Page 1 of 58 Diabetes
DB16-0887
Mafa enables Pdx1 to effectively convert pancreatic islet progenitors and committed
islet ααα-cells into βββ-cells in vivo
Taka-aki Matsuoka1,＊＊＊, Satoshi Kawashima1, Takeshi Miyatsuka1,2, Shugo Sasaki1,
Naoki Shimo1, Naoto Katakami1, Dan Kawamori1, Satomi Takebe1, Pedro L. Herrera3,
Hideaki Kaneto4, Roland Stein5, Iichiro Shimomura1
1 Department of Metabolic Medicine, Osaka University Graduate School of Medicine, 2 2 Yamadaoka, Suita, Osaka 565 0871, Japan
2 Center for Molecular Diabetology, Juntendo University Graduate School of Medicine, Tokyo, Japan
3 Department of Genetic Medicine and Development, University of Geneva Faculty of Medicine, CH 1211 Geneva 4, Switzerland
4 Department of Diabetes, Endocrinology and Metabolism, Kawasaki Medical School, Okayama, Japan
5 Department of Molecular Physiology & Biophysics, Vanderbilt University Medical School, 723 Light Hall, Nashville, TN 37232, U.S.A.
＊Corresponding author. Address: 2 2 Yamadaoka, Suita, Osaka 565 0871, Japan Tel: 81 6 6879 3743 Fax: 81 6 6879 3739 e mail: [email protected] u.ac.jp
Key words: islet α cells, β cells, transdifferentiation, Mafa, Pdx1, diabetes
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Diabetes Publish Ahead of Print, published online February 21, 2017 Diabetes Page 2 of 58
Abstract
Among the therapeutic avenues being explored for replacement of the functional islet
β cell mass lost in Type 1 diabetes (T1D), reprogramming of adult cell types into new β cells has been actively pursued. Notably, mouse islet α cells will transdifferentiate into β cells under conditions of near β cell loss, a condition similar to T1D. Moreover, human islet α cells also appear to poised for reprogramming into insulin+ cells. Here we have generated transgenic mice conditionally expressing the islet β cell enriched Mafa and/or Pdx1 transcription factors to examine their potential to transdifferentiate embryonic pan islet cell Ngn3+ progenitors and the later glucagon+ α cell population into β cells. Mafa was found to both potentiate the ability of
Pdx1 to induce β cell formation from Ngn3+ endocrine precursors, and enable Pdx1 to produce
β cells from α cells. These results provide valuable insight into the fundamental mechanisms influencing islet cell plasticity in vivo.
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Introduction
While current therapies of insulin treatment afford glycemic control to T1D patients,
these relatively static methods do not completely recapitulate the acute regulation of the
endogenous islet β cells destroyed by autoimmune destruction. Consequently, T1D patients
have a dramatically shortened life expectancy due to serious long term diabetic complications,
including coronary and renal disease. A variety of innovative approaches are being explored to
produce β cells from embryonic stem cells (1, 2) and adult cell types (3 5). A supposition in
these efforts involves producing conditions that correctly regulate the transcription factor
networks required in programming pancreatic progenitor cells into β cells, and subsequently
controlling mature islet cell function. These include transcription factors like Pdx1 (6 10) that is
essential in the formation of early pancreatic epithelium, developing β cells and adult islet
β cells, as well as Neurogenin3 (i.e. Ngn3) (11 13) that is required during embryogenesis for
specification of all islet cells types (i.e. β, glucagon hormone producing α, somatostatin δ, and
pancreatic polypeptide PP, ghrelin ε). In addition, there are transcription factors like Mafa (14,
15) that are influential later during postnatal β cell maturation and adult cell function. Indeed,
ectopic expression of Pdx1, Ngn3, and Mafa can reprogram pancreatic exocrine cells (3) and
intestinal cells (4) into functional β like cells in vivo.
T1D results from the specific loss of islet β cells. Interestingly, functional β like cells
are produced from endogenous mouse islet α (16) or δ cells (17) after near total targeted
destruction of this cell population, a model mimicking the disease state (16). Furthermore,
epigenomic findings suggests that human α cells are poised for reprogramming, with treatment to
prevent histone 3 repressor site marking at lysine 27 leads to the appearance of insulin+
glucagon+ bihormonal cells in human islets (18).
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Here, we generated transgenic mice that allow conditionally and targeted expression of
Mafa and/or Pdx1 to determine their contribution to β cell generation from embryonic endocrine Ngn3+ and committed glucagon+ progenitors. Earlier studies had established that forced Pdx1 expression in this endocrine precursor population results in greater β cell production at the expense of α cells, with no effect on δ or PP cells (19). We found that Mafa was not only found to potentiate the ability of Pdx1 to reprogram Ngn3+ endocrine progenitor cells to insulin+ cells, but also empowered Pdx1 to transdifferentiate committed glucagon+
α cells to this cell fate. These results provide further support for the essential role of Mafa and
Pdx1 in the production of therapeutic β cells for treatment of T1D patients.
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Results
Mafa + Pdx1 more efficiently program endocrine Ngn3+ precursors to β cells than Pdx1 alone.
To explore how Mafa influences the ability of Pdx1 to convert α cells into β cells, we
developed mouse lines that allow for conditional and heritably over production of either Mafa,
Pdx1, or Mafa + Pdx1 upon expression of Cre recombinase (Fig. 1A). Antibodies to Mafa, Pdx1
and their incorporated antigenic tags were used to immunohistochemically detect the levels of
the transgenic proteins, termed Mafamyc and Pdx1flag. Both of these islet β cell enriched proteins
(20 22) were now found throughout the islet cell population upon activation in embryonic
endocrine precursors with Ngn3 driven Cre, which were termed the Ngn3-Cre;CAT-Mafa and
Ngn3-Cre;CAT-Pdx1 lines. For example, flag tagged Pdx1 was produced in essentially all islet
cell in P0.5 and 6 week old Ngn3-Cre;CAT-Pdx1 mice; the penetrance rate was approximately
95% in the insulin+ and/or glucagon+ cell populations (Supplemental Fig. 1). Notably, neither
Mafamyc nor Pdx1flag were expressed in the surrounding acinar or ductal cells of the pancreas
(Fig. 1B). While there was no overt effect on α cell fate in Ngn3-Cre;CAT-Mafa islets (Figs. 1C,
D), insulin was expressed in around 10% of Ngn3-Cre;CAT-Pdx1 glucagon+ cells at P0.5. These
double hormone+ cells resolved to become only insulin+ by 6 weeks, with essentially no islet
glucagon+ cells remaining (Fig. 1C) as reported previously (19). Moreover, there was no
evidence for apoptosis within Mafamyc and Pdx1flag expressing islet glucagon+ or insulin+ cells,
with the TUNEL+ signal detected exclusively from red blood cells (Supplemental Fig. 2). These
results suggested that resolution of the Mafamyc + Pdx1flag positive cells to mono hormonal
insulin production reflects transdifferentiation of α to β like cells.
Mafa expression in endocrine precursor cells augmented the actions of Pdx1 in the
Ngn3-Cre;CAT-Mafa;CAT-Pdx1 line. Thus, there was a roughly 9 fold increase in the number of
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insulin and glucagon co positive cells in these islets at birth when compared to
Ngn3-Cre;CAT-Pdx1 islets (17.9 to 2.0%, Fig. 1D). Insulin was also clearly detected in
Ngn3-Cre;CAT-Mafa;CAT-Pdx1 PP cells at P0.5, while no induction was found in
Ngn3-Cre;CAT-Mafa or Ngn3-Cre;CAT-Pdx1 islets (Fig. 2A, Supplemental Fig. 3A). Notably, insulin and PP co positive cells were still detectible in 6 week old
Ngn3-Cre;CAT-Mafa;CAT-Pdx1 islets (Fig. 2A). In addition and as reported earlier, insulin was produced in very few somatostatin hormone expressing Ngn3-Cre;CAT-Pdx1 islet δ cells (19), or Ngn3-Cre;CAT-Mafa;CAT-Pdx1 (Supplemental Fig. 3). These results are consistent with lineage tracing experiments indicating that islet β and PP cells are derived from a common PP+ precursor (23), and illustrate the differential plasticity of islet lineages to Mafa and Pdx1 reprogramming.
Mafa and Pdx1 can effectively convert α cells into islet β cells.
Pdx1 alone is incapable of converting α cells into islet β cells in vivo (19). However, since co synthesized Mafa + Pdx1 favored the production of β cells at the expense of α cells when coproduced in Ngn3+ progenitors (Fig. 1D), we wondered whether these factors would have such facilitating properties if exclusively expressed in α cells. Glucagon enhancer/promoter driven Cre transgenic mice (24) (i.e. termed Gcg-Cre) were used to overexpress Mafa, Pdx1, and Mafa + Pdx1 in α cells, with concomitant excision of the loxP site flanked lacZ or green fluorescent protein (GFP) expression cassettes in the ROSA26 locus used for cell lineage labeling. Cre mediated recombination was observed in roughly 35% of islet glucagon+ cells in Gcg-Cre;CAT-Mafa (Fig. 3A) and Gcg-Cre;CAT-Pdx1 (Fig. 3B) islets.
Moreover, neither insulin, somatostatin nor PP positive cells were β gal positive (Supplemental
Fig. 4; data not shown).
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The effect on islet insulin+ cell formation of forced Mafa + Pdx1 expression in α cells
was compared between Gcg-Cre;CAT-Mafa;CAT-Pdx1, Gcg-Cre;CAT-Mafa and
Gcg-Cre;CAT-Pdx1 islets. Neither Mafa nor Pdx1 could independently produce insulin in
α cells (Fig. 3A, B), a result consistent with earlier data examining forced Pdx1 expression (19).
In contrast, insulin staining was clearly detectable in Gcg-Cre;CAT-Mafa;CAT-Pdx1 α cells
marked by β gal, myc tag, and flag tag by 2 weeks; the analysis was performed at this time
point rather than P0.5 due to the later developmental expression of Gcg Cre relative to
Ngn3 Cre. Notably, glucagon was no longer detectible in the 2 week old lineage labeled
insulin positive cell population (Fig. 3C, D, and Supplemental Fig. 5A, B) nor was there any
overt change in number of these cells in 6 week old Gcg-Cre;CAT-Mafa;CAT-Pdx1 islets
(Supplemental Fig. 6, data not shown). As observed in the insulin+ cell population generated by
Mafamyc and Pdx1flag expression in Ngn3 islet progenitors, there was no evidence of cell death
within Gcg-Cre;CAT-Mafa;CAT-Pdx1 islets (Supplemental Figs. 2, 7). The remaining
glucagon+ cells in Gcg-Cre;CAT-Mafa;CAT-Pdx1 islets never expressed Mafamyc or Pdx1flag, a
reflection of the poor penetrance of Gcg Cre (Supplemental Fig. 8).
To further assess the change to β cell identity of the α cells made to co express Mafa
and Pdx1, immunostaining was performed with various islet α cell (i.e. Mafb, Arx) and β cell
(i.e. Glut2, Nkx6.1, Urocortin3 (Ucn3) (25)) enriched protein antibodies on this new insulin+
cell population (Fig. 4 and Supplemental Fig. 9). Significantly, many of the β cell enriched
products were made in these cells, including the principal β cell glucose transporter, Glut2 (i.e.
80 % of GFP traced recombinated cells), the Nkx6.1 transcription factor (36% of β Gal traced
recombinated cells), and UCN3 (46% of Flag traced recombinated cells). In contrast, Mafb (20)
was not present in Mafamyc + Pdx1flag produced insulin+ cells (Fig. 4C), a transcription factor
normally produced in immature islet β cells (15). Similarly, Arx (26), a transcription factor
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essential for α cell formation (26), was expressed poorly in α cell derived insulin+ cells (i.e. approximately 21%, Supplemental Fig. 9B). Taken together, these findings demonstrate that the
Pdx1 + Mafa can transdifferentiate α cells into β like cells, by inducing key features of the β cell and suppressing those of the α cell.
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Discussion
Several distinct islet enriched transcription factors are capable reprogramming other
pancreatic cell types into β like cells, including Pdx1, Ngn3 and Mafa in exocrine acinar cells
(3), Pdx1 (19) as well as Nkx6.1 in Ngn3+ precursors (27), and Pax4 in ductal cells (28). Such
observations illustrate the tremendous cellular plasticity within the pancreas that could be
exploited therapeutically to sustain islet β cell activity in T1D patients. Interestingly, islet
α cells, the principal remaining pancreatic islet cell population in these individuals, convert to
β cells upon ablation of this insulin+ cell population in adult mice. However, little is known
about the factors that initiate this cell fate change. Here we show that islet β cell enriched Mafa
not only potentiates the ability of Pdx1 to convert of Ngn3+ cells to insulin+ cells, but also
permits Pdx1 to reprogram α cells into β cells. These findings illustrate the mutually supportive
abilities of these transcription factors to promote the β cell fate choice.
Heritably expressing Mafa and Pdx1 into Ngn3+ cells profoundly impacted the fraction
of α cells converted to insulin and glucagon coproducing cells at birth, with essentially all
α cells undergoing a cell fate switch in Ngn3-Cre;CAT-Mafa;CAT-Pdx1 islets (Fig. 1D). This
population appears to be completely reprogrammed to β cells by 6 weeks in
Ngn3-Cre;CAT-Pdx1 (19) and Ngn3-Cre;CAT-Mafa;CAT-Pdx1 islets (Fig. 1D), as illustrated
by the gain in β cell identify marker expression and loss of α (Fig. 4 and Supplementary Fig. 9).
The significant enhancement by Mafa of Pdx1 mediated transdifferentiation at P0.5 implies that
both factors act simultaneously to change the chromatin architecture of α cell progenitors to a
β like fate. In addition, Mafa + Pdx1 induced insulin production in islet PP cells, and silenced
PP hormone expression (Fig. 2A). Unfortunately, the lack of distinctive maturation markers for
PP cell markers precludes us from determining the extent of reprogramming.
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Notably, while Nkx6.1 also effectively converts Ngn3+ progenitors to insulin+ cells, there are clear difference in how this transcription factor acts in relation to Mafa + Pdx1. For example, Nkx6.1 reprogrammed all neonatal islet cell types to express the insulin hormone (i.e.
α, δ, ε, and PP), and not simply the α and PP cells (27). However, developmental Nkx6.1 expression is also unable to effectively convert α cell progenitors to insulin producing, and it would be interest to determine if Mafa has the same enabling capabilities. Even more profound mechanistic differences were observed upon expression of Pax4 in embryonic Ngn3+ cells or
α cells (28), which produced β like cells from pancreatic ductal cells that resulted in oversized islets, β cell dysfunction and diabetes. Each of these reprogramming factors presumably initiates the β cell fate switch by binding to target sequences in silent chromatin (29). The differences in their potential presumably reflects their ability to embed in nucleosomal DNA and the functional properties of recruited coregulators, as illustrated for the Oct4, Sox2, Klf4 and c Myc transcription in generating pluripotent stem cells (30).
Gene induction involves the recruitment of transcriptional coregulators by enhancer bound transcription factors, like Mafa and Pdx1. These protein protein interactions ultimately lead to assembly at the promoter of the RNA polymerase II transcriptional machinery.
Coregulators can have positive (coactivator) and/or negative (corepressor) actions on target gene transcription, thus conferring a second level of specificity to the transcriptional response.
For example, the islet cell enriched Nkx2.2 transcription recruits the DNMT3a, Grg3, and
HDAC1 corepressors to silence transcription of the Arx gene in developing β cell (31). It will be interesting to determine how various recently described coactivators of Pdx1 and Mafa impact their reprogramming abilities, as one would predict that the chromatin modifying activities of
Swi/Snf4 (i.e. Pdx1 (32)) and MLL4 (i.e. Mafa (33)) to be of importance.
Collectively, these results strongly suggest that Pdx1 and Mafa are principal mediators
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of β cell production from non β cells, including in the context of developing α cells as well as
adult acinar (3), stomach (5), and intestinal cells (4). Upon considering these observations, we
predict that these transcription factors also play a principal role in the induction of the newly
formed β cells produced from α (16) and δ (17) cells after almost complete ablation of the islet
β cell population by diphtheria toxin expression. Importantly, islet α, PP, and δ cells appear to
be preserved in the context of the depleted β cell mass in T1D (34 36) and T2D (37, 38)
patients, and, consequently potentially reprogrammable by transcription factor expression (e.g.
Mafa + Pdx1, Pax4 (28)) or drug treatment (18). Collectively, these findings further illuminate
the significance of understanding the mechanisms involved in regulating islet cell identify and
function in diabetes treatment.
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Experimental Procedures
CAG-CAT-Pdx1flag, CAG-CAT-Mafamyc , Cre, and ROSA26 mice
CAG-CAT-Mafamyc (39), CAG-CAT-Pdx1flag (40), Ngn3 Cre (12), Gcg Cre (23),
Rosa26-LacZ (41), and Rosa26-GFP (42) mice have be described previously. All animal procedures were approved by the Ethics Review Committee for Animal Experimentation of the
Osaka University Graduate School of Medicine.
Immunohistochemistry and cell quantification
Pancreata were dissected and fixed in 4% paraformaldehyde in PBS at 4 °C, washed in
PBS, immersed in sucrose solution, embedded and frozen in Tissue Tek (OCT Compound;
Sakura) or processed routinely for paraffin embedding. Frozen and paraffin blocks were sectioned at 6 m thickness and immunostained. The following primary antibodies were used at the given dilutions: rabbit anti MafA (Bethyl Laboratories, Inc., Montgomery, TX), 1:500; goat anti MafA (43) 1:200; rabbit anti Pdx1 (44), 1:1000; rabbit anti Nkx6.1 (Sigma Aldrich, Inc., St
Louis, MO), 1:200; rabbit anti MafB (Bethyl Laboratories, Inc.), 1:200; goat anti Arx (Santa
Cruz Biotechnology, Inc. Dallas, TX), 1:200; rabbit anti myc (Cell Signaling Technology, Inc.,
Danvers, MA), 1:200; rabbit anti flag (Affinity BioReagents, Golden, CO), 1:100; mouse anti flag (Trans Genic Inc., Kobe, Japan), 1:500; rabbit anti Glut2 (abcam, Cambridge, United
Kingdom), 1:200; guinea pig anti insulin (DAKO, Glostrup, Denmark), 1:2000; rabbit anti glucagon (DAKO) 1:500; guinea pig anti glucagon (Millipore, St. Charles, MO) 1:200; rabbit anti β gal antibody (Medical & Biological Laboratories, Nagoya, Japan) 1: 200; chicken anti β gal antibody (abcam) 1: 200; chicken anti GFP antibody (abcam) 1:500; Primary antibodies were detected with donkey raised secondary antibodies conjugated fluorescein at a
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1:500 dilution. Fluorescent images were captured using an OLYMPUS FV1000 D confocal
microscope. The images shown are representative of our analysis of at least 3 independent
derived mice, unless otherwise specified.
The total number of insulin+, glucagon+, PP+, and β gal+ cells in 5 sections per
pancreas from at least 3 mice per genotype were manually counted in the Ngn3 Cre (Fig. 1D,
2B) and Gcg Cre (Fig. 3D) lines. Approxmiately 500 cells were counted in the 6 week old
pancreas and 200 cells in the p0.5. The data is presented at the ratio of each hormone+ cells, or
hormone+ to β gal+ cells for each genotype.
Statistical analysis
Statistical analysis was performed using one way ANOVA followed by the Fisher’s test.
A value of p < 0.05 was considered to be statistically significant.
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Author Contributions
T. Ma. designed experiments. T.Ma., T.Mi, and P.L.H. generated the mouse. T.Ma., and S.K. performed immunohistochemistry. T.Ma., S.K., S.S., N.S., and S.T. performed mouse experiments. T.Ma., T.Mi. and R.S. wrote the manuscript. I.S., H.K., N.K., D.K., and R.S. supervised the project.
Acknowledgments
We thank Ms. Satomi Takebe and Ms. Yuko Sasaki for excellent technical assistance and Ms. Chikayo Yokogawa for excellent secretarial assistance. This work was supported by grants from the Juvenile Diabetes Research Foundation (Career Development Award
2 2005 946 to T. Matsuoka; #46 2010 755, #26 2007 928, and #17 2012 389 to P.L. Herrera),
KAKENHI (No. 10379258 to T. Matsuoka), the NIH/NIDDK (DK50203 and DK090570 to R.
Stein; DK089566 and DK089572 to P.L. Herrera), and the Swiss National Science Foundation
(to P.L. Herrera). The authors have no conflict of interest directly relevant to the content of this article.
T. Matsuoka 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.
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Gomis R, Bragado R, Marti M, Jaraquemada D, et al. Pancreas in recent onset insulin dependent diabetes mellitus. Changes in HLA, adhesion molecules and autoantigens, restricted T cell receptor V beta usage, and cytokine profile. J Immunol. 1994;153(3):1360 77. 36. Gomez Dumm CL, Console GM, Luna GC, Dardenne M, and Goya RG. Quantitative immunohistochemical changes in the endocrine pancreas of nonobese diabetic (NOD) mice. Pancreas. 1995;11(4):396 401. 37. Yoon KH, Ko SH, Cho JH, Lee JM, Ahn YB, Song KH, Yoo SJ, Kang MI, Cha BY, Lee KW, et al. Selective beta cell loss and alpha cell expansion in patients with type 2 diabetes mellitus in Korea. The Journal of clinical endocrinology and metabolism. 2003;88(5):2300 8. 38. Henquin JC, and Rahier J. Pancreatic alpha cell mass in European subjects with type 2 diabetes. Diabetologia. 2011;54(7):1720 5. 39. Matsuoka TA, Kaneto H, Kawashima S, Miyatsuka T, Tochino Y, Yoshikawa A, Imagawa A, Miyazaki J, Gannon M, Stein R, et al. Preserving Mafa expression in diabetic islet beta cells improves glycemic control in vivo. The Journal of biological chemistry. 2015;290(12):7647 57. 40. Miyatsuka T, Kaneto H, Kajimoto Y, Hirota S, Arakawa Y, Fujitani Y, Umayahara Y, Watada H, Yamasaki Y, Magnuson MA, et al. Ectopically expressed PDX 1 in liver initiates endocrine and exocrine pancreas differentiation but causes dysmorphogenesis. Biochemical and biophysical research communications. 2003;310(3):1017 25. 41. Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nature genetics. 1999;21(1):70 1. 42. Mao X, Fujiwara Y, Chapdelaine A, Yang H, and Orkin SH. Activation of EGFP expression by Cre mediated excision in a new ROSA26 reporter mouse strain. Blood. 2001;97(1):324 6. 43. Matsuoka TA, Kaneto H, Miyatsuka T, Yamamoto T, Yamamoto K, Kato K, Shimomura I, Stein R, and Matsuhisa M. Regulation of MafA expression in pancreatic beta cells in db/db mice with diabetes. Diabetes. 2010;59(7):1709 20. 44. Matsuoka T, Kajimoto Y, Watada H, Kaneto H, Kishimoto M, Umayahara Y, Fujitani Y, Kamada T, Kawamori R, and Yamasaki Y. Glycation dependent, reactive oxygen species mediated suppression of the insulin gene promoter activity in HIT cells. The Journal of clinical investigation. 1997;99(1):144 50.
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Figure legends
Figure 1. Mafa and Pdx1 enhance production of insulin+ from glucagon+ cells when
co expressed in pancreatic endocrine precursor cells.
(A) Schematic representation of Mafamyc, Pdx1flag, and Cre mediated recombination. Exogenous
myc tagged Mafa or flag tagged Pdx1 are expressed via the excision of the stuffer CAT cassette
upon production in islet cell precursors of Ngn3 Cre or of Gcg Cre in islet α cells. (B) MafA
and Pdx1 antibodies were used to show ectopically expressed Mafamyc and Pdx1flag in essentially
all Ngn3-Cre;CAT-Mafa (left panel) or Ngn3-Cre;CAT-Pdx1 (right) islet glucagon (Gcg)+ cells
at P0.5. The arrows illustrate Mafa or Pdx1 expressing glucagon+ cells. (C) Insulin (Ins)+ and
glucagon+ cells in representative islets of the Neurog3-Cre, Ngn3-Cre;CAT-Mafa,
Ngn3-Cre;CAT-Pdx1 and Ngn3-Cre;CAT-Mafa;CAT-Pdx1 pancreas at P0.5 or 6W. The arrows
depict insulin and glucagon co positive cells, which are only detected in the
Ngn3-Cre;CAT-Mafa;CAT-Pdx1 pancreas at P0.5. (D) Quantitative analysis of the islet insulin,
glucagon, or co hormone positive production at P0.5 and 6W cells in the Neurog3-Cre,
Ngn3-Cre;CAT-Mafa, Ngn3-Cre;CAT-Pdx1 and Ngn3-Cre;CAT-Mafa;CAT-Pdx1 pancreas. The
ratio of the insulin+, glucagon+, or double positive cells is presented relative to the total
hormone+ cell number. The ratio of the double hormone+ cells was compared at P0.5 to
glucagon+ cells, and glucagon + cells to insulin+ cells at 6W. n = 3 to 6, *P<0.001. Scale bar: 50
m.
Figure 2. Insulin+ cells are produced from PP cells upon co expression of Mafa and Pdx1 in
pancreatic endocrine precursor cells.
(A) Immunohistochemical analysis of pancreata from Neurog3-Cre, Ngn3-Cre;CAT-Mafa,
- 19 - Diabetes Page 20 of 58
Ngn3-Cre;CAT-Pdx1, Ngn3-Cre;CAT-Mafa;CAT-Pdx1 mice at P0.5 and 6W for insulin, pancreatic polypeptide (PP), and somatostatin (Sst). Nuclei were stained with DAPI. There were
PP cells co expressing PP and insulin at P0.5 and 6W. Arrow depicts insulin + PP co positive cells. Scale bars: 50 m. (B) The ratio of insulin + somatostatin positive cells to somatostatin positive cells were compared at P0.5; the somatostatin positive cells to insulin positive cell ratio was compared at 6W. n = 3 to 6, *P<0.001.
Figure 3. Mafa + Pdx1 produce insulin+ cells from α cells.
Insulin, glucagon and β gal staining were performed in the (A) Gcg-Cre;CAT-Mafa;ROSA-LacZ,
(B) Gcg-Cre;CAT-Pdx1;ROSA-LacZ and (C) Gcg-Cre;CAT-Mafa;CAT-Pdx1;ROSA-LacZ pancreas. The arrows in (A) and (B) illustrate that Gcg Cre activates ROSA lacZ reporter expression in only α cells. Because Gcg-Cre was only expressed in approximately 35% of
α cells, a non representative islet image is shown to more clearly illustrate the extent of glucagon+ to insulin+ cell conversion in Gcg-Cre;CAT-Mafa;CAT-Pdx1;ROSA-LacZ islets.
The arrows show that a large fraction of β gal+ cells express insulin by 2W in the (C)
Gcg-Cre;CAT-Mafa;CAT-Pdx1;ROSA-LacZ pancreas. (D) The number of insulin+, glucagon+, and insulin + glucagon+ cells at 2W and 6W is displayed as number of hormone+ cells to total
β gal+ cells in Gcg-Cre;ROSA-LacZ, Gcg-Cre;CAT-Mafa;ROSA-LacZ,
Gcg-Cre;CAT-Pdx1;ROSA-LacZ, and Gcg-Cre;CAT-Mafa;CAT-Pdx1;ROSA-LacZ mice. n = 4.
Scale bars: 50 m.
Figure 4. Islet β cell identity marker expression is gained at the expense of α in
Gcg-Cre;CAT-Mafa;CAT-Pdx1 islets.
Islet β cell enriched (A) Glut2 and (B) Nkx6.1 production is specifically induced in
- 20 - Page 21 of 58 Diabetes
Gcg-Cre;CAT-Mafa;CAT-Pdx1;ROSA α cell lineage labelled (A) GFP and (B) β gal cells, at the
rate of 80% and 36% among labelled cells, respectively. In contrast, the production of α cell
identity (C) Mafb is lost in this α lineage marked cell population. The arrows illustrate Glut2+,
Nkx6.1+, or Mafb cells labeled with the lineage marker, and presumably expressing Mafamyc
and/or Pdx1flag. Scale bars: 50 m.
- 21 - Diabetes Page 22 of 58 Figure 1A
loxP loxP myc CAG promoter CAT-Mafa CAT Mafa pA pA loxP loxP CAG promoter CAT-Pdx1 CAT Pdx1 pA flag pA Cell specific promoter Cre recombinase Cell specific-Cre Cre-mediated recombination
Cell specific misexpression loxP myc CAG promoter Mafa Mafa (myc-tagged) pA loxP CAG promoter Pdx1 Pdx1 (flag-tagged) flag pA Page 23 of 58 Diabetes Figure 1B
Ngn3-Cre Ngn3-Cre;CAT-Mafa Ngn3-Cre Ngn3-Cre;CAT-Pdx1 MAFA MAFA PDX1 PDX1 GCG GCG GCG GCG P 0.5 P 6W P 0.5 Figure GCG GCG INS INS Ngn3 1C - Cre GCG GCG INS INS Ngn3 - Cre;CAT - Mafa Diabetes GCG GCG INS INS INS Ngn3 - Cre;CAT - Pdx1 GCG GCG INS INS CAT - Mafa;CAT Ngn3 - Cre; Page 24of58 - Pdx1 Page 25 of 58 Diabetes Figure 1D Insulin positive cells Glucagon positive cells Insulin & Glucagon positive cells
* * * P 0.5 * 6W * * * * 1 1 *
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2 Ratio of Ins and/or Gcg cells cells Gcg and/or Ins of Ratio 0 0 Figure
6W P 0.5 2A DAPI DAPI PP INS PP INS Ngn3 - Cre DAPI DAPI PP INS PP INS Ngn3 - Cre;CAT Diabetes - Mafa DAPI DAPI PP INS PP INS Ngn3 - Cre;CAT - Pdx1 Page 26of58 Page 27 of 58 Diabetes Figure 2A
Ngn3-Cre Ngn3-Cre;CAT-Mafa Ngn3-Cre;CAT-Pdx1 INS INS INS Sst Sst Sst DAPI DAPI DAPI P 0.5 P
INS INS INS Sst Sst Sst DAPI DAPI DAPI 6W Figure 2A
6W P 0.5 Ngn3 - Cre;CAT Diabetes DAPI PP DAPI PP INS INS - Mafa;CAT - Pdx1 DAPI Sst INS DAPI Sst INS Page 28of58 Page 29 of 58 Diabetes Figure 2B Insulin positive cells PP positive cells Insulin & PP positive cells
P 0.5 * 6W * * * * * 1 1
cells cells 0.8 0.8 PP PP
0.6 0.6
0.4 0.4
0.2 0.2 Ratio of Ins and/or Ins of Ratio 0 0 Diabetes Page 30 of 58 Figure 3A Gcg-Cre;CAT-Mafa;ROSA-lacZ INS b-GAL merge
2W GCG b-GAL merge Page 31 of 58 Diabetes Figure 3B Gcg-Cre;CAT-Pdx1;ROSA-lacZ INS b-GAL merge
2W GCG b-GAL merge Diabetes Page 32 of 58 Figure 3C Gcg-Cre;CAT-Mafa;CAT-Pdx1;ROSA-lacZ INS b-GAL merge
2W GCG b-GAL merge Page 33 of 58 Diabetes Figure 3D Insulin positive cells Glucagon positive cells
* 2W * * 1
0.8 gal cells cells gal - b / 0.6 Gcg 0.4 Ins or Ins 0.2
Ratio of Ratio 0 Diabetes Page 34 of 58 Figure 4A
Gcg-Cre;ROSA-GFP GFP GLUT2 DAPI merge 2W
Gcg-Cre;CAT-Mafa;CAT-Pdx1;ROSA-GFP GFP GLUT2 DAPI merge 2W Page 35 of 58 Diabetes Figure 4B
Gcg-Cre;ROSA-lacZ b-GAL NKX6.1 merge 2W
Gcg-Cre;CAT-Mafa;CAT-Pdx1;ROSA-lacZ b-GAL NKX6.1 merge 2W Diabetes Page 36 of 58 Figure 4C
Gcg-Cre FLAG MAFB INS merge 2W
Gcg-Cre;CAT-Mafa;CAT-Pdx1 FLAG MAFB INS merge 2W Page 37 of 58 Diabetes DB16-0887 Mafa enables Pdx1 to effectively convert pancreatic islet progenitors and committed islet a-cells into b-cells in vivo
Taka-aki Matsuoka1,＊, Satoshi Kawashima1, Takeshi Miyatsuka1,2, Shugo Sasaki1, Naoki Shimo1, Naoto Katakami1, Dan Kawamori1, Satomi Takebe1, Pedro L. Herrera3,
Hideaki Kaneto4, Roland Stein5, Iichiro Shimomura1
1 Department of Metabolic Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
2 Center for Molecular Diabetology, Juntendo University Graduate School of Medicine, Tokyo, Japan
3 Department of Genetic Medicine and Development, University of Geneva Faculty of Medicine, CH-1211 Geneva 4, Switzerland
4 Department of Diabetes, Endocrinology and Metabolism, Kawasaki Medical School, Okayama, Japan
5 Department of Molecular Physiology & Biophysics, Vanderbilt University Medical School, 723 Light Hall, Nashville, TN 37232, U.S.A.
＊Corresponding author Diabetes Page 38 of 58 Supplemental Figure 1
Ngn3-Cre;CAT-Pdx1 FLAG GCG INS merge P0.5
FLAG GCG INS merge W 6 Page 39 of 58 Diabetes
Supplemental Figure 1. Pdx1flag is expressed in most P0.5 and 6W Ngn3- Cre;CAT-Pdx1 islet cells. Flag staining (red) was observed in most of Ngn3- Cre;CAT-Pdx1 insulin+ (blue) and glucagon+ (green) cells at P0.5 and 6W; the Cre penetrance rate was a maintained at approximately 95%. Scale bars: 50 mm. P0.5 P0.5 Supplemental TUNEL TUNEL Figure 2A Ngn3 Ngn3 - Cre;CAT - GC GC Cre;CAT Diabetes G G - Mafa;CAT - Pdx1 - Pdx1 INS INS Page 40of58 merge merge Page 41of58 6W 6W Supplemental TUNEL TUNEL Figure 2B Ngn3 Ngn3 - Cre;CAT - GC GC Cre;CAT Diabetes G G - Mafa;CAT - Pdx1 - Pdx1 INS INS merge merge Diabetes Page 42 of 58 Supplemental Figure 2C
Akita TUNEL INS merge W 8 Page 43 of 58 Diabetes
Supplemental Figure 2. There were not any TUNEL+ apoptosis observed within Mafamyc and/or Pdx1flag expressing islet cells. TUNEL staining was performed with (A) P0.5, and (B) 6W Ngn3-Cre;CAT-Mafa;CAT-Pdx1 and Ngn3- Cre;CAT-Pdx1 mice. The arrows mark the insulin and glucagon double positive cells. No TUNEL+ cells (green) were found within islet glucagon+ (red) or insulin+ (blue) cells. Note: the green staining was shown to be within red blood cells. (C) The C57BL/6 Ins2Akita (Akita) mouse, an ER stress induced apoptosis model, was used as a control to observe TUNEL+ β-cells. Scale bars: 50 mm. Diabetes Page 44 of 58 Supplemental Figure 3A
Ngn3-Cre;CAT-Pdx1 PP FLAG INS merge W
6 SST FLAG INS merge Page 45 of 58 Diabetes Supplemental Figure 3B
Ngn3-Cre;CAT-Mafa;CAT-Pdx1 PP FLAG INS merge W
6 SST FLAG INS merge Diabetes Page 46 of 58
Supplemental Figure 3. PP and d cells produce Pdx1flag in Ngn3-Cre;CAT-Pdx1 and Ngn3-Cre;CAT-Mafa;CAT-Pdx1 islets. Pdx1flag epitope staining (red) was observed in most A) Ngn3-Cre;CAT-Pdx1 and B) Ngn3-Cre;CAT-Mafa;CAT-Pdx1 insulin (blue), PP (green), and somatostatin (green) cells in 6W islets. However, PP+ cells are seldom observed in 6W Ngn3-Cre;CAT-Pdx1;CAT-Mafa islets. The green staining for PP in 6W Ngn3-Cre;CAT-Pdx1;CAT-Mafa islets was only found in red blood cells. The arrows mark insulin and somatostatin double positive cell. Scale bars: 50 mm. Page 47 of 58 Diabetes Supplemental Figure 4
Gcg-Cre;CAT-Mafa;CAT-Pdx1;ROSA-lacZ PP SST b-GAL b-GAL 2W
Supplemental Figure 4. Mafamyc + Pdx1flag expression does not affect islet PP- and δ- cell levels in Gcg-Cre;CAT-Mafa;CAT-Pdx1 islets. The ROSA26-driven β-gal (red) was used to trance the α-cell fate in PP (green) and somatostatin (SST, green) cells. Notably, there was no evidence of β-gal expression in these 2 week-old Gcg-Cre;CAT-Mafa;CAT- Pdx1 islet cells.Scale bars: 50 mm. Diabetes Page 48 of 58 Supplemental Figure 5A
Gcg-Cre;CAT-Mafa;CAT-Pdx1 FLAG GCG INS merge
2W MYC GCG INS merge Page 49 of 58 Diabetes Supplemental Figure 5B
Gcg-Cre;CAT-Mafa;CAT-Pdx1;ROSA-GFP GFP INS GCG merge 2W
Supplemental Figure 5. MafAmyc + Pdx1flag induces insulin production in pancreatic α- cells. To trace the cell fate of the α-cells misexpressing MafAmyc + Pdx1flag in Gcg- Cre;CAT-Mafa;CAT-Pdx1 mice, (A) flag- and myc-tagged staining (red) was compared in glucagon (blue) and insulin (green). (B) Tagged Mafa- and Pdx1-expressing α-cells were also traced by immunostaining for GFP (green), insulin (blue), and glucagon (red) in Gcg- Cre;CAT-Mafa;CAT-Pdx1;ROSA-GFP mice. Significantly, most of Pdx1flag, MafAmyc, and GFP co-positive cells are only insulin+ in 2 week-old Gcg-Cre;CAT-Mafa;CAT-Pdx1 islets (indicated by arrows). Scale bars: 50 mm. Diabetes Page 50 of 58 Supplemental Figure 6
Gcg-Cre;CAT-Mafa;CAT-Pdx1 INS FLAG DAPI merge 2W
INS FLAG DAPI merge W 6 Page 51 of 58 Diabetes
Supplemental Figure 6. There was not a marked change in the level of a-cell transdifferentiation between 2W and 6W Gcg-Cre;CAT-Mafa;CAT-Pdx1 islets. Approximately 75% of Pdx1flag expressing cells were insulin+ in Gcg-Cre;CAT- Mafa;CAT-Pdx1 islets. The arrows indicate flag and insulin double positive cells. Scale bars: 50 mm. Diabetes Page 52 of 58 Supplemental Figure 7
Gcg-Cre;CAT-Mafa;CAT-Pdx1 TUNEL FLAG INS merge 2W
TUNEL FLAG INS merge W 6 Page 53 of 58 Diabetes
Supplemental Figure 7. There were no TUNEL+ Pdx1flag expressing cells in 2W and 6WGcg-Cre;CAT-Mafa;Cat-Pdx1 islets. TUNEL+ (green), flag+ (red), and insulin+ (blue) staining is shown. Flag+ cells were indicated by arrows. The green staining cells was only found in red blood cells. Scale bars: 50 mm. Diabetes Page 54 of 58 Supplemental Figure 8
Gcg-Cre;CAT-Mafa;CAT-Pdx1 FLAG MYC INS merge
2W FLAG MYC GCG merge Page 55 of 58 Diabetes
Supplemental Figure 8. Only monohormonal islet insulin+ cells are generated upon MafAmyc + Pdx1flag expression in embryonic α-cells. Flag (green), myc (red), insulin (blue), and glucagon (blue) immunostaining was performed in Gcg- Cre;CAT-Mafa;CAT-Pdx1 islets. Only insulin+ cells were produced upon simultaneously production of the tagged transcription factors (indicated by arrows) in developing α-cells. Scale bars: 50 mm. Diabetes Page 56 of 58 Supplemental Figure 9A
Gcg-Cre FLAG UCN3 INS merge 2W
Gcg-Cre;CAT-Mafa;CAT-Pdx1 FLAG UCN3 INS merge 2W Page 57 of 58 Diabetes Supplemental Figure 9B Gcg-Cre;CAT-Mafa;CAT-Pdx1 (2W) FLAG ARX INS
merge Diabetes Page 58 of 58
Supplemental Figure 9. Ucn3 expression is gained and Arx compromised upon α-cell conversion to β-like cells in Gcg-Cre;CAT-Mafa;CAT-Pdx1 islets. (A) The islet Ucn3 (red) β-cell-enriched and GSIS-regulating protein is detectable in 46% of Pdx1flag (green) cells within 2W Gcg-Cre;CAT-Mafa;CAT-Pdx1 islets (indicated by arrows). (B) The α-cell-specific Arx transcription factor (green) was present in roughly 21% of FLAG+ (red) cells. FLAG+ and Arx- cells are marked by arrows. Notably, Arx expression was eventually silenced in the islet β-cells produced upon misexpressing Pdx1 in Ngn3+ progenitors (19). Scale bars: 50 mm.