Pancreatic Mesenchyme Regulates Epithelial
Organogenesis throughout Development
Limor Landsman1, Amar Nijagal2, Theresa J. Whitchurch1, Renee L. VanderLaan1, Warren E. Zimmer3,
Tippi C. MacKenzie2, Matthias Hebrok1*
1Diabetes Center, Department of Medicine, University of California, San Francisco, San Francisco, California, United States of America, 2Eli and Edythe Broad Center of
Regeneration Medicine and Stem Cell Research, Department of Surgery, University of California, San Francisco, San Francisco, California, United States of America,
3Department of Systems Biology and Translational Medicine, Texas A&M Health Science Center, College Station, Texas, United States of America
The developing pancreatic epithelium gives rise to all endocrine and exocrine cells of the mature organ. During
organogenesis, the epithelial cells receive essential signals from the overlying mesenchyme. Previous studies, focusing on ex
vivo tissue explants or complete knockout mice, have identified an important role for the mesenchyme in regulating the
expansion of progenitor cells in the early pancreas epithelium. However, due to the lack of genetic tools directing
expression specifically to the mesenchyme, the potential roles of this supporting tissue in vivo, especially in guiding later
stages of pancreas organogenesis, have not been elucidated. We employed transgenic tools and fetal surgical techniques to
ablate mesenchyme via Cre-mediated mesenchymal expression of Diphtheria Toxin (DT) at the onset of pancreas formation,
and at later developmental stages via in utero injection of DT into transgenic mice expressing the Diphtheria Toxin receptor
(DTR) in this tissue. Our results demonstrate that mesenchymal cells regulate pancreatic growth and branching at both early
and late developmental stages by supporting proliferation of precursors and differentiated cells, respectively. Interestingly,
while cell differentiation was not affected, the expansion of both the endocrine and exocrine compartments was equally
impaired. To further elucidate signals required for mesenchymal cell function, we eliminated b-catenin signaling and
determined that it is a critical pathway in regulating mesenchyme survival and growth. Our study presents the first in vivo
evidence that the embryonic mesenchyme provides critical signals to the epithelium throughout pancreas organogenesis.
The findings are novel and relevant as they indicate a critical role for the mesenchyme during late expansion of endocrine
and exocrine compartments. In addition, our results provide a molecular mechanism for mesenchymal expansion and
survival by identifying b-catenin signaling as an essential mediator of this process. These results have implications for
developing strategies to expand pancreas progenitors and b-cells for clinical transplantation.
Citation: Landsman L, Nijagal A, Whitchurch TJ, VanderLaan RL, Zimmer WE, et al. (2011) Pancreatic Mesenchyme Regulates Epithelial Organogenesis throughout
Development. PLoS Biol 9(9): e1001143. doi:10.1371/journal.pbio.1001143
Academic Editor: Antonio J. Vidal-Puig, University of Cambridge, United Kingdom
Received January 19, 2011; Accepted July 28, 2011; Published September 6, 2011
Copyright: ? 2011 Landsman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: LL was supported by a Juvenile Diabetes Research Foundation postdoctoral fellowship (www.jdrf.org) and an American Diabetes Association
Mentorship Award (www.diabetes.org). Work in MH’s laboratory was supported by a grant from the US National Institutes of Health (www.nih.gov) (DK60533).
Image acquisition was supported by the UCSF Diabetes and Endocrinology Research Center microscopy core (www.nih.gov) (P30 DK63720). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: DT, Diphtheria Toxin; DTA, Diphtheria Toxin active A subunit; DTR, Diphtheria Toxin receptor; hbEGF, heparin binding epidermal growth factor;
hESC, human embryonic stem cell; Ngn3, Neurogenin 3; RA, Retinoic Acid
* E-mail: email@example.com
Organogenesis is a complex and dynamic process that requires
tight spatial and temporalregulationofdifferentiation,proliferation,
and morphogenesis. The pancreas serves as an interesting model for
thestudyofthese processesas itsepitheliumgivesriseto functionally
distinct cells: endocrine cells, including insulin-producing b-cells
that release hormones into the blood stream to regulate glucose
homeostasis, and exocrine cells that produce, secrete, and transport
digestive enzymes. These diverse cell types derive from common
progenitors residing in the embryonic pancreatic epithelium
through a well-orchestrated multi-step process. While numerous
studies have delineated the cascades of transcription factors within
the epithelium that guide epithelial cell development (reviewed in
[1,2]), the role of the surrounding mesenchyme in governing
pancreas organogenesis atdifferent stagesremains largely unknown.
Mesenchymal cells start to coalesce around the nascent gut tube
shortly before pancreas epithelial cells evaginate around mouse
embryonic day 9.5 (e9.5) to form the dorsal and ventral buds .
At e13.5–e14.5 Pdx1+epithelial precursor cells become committed
to either the endocrine or the exocrine lineage, and from e15.5
until the end of gestation, pancreatic cells undergo final
differentiation to give rise to all pancreatic cell types found in
the adult organ. The first evidence that mesenchymal cells were
required for pancreatic epithelial growth was provided in the
1960s by seminal work by Golosow and Grobstein , in which it
was shown that e11 mouse pancreatic epithelium rudiments
stripped of their overlying mesenchyme failed to grow in culture.
However, further studies addressing the role of the mesenchyme at
later stages have been difficult as the expanding pancreas
epithelium quickly branches into the surrounding mesenchyme,
thus preventing clean physical separation of these two layers after
PLoS Biology | www.plosbiology.org1 September 2011 | Volume 9 | Issue 9 | e1001143
,e12 in the mouse. Additionally, while improved culture
conditions for organ rudiments mimic embryonic development
during early stages quite well , full replication of all in vivo
aspects of later pancreas organogenesis have not been achieved ex
vivo . As a consequence, studying the role of the mesenchyme at
advanced stages of pancreas development using explant systems
resulted in controversial findings. A number of studies have shown
that while mesenchymal cells have a positive effect on exocrine
differentiation and growth in culture, they impair endocrine cell
development [6–10]. Other studies have observed that close
proximity between mesenchyme and epithelium promotes exo-
crine differentiation, while secreted mesenchymal factors enhance
endocrine differentiation over a distance . More recently, a
study by Attali and colleagues showed that co-culture of
epithelium with mesenchyme promotes the production of
insulin-expressing cells, an effect largely due to the expansion of
Pdx1+precursor cells rather than maturation or proliferation of
insulin-positive cells . Importantly, endocrine development
was highly variable and dependent on the culture conditions such
as oxygen levels , further indicating that in vivo manipulation
of mesenchymal gene expression is necessary to fully uncover all
mesenchymal functions throughout pancreas development.
Starting in the 1970s, extensive efforts were made to identify
mesenchymal factors responsible for these effects on the epithelial
compartment [12,13]. A decade ago, Bhushan and colleagues
demonstrated that fibroblast growth factor 10 (Fgf10), expressed
by mesenchymal cells from e9.5 until e11.5, is essential for
pancreas growth and differentiation as it stimulates proliferation of
Pdx1-expressing precursor cells . Since then, germ-line knock
out mouse lines, genetically manipulated zebra fish, and
transfected chick embryos have been used to study a limited
number of additional mesenchymal signaling pathways for their
role in guiding pancreas formation (summarized in ). These
studies provided evidence for Retinoic Acid (RA), Wnt, FGF,
BMP, TGFb, and EGF signaling pathways as important regulators
of pancreas formation [1,10,14–17]. However, detailed studies of
the requirement for individual mesenchymal factors in pancreas
development have been hampered by the lack of transgenic tools
that permit manipulation of gene expression specifically in the
Here, we present experiments that take advantage of Nkx3.2
(Bapx1)-Cre transgenic mice in which Cre-expression is directed to
the embryonic pancreatic mesenchyme, but not the epithelium.
Using this Cre line in conjunction with mouse lines allowing
Diphtheria Toxin (DT) induced apoptosis, we depleted mesen-
chymal cell during various stages of in vivo pancreas development.
As expected, elimination of mesenchymal cells at the onset of
pancreas development completely blocked pancreas organogene-
sis. Surprisingly, mesenchymal requirement was not restricted to
this early stage, as ablation at later developmental stages also led to
severe epithelial hypoplasia, reduced branching, and impaired b-
cell and exocrine cell expansion. To elucidate the signaling
pathways essential for mesenchyme function, we eliminated
canonical Wnt signaling from the tissue. Loss of Wnt signaling
within the mesenchyme resulted in mesenchymal cell ablation—
subsequently leading to reduction in both exocrine and endocrine
cell mass. Summarily, our results demonstrate that the pancreatic
epithelium depends on mesenchymal signals for proper expansion
and morphogenesis throughout development.
Nkx3.2-Cre Directs Gene Expression to the Pancreatic
Mesenchyme, but Not the Epithelium
In order to manipulate gene expression in pancreatic mesen-
chyme, but not epithelium, we looked for genes whose expression
matches this pattern. Previous studies pointed to the homeobox
gene Nkx3.2 (also known as Bapx1), whose expression was found in
the forming somites as well as in the mesenchyme of developing
pancreas, stomach, and gut [18–23]. In contrast, Nkx3.2
expression was not detected in endodermally derived cells in
these tissues [18,20,23]. In the pancreatic mesenchyme Nkx3.2 is
expressed as early as e9.5, and by e12.5 its expression becomes
restricted to the mesenchymal area, which will give rise to the
splenic bud [18–20,23]. An Nkx3.2 (Bapx1)-Cre line, in which one
copy of the endogenous Nkx3.2 gene was replaced by a transgene
encoding the Cre recombinase, had previously been generated
[24,25]. This transgenic mouse line faithfully replicates the
endogenous expression of Nkx3.2 and directs Cre activity to the
foregut mesenchyme and skeletal somites starting at e9.5 .
Given that pancreatic expression of the Nkx3.2-Cre transgene
was not thoroughly analyzed in prior studies, we first crossed the
transgenic mice to two reporter strains, the R26-LacZf/+and the
R26-YFPf/+lines, which express LacZ or YFP, respectively, upon
Cre-mediated recombination. YFP expression in Nkx3.2-Cre;R26-
YFPf/+embryos (from here on referred to as Nkx3.2/YFP) was not
found in the endodermally-derived pancreatic epithelium marked
by E-Cadherin and Pdx1 at e9.5 , but was ubiquitously
detected in the surrounding mesenchyme (Figure 1A). Similarly,
X-gal staining in Nkx3.2-Cre;R26-LacZf/+(from here on referred
to as Nkx3.2/LacZ) indicated LacZ expression was confined to the
surrounding mesenchyme at e11.5 (Figure 1B). At p0, Nkx3.2/
LacZ and Nkx3.2/YFP expressing cells with fibroblast-like
morphology were observed around islets, ducts, and blood vessels
(Figure 1C,C9,D). Importantly, we could not detect reporter genes’
expression in either epithelial (Figure 1C,C9,D), endothelial, or
neuronal cells (Figure S1), indicating that Nkx3.2-Cre activity is
excluded from those compartments throughout pancreatic devel-
opment. Thus, the Nkx3.2-Cre line directs Cre-activity exclusively
to the mesenchyme during pancreas development and serves as a
Embryonic development is a highly complex process that
requires tight orchestration
differentiation, and migration as cells grow within loosely
aggregated mesenchyme and more organized epithelial
sheets to form organs and tissues. In addition to intrinsic
cell-autonomous signals, these events are further regulat-
ed by environmental cues provided by neighboring cells.
Prior work demonstrated a critical role for the surrounding
mesenchyme in guiding epithelial growth during the early
stages of pancreas development. However, it remained
unclear whether the mesenchyme also guided the later
stages of pancreas organogenesis when the functional
exocrine and endocrine cells are formed. Here, we show
that specific genetic ablation of the mesenchyme at
distinct developmental stages in vivo results in the
formation of a smaller, misshapen pancreas. Loss of the
mesenchyme profoundly impairs the expansion of both
endocrine and exocrine pancreatic progenitors, as well as
the proliferative capacity of maturing cells, including
insulin-producing beta-cells. Thus, our studies reveal
unappreciated roles for the mesenchyme in guiding the
formation of the epithelial pancreas throughout develop-
ment. The results suggest that identifying the specific
mesenchymal signals might help to optimize cell culture
protocols that aim to achieve the differentiation of stem
cells into insulin-producing beta cells.
of cellular proliferation,
Mesenchyme Is Essential for Pancreas Formation
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novel tool to specifically manipulate embryonic gene expression in
General histological analysis implied that the relative proportion
of mesenchyme to epithelium shifts during pancreas organogenesis
as epithelial cell numbers expand. We therefore took advantage of
Nkx3.2-Cre transgenic mice to quantify the mesenchymal area
during different developmental stages. By measuring the percent-
age of the pancreatic area marked by Nkx3.2/LacZ and Nkx3.2/
YFP cells at various developmental stages, we determined that
while the relative mesenchymal area is significantly reduced during
pancreas organogenesis, it still comprised 11% and 6% of the
pancreatic area at e15.5 and e18.5, respectively (Figure 1E). Thus,
although there is a dramatic reduction in their portion over time,
embryonic mesenchymal cells are present throughout pancreas
Mesenchymal Cells Are Required to Support Early Stages
of Pancreas Development In Vivo
Next, we tested the requirement for mesenchyme during
pancreas organogenesis in vivo. Studies using cultured pancreatic
rudiments as well as Fgf10 knockout mice demonstrated a crucial
role for the mesenchyme in expanding the pool of epithelial
pancreatic precursor cells at early developmental stages (e9.5–
e11.5) [3,14,27,28]. In order to determine the role of the
mesenchyme during pancreas development in vivo, we decided
to ablate this tissue by employing transgenic mice carrying the
Diphtheria Toxin (DT) active A subunit (DTA) flanked by flox
sites (R26-eGFP-DTA mice , from here on referred to as DTA).
Upon Cre-mediated recombination, the DTA produced by the
transgene inhibits protein synthesis, resulting in rapid apoptosis of
Cre-positive cells within less than 24 h . Given that Nkx3.2-
Cre is expressed in mesenchymal cells surrounding the pancreas
from the time organ morphogenesis is initiated (e9.5, Figure 1A),
Nkx3.2-Cre;DTA embryos permit the study of mesenchymal
requirement at early stages of pancreas development (illustrated
in Figure 2A).
We first analyzed potential defects in e10.5 embryos. At this
stage, Nkx3.2-Cre;DTA embryos presented with Pdx1+E-Cadherin+
epithelial pancreatic cells (Figure 2B,C). However, while non-
transgenic control pancreatic epithelial cells were completely
surrounded by E-Cadherin2mesenchymal cells, Nkx3.2-Cre;DTA
embryos lacked most of the adjacent mesenchymal cell layer
(Figure 2B,C). To assess potential defects in pancreatic bud
morphology, we performed whole mount staining with the
epithelial marker E-Cadherin. In wild-type embryos this staining
revealed the expected organization of stomach, liver, and ventral
and dorsal pancreatic buds (Figure 2D). In contrast, pancreatic
buds of Nkx3.2-Cre;DTA embryos were severely reduced in size and
did not evaginate from the foregut epithelium (Figure 2E).
Nkx3.2-Cre;DTA transgenic mice suffered from embryonic
lethality starting at e15.5 as well as severe skeletal defects
(Figure 2F,G) resulting from Nkx3.2-Cre activity in the somites
[21,22,24]. Although the few viable Nkx3.2-Cre;DTA embryos
recovered at e15.5 were only slightly smaller than non-transgenic
littermates (Figure 2F,G), their gastrointestinal tract was dramat-
ically reduced in size (Figure 2H,I), likely due to Nkx3.2-Cre-
mediated expression of DTA in the mesenchyme surrounding
these tissues [23,24]. Notably, while pancreatic tissue was clearly
detected in non-transgenic embryos at this stage (Figure 2H,
demarcated by the white line), Nkx3.2-Cre;DTA embryos had no
visible pancreatic tissue (Figure 2I). Histological analysis of gut
rudiments confirmed the gross morphology observation and
showed only intestine-like tissue in Nkx3.2-Cre;DTA embryos, with
no discernable stomach, spleen, or pancreatic tissues (Figure 2J,K).
Thus, elimination of mesenchyme at the earliest stages of pancreas
formation leads to complete agenesis caused by the inability of
pancreatic epithelium to evaginate from the forming gut and to
Mesenchymal Cell Ablation at Later Embryonic Stages
Impairs Epithelial Pancreas Development
Pancreas development is a multistep process during which the
epithelium undergoes complex morphological changes while
common precursor cells differentiate into the various cells types
that form the adult pancreas . To test whether mesenchymal
Figure 1. Nkx3.2-Cre drives gene expression in the embryonic
pancreatic mesenchyme. (A) e9.5 Nkx3.2-Cre;R26-YFPflox/+embryos
were stained with antibodies against YFP (green), Pdx1 (red), and E-
Cadherin (blue). YFP positive cells surround both the dorsal and ventral
pancreatic epithelia and do not co-stain with the epithelial markers
Pdx1 and E-Cadherin. Insert shows higher magnification of E-
Cadherin+Pdx1+and YFP+cells (B,C,C9) Nkx3.2-Cre;R26-LacZf/+embryos
stained with X-gal (blue) and counterstained with Fast Red (pink). (B)
LacZ positive cells were found in the mesenchymal but not in the
epithelial layer of the e11.5 pancreatic bud (B) and p0 pancreatic tissue
(C, C9). (C9) A higher magnification of the areas marked with a box in (C).
(D) p0 pancreatic tissues of Nkx3.2-Cre;R26-YFPf/+stained for YFP
(green) and E-Cadherin (red) to reveal clear separation between Nkx3.2/
YFP+cells and E-Cadherin+epithelial cells. (E) Bar diagram shows the
mesenchyme area as a percentage of total pancreatic area at the
indicated days. Nkx3.2-Cre;R26-LacZf/+e11.5 pancreatic dorsal buds
were stained as described in (B) and Nkx3.2-Cre;R26-YFPf/+e15.5 and
e18.5 pancreata were stained for YFP. The portion of Nkx3.2/YFP and
Nkx3.2/LacZ areas were then measured as described in the Materials
and Methods. n=3. M, mesenchyme; E, endodermal epithelium; #, islet
of Langerhans; *, duct; V, blood vessel. p values: **p,0.01, ***p,0.005.
Mesenchyme Is Essential for Pancreas Formation
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cells play distinct roles during different stages of pancreas
development, we depleted the mesenchyme at various time points
by injecting DT into developing embryos. Unlike primates, rodent
cells lack a high affinity receptor for DT and therefore do not
endocytose the toxin . Since DT internalization into the cell
cytoplasm is crucial for its ability to trigger the apoptotic
machinery, rodent cells are resistant to ectopically administrated
DT. However, mouse cells expressing a human DT Receptor
(DTR) transgene, encoding for the human heparin binding
epidermal growth factor (hbEGF), gain sensitivity to DT and are
rapidly eliminated upon exposure to the toxin . Prior studies
have established that cell specific expression of human DTR in
transgenic mice allows the ablation of targeted cells within 6 h
following DT administration . By crossing transgenic mice in
which DTR expression is activated upon Cre-mediated recombi-
nation (iDTR , from here on referred to as DTR) with the
Nkx3.2-Cre mice (Nkx3.2-Cre;DTR) we were able to specifically
ablate the mesenchyme at different embryonic time points during
pancreas development upon DT injection.
To ensure efficient delivery of DT to the developing pancreas,
we injected the agent directly into embryos intraperitoneally (i.p.;
the experimental procedure is illustrated in Figure 3A and Figure
S2A–D) [35,36]. As early as 4 h following DT injection into e13.5
Nkx3.2-Cre;DTR embryos, we observed an increase in apoptotic
mesenchymal cells compared to controls (Figure S2E,F). One day
after DT injection we detected only E-Cadherin expressing cells in
Nkx3.2-Cre;DTR pancreata (Figure S2G,H), strongly indicating
that E-Cadherin-negative mesenchymal cells were eliminated. The
loss of Nkx3.2-Cre;DTR-positive mesenchymal cells was further
confirmed by direct staining for human DTR expression (Figure
At the end of gestation (e18.5), Nkx3.2-Cre;DTR embryos
injected with DT at e13.5 were viable and appeared grossly
normal, with normal body weight (Figure 3B–D). At e18.5 the
transgenic embryos displayed skeletal dysplasia, gastrointestinal
defects, and asplenia (Figure 3B,C and Figure S3), likely a result of
ablation of Nkx3.2-Cre expressing cells in these organs, and they
died at birth. Therefore, in utero injections of DT into Nkx3.2-
Cre;DTR do not cause embryonic lethality and permit studying the
effects of mesenchyme ablation on epithelial pancreas develop-
ment during embryogenesis.
To elucidate the requirement of mesenchyme at different stages
we injected Nkx3.2-Cre;DTR embryos and non-transgenic litter-
mates in utero with a single dose of DT at embryonic days 11.5,
12.5, 13.5, 14.5 ,15.5, or 16.5 (illustrated in Figure 3E). Embryos
were then allowed to develop in situ until e18.5 when pancreata
were dissected and weighed. Surprisingly, both dorsal and ventral
pancreatic regions were significantly reduced in size in treated
Figure 2. Depletion of pancreatic mesenchyme in Nkx3.2-Cre;DTA embryos inhibits epithelial growth. (A) A schemeillustratingembryonic
development from e9.5 to e18.5. Arrow marks the time from the onset of mesenchymal Diphtheria Toxin A subunit (DTA) expression in pancreas to the
time of analysis. (B–E) Analysis of e10.5 Nkx3.2-Cre;DTA embryos and non-transgenic littermates. (B,C) Pancreatic bud stained for Pdx1 (green), E-
Cadherin (red), and DAPI (blue). The E-Cadherin2mesenchymal layer completely surrounds Pdx1+E-Cadherin+epithelial cells (marked with arrow) in
control (B) but not in transgenic embryos (C). Inserts show higher magnification of the epithelial bud. (D,E) Whole mount staining against E-Cadherin
marks pancreatic dorsal (arrowhead) and ventral (arrow) buds that are both smaller in transgenic embryos (E) as compared to control (D). St, Stomach.
(F–K) Analysis of e15.5 Nkx3.2-Cre;DTA embryos and non-transgenic littermates. (F,G) Images show skeletal abnormalities in transgenic embryos (G) as
comparedto controls(F).(H–K) Gross morphology andhistologicalanalysisof embryonic gastrointestinal tract.(H,I) Isolated whole gastrointestinal tract.
(J,K) Cross-sections stained with Hematoxylin and Eosin (H&E). Pancreatic tissue (Pan, outlined with a white dashed line in H and with arrows in J),
stomach (St), spleen (Sp), and gut (G) are detected in control (H,J) but only indeterminate gut-like structures are found in transgenic (I, K) embryos.
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transgenic embryos independent of time of DT administration
(Figure 3F–H). The most dramatic reduction in pancreas mass, up
to 80%, was observed when transgenic embryos were injected
between e11.5 and e13.5 (Figure 3H). DT injection at later stages,
e14.5 and e15.5, resulted in an approximately 50% loss of
pancreas mass. Notably, mesenchymal elimination as late as e16.5
led to a marked reduction in pancreas size to about two-thirds of
non-transgenic littermates (Figure 3H). These results demonstrate
that the mesenchyme is continuously required for proper pancreas
development and organogenesis.
Depletion of Mesenchymal Cells Affects Pancreas
Morphology, but Not Pancreatic Cell Differentiation
Next, we performed an in-depth analysis of pancreas morpho-
genesis and cell differentiation in transgenic animals in which
mesenchyme was depleted mid-way through organogenesis (DT
injections into e13.5 Nkx3.2-Cre;DTR embryos followed by analysis
at e18.5; DT e13.5Re18.5).
When compared to normal tissues , mesenchyme-ablated
pancreata displayed an abnormal globular morphology. DT-
treated Nkx3.2-Cre;DTR pancreata were smooth and lacked the
typical extension of the left branches (Figure 4A,B) as well as the
gastric lobe (Figure 3F,G). In addition, DT-treated transgenic
pancreata presented with a rounded tail instead of the stereotyp-
(Figure 4A,B) . Histological analysis further revealed more
compacted cellular distribution in mesenchyme-ablated pancreata
as compared to control, as shown by severe reduction of typical
acellularareas normally found
Figure 3. Pancreatic mesenchyme depletion at various developmental stages impairs organ development. Nkx3.2-Cre;DTR and non-
transgenic littermates embryos were injected i.p. with a single dose of Diphtheria Toxin (DT) (8 ng/mg body weight) while in utero at indicated
embryonic days. Embryos were then allowed to develop in situ until analyzed at e18.5, as illustrated in (A). (B–D) Embryos were injected with DT at
e13.5 and analyzed at e18.5. (B,C) Whole body images reveal no gross defects in transgenic embryo (C) as compared to control littermate (B). (D) Body
weight of transgenic (black bar) to non-transgenic (non tg, gray bar, set to 100%) littermates is equivalent. n=5. (E) A scheme illustrating embryonic
development from e9.5 to e18.5. Arrows mark the time between in utero DT injection to the day of analysis (e18.5). Different arrow colors represent
different injection days. (F,G) Imaging of whole e18.5 pancreata, injected with DT at e13.5, reveals profound loss of pancreas tissue in DT transgenic
embryos (G). (H) Bar diagram summarizing the relative pancreatic weight at e18.5 of Nkx3.2-Cre;DTR embryos either uninjected (-DT, empty bar) or
DT-injected at e11.5 (yellow bar), e12.5 (orange bar), e13.5 (red bar), e14.5 (magenta bar), e15.5 (blue bar), or e16.5 (green bar). Non-transgenic
littermates injected with DT at the corresponding days serve as controls (non tg, gray bar, set to 100%). Pancreas weight of uninjected transgenic
embryos is comparable to control, whereas DT-injected transgenic pancreata weighed significantly less at all time points analyzed. n.5 for each
group (from at least two independent litters). Student t test was used to compare the average weight of transgenic pancreata to non-transgenic
littermates as well as to those injected with DT at e13.5 (indicated by horizontal lines). p value: *p,0.05, ***p,0.0001.
Mesenchyme Is Essential for Pancreas Formation
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Staining for the endothelial cell marker PECAM1 revealed that
pancreatic vasculature, known to be crucial for organ development
[38,39], was not overtly disrupted in DT-treated transgenic
pancreata (Figure S4A–D). Similarly, Tuj1 (b-III Tubulin)-
expressing neuronal cells, known to be required for proper
endocrine differentiation [40,41], could be found in pancreata of
DT-treated Nkx3.2-Cre;DTR embryos (Figure S4E,F).
Previous in vitro studies have implied that mesenchymal cells
may control the differentiation of pancreatic epithelial cells [6,9].
In order to study the in vivo effect of the mesenchyme on epithelial
cell differentiation, we analyzed pancreatic tissues from DT-
treated Nkx3.2-Cre;DTR for the expression of exocrine and
endocrine markers at the end of gestation (DT e13.5Re18.5).
Normal expression patterns for both the duct cell marker Mucin1+
and the acinar cell marker Amylase+in treated transgenic
pancreas indicated normal exocrine differentiation (Figure 4E,F).
Furthermore, endocrine differentiation was not disturbed by
mesenchymal ablation as Insulin, Glucagon, and Somatostatin
expressing cells could be detected in transgenic pancreata
(Figure 4G,H). Endocrine cells were single-hormone positive,
clustered in islet-like structures typical for this developmental
stage, and were distributed throughout the pancreas in a normal
pattern (Figure 4C,D,G,H). Moreover, b-cells from transgenic
embryos expressed the transcription factor MafA (Figure 4I,J),
which is critical for full maturation and glucose responsiveness
, strongly indicating that mesenchyme ablation does not block
their differentiation potential. Thus, while pancreas morphogen-
esis is impaired upon mesenchyme elimination after the first stages
of pancreas formation, differentiation of the major cell types was
Pancreatic Mesenchyme Is Required for Precursor Cell
Although each of the specific pancreatic epithelium lineages
formed in mesenchyme-depleted pancreata, the dramatic reduc-
tion in pancreatic organ size suggests a decrease in the overall
number of pancreatic epithelial cells. In order to understand
whether mesenchymal ablation affects either endocrine or
exocrine mass, Nkx3.2-Cre;DTR mice treated with DT at e13.5
(illustrated in Figure 5A) were analyzed at e18.5 for b- and acinar
cell masses. Both Insulin+b-cell and Amylase+acinar-cell mass
were significantly reduced in transgenic mice when compared to
non-transgenic littermates (Figure 5B,C), suggesting a requirement
for mesenchymal cells during the expansion of both exocrine and
Figure 4. Depletion of pancreatic mesenchyme affects epithelial branching, but not cell differentiation. Morphological and histological
analysis of Nkx3.2-Cre;DTR and non-transgenic littermates (non tg) in utero injected with DT at e13.5 and analyzed at e18.5. (A,B) Imaging of dorsal
pancreata shows abnormal gross morphology of transgenic tissue (B). Typical left branches (arrowhead) found in non-transgenic pancreas are absent
from the DT-treated transgenic tissue. In addition, DT-treated non-transgenic control present with an anvil-shaped tail, while transgenic pancreas
have a rounded tail (red dashed lines). Pan, pancreas; St, stomach; Sp, spleen. (C,D) Histological analysis of pancreatic sections stained with H&E
indicates abnormal and more condensed cellular distribution in transgenic pancreata (D), as compared to non-transgenic control (C). (E,F) Analysis for
the acinar marker Amylase (green) and duct cell marker Mucin1 (red) reveals the presence of these two cell types in treated transgenic pancreata (F),
similar to control (E). (G,H) Pancreatic sections were stained with antibodies against Insulin (green) as a b-cell marker, Glucagon (red) as a a-cell
marker, and Somatostatin (blue) as d-cell marker. Islet-like structures containing all these three endocrine cell types are found in the DT-treated
transgenic embryos (H), similar to control (G). (I,J) Analysis for MafA (red) expression in b-cells (insulin+cells, green) indicating normal cell maturation
in transgenic pancreata (J).
Mesenchyme Is Essential for Pancreas Formation
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endocrine compartments/precursors. The observation that trans-
genic pancreata maintained a normal acinar to b-cell ratio
(Figure 5D) indicates that both cell types depend in equal measures
on mesenchymal signals for their proliferation.
To determine the developmental stage during which mesen-
chyme ablation affects pancreatic mass, we injected embryos at
e13.5 and investigated pancreata 2 d later at e15.5 (DT
e13.5Re15.5). At that stage, pancreas mass in transgenic embryos
was already reduced by 80% as compared to controls (Figure 5E),
similar to the reduction observed in pancreata injected at e13.5
and analyzed at e18.5 (Figure 3H). Since mesenchymal cells
comprise only 11% of pancreatic tissue at e15.5 (Figure 1E), the
observed reduction in pancreatic weight was likely due to a rapid
and significant loss of the epithelial compartment of the organ.
Next, we investigated whether cells of either the endocrine or
exocrine compartments were already affected in e15.5 Nkx3.2-
Figure 5. Mesenchymal ablation at e13.5 leads to reduced b- and acinar-cell mass due to impaired proliferation of precursor cells.
Nkx3.2-Cre;DTR embryos and non-transgenic (non tg) littermates were injected with DT at e13.5 and analyzed at the embryonic days indicated. (A) A
scheme illustrating embryonic development from e9.5 to e18.5. Red arrow marks the time from DT injection (e13.5) to analysis endpoint (e18.5). (B)
Bar diagram shows marked reduction in b-cell mass in transgenic pancreata at e18.5 (black bar) compared to control tissue (gray bar). b-cell mass was
calculated as the fraction of Insulin+area out of the total pancreatic area multiplied by gross pancreatic mass. n=3. (C) Analysis of acinar cells at e18.5
indicates a significant loss of acinar mass in transgenic pancreata (black bar). Acinar cell mass was calculated as described above for b-cell mass. n=3.
(D) Bar diagram depicting similar ratios between b (insulin+) and acinar (amylase+) -cell areas in transgenic samples (black bar) and control embryos
(gray bar) at e18.5. Amylase+and Insulin+areas were calculated for each embryo (as described in the Material and Methods), and the Insulin+area was
divided by the Amylase+area to obtain the relative ratio between the two components. For clarity, the ratios were normalized to those obtained from
non-transgenic controls, which were set to ‘‘1.’’ n=3. (E) Pancreatic weight of e15.5 embryos injected with DT at e13.5. Transgenic embryos (black
bar) show reduced pancreatic weight in comparison to non-transgenic control littermates (gray bar, set to 100%). n=4. (F–H) The number of
Neurogenin 3 (Ngn3)-expressing cells is reduced in e15.5 transgenic pancreata. (F,G) Pancreatic tissues from DT injected non-transgenic (F) and
Nkx3.2-Cre;DTR (G) embryos were stained for Ngn3 (green) and Sox9 (red), revealing normal expression pattern in the transgenic tissue at e15.5. (H)
Ngn3-expressing cells were counted and their numbers were normalized to that found in non-transgenic pancreata. Number of Ngn3+cells in Nkx3.2-
Cre;DTR pancreata (black bar) was reduced by 50% when compared to non-transgenic littermates (non-tg, gray bar; total number of Ngn3+cells was
set to ‘‘1’’). n=3. (I–K) Reduced number of Ptf1a+cells in transgenic pancreata. DT-treated non-transgenic (I) and transgenic (J) pancreata were
stained for Ptf1a (green) and Cpa1 (red). (K) The number of Ptf1a+cells in transgenic embryos (black bar) was reduced significantly by 40% when
normalized to controls (non-tg, gray bar; total number of Ptf1a+cells was set to ‘‘1’’). n=3. (L–N) Measurement of Cpa1+cell proliferation
demonstrates reduced rates in transgenic pancreata. e14.5 pancreatic tissues were stained against Cpa1 (green) and phosphorylated Histone H3
(pHH3, red) (L,M), and the percentage of Cpa1+pHH3+cell as part of the Cpa1+cell population was counted (N). Cpa1+cells in transgenic pancreata
(black bar) showed decreased proliferation as compared to non-transgenic control (non-tg, gray bar). n=3. (O–Q) Reduced proliferation rate of Sox9+
precursor cells in transgenic embryos. e14.5 pancreatic tissues were stained against Sox9 (red) and Ki67 (green) (O,P). The percentage of proliferating
Sox9+Ki67+cells as part of the Sox9+cell population (black bar) was reduced when compared to non-transgenic controls (non-tg, gray bar) (Q). n=3.
p value: *p,0.05, **p,0.01, ***p,0.005.
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Cre;DTR embryos that were DT-treated 2 d before (i.e., at e13.5).
While present in DT-treated transgenic pancreata (Figure 5F,G),
the number of cells positive for Neurogenin 3 (Ngn3), a
transcription factor that marks endocrine precursor cells ,
was significantly reduced compared to littermate control mice
(Figure 5H). Similarly, the number of cells expressing Ptf1a, a
transcription factor found in exocrine precursor and differentiated
acinar cells , was significantly reduced 2-fold in DT
e13.5Re15.5 Nkx3.2-Cre;DTR pancreata as compared to non-
transgenic controls (Figure 5I–K). Therefore, the reduction in b-
cell and acinar cell mass detected at DT e13.5Re18.5 Nkx3.2-
Cre;DTR embryos (Figure 5B,C) is, at least in part, due to the
decreased number of Ngn3+precursor cells and Ptf1a+exocrine
cells at earlier developmental stages.
Since pancreatic growth between e13.5 and e15.5 relies heavily
on proliferation of precursor cells , we next analyzed the effect
of mesenchymal depletion on the proliferation rate of these cell
populations in e14.5 Nkx3.2-Cre;DTR embryos treated at e13.5
(DT e13.5Re14.5). Epithelial tip cells serve as multi-potent
progenitors before they become committed to the exocrine lineage
around e14.5 . Staining these cells, identified as Carboxypep-
tidase 1 (Cpa1) expressing cells, with an antibody against
phosphorylated Histone H3, a marker of cell proliferation,
revealed a 50% reduction in proliferating tip cells in Nkx3.2-
Cre;DTR embryos compared to controls (Figure 5L–N). In
addition, we analyzed proliferation of Sox9 expressing cells, a
transcription factor that marks epithelial precursor cells giving rise
to exocrine cells as well as to Ngn3+endocrine precursors [46–48].
The percentage of Sox9+proliferating cells was slightly but
significantly smaller in transgenic embryos (Figure 5O–Q). We
could not detect apoptotic epithelial cells by TUNEL (terminal
deoxynucleotidyl transferase dUTP biotin nick end labeling) assays
(unpublished data), concluding that depletion of mesenchymal
cells affects both endocrine and exocrine mass through reduced
proliferative capacity of epithelial progenitor cells rather than their
Mesenchymal Cells Are Required for Proliferation of
Differentiated Epithelial Cells
The reduced proliferative potential of progenitor cells at e14.5
explained, at least in part, the reduction in pancreas mass in
embryos treated with DT at e13.5. However, when mesenchyme
was eliminated at e16.5 we also observed a significant reduction of
about 35% in pancreas mass at e18.5, affecting both the endocrine
and exocrinecompartments (DT e16.5Re18.5;Figures 3H,6A–D).
By e16.5, the various pancreatic cell types are committed towards
their final differentiation fate and present with many of their mature
cell characteristics. Since pancreatic growth at those late stages of
developmentis attributed toproliferationofthesedifferentiated cells
, the decrease in pancreatic mass could not be due to reduced
proliferation of progenitor cells. While previous studies did not
detect effects of mesenchymal cells on b-cell proliferation in culture
, invivo analysis ofNkx3.2-Cre;DTR embryos treated withDTat
e16.5 and analyzed at e17.5 revealed decreased proliferative
potential of both insulin and amylase expressing cells (Figure 6E–
J). In agreement with what we had found at earlier stages, the ratio
between Insulin+/Amylase+areas was not affected in the DT-
treated embryos (Figure 6D), suggesting mesenchymal factors have
similar effect on cells of these two compartments.
Figure 6. Mesenchymal depletion toward the end of gestation
impairs acinar and b-cell proliferation. Nkx3.2-Cre;DTR and non-
transgenic littermates were injected with DT in utero at e16.5 and
analyzed at indicated embryonic days. (A) A scheme illustrating
embryonic development from e9.5 to e18.5. Green arrow marks the
time from DT injection (e16.5) to analysis endpoint (e18.5). (B) Analysis
for b-cell mass (as described for Figure 5) at e18.5 shows significant
reduction in transgenic pancreata (black bar) compared to controls
(non-tg, gray bar). n=3. (C) Bar diagrams show reduced acinar cell (as
described for Figure 5) mass at e18.5 in transgenic pancreata (black bar)
compared to non-transgenic littermates (non-tg, gray bar). n=3. (D) b-
to acinar-cell ratio is similar in transgenic and non-transgenic controls.
Bar diagram presents the normalized ratio between b- and acinar-cell
area at e18.5, as described for Figure 5. n=3. (E–G) Reduced b-cell
proliferation in transgenic pancreata (F,G, black bars) as compared to
controls (E,G, gray bars). E17.5 pancreatic tissues were stained against
Insulin (green) and Ki67 (red), and the percentage of double positive
cell within the Insulin+cell population was counted. n=3. (H–J)
Reduced Acinar cell proliferation in transgenic embryos (I,J, black bars)
compared to control embryos (H,J, gray bars). (H,I) e17.5 pancreatic
tissues stained against Amylase (green) and Ki67 (red). The percentage
of Amylase+Ki67+cell as part of the Amylase+cell population was
counted (J). n=3. p value: *p,0.05, **p,0.01, ***p,0.005.
Mesenchyme Is Essential for Pancreas Formation
PLoS Biology | www.plosbiology.org8 September 2011 | Volume 9 | Issue 9 | e1001143
Mesenchymal Wnt Signaling Is Required for Epithelial
Growth by Maintaining the Mesenchymal Layer
Upon determining the requirement for mesenchymal cells to
guide epithelial organ formation throughout development, we set
out to identify signals and pathways critical for the mesenchymal
effects. Canonical Wnt signaling is active in the developing
pancreas, and both the mesenchyme and the epithelium express
various Wnt ligands and receptors in a dynamic fashion . At
e11.5, Wnt signaling is observed in epithelial cells, and its level of
activation declines in the following embryonic days , while its
activity in the mesenchymal layer has been first reported around
In order to directly investigate the role of mesenchymal Wnt
signaling in pancreas development, we decided to block this
pathway specifically in the mesenchyme by crossing transgenic
mice carrying floxed alleles of b-catenin (bcatf/f), an essential
mediator of canonical Wnt signaling, with Nkx3.2-Cre mice. In
addition to its critical role in Wnt signaling, b-catenin has other
functions within cells, most notably in maintaining cell-cell
interactions as part of a complex with E-Cadherin. However, in
pancreatic mesenchymal cells we failed to observe membrane-
associated localization of the b-catenin protein (Figure S5A).
Therefore, elimination of this gene in Nkx3.2-Cre;b-catf/fpancre-
ata is unlikely to perturb cell-cell interactions but should reveal the
requirement for b-catenin mediated Wnt signaling in mesen-
As expected, elimination of b-catenin did not affect epithelial
size at e12.5 (Figure S5B) prior to the reported onset of
mesenchymal Wnt signaling. In contrast, Nkx3.2-Cre;b-catf/f
pancreata were markedly reduced in size at e15.5 and e18.5
(Figure 7A,B), indicating that mesenchymal b-catenin signaling is
critical for organ formation at later stages. In addition, Nkx3.2-
Cre;bcatf/fpancreata exhibited aberrant morphology with dimin-
ished branching when compared to controls (Figure 7A,C,D).
In order to identify the potential effects on pancreatic epithelial
development in Nkx3.2-Cre;bcatf/fembryos, we stained e18.5
knock-out pancreata for various cell markers and assessed acinar-
and b-cell mass. All major pancreatic cell types, both of the
exocrine (acinar and duct cells, Figure 7E,F) and of the endocrine
compartments (a-, b-, and d-cells, Figure 7G,H), were detected in
the Nkx3.2-Cre;bcatf/fe18.5 pancreata. However, both b-cell and
acinar-cell mass was significantly reduced in knock-out embryos
(Figure 7I,J). Interestingly, the ratio between b- and acinar cells
was maintained in Nkx3.2-Cre;bcatf/fpancreata (Figure 7K).
The Wnt signaling pathway was shown to becomeactivated inthe
pancreatic mesenchyme around e13.5 [15,52]. To address whether
the reduction in pancreatic mass observed in Nkx3.2-Cre;bcatf/fat
e15.5 and e18.5 is due to effects on epithelial growth at earlier stages,
we studied epithelial proliferation in these mice at e13.5. At this
stage, proliferating Cpa1+tips cells serve as multipotent pancreatic
shown in Figure 7L, the proliferation rate of Cpa1+cells was
significantly lower in Nkx3.2-Cre;bcatf/fembryos as compared to
controls. Cell death was not apparent as we could not detect
apoptotic epithelial cells by TUNEL assays or by staining for cleaved
Caspase3 (unpublished data). Therefore, blocking mesenchymal
Wnt signaling leads to reduced pancreatic mass by affecting the
proliferation capacity of epithelial precursor cells.
Wnt signaling is known to regulate cell survival and proliferation
. Since pancreata from Nkx3.2-Cre;bcatf/fmice phenocopied
those from DT-treated Nkx3.2-Cre;DTR mice, we wondered
whether Wnt signaling is required for mesenchymal cell survival.
Indeed, while e13.5 pancreatic tissue from wild type embryos
contained both E-Cadherin-positive epithelial cells and E-Cad-
herin-negative mesenchymal cells (Figure 7M), we could detect only
indicating ablation of the pancreatic mesenchymal layer in
transgenic mice. Thus, our results point to mesenchymal Wnt
signaling as a critical mediator of mesenchymal cell survival in vivo
and therefore of epithelial growth and patterning.
Despite extensive efforts, the role of the mesenchyme during in
vivo pancreas development has remained elusive. Due to the
absence of suitable genetic tools, probing the role of the pancreatic
mesenchyme during organogenesis has been mainly restricted to
organ rudiment culture experiments. In addition to the inability to
faithfully mimic the in vivo conditions, clean separation of
mesenchyme and epithelium for culture experiments is limited to
early stages of pancreas organogenesis before the epithelial layer
has integrated into the overlying mesenchyme (prior to e12.5 in
the mouse). Here, we show that Nkx3.2-Cre mice permit transgene
manipulation specifically in mesenchyme, but not in other
pancreatic tissues. By using this tool to eliminate pancreatic
mesenchymal cells at will, we expand upon classical tissue culture
studies and for the first time present a model system suitable for
detailed analysis of the various functions of this supporting tissue in
vivo at multiple gestational ages.
A key finding of our studies is the observation that pancreatic
mesenchyme provides critical functions for the proper develop-
ment of the epithelial compartment throughout organogenesis. As
expected from previous studies [3,14], depletion of mesenchymal
cells at the onset of pancreas development using Nkx3.2-Cre;DTA
mice arrests pancreas organogenesis. Mesenchymal ablation at
later stages, by injecting DT into Nkx3.2-Cre;DTR transgenic
embryos at various stages or by blocking mesenchymal Wnt
signaling in Nkx3.2-Cre;bcatf/fmice, impairs pancreatic epithelium
growth and branching. Notably, while the morphological changes
observed are profound, we did not observe alterations in cell
differentiation capacity of the three main pancreatic cell types, the
endocrine, acinar, and duct cells. However, our results clearly
demonstrate a requirement of the mesenchyme for the expansion
of epithelial progenitor cells, as well as proliferation of differen-
tiated pancreatic cells.
Requirement of Secreted Mesenchymal Factors During
Factors secreted by the pancreas mesenchyme have previously
been shown to regulate pancreas organogenesis , including
Fgf10 whose function is required for the expansion of common
epithelial progenitor cells during early stages of pancreas
development . The pancreatic defects we observe in Nkx3.2-
Cre;DTA embryos are more severe than those previously reported
for Fgf102/2pancreata , a finding likely explained by the
absence of mesenchymal cells, and thus reduction of all
mesenchymal factors, in transgenic mice.
Our results further demonstrate a requirement for mesenchymal
cells in promoting proliferation of various epithelial cell types,
including precursors and differentiated cells. While it is theoret-
ically possible that these functions are mediated by a limited
number of factors throughout all stages of development, the
dynamic activation of mesenchymal signaling pathways (summa-
rized in ) would suggest a more complex interplay of a diverse
set of molecules that changes over time. Our findings also suggest
that mesenchyme supports proliferation of multiple distinct cell
types, even during the same developmental stage. For instance,
mesenchyme ablation has similar effects on proliferation of mature
Mesenchyme Is Essential for Pancreas Formation
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acinar and b-cells towards the end of gestation. This observation
poses the question as to whether different epithelial cell types rely
on the same mesenchymal factor(s) for their proliferation, or
whether these processes are mediated by distinct signals. Future
analysis is required to identify secreted factors expressed by the
pancreatic mesenchyme at different developmental stages. The use
of the Nkx3.2-Cre line will allow specific manipulation of the genes
coding for these signals to ascertain their role during pancreas
Mesenchyme Governs Endocrine and Exocrine
Development and Growth
Another important finding concerns the observation that the
pancreatic mesenchyme is required for both endocrine and
exocrine development in vivo. Previous reports had reached
differing conclusions, with some demonstrating a positive role for
the mesenchyme on exocrine formation but not endocrine cell
development [6,7,9], and others indicating that mesenchymal
factors promote proliferation of multi-potent pancreas progenitors
that subsequently increase the formation of endocrine cells .
Some of these conflicting results can be explained by the different
culture conditions used in each experiment.
In contrast to the cultured studies, in vivo depletion of the
mesenchyme investigated here revealed similar requirements for this
tissue with regard to the endocrine and exocrine cytodifferentiation. At
this point, we cannot exclude that other cells types, including
endothelial and neural-crest derived cells [38–41], or cells residing in
the adjacent liver, stomach, gut, or kidneys might provide signals that
vivo. In addition, mesenchymal cells that did not originate from
Nkx3.2-Cre expressing cells might still be present in our in vivo model
and could provide either instructive or permissive signals.
Figure 7. Elimination of mesenchymal Wnt signaling impairs pancreas formation. Pancreatic tissues from Nkx3.2-Cre;bcatf/fand non-
transgenic (non-tg) littermates were analyzed at indicated time points. (A) Gross morphology shows smaller transgenic pancreas (right) with aberrant
branching morphology. (B) Reduced pancreatic mass in e15.5 and e18.5 mutant embryos (black bars) as compared to non-transgenic littermates
(non-tg, gray bars). For clarity, pancreatic mass in control mice was set to 100%. n=5. (C,D) Histological analysis of pancreatic sections stained with
H&E reveals abnormal tissue morphology in e18.5 mutant embryos (D) when compared to control (C). (E,F) Amylase+(green) acinar cells and Mucin1+
(red) duct cells can be found in mutant pancreata at e18.5 (F). (G,H) Endocrine cells are present and express mature markers in e18.5 mutant
pancreata (H), similar to control (G). Tissues were stained with antibodies against Insulin (green), Glucagon (red), and Somatostatin (blue). (I) Reduced
b-cell mass (quantification described in Figure 5) in e18.5 mutant pancreata (black bar) as compared to control (gray bar). n=3. (J) Reduced Acinar cell
mass (quantification described in Figure 5) in mutant pancreata at e18.5 (black bar) as compared to non-transgenic littermates (gray bar). n=3. (K)
The ratio between b- and acinar cell areas (as described in Figure 5) at e18.5 is maintained in mutant pancreata (black bar) when compared to non-
transgenic littermates (gray bar, set to ‘‘1’’). n=3. (L) Decreased proliferation of Cpa1+precursor cells in mutant pancreata at e13.5. Bar diagram shows
the percentage of pHH3+Cpa1+cells out of the whole Cpa1+population in mutants (black bar) and non-transgenic pancreata (gray bar). n=3. (M,N)
e13.5 pancreatic tissue stained with E-Cadherin (red) and counter-stained with DAPI (blue) shows absence of E-Cadherin2mesenchymal cells in
mutant tissues (N). Inserts represent higher magnification of the areas marked with white frames. p values: *p,0.05, **p,0.01, ***p,0.005.
Mesenchyme Is Essential for Pancreas Formation
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Prior organ culture studies proposed another model to explain
the various effects of the mesenchyme on the epithelial
compartments by demonstrating distinct effects of the mesen-
chyme on epithelial cells depending on the physical distance and
contact between these tissues . In these experiments, close
proximity between epithelial and mesenchymal cells promoted
exocrine differentiation while at the same time blocked endocrine
formation. In contrast, mesenchyme factors supported endocrine
differentiation at a distance, indicating that the physical relation
between mesenchymal and epithelial cells is critical for endocrine
versus exocrine differentiation. Our studies support the notion of
mesenchymal signals being important for both endocrine and
exocrine development. However, our lineage tracing experiments
provide evidence of close physical contact between Nkx3.2/LacZ
and Nkx3.2/YFP expressing cells with endocrine cells, indicating
that close proximity between mesenchymal and epithelial cells
does not necessarily interfere with endocrine differentiation.
However, since mesenchymal cells surround islets, they are likely
in close contact only with peripheral endocrine cells, such as a-
cells, while direct interactions with centrally located b-cells might
not be common. Whether the mesenchyme contributes to b-cell
expansion by releasing secreted factors or through cell-cell
interactions as well as how the mesenchyme affects other
endocrine cells are questions that need to be addressed in future
experiments. Furthermore, isolation and characterization of
mesenchymal cells throughout development might reveal cell
heterogeneity that could explain differential functions with regard
to promoting endocrine versus exocrine development.
Requirement of Mesenchymal Signaling Pathways in
Our results also point to sustained mesenchyme function as a
critical regulator of epithelial pancreas development and identify
Wnt signaling as an essential mediator of mesenchyme survival. It
is not clear as to whether Wnt signaling is activated in an autocrine
or paracrine manner, as several Wnt ligands are expressed by both
pancreatic epithelial and mesenchymal cells during development
. It is noteworthy that the defects we observe in Nkx3.2-
Cre;bcatf/fonly occur after the onset of canonical Wnt signaling in
pancreas mesenchyme as measured by expression of transgenic
Wnt-reporters (i.e., e13.5 [15,52]). The implication of canonical
Wnt signaling as the cause for the observed phenotypes is
indirectly supported by a previous study using germ-line knock-out
mice in which mPygo2, a critical component of the nuclear b–
catenin/Tcf complex required for b-catenin transcriptional
activity, has been eliminated . mPygo2/2mice show pancreas
hypoplasia and a reduction in endocrine mass , phenotypes
that are not observed when this gene is specifically eliminated in
pancreas epithelium. Thus, while mesenchyme specific depletion
of mPygo2 has not been reported, the absence of pancreas
hypoplasia upon epithelial-specific mPygo2 elimination suggests
that at least some of the pancreatic defects are caused by reduced
mesenchymal Wnt signaling. However, and in contrast to Nkx3.2-
Cre;bcatf/fpancreata, the exocrine compartment is not affected in
mPygo22/2mutants and mesenchyme depletion was not reported
in those mice. Since Wnt signaling is significantly reduced, but not
completely blocked in the absence of mPygo2 , it is possible that
low level of canonical Wnt signaling is sufficient for mesenchymal
cell survival and the production of factors that promote exocrine
cell development. Alternatively, b-catenin is known to regulate
cell-cell interactions as part of Cadherin complexes and these
additional functions might be crucial for the maintenance of the
pancreatic mesenchyme. However, we did not observe b-catenin
localized to membranes in mesenchymal cells. In order to study
whether different levels of mesenchymal Wnt signaling have a
different effect on endocrine and exocrine expansion, mice
specifically lacking mesenchymal expression of various compo-
nents of this pathway (such as mPygo2) would need to be
In addition to Wnt signaling, other signaling pathways, such as
the RA, BMP, and Hedgehog, have been implicated as
mesenchymal factors regulating pancreas development [15–
17,54,55]. Using Nkx3.2-Cre line as a novel tool to manipulate
gene expression in the pancreatic mesenchyme will allow direct
study of the role of these and potentially other pathways in
In summary, data presented here indicate continuous require-
ment of mesenchymal cells and/or mesenchyme-derived signals to
regulate epithelial pancreas formation from the onset of organ
morphogenesis until the end of gestation. Isolation of mesenchy-
mal cells at different stages of pancreas formation might allow
identification of candidate factors that regulate expansion of
common and endocrine progenitors as well as of differentiated b-
cells. Future therapies for both type I and II diabetes rely on
renewable sources of functional insulin-producing b-cells .
Current protocols allow the formation of pancreas progenitor cells
from human embryonic stem cells (hESC) in vitro, but not fully
differentiated b-cells. Our results demonstrate that mesenchymal
factors provide critical signals for the expansion of both precursors
and differentiated endocrine and exocrine cells. Thus, mesenchy-
mal signaling factors not yet identified will likely be useful for
expansion of hESC derived pancreas progenitor and differentiated
Material and Methods
Mice used in this study were maintained according to protocols
approved by the Committee on Animal Research at the University
of California, San Francisco. Nkx3.2 (Bapx1)-Cre mice were
described previously . R26-YFPflox(Gt(ROSA)26Sortm1(EYFP)Cos),
SA)26Sortm1(DTA)Jpmb), DTR (iDTR, Gt(ROSA)26Sortm1(HBEGF)Awai),
and b-cateninflox(Ctnnb1tm2Kem) mice were obtained from Jackson
Laboratories. Noon on the day a vaginal plug was detected was
considered as embryonic day 0.5.
Diphtheria Toxin (DT) Injections
Injections were preformed as previously described [35,36].
Briefly, pregnant females were anesthetized, a laparotomy was
performed, and the uterus was delivered through the incision (as
illustrated in Figure S2A–D). Each embryo was micro-injected
with 8 ng/gr body weight Diphtheria Toxin (Sigma) diluted in
5 ml PBS. The uterus was placed back into the abdominal cavity
and the laparotomy was closed. Embryos were allowed to develop
in situ until indicated stages.
For immunofluorescence, dissected embryos and pancreatic
tissues were fixed with Z-fix (Anatech) for 2–16 h, embedded in
paraffin wax, and sectioned. For Ptf1a staining, tissues were fixed
with Z-fix for 2 h, embedded in OCT (Tissue Tek), and
cryosectioned. Tissue sections were stained using the following
primary antibodies: rabbit anti-Amylase (1:200, Sigma), goat anti-
Cpa1 (1:200, R&D), mouse anti-E-Cadherin (1:200, BD), rabbit
anti-Glucagon (1:200, Linco), guinea pig anti-Insulin (1:200,
Linco), mouse anti-Ki67 (1:200, BD), rabbit anti-MafA (1:200,
Bethyl), armenian hamster anti-Mucin1 (1:200, Neomarker),
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guinea pig anti-Neurogenin 3 (1:400, Millipore), rabbit anti-
phosphorylated Histone H3 (1:200, Millipore), rabbit anti-Pdx1
(1:200, Millipore), rabbit anti-Ptf1a (1:600, a gift from Dr. Helena
Edlund), rat anti-Somatostatin (1:200, Chemicon), rabbit anti-
Sox9 (1:200, Chemicon), and chicken anti-YFP/GFP (1:400,
Abcam) followed by staining with Alexa Fluor tagged secondary
antibodies (1:500, Invitrogen) and mounting with DAPI-contain-
ing Vectashield media (Vector). For TUNEL analysis, ApopTag
Plus Fluorescein In Situ Apoptosis Detection kit (Millipore) was
used according to the manufacturer’s protocol.
For embryo wholemount staining, tissues were processed as
previously described  and stained with rat anti-E-Cadherin
(1:1,000, CalBiochem), followed by staining with Alexa Fluor 555
anti-rat secondary antibody (1:500, Invitrogen).
For x-gal staining, tissues were fixed with 2% PFA and 0.25%
Glutaraldehyde for 2 h and incubated overnight with 0.5 mg/ml
x-gal solution (Roche), followed by a second round of fixation in
4% PFA overnight. Tissues were then embedded in paraffin,
sectioned, and counter-stained with nuclear Fast Red (Vector).
For histological analysis, dissected tissues were fixed with Z-fix
(Anatech), for 4 h, and embedded in paraffin wax. Tissue sections
were stained with Meyer’s Hematoxylin (Sigma) followed by
staining with Eosin (Protocol).
Images were acquired using Zeiss ApoTome, Leica MZ FL3
and SP5, and Olympus IX70 microscopes.
For all quantifications presented in this study, each transgenic
tissue was processed and stained in parallel with a littermate
control, with each analyzed group comprising at least three pairs
of transgenic and control embryos (i.e., n$3) as indicated in the
figure legends. Throughout each analysis, images were acquired
using the same exposure time and magnification. When
MetaMorph software was used for image analysis, the same
signal-to-noise threshold was applied throughout the experiment.
For all measurements presented in this study, with the exception of
the measurement of the mesenchymal area at e11.5 and Ptf1a+cell
numbers at e15.5, the following regimen was applied: the entire
pancreatic tissue, including both dorsal and ventral buds, was
embedded in paraffin wax and cut into 5 mm thick sections. Every
fifth section (20% of total tissue) was then immuno-stained with
indicated antibodies as described above. Images were acquired as
detailed below and analyzed blindly.
For measurement of mesenchymal areas at e15.5 and e18.5,
isolated pancreatic tissues from Nkx3.2-Cre;R26-YFPfloxembryos
were stained with an anti-YFP antibody and a fluorescent
secondary antibody and entire sections were automatically imaged
using Olympus IX70 widefield microscope and MetaMorph
software. Over-exposure of the tissue and DAPI staining were
used to determine the edges of the section. Images were analyzed
using MetaMorph software, which automatically measured the
positive area in each channel. To determine the percentage of
mesenchymal area, total YFP-positive area was divided by total
tissue area of each section.
For b- and acinar cell mass, isolated e18.5 tissues (including
both dorsal and ventral tissues) were dissected and weighed.
Following fixation, tissues were embedded in paraffin wax,
sectioned as described above, and immuno-stained with anti-
Insulin and anti-Amylase antibodies. Images were acquired as
described above for mesenchymal area measurement, and areas
positive for either Amylase or Insulin, as well as the total
pancreatic area, were automatically measured using MetaMorph
software. To determine the fractions of the b- and acinar cell
areas, total Insulin or Amylase positive area was divided by total
tissue area. Cell mass was calculated as the fraction of Amylase+or
Insulin+areas of the total pancreatic area multiplied by gross
To calculate the b-cell/acinar cell ratio, for each embryo
Insulin+and Amylase+area was determined as described above for
cell mass measurement, and Insulin+area was divided by
Amylase+area. For clarity, the ratio obtained in non-transgenic
controls was set to ‘‘1.’’
For quantification of Ngn3-expressing cells, whole e15.5
pancreatic tissues were isolated and processed as described above.
Sections were stained with anti-Ngn3 antibody followed by
fluorescent secondary antibody and images were then acquired
as described above for mesenchymal area measurement, but
positive cells were counted manually. To accommodate for
potential differences in the developmental stage of the various
litters analyzed, the number of transgenic Ngn3-positive cells was
normalized to the number of Ngn3 cells counted in the
corresponding non-transgenic littermate controls.
For cell proliferation, whole pancreatic tissue (including both
dorsal and ventral tissues) was isolated from embryos e15.5 and
older. From embryos at e13.5 or e14.5, pancreatic tissue was
isolated together with the adjacent stomach and duodenum.
Following fixation, tissues were paraffin-embedded and sectioned
as described above. Tissue sections were stained with indicated
antibodies and imaged using Zeiss ApoTome or Leica SP5
microscopes. For each section, the percentage of proliferating cells
was determined via manual counting of either Ki67 or pHH3
positive cells divided by the number of total target cells.
To determine mesenchymal area at e11.5, Nkx3.2-Cre;R26-
LacZf/+embryos were stained with X-gal as described above.
Entire embryos were then cut to obtain 5 mm thick sections, and
all sections were counterstained with FastRed dye and imaged
using Zeiss ApoTome. The total dorsal pancreatic bud area,
identified by its typical localization and morphology, and
pancreatic mesenchyme area, identified by blue x-gal staining,
were manually selected and measured using MetaMorph software.
For Ptf1a+cell quantification, isolated e15.5 pancreatic tissue
were fixed, embedded in OCT, frozen, and cryosectioned. 10 mm
thick sections were used and every 10th section was stained (10%
of total tissue). Whole sections were imaged using Leica SP5
confocal microscope and the number of positive cells was counted
manually. To account for potential differences in developmental
stage of each litter, the number of positive cells obtained for each
transgenic animal was normalized to the number obtained from
the non-transgenic littermate control.
P values were determined using unpaired, two-tailed student t
test. Error bars in bar diagrams represent standard deviation of the
and endothelial cells. Analysis of p0 pancreatic tissues of Nkx3.2-
Cre;R26-YFPf/+shows that YFP expressing cells do not express the
neuronal marker Tuj1 or the endothelial marker PECAM1. (A)
Immunofluorescence analysis for YFP (Green), PECAM1 (Red),
and DAPI (Blue). (B) Flow cytometry analysis showing staining for
PECAM1 of YFP-expressing (green histogram) and non-express-
ing cells (black line). For clarity, acinar (negative for YFP) and
dead cells were excluded from the analysis based on size and DAPI
staining, respectively. (C) Tissues were stained with antibodies
Nkx3.2-Cre is not expressed by pancreatic neurons
Mesenchyme Is Essential for Pancreas Formation
PLoS Biology | www.plosbiology.org 12September 2011 | Volume 9 | Issue 9 | e1001143
against YFP (green) and Tuj1 (Red) and were counterstained with
Nkx3.2-Cre;DTR embryos leads to death of pancreatic mesenchy-
mal cell. (A–D) Graphic illustration of the injection procedure. A
laparotomy was made (A) and the uterus, containing embryos, was
delivered through the incision (B). Each embryo was injected with
5 ml of a solution containing varying concentrations of DT
designed to result in a final concentration of 8 ng DT/gr embryo
weight into the visible liver area (C). The uterus and embryos were
placed back into the abdomen (D) and the incision was closed.
Adapted from . (E,F) Apoptotic pancreatic mesenchymal cell
can be detected 4 h after DT injection to transgenic embryos.
Nkx3.2-Cre;DTR embryos (F) and non-transgenic controls (E)
injected with DT at e13.5 and analyzed 4 h after injection.
Immunofluorescence staining for cleaved Caspase 3 (Green) as a
marker for activation of the apoptotic machinery, for the epithelial
marker E-Cadherin (Red) and for DAPI (blue) was performed.
(G,H) Elimination of E-Cadherin-negative mesenchymal cells a
day after DT injection. Nkx3.2-Cre;DTR (H) and non-transgenic
embryos (G) were injected with DT at e13.5 and analyzed 24 h
later for E-Cadherin (red) and DAPI (blue). (I,J) Elimination of
DTR-expressing cells a day after DT injection. Nkx3.2-Cre;DTR
were either injected with DT at e13.5 (J) or were left untreated (I).
Tissues were harvested at e14.5 (24 h after DT injection) and
stained for DTR (human hbEGF, red) and Pdx1 (green).
In utero i.p. injection of Diphtheria Toxin (DT) to
mesenchymal ablation. Images of e18.5 stomach, pancreas, spleen,
and gut of Nkx3.2-Cre;DTR embryos and non-transgenic controls
injected with DT at e13.5.
Gastrointestinal tract development is affected by
mesenchyme-depleted pancreata. Nkx3.2-Cre;DTR and non-
transgenic embryos were injected with DT at e13.5 and analyzed
at e18.5. (A,B) Staining for the endothelial marker PECAM1 (red)
indicates presence of blood vessels in transgenic pancreata (B).
(C,D) Whole mount staining against the endothelial marker
Neurons and endothelial cells are present in
PECAM1 (brown) was performed. Images reveal dense vascula-
ture in DT-treated transgenic pancreata (D) and control (C).
(C9,D9) A higher magnification of the areas marked with a white
box in (C) and (D), respectively. (E,F) Staining for the neuronal
marker Tuj1 (green) indicates presence of neurons in transgenic
pancreata (F), similar to non-transgenic control (F).
pancreatic mesenchymal cells, and its elimination in the
mesenchyme does not affect epithelial growth prior to the onset
of mesenchymal Wnt signaling. (A) b-catenin is localized to the
membrane of Pdx1+epithelial cells but not to the membrane of
Nkx3.2/YFP+mesenchymal cells. Nkx3.2-Cre;R26-YFPf/+e14.5
pancreatic tissue was stained for YFP (green), b-catenin (red), and
Pdx1 (blue). Left panel shows all three markers, while middle and
right panels show only indicated markers. (B) Normal epithelial
size in Nkx3.2-Cre;bcatf/fat e12.5. Embryos were stained with
H&E and epithelial area was measured and compared to non-
transgenic littermates (which was set to ‘‘1’’). Epithelial area in
mutant embryos (black bar) was comparable to that of non-
transgenic animals (non-tg, gray bar, set to ‘‘1’’). n=3.
b-catenin is not localized to the membrane of
reagents used to generate the data presented in the supporting
figures are described in detail.
Supporting materials and methods. The procedures and
We would like to thank John P. Morris IV and Drs. Michael German,
Grace Wei, Sapna Puri, Alethia Villasenor, and Christophe Pierreux for
helpful discussion and Dr. Helena Edlund for sharing a critical reagent.
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: LL MH.
Performed the experiments: LL AN RLV TJW. Analyzed the data: LL
TJW. Contributed reagents/materials/analysis tools: WEZ TCM. Wrote
the paper: LL MH.
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