Parsons MJ, Leach SD: Aldh1-expressing endocrine progenitor cells regulate secondary islet formation in larval zebrafish pancreas. PloS one 2013;8:e74350

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49. Blackman SM, Commander CW, Watson C, Arcara KM, Strug LJ, Stonebraker JR, Wright FA, Rommens JM, Sun L, Pace RG, Norris SA, Durie PR, Drumm ML, Knowles MR, Cutting GR: Genetic modifiers of cystic fibrosis-related diabetes. Diabetes 2013;62:3627-3635 50. Wilke M, Buijs-Offerman RM, Aarbiou J, Colledge WH, Sheppard DN, Touqui L, Bot A, Jorna H, de Jonge HR, Scholte BJ: Mouse models of cystic fibrosis: phenotypic analysis and research applications. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society 2011;10 Suppl 2:S152-171

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Figure 1. RNAseq of the CAC transcriptome. (A) PNCs (arrows) form the entire

(green) in 5 dpf larvae. gall bladder (gb), scale bar = 50 m. (B) In the

adult pancreas, CACs (arrows) represent a subset of the ductal epithelium (green),

which contains mostly nonNotchresponsive cells (arrowheads). CACs are the only

epithelial pancreas cells that remain Notchresponsive. scale bar = 20 m. anti2F11

(green), Tp1:hmgbmCherry (red), DAPI (blue). (C) Differential gene expression

(fold change > 1.5) between CACs and nonCACs from two separate preparations.

Expression of 236 genes were enriched in CACs in both preparations. (D) Genes most

highlyenriched in CACs vs nonCACs. P = probability of a gene being equally

expressed in CAC and nonCAC samples. (E) qPCR for cftr, clcn1b, her15 from an

independent cell preparation confirm their enrichment in CACs (**p ≤ 0.005, ttest).

(F) Functional annotation clustering of genes upregulated in CACs. DAVID analysis

of biological process, cellular component, and molecular function gene ontology

terms reveals significant enrichment (enrichment score > 1.3) of four clusters of terms

associated with the identified 236 CAC markers.

Figure 2. CACenriched gene expression in adult pancreas. (A) Nkx6.1 (green) is

expressed in CACs visualized using the Notch reporter Tp1:hmgb1mCherry (red).

(B) Endogenous expression pattern and subcellular location of a Cftr in TgBAC(cftr

GFP) fish. CftrGFP fusion protein (green) is expressed in cell extensions of Nkx6.1

expressing cells (red). (C) Endogenous expression pattern of nkx2.2a in

TgBAC(nkx2.2a:meGFP) fish; membraneGFP (green) is expressed in Nkx6.1

expressing cells (red). membraneGFP expression is occasionally observed in non

CACs cells within islets (yellow asterisk). CACs (arrows), scale bars = 20 m.

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Figure 3. PNCs express CAC markers. (AB) Whole mount in situ hybridization for clcn1b, and her15 mRNA (purple) in Tp1:GFP (green) pancreata at 5 dpf. (CD)

Nkx6.1 and TgBAC(cftrGFP) expression (green) in whole mount Tp1:hmgb mCherry (red nuclei) pancreata at 5 dpf. (E) TgBAC(nkx2.2a:meGFP) expression in

Nkx6.1 expressing cells in 5 dpf pancreas. scale bars = 50 m.

Figure 4. βcell ablation and regeneration. (A) ins:NTR transgene (BC) Compared to controls, at MTZ +3 days Insulinexpressing β cells (green) were nearly absent from islets. AntiInsulin (green), antiGlucagon (red), DAPI (blue). scale bars = 50 m. (D)

The percentage of β cells per islet returns to control levels by MTZ +17 days. (E)

Blood glucose levels in MTZ and vehicle treated fish. βcell ablation results in hyperglycemia, which returns to control levels by MTZ +17 days. N = number of fish analyzed, *p < 0.05, ns = not significant, ttest compared to control.

Figure 5. CACs respond dynamically and proliferate during βcell regeneration. (A

C) ins:NTR fish contain single β cells (arrowheads, A), which become more closely associated with CACs by MTZ +7 days (B). GFP/Insulin doublepositive single cells are seen at this time (C). (D) Quantification of the distance between single β cells and the nearest two CACs. (E) Large islets are also associated with CACs. (F) By MTZ

+7 days CACs (arrowhead) are also found within islets. (G) GFP/Insulin double positive cells are observed in islets at MTZ +10 days (inset). (H) Quantification of the distance between islets and the nearest two CACs. (I) Quantification of the percentage of islets containing Notchresponsive cells and the percentage of islets containing

GFP/Insulin doublepositive cells. Within those islets containing GFP+ cells,

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quantification of GFP+ β cells and Insulin+ CACs. (JK) At MTZ +17 days, β cells

(open arrow) and CACs (arrowhead) have incorporated EdU. Tp1:GFP (green), anti

Glucagon (red), antiInsulin (white), antiEdU (magenta), DAPI (blue). scale bars =

50 m. *p < 0.05, ttest compared to control.

Figure 6. CACs are progenitors of endocrine and ductal cell types. (AC) 4OHT

injection labels 75% of Notchresponsive cells (green) with nucmCherry (red nuclei)

in LT; Tp1:GFP fish. (DF) LT; ins:NTR regenerating islets at MTZ +7 days, nuc

mCherry labels Insulinexpressing CAC progeny (white, yellow arrowheads). (GI)

LT; Ptf1a:gfp fish at PPx +7 days, nucmCherry (red nuclei) labels regenerating duct

and Insulinexpressing islet cells (white), but not eGFPpositive acinar cells (green).

AntiInsulin (white), antiGlucagon (green in B), antimCherry (red), DAPI (blue).

scale bars = 25 m.

Figure 7. Cftr plays an important role in the pancreas. (AC) CACs (antiNkx6.1,

green) and (E) secondary islet cells (antiInsulin + antiGlucagon + antiSomatostatin,

arrows, red) induced by DAPT treatment in cftr/ 5 dpf pancreata compared to wild

type and cftr+/. scale bar = 100 m. (GI) Quantification of the number of CACs (G),

surface area (H), and number of induced secondary islet cells (I) in wildtype, cftr+/

and cftr/ 5 dpf pancreata. N = number of pancreata quantified, **p<0.05.

***p<0.001, ttest.

Supplemental Figure 1. Isolation of CACs. (A) Confocal image of Tp1:GFP; fli:rfp

fish used for Method 2. Notchresponsive cells (green, asterisks), endothelial cells

(red, arrowheads), Notchresponsive endothelial cells (arrows). (B) FACS plots for

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Preparation 1 sorted using Method 1, and Preparation 2 using Method 2. (C) qPCR from sorted cells for the acinar cell marker amylase, the endothelial cell marker cdh5, gfp, and sox9b. Enrichment for amylase was not detected in any of the cell fractions.

Supplemental Figure 2. Functional annotation clustering of CACenriched genes.

Clusters represent GO terms associated with developmental programming (Cluster 1), epithelial cell biology (Cluster 2), epithelial development (Cluster 3), and cell motility/cytoskeletal organization (Cluster 4).

Supplemental Figure 3. Quantification of βcell regeneration. (A) Percentage of

Glucagon+ volume in islets. (B) Number of single β cells and (C) βcell clusters.

Glucagon+ volume was calculated using Nikon NIS Elements 4.1 software. *p < 0.05, ttest compared to control. ns = not significant.

Supplemental Figure 4. Proliferation following PPx. (A) EdU incorporation in adult pancreas at PPx +7 days. Ptf1a:GFP (green), EdU (red), DAPI (blue). scale bar = 20

m.

Supplemental Figure 5. Glycemia in cftr mutant larvae. (AB) Kupffer’s vesicle

(arrow) is clearly visible in wildtype embryos at the 10 somite stage (14 hpf), but is absent from cftr/ embryos. (C) Glucose per embryo at 5 dpf is not significantly different in wildtype and cftr/ embryos. As a control, ins:NTR embryos treated with

MTZ contain more glucose than ins:NTR embryos treated with vehicle. *p < 0.05, ns

= not significant, ttest.

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Supplemental Table 1: Genes Differentially Expressed in CACs vs non-CACs Preparation 1 Preparation 2 ensembl id gene name fold change P fold change P ENSDARG00000089382 zgc:158463 250589.627 0.000E+00 15.678 2.478E-13 ENSDARG00000012269 clcn1b 2144.024 1.862E-12 68.854 0.000E+00 ENSDARG00000054562 her15.1 1336.912 1.789E-11 18.995 6.845E-11 ENSDARG00000090140 TMEM196 645.479 6.535E-10 13.374 5.933E-05 ENSDARG00000009215 zgc:112437 569.971 2.173E-09 46.662 1.045E-06 ENSDARG00000045141 aqp8a.1 469.959 4.636E-09 13.017 5.240E-14 ENSDARG00000041107 cftr 428.940 7.645E-09 29.001 0.000E+00 ENSDARG00000054560 her15.2 320.080 1.700E-08 20.747 1.119E-10 ENSDARG00000069332 TMEM72 252.812 4.994E-08 15.610 4.595E-07 ENSDARG00000044975 zgc:153629 230.014 2.063E-07 11.871 1.454E-14 ENSDARG00000078638 zgc:171750 226.078 1.621E-07 456.112 0.000E+00 ENSDARG00000086857 MUC21 207.098 1.555E-07 18.032 9.818E-07 ENSDARG00000051711 CR339041.3 194.463 1.732E-07 13.413 5.974E-07 ENSDARG00000024032 coch 190.783 2.286E-07 19.583 0.000E+00 ENSDARG00000088911 CR774179.2 187.570 2.118E-07 34.981 3.763E-05 ENSDARG00000019130 plk2b 179.048 3.600E-07 6.923 9.116E-08 ENSDARG00000088306 CABZ01088737.1 176.823 3.049E-07 226.105 0.000E+00 ENSDARG00000090738 mylka 164.362 3.819E-07 9.386 4.810E-04 ENSDARG00000086057 PLXND1 160.300 5.023E-07 12.768 2.413E-03 ENSDARG00000069103 si:dkeyp-82a1.4 160.247 5.181E-07 33.095 5.058E-13 ENSDARG00000069608 PALM2-AKAP2 156.521 5.944E-07 12.080 4.909E-12 ENSDARG00000091014 CU138542.4 155.084 2.286E-06 56.772 6.539E-08 ENSDARG00000057606 arl4ca 150.142 8.712E-07 7.592 1.668E-04 ENSDARG00000077424 im:7137006 142.012 7.761E-07 20.080 1.501E-05 ENSDARG00000074509 TACR2 139.390 1.053E-06 11.045 5.764E-04 ENSDARG00000053358 BASP1 138.295 1.285E-06 20.894 0.000E+00 ENSDARG00000058389 si:ch211-89f7.4 131.314 2.522E-06 4.223 6.692E-05 ENSDARG00000016691 cd9b 127.967 1.680E-06 13.909 1.409E-11 ENSDARG00000036820 mgll 127.004 2.418E-06 8.401 8.443E-11 ENSDARG00000074319 sall1a 123.492 2.415E-06 8.936 5.136E-11 ENSDARG00000042707 cx30.3 123.098 2.356E-06 20.866 0.000E+00 ENSDARG00000075112 si:dkey-162b23.3 122.107 4.174E-06 25.424 1.034E-03 ENSDARG00000059507 BX323555.3 118.947 3.460E-06 6.889 4.527E-09 ENSDARG00000014047 cldn7b 115.910 5.149E-06 13.483 5.329E-15 ENSDARG00000089038 PLEKHG5 111.428 4.191E-06 6.852 4.849E-09 ENSDARG00000042106 hlxb9la 109.827 3.095E-06 33.522 3.878E-13 ENSDARG00000069504 IL12B 108.944 2.627E-06 7.677 4.317E-03 ENSDARG00000022569 nkx6.1 108.220 3.885E-06 4.977 1.607E-05 ENSDARG00000013576 gadd45bb 105.162 6.561E-06 8.807 9.566E-11 Diabetes Page 40 of 50

ENSDARG00000078258 GGT5 101.729 4.034E-06 18.357 3.317E-08 ENSDARG00000044808 slc4a4b 101.182 6.524E-06 13.089 1.152E-13 ENSDARG00000006514 her6 99.010 9.793E-06 7.359 1.503E-09 ENSDARG00000041051 mid1ip1a 98.291 4.524E-06 31.566 3.126E-11 ENSDARG00000075406 PVRL4 94.594 8.544E-06 21.620 0.000E+00 ENSDARG00000069027 ADM 92.081 5.747E-06 30.734 1.565E-04 ENSDARG00000018383 frzb 91.947 1.151E-05 5.288 1.002E-06 ENSDARG00000005648 olfml2a 91.709 7.760E-06 4.562 8.225E-05 ENSDARG00000089858 COBLL1 89.802 9.282E-06 22.065 4.441E-16 ENSDARG00000079907 si:ch211- 88.993 6.896E-06 40.291 6.975E-06 234e14.2 ENSDARG00000079155 si:ch211-67f13.8 88.382 7.301E-06 22.398 1.016E-09 ENSDARG00000060148 sh3pxd2aa 86.659 1.847E-05 18.330 0.000E+00 ENSDARG00000053298 nkx2.2a 85.872 9.579E-06 7.390 2.351E-06 ENSDARG00000016695 p2rx1 85.602 8.062E-06 25.424 1.034E-03 ENSDARG00000087607 CU896640.1 84.882 9.377E-06 3.164 6.617E-03 ENSDARG00000068219 B3GNT8 84.244 1.402E-05 11.201 1.943E-12 ENSDARG00000089441 si:ch211- 83.483 1.304E-05 3.727 1.894E-03 105c13.3 ENSDARG00000027310 BX004812.1 83.150 1.455E-05 21.751 1.221E-15 ENSDARG00000012078 meis1 82.285 1.789E-05 5.313 1.902E-06 ENSDARG00000020133 JDP2 81.973 2.599E-05 5.181 3.206E-06 ENSDARG00000005565 entpd8 81.179 2.249E-05 7.345 2.197E-09 ENSDARG00000003411 foxa2 81.085 1.579E-05 12.298 4.148E-12 ENSDARG00000090953 IGSF5 77.740 1.660E-05 12.923 1.405E-08 ENSDARG00000036463 cldn15a 76.942 3.362E-05 6.622 1.000E-08 ENSDARG00000000002 ccdc80 75.096 1.869E-05 5.705 1.722E-05 ENSDARG00000042586 CABZ01084655.1 74.509 1.617E-05 8.218 5.217E-06 ENSDARG00000010625 clic2 73.702 3.500E-05 4.029 1.382E-04 ENSDARG00000089544 BX572619.6 73.390 2.914E-05 8.159 8.030E-08 ENSDARG00000034210 si:dkey-240h12.4 72.972 2.479E-05 9.362 8.980E-10 ENSDARG00000038969 zgc:113142 72.420 2.798E-05 20.416 7.988E-12 ENSDARG00000039232 DUSP8 71.536 2.805E-05 3.739 1.220E-03 ENSDARG00000055158 prox1 70.712 3.672E-05 7.046 5.255E-09 ENSDARG00000091539 ptprjb 69.448 3.012E-05 10.916 1.950E-09 ENSDARG00000095866 fabp1b.2 68.651 2.660E-05 7.065 2.928E-03 ENSDARG00000059680 fscn1 68.535 3.298E-05 11.401 5.178E-10 ENSDARG00000003046 sorbs2a 68.095 3.445E-05 6.648 1.167E-07 ENSDARG00000018787 efna1b 65.443 4.426E-05 7.423 9.052E-09 ENSDARG00000052139 notch3 65.083 7.371E-05 6.914 3.453E-09 ENSDARG00000035875 tmem54 64.561 3.626E-05 5.095 4.970E-04 ENSDARG00000037960 lrrc17 64.549 3.560E-05 8.863 2.558E-07 ENSDARG00000040534 EPCAM 63.984 9.587E-05 7.391 1.203E-09 ENSDARG00000004433 btk 62.583 4.427E-05 3.989 1.660E-03 Page 41 of 50 Diabetes

ENSDARG00000032724 pmp22a 62.186 3.586E-05 18.709 3.655E-05 ENSDARG00000095453 si:rp71-17i16.6 61.656 4.330E-05 13.147 7.885E-07 ENSDARG00000002779 pdx1 60.822 6.314E-05 3.967 3.777E-04 ENSDARG00000067831 C10orf27 59.847 4.378E-05 11.337 4.300E-04 ENSDARG00000076981 zgc:198329 59.542 7.809E-05 10.992 1.631E-12 ENSDARG00000077293 SYNPO2L 59.229 4.518E-05 20.114 7.792E-03 ENSDARG00000005673 f3b 58.940 1.383E-04 4.880 4.007E-06 ENSDARG00000079092 gprc5c 58.873 1.111E-04 10.359 4.837E-13 ENSDARG00000060610 PCDH7 58.551 5.399E-05 5.945 3.217E-06 ENSDARG00000055100 cxcl12b 58.187 5.404E-05 3.820 8.592E-04 ENSDARG00000090614 PCDHAC1 58.101 5.726E-05 46.896 9.774E-07 ENSDARG00000038709 MYADM 56.946 5.325E-05 214.445 0.000E+00 ENSDARG00000077540 f2rl1.2 56.761 1.301E-04 12.137 2.154E-14 ENSDARG00000018921 fam84a 56.575 6.751E-05 10.494 1.252E-10 ENSDARG00000044532 nr4a2b 56.467 1.142E-04 7.453 2.000E-09 ENSDARG00000007982 onecut1 56.305 8.209E-05 34.277 6.661E-16 ENSDARG00000055632 smtnl 56.031 6.591E-05 4.102 1.036E-03 ENSDARG00000009544 cldnb 55.105 1.084E-04 10.701 1.361E-11 ENSDARG00000079549 CDC42EP1 53.810 1.031E-04 4.247 1.793E-04 ENSDARG00000052630 pvrl1b 53.647 7.809E-05 5.683 2.865E-03 ENSDARG00000073870 si:dkey-31j3.9 51.765 1.272E-04 8.759 7.055E-10 ENSDARG00000076431 AL732629.1 51.579 1.539E-04 4.799 9.745E-06 ENSDARG00000076496 FRMD4B 51.218 1.018E-04 4.918 3.694E-03 ENSDARG00000020298 btg2 49.502 4.598E-04 3.486 2.708E-03 ENSDARG00000008030 myl9b 49.498 2.598E-04 4.476 2.298E-05 ENSDARG00000042722 blnk 49.106 2.140E-04 3.583 1.028E-03 ENSDARG00000018643 igf2a 48.903 1.591E-04 6.920 1.166E-07 ENSDARG00000015955 cldnc 47.233 2.787E-04 10.700 6.060E-13 ENSDARG00000089354 TSPAN4 46.910 1.899E-04 7.900 1.417E-08 ENSDARG00000014488 ca2 46.779 3.010E-04 5.410 5.495E-07 ENSDARG00000093233 si:ch211-215d8.2 46.576 1.556E-04 25.424 1.034E-03 ENSDARG00000076799 rnd3a 46.070 2.081E-04 5.098 8.649E-06 ENSDARG00000088122 CT583709.2 45.553 1.606E-04 27.824 4.337E-04 ENSDARG00000062788 irg1l 44.877 3.111E-04 19.976 2.548E-13 ENSDARG00000091231 BX572619.7 44.220 4.754E-04 3.930 2.006E-04 ENSDARG00000006202 erbb3a 44.202 3.099E-04 15.594 6.117E-14 ENSDARG00000044569 cldn19 43.392 1.859E-04 0.088 6.059E-03 ENSDARG00000015033 slc23a1 41.635 2.822E-04 6.866 5.525E-06 ENSDARG00000093331 si:ch211-192f15.3 41.267 2.254E-04 9.172 6.284E-04 ENSDARG00000062707 PLAT 41.151 3.077E-04 13.822 3.566E-07 ENSDARG00000086742 ECM1 40.980 5.106E-04 6.426 2.552E-08 ENSDARG00000003169 magi1 40.972 5.546E-04 4.924 4.061E-06 ENSDARG00000011821 plod2 40.757 3.705E-04 3.287 8.299E-03 Diabetes Page 42 of 50

ENSDARG00000070702 s100v2 40.530 5.117E-04 5.007 3.797E-06 ENSDARG00000077084 col28a1 40.378 2.540E-04 112.820 4.931E-13 ENSDARG00000036548 s1pr2 40.343 3.470E-04 5.602 3.972E-05 ENSDARG00000094511 ccl20 40.236 2.751E-04 8.469 3.748E-05 ENSDARG00000070174 PLEKHH3 39.949 4.505E-04 4.317 2.840E-04 ENSDARG00000022650 cyp2ad3 39.883 5.111E-04 5.146 4.848E-06 ENSDARG00000077811 sox11a 39.860 2.711E-04 20.063 3.609E-10 ENSDARG00000009978 icn 39.827 4.946E-04 7.140 6.946E-07 ENSDARG00000059196 si:ch73-177h5.4 39.472 3.712E-04 13.351 3.752E-09 ENSDARG00000076548 si:dkey-266m15.5 39.282 5.133E-04 6.415 4.366E-07 ENSDARG00000042285 ATP8B3 38.954 4.219E-04 5.235 6.031E-04 ENSDARG00000092870 si:dkey-100n23.4 37.302 4.168E-04 5.467 1.637E-03 ENSDARG00000021004 C5 37.146 9.851E-04 16.633 2.220E-16 ENSDARG00000010654 ARHGAP42 37.009 6.154E-04 5.486 3.805E-06 ENSDARG00000026329 ARHGAP29 36.796 8.334E-04 3.925 2.054E-04 ENSDARG00000042293 ca4b 36.223 5.673E-04 11.360 8.629E-11 ENSDARG00000052954 RAB25 35.506 6.016E-04 4.841 3.121E-03 ENSDARG00000036245 WIPF3 34.927 5.874E-04 5.786 2.326E-03 ENSDARG00000057714 cmah 34.527 8.830E-04 4.305 2.649E-04 ENSDARG00000031622 nrp2a 34.074 8.567E-04 5.690 6.053E-05 ENSDARG00000069476 spint2 34.001 1.420E-03 5.558 3.860E-07 ENSDARG00000039941 OLFML2A 33.830 1.337E-03 6.282 2.724E-08 ENSDARG00000007108 zgc:91985 32.765 7.613E-04 19.779 2.411E-07 ENSDARG00000021806 zfp36l2 32.735 1.624E-03 3.152 7.030E-03 ENSDARG00000079355 FLRT2 32.584 8.199E-04 9.366 2.365E-08 ENSDARG00000094056 si:dkey-235d18.6 32.235 6.753E-04 0.018 7.286E-09 ENSDARG00000077982 elf3 31.336 2.449E-03 8.718 3.652E-11 ENSDARG00000006705 pkn2 31.225 1.424E-03 4.268 9.035E-05 ENSDARG00000068874 PTCHD3 31.187 9.811E-04 5.693 5.310E-03 ENSDARG00000094123 si:dkey-210i3.1 31.024 2.969E-03 13.973 7.641E-03 ENSDARG00000017127 pdzk1ip1l 30.804 1.892E-03 4.985 3.213E-06 ENSDARG00000056206 CAMK2G 30.505 1.314E-03 5.795 2.324E-05 ENSDARG00000034801 MYLK 29.918 1.416E-03 10.848 4.896E-08 ENSDARG00000062204 sigirr 29.871 2.013E-03 3.959 2.427E-04 ENSDARG00000070162 stox2 29.752 2.108E-03 5.275 9.019E-07 ENSDARG00000075718 rpz5 29.708 1.912E-03 3.769 6.564E-04 ENSDARG00000002634 b4galt1 29.422 1.378E-03 4.924 3.766E-05 ENSDARG00000074381 FARP1 29.371 1.901E-03 6.017 1.872E-06 ENSDARG00000044688 dusp4 29.189 2.161E-03 3.658 1.063E-03 ENSDARG00000069378 pard6a 28.900 1.605E-03 4.374 1.615E-04 ENSDARG00000069956 pdlim2 28.224 2.349E-03 5.513 1.014E-06 ENSDARG00000087508 FP236869.1 28.190 3.142E-03 3.439 2.321E-03 ENSDARG00000006615 hnf1ba 28.155 2.755E-03 7.865 4.387E-10 Page 43 of 50 Diabetes

ENSDARG00000004782 fgfr3 27.893 1.536E-03 4.496 8.538E-04 ENSDARG00000017165 slc3a1 26.880 3.311E-03 5.913 6.046E-07 ENSDARG00000040513 zgc:92313 26.821 2.837E-03 3.465 2.164E-03 ENSDARG00000061827 zbtb4 26.763 3.384E-03 3.851 2.904E-04 ENSDARG00000086578 zgc:158345 26.743 1.810E-03 22.238 3.419E-03 ENSDARG00000074390 tmem176l.4 26.409 2.348E-03 6.338 1.179E-06 ENSDARG00000079307 si:dkey-205h13.1 26.395 2.554E-03 3.833 2.648E-03 ENSDARG00000079317 DLC1 26.234 3.203E-03 3.615 8.677E-04 ENSDARG00000053344 gpr133 25.817 4.510E-03 3.861 2.680E-04 ENSDARG00000019709 rhoua 25.726 2.717E-03 3.374 9.855E-03 ENSDARG00000075121 hbegfa 25.651 4.304E-03 3.904 3.166E-04 ENSDARG00000057743 TMEM56 25.444 1.870E-03 8.425 9.019E-03 ENSDARG00000003963 traf4a 24.721 4.024E-03 3.733 9.250E-04 ENSDARG00000042939 gpr39 24.546 5.124E-03 4.269 8.059E-05 ENSDARG00000077169 wu:fj08f03 24.532 4.276E-03 19.638 1.049E-08 ENSDARG00000039351 si:dkey-200l5.2 24.476 5.239E-03 4.909 4.573E-05 ENSDARG00000027744 gadd45ba 24.399 6.432E-03 3.164 6.208E-03 ENSDARG00000029668 crim1 24.357 3.284E-03 3.585 6.369E-03 ENSDARG00000041869 tnfrsf19 24.171 4.073E-03 6.471 2.625E-07 ENSDARG00000046124 rab11fip1a 24.159 4.016E-03 3.507 1.805E-03 ENSDARG00000078108 dok1a 23.877 3.137E-03 7.889 9.846E-06 ENSDARG00000006693 tmprss4a 23.480 3.119E-03 6.901 7.230E-06 ENSDARG00000087844 plekhn1 23.300 6.373E-03 5.067 3.325E-06 ENSDARG00000089414 CU463857.1 23.159 2.826E-03 7.674 2.949E-06 ENSDARG00000069912 hmga2 23.086 3.475E-03 5.455 6.962E-04 ENSDARG00000042518 hipk2 22.692 8.177E-03 3.864 2.611E-04 ENSDARG00000067984 gas1b 22.642 5.169E-03 3.252 9.623E-03 ENSDARG00000076120 foxp4 22.642 7.238E-03 6.024 6.652E-08 ENSDARG00000036135 bbox1 22.523 8.413E-03 6.395 3.572E-08 ENSDARG00000042071 PAM 22.414 9.834E-03 3.458 1.787E-03 ENSDARG00000091408 CCDC165 22.320 4.612E-03 4.993 3.092E-03 ENSDARG00000057706 si:ch211- 22.207 9.377E-03 12.436 9.004E-14 137i24.10 ENSDARG00000089449 KAZN 21.807 3.787E-03 9.299 3.529E-03 ENSDARG00000090526 C12H10orf81 21.755 4.151E-03 5.930 3.392E-03 ENSDARG00000042956 cyp2ad6 21.723 7.224E-03 4.538 1.596E-04 ENSDARG00000021366 fbp1a 21.551 6.662E-03 3.830 2.187E-03 ENSDARG00000087431 zgc:173962 21.321 4.353E-03 9.680 8.767E-03 ENSDARG00000088630 ARHGEF10L 21.016 6.029E-03 4.397 5.161E-04 ENSDARG00000075764 grik5 20.520 5.753E-03 7.541 3.718E-06 ENSDARG00000091766 ARHGEF17 20.366 6.119E-03 7.606 3.401E-06 ENSDARG00000027609 HPN 19.929 8.226E-03 5.479 1.462E-05 ENSDARG00000094428 si:dkey-31f5.8 19.698 5.513E-03 11.560 1.178E-06 Diabetes Page 44 of 50

ENSDARG00000061672 si:ch73-334d15.1 19.670 6.171E-03 11.735 2.902E-05 ENSDARG00000090967 HPN 19.576 9.664E-03 5.392 5.841E-06 ENSDARG00000026166 emilin1b 19.479 7.616E-03 3.692 5.047E-03 ENSDARG00000058323 tmbim1 19.126 9.994E-03 3.212 8.159E-03 ENSDARG00000092899 si:dkey-33i11.4 18.838 6.553E-03 11.854 4.837E-03 ENSDARG00000040469 enpp6 18.837 8.225E-03 6.049 2.715E-03 ENSDARG00000079898 cd248a 18.403 7.695E-03 4.764 7.805E-03 ENSDARG00000074721 RASSF9 17.982 9.959E-03 5.085 1.510E-05 ENSDARG00000090665 CABZ01063203.1 17.528 9.384E-03 6.156 1.431E-05 ENSDARG00000078279 foxp3b 0.028 9.918E-03 0.183 1.006E-04 ENSDARG00000089731 BX511311.7 0.026 7.639E-03 0.231 7.130E-05 ENSDARG00000075227 GRM5 0.026 9.221E-03 0.244 7.401E-03 ENSDARG00000058775 SLC22A3 0.026 7.714E-03 0.246 1.217E-03 ENSDARG00000060053 KCNH2 0.025 6.765E-03 0.247 1.076E-03 ENSDARG00000070627 BX908806.2 0.024 8.794E-03 0.230 9.646E-06 ENSDARG00000043475 tagap 0.023 6.799E-03 0.120 5.114E-08 ENSDARG00000037995 dvr1 0.023 5.717E-03 3.542 8.557E-03 ENSDARG00000071745 CR456631.1 0.023 9.980E-03 0.144 4.249E-03 ENSDARG00000093004 BX548044.8 0.022 7.485E-03 0.025 3.441E-12 ENSDARG00000053535 lmo7b 0.021 4.748E-03 8.695 1.111E-08 ENSDARG00000078368 si:dkey-199f5.4 0.020 7.296E-03 0.152 5.622E-06 ENSDARG00000094132 igf1 0.019 6.568E-03 0.100 2.385E-04 ENSDARG00000090127 CR855997.3 0.019 5.026E-03 161.052 3.331E-16 ENSDARG00000057769 si:dkey-10f21.3 0.019 5.278E-03 0.197 1.936E-05 ENSDARG00000070885 si:ch211-258l4.4 0.019 3.691E-03 0.096 1.397E-08 ENSDARG00000094927 si:dkey-93l1.4 0.018 4.195E-03 0.238 7.213E-04 ENSDARG00000009550 foxi3b 0.017 9.434E-03 0.104 2.668E-03 ENSDARG00000094917 BX324155.2 0.017 8.970E-03 28.907 2.955E-04 ENSDARG00000093631 BX470083.8 0.017 2.540E-03 0.198 3.379E-05 ENSDARG00000071020 si:dkey-42l16.1 0.017 3.036E-03 4.090 3.023E-03 ENSDARG00000014420 elavl3 0.017 3.267E-03 0.268 6.900E-03 ENSDARG00000079556 C2CD4C 0.016 4.132E-03 0.058 7.064E-05 ENSDARG00000094552 CR450805.3 0.016 7.557E-03 0.011 9.717E-13 ENSDARG00000075598 scpp6 0.016 7.151E-03 0.016 6.212E-10 ENSDARG00000077665 si:dkey-236a14.6 0.016 1.952E-03 0.278 1.277E-03 ENSDARG00000094348 CR848663.2 0.016 6.878E-03 0.012 5.984E-12 ENSDARG00000070340 hoxc5a 0.016 6.873E-03 0.025 6.741E-11 ENSDARG00000092514 BX510654.1 0.015 6.573E-03 0.033 3.587E-10 ENSDARG00000092280 si:ch211- 0.015 6.452E-03 0.095 6.573E-08 215m21.15 ENSDARG00000095577 cybrd1 0.015 1.982E-03 0.139 5.924E-03 ENSDARG00000094827 si:ch211-255l14.4 0.015 6.276E-03 0.120 9.492E-06 ENSDARG00000021032 foxd3 0.015 6.021E-03 0.024 7.620E-07 Page 45 of 50 Diabetes

ENSDARG00000089640 si:dkey-117n7.5 0.015 6.021E-03 0.040 3.658E-04 ENSDARG00000021936 zgc:109949 0.014 2.019E-03 0.291 7.990E-04 ENSDARG00000094014 si:ch211- 0.014 3.524E-03 0.126 2.827E-08 165a10.4 ENSDARG00000063269 DUPD1 0.014 2.761E-03 0.184 5.679E-06 ENSDARG00000090413 BX470083.5 0.014 5.259E-03 0.123 6.743E-04 ENSDARG00000043907 FGF11 0.014 2.219E-03 0.255 1.728E-03 ENSDARG00000070419 nkd3l 0.014 1.536E-03 0.206 4.555E-06 ENSDARG00000040938 C22H3orf32 0.014 5.019E-03 0.114 1.256E-03 ENSDARG00000052122 rag1 0.014 5.019E-03 0.277 7.219E-03 ENSDARG00000010384 lpar6l 0.014 5.007E-03 0.130 4.463E-04 ENSDARG00000094200 si:dkey-2j6.2 0.014 4.953E-03 0.104 2.668E-03 ENSDARG00000092486 si:dkey-152b24.8 0.013 4.644E-03 0.056 7.440E-03 ENSDARG00000095771 BX511311.11 0.013 1.440E-03 0.219 9.020E-06 ENSDARG00000070346 hoxc8a 0.013 2.385E-03 0.151 1.231E-03 ENSDARG00000092456 si:ch211-188i4.4 0.013 1.405E-03 0.297 1.314E-03 ENSDARG00000014391 CSMD3 0.013 1.770E-03 0.173 2.962E-06 ENSDARG00000035169 layna 0.012 3.549E-03 0.114 1.220E-03 ENSDARG00000094999 si:ch211- 0.012 3.519E-03 0.138 3.802E-05 254p10.3 ENSDARG00000076789 cx32.2 0.012 1.048E-03 0.212 2.789E-04 ENSDARG00000069025 si:ch211-283g2.5 0.012 3.304E-03 0.114 6.744E-03 ENSDARG00000077938 cd248b 0.012 3.304E-03 0.095 2.206E-05 ENSDARG00000095450 si:ch211-213i16.2 0.012 3.280E-03 0.273 2.193E-03 ENSDARG00000009511 tnfa 0.012 3.195E-03 0.036 1.220E-04 ENSDARG00000090396 CABZ01074946.1 0.011 1.069E-03 0.148 1.251E-05 ENSDARG00000054497 chrna3 0.011 2.896E-03 0.034 2.442E-08 ENSDARG00000002502 VAT1L 0.011 2.838E-03 0.198 3.403E-04 ENSDARG00000091445 CU137681.8 0.011 2.811E-03 0.043 7.772E-16 ENSDARG00000067767 BX510309.1 0.011 9.487E-04 0.213 1.781E-06 ENSDARG00000070121 rhoh 0.011 2.717E-03 0.072 7.896E-04 ENSDARG00000045282 BX322605.1 0.011 2.715E-03 0.160 4.804E-06 ENSDARG00000076873 C9orf172 0.011 1.420E-03 4.296 9.548E-04 ENSDARG00000089567 PATL2 0.011 1.361E-03 5.087 2.534E-04 ENSDARG00000041041 cxcr3.2 0.011 2.513E-03 0.023 1.045E-11 ENSDARG00000076862 FAM198A 0.011 2.447E-03 8.424 4.041E-05 ENSDARG00000036139 lctla 0.011 2.444E-03 0.156 1.727E-03 ENSDARG00000013649 CASR 0.011 2.426E-03 0.160 2.247E-05 ENSDARG00000091737 zp2.2 0.011 2.421E-03 37.842 0.000E+00 ENSDARG00000092653 si:dkey-1h24.6 0.010 1.892E-03 0.076 5.121E-06 ENSDARG00000074082 CU467828.1 0.009 1.731E-03 8.423 9.047E-03 ENSDARG00000016348 DIP2B 0.009 6.872E-04 3.644 1.423E-03 ENSDARG00000069984 CR786578.1 0.009 1.600E-03 0.203 7.771E-06 ENSDARG00000053966 cyp17a2 0.009 8.168E-04 0.262 2.206E-03 Diabetes Page 46 of 50

ENSDARG00000090613 CABZ01042513.3 0.009 1.551E-03 0.154 3.788E-04 ENSDARG00000076262 pou3f2 0.009 1.443E-03 0.043 1.045E-11 ENSDARG00000074782 GPR97 0.009 1.425E-03 0.050 4.734E-09 ENSDARG00000042130 zp3a.2 0.009 1.423E-03 47.510 0.000E+00 ENSDARG00000087717 TMPRSS5 0.008 1.340E-03 0.224 2.468E-03 ENSDARG00000092223 si:dkey-211e20.1 0.008 8.472E-04 0.304 9.086E-03 ENSDARG00000086084 CU571170.2 0.008 1.175E-03 0.201 9.453E-03 ENSDARG00000090151 CT033790.3 0.008 1.120E-03 0.108 1.689E-09 ENSDARG00000086688 AL772241.1 0.007 9.173E-04 0.138 9.252E-03 ENSDARG00000086403 CR381537.1 0.007 8.937E-04 0.243 6.469E-04 ENSDARG00000093408 si:dkey-1p9.3 0.007 8.546E-04 0.118 2.328E-06 ENSDARG00000090777 PKD2L2 0.007 8.540E-04 0.326 8.577E-03 ENSDARG00000040433 cyp2k22 0.007 4.312E-04 14.139 8.721E-04 ENSDARG00000089439 TMEM132A 0.007 8.173E-04 0.079 1.184E-04 ENSDARG00000073809 CR936484.2 0.007 7.992E-04 0.098 1.427E-13 ENSDARG00000079034 wu:fi38e01 0.007 7.320E-04 4.080 1.388E-04 ENSDARG00000011633 dnah9 0.007 1.823E-04 0.279 2.515E-03 ENSDARG00000043436 si:dkey-5n18.1 0.006 3.060E-04 0.153 6.237E-04 ENSDARG00000092830 NLRX1 0.006 5.575E-04 0.216 1.690E-03 ENSDARG00000059616 hs3st2 0.006 5.472E-04 5.536 2.804E-04 ENSDARG00000076897 CU570782.1 0.006 4.970E-04 0.090 1.494E-07 ENSDARG00000002816 rasgrf2 0.006 1.548E-04 0.290 1.190E-03 ENSDARG00000089747 FP565451.1 0.006 4.475E-04 0.310 7.278E-03 ENSDARG00000069251 si:dkeyp-50f7.2 0.005 3.997E-04 27.834 0.000E+00 ENSDARG00000091232 kcnj1 0.005 3.467E-04 0.183 1.789E-05 ENSDARG00000094631 si:ch211-81a5.2 0.005 3.457E-04 0.159 2.772E-05 ENSDARG00000069692 col7a1l 0.005 3.093E-04 0.176 2.038E-03 ENSDARG00000026049 mxf 0.004 2.345E-04 0.164 2.382E-05 ENSDARG00000095715 si:ch211- 0.004 2.329E-04 0.256 2.417E-03 202e12.3 ENSDARG00000092016 BX296549.2 0.004 2.174E-04 0.154 1.678E-07 ENSDARG00000040178 zgc:92647 0.004 2.049E-04 0.117 1.089E-04 ENSDARG00000075551 si:ch211- 0.004 1.729E-04 0.205 3.468E-05 274p24.3 ENSDARG00000075342 BX649628.1 0.004 1.670E-04 0.072 3.905E-07 ENSDARG00000086522 zp2.5 0.004 1.630E-04 1128.149 0.000E+00 ENSDARG00000086096 CT033790.1 0.004 1.592E-04 0.297 2.571E-03 ENSDARG00000058045 tlr21 0.004 1.469E-04 0.207 5.627E-05 ENSDARG00000058386 BX323449.1 0.004 1.277E-04 0.086 2.438E-10 ENSDARG00000057253 si:dkey- 0.003 1.044E-04 0.218 9.963E-05 236a14.13 ENSDARG00000095627 c1qc 0.003 9.306E-05 0.142 1.746E-07 ENSDARG00000089682 BX323556.1 0.003 4.082E-05 0.187 8.875E-07 ENSDARG00000092701 FP016063.1 0.003 8.602E-05 0.061 1.273E-13 Page 47 of 50 Diabetes

ENSDARG00000038226 CABZ01074994.1 0.003 7.198E-05 0.158 1.001E-07 ENSDARG00000053833 mtnr1ba 0.003 6.747E-05 0.107 2.382E-07 ENSDARG00000042712 timm44 0.003 5.832E-05 45.186 1.604E-06 ENSDARG00000095804 CH211-14C17.6 0.003 5.718E-05 0.238 2.619E-03 ENSDARG00000088626 BX927341.2 0.003 5.427E-05 0.082 3.964E-09 ENSDARG00000075091 CU571064.1 0.003 5.175E-05 0.098 1.378E-12 ENSDARG00000095591 si:ch73-138e16.7 0.003 5.095E-05 239.188 0.000E+00 ENSDARG00000088658 CU041380.1 0.003 4.669E-05 0.211 1.070E-05 ENSDARG00000079792 BX511311.1 0.002 3.867E-05 0.105 2.005E-03 ENSDARG00000088745 MFAP4 0.002 3.287E-05 0.119 1.420E-06 ENSDARG00000077344 BX000700.1 0.002 2.907E-05 0.180 5.837E-04 ENSDARG00000056723 si:dkey-24f17.5 0.002 1.073E-05 0.313 3.691E-03 ENSDARG00000057265 si:dkey- 0.002 2.015E-05 0.148 3.987E-06 236a14.11 ENSDARG00000057284 si:dkey-1p9.2 0.002 1.679E-05 0.214 2.243E-05 ENSDARG00000052935 BX629350.2 0.002 1.621E-05 0.097 1.193E-13 ENSDARG00000091103 CABZ01068499.1 0.002 1.463E-05 0.250 5.873E-04 ENSDARG00000023236 KCNH1 0.001 3.561E-06 0.183 1.642E-06 ENSDARG00000094144 CU137681.9 0.001 1.161E-06 0.173 6.495E-08

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Supplemental Table 2: Transgenic Lines line abbreviation reference Tg(HS4sst2:CFP;ins:PhiYFPmdest12TA ins:NTR this work nfsB)lmc009 Tg(Tp1glob:creERT2)jh1 cre driver Wang, et al. (2011) Tg(T2Kβactin:loxPstoploxPhmgb1 cre responder Wang, et al. mCherry)jh15 (2011) Tg(Tp1bglob:eGFP)um14 Tp1:GFP Wang, et al. (2011) Tg(Tp1glob:hmgb1mCherry)fh32 Tp1:hmgb1- Wang, et al. mCherry (2011) Tg(fli1aep:DsRed) fli:dsRed Villefranc, et al. (2007) TgBAC(cftrGFP)pd1041 Navis, et al. (2013) cftrpd1049 Navis, et al. (2013) TgBAC(nkx2.2a:meGFP)vu17 Ng, et al. (2005)

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Supplemental Table 3: Primer Sequences qPCR gene fwd rvs cftr CCGCATTTTCACCACTCTCT CTCATCCCATGAAGCATGCA clcn1b CCGTCTGATTTACCCTGGTG TGAACCCCAGATCTTTGTCC her15 CTCCAAGCTTTCCAAGG AAAACCACCGTCATCTCCAG nkx6.1 CGTGCTCACCACATCAAAAC CGGTTTTGAAACCACACCTT sox9b AGGGCGAGAAGCGTCCGTTTGTG CGGTGCTGGAGTCGGGTGTTTCTG G bactin CGAGCAGGAGATGGGAACC CAACGGAAACGCTCATTGC

in situ probes gene fwd rvs cftr AGATCACCTGTGGAGGATGC CGGGCAGTAAACAACAAACC clcn1b AGTAGGATGTGCTGTTGGGG AGCTGCTGCCCCTATCACTG her15 TACTCCAAGCTTTCCAACAAGGAG GCATTGATAATGCACAAATGATCAC A A

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Supplemental Table 4: Antibodies antigen concentration species source Nkx6.1 1:100 mouse monoclonal DSHB GFP 1:400 rabbit polyclonal Life Technologies dsRed 1:400 mouse monoclonal Clontech Insulin 1:400 guinea pig Dako polyclonal Glucagon 1:400 rabbit polyclonal Dako fluorescent 1:400 Jackson secondaries ImmunoResearch Labs