© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 2939-2949 doi:10.1242/dev.104810
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Insm1 promotes endocrine cell differentiation by modulating the
expression of a network of genes that includes Neurog3 and
Ripply3
ABSTRACT
Insulinoma associated 1 (Insm1) plays an important role in regulating
the development of cells in the central and peripheral nervous
systems, olfactory epithelium and endocrine pancreas. To better
define the role of Insm1 in pancreatic endocrine cell development we
generated mice with an Insm1GFPCre reporter allele and used them to
study Insm1-expressing and null populations. Endocrine progenitor
cells lacking Insm1 were less differentiated and exhibited broad
defects in hormone production, cell proliferation and cell migration.
Embryos lacking Insm1 contained greater amounts of a non-coding
Neurog3 mRNA splice variant and had fewer Neurog3/Insm1
co-expressing progenitor cells, suggesting that Insm1 positively
regulates Neurog3. Moreover, endocrine progenitor cells that
express either high or low levels of Pdx1, and thus may be biased
towards the formation of specific cell lineages, exhibited cell typespecific differences in the genes regulated by Insm1. Analysis of the
function of Ripply3, an Insm1-regulated gene enriched in the Pdx1high cell population, revealed that it negatively regulates the
proliferation of early endocrine cells. Taken together, these findings
indicate that in developing pancreatic endocrine cells Insm1 promotes
the transition from a ductal progenitor to a committed endocrine cell
by repressing a progenitor cell program and activating genes essential
for RNA splicing, cell migration, controlled cellular proliferation,
vasculogenesis, extracellular matrix and hormone secretion.
KEY WORDS: Pancreas development, Endocrine progenitor cells,
Gene expression, Transcription factors, Mouse
INTRODUCTION
The genetic program responsible for the generation of pancreatic
endocrine cells from endocrine progenitors remains incompletely
understood (Oliver-Krasinski and Stoffers, 2008; Rieck et al., 2012;
Stanger and Hebrok, 2013). Beginning at around embryonic day (E)
9-9.5 in the mouse, and peaking around E15.5, pancreatic epithelial
1
Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville,
2
TN 37232, USA. Department of Molecular Physiology and Biophysics, Vanderbilt
3
University School of Medicine, Nashville, TN 37232, USA. Department of Animal
4
Science, Cornell University, Ithaca, NY 14850, USA. Penn Center for
Bioinformatics, Department of Genetics, University of Pennsylvania School of
5
Medicine, Philadelphia, PA 19104, USA. Department of Laboratory Animal
Science, Kitasato University School of Medicine, Sagamihara, 252-0374, Japan.
6
Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences,
Okazaki, Aichi, 444-8787, Japan.
*Author for correspondence ([email protected])
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
Received 10 October 2013; Accepted 26 May 2014
cells express Neurog3, which encodes a master regulatory factor that
activates a cascade of secondary transcription factor genes, including
Insm1, Neurod1, Pax4, Arx and Rfx6. Together, these and other
transcription factors orchestrate the formation of the five endocrine
cell types found in adult islets (Huang et al., 2000; Schwitzgebel
et al., 2000; Gu et al., 2002; Collombat et al., 2003; Smith et al.,
2003; Mellitzer et al., 2006; Soyer et al., 2010). Neurog3 triggers preendocrine cells to delaminate from a pancreatic ductal epithelium by
an epithelial-to-mesenchymal transition (EMT), lose basal-apical
cell polarity and epithelial adhesions, then migrate and aggregate to
form islets of Langerhans (Cole et al., 2009; Gouzi et al., 2011).
Moreover, Neurog3 expression coincides both with a markedly
lower rate of cell proliferation and an increase in the expression of
cyclin-dependent kinase inhibitors such as Cdkn1c ( p57Kip2) and
Cdkn1a ( p21Cip1) (Georgia et al., 2006; Miyatsuka et al., 2011).
Multiple transcription factors regulate pancreatic endocrine cell
development, and they have interacting and sometimes opposing
functions. For instance, Arx drives the formation of glucagonproducing α-cells. In its absence, there is a preponderance of insulinproducing β-cells and somatostatin-producing δ-cells. Similarly,
Pax4 opposes the effect of Arx and is essential for the formation of βcells, since mice lacking this factor are characterized by an
expansion in α-cells (Sosa-Pineda et al., 1997; Collombat et al.,
2003). Moreover, nascent β-cells express higher amounts of Pdx1, a
transcription factor crucial for the early specification of pancreatic
epithelium, compared with other pre-endocrine cells (Ohlsson
et al., 1993; Ahlgren et al., 1998; Fujitani et al., 2006; Nishimura
et al., 2006; Gannon et al., 2008). Other transcription factors
important for β-cell specification and development, such as Nkx2.2,
Neurod1, Nkx6.1, Mafb and Mafa, also function in an interrelated
manner (Sosa-Pineda et al., 1997; Sussel et al., 1998; Nishimura
et al., 2006; Nelson et al., 2007; Schaffer et al., 2013).
The expression of Insm1, which encodes a zinc-finger transcription
factor, is activated by the binding of Neurog3 to sites within its
promoter (Mellitzer et al., 2006). Endocrine cell differentiation is
markedly perturbed in mice lacking Insm1 (Gierl et al., 2006). In the
absence of this factor, there is a reduction in the number of insulinexpressing cells, with many cells lacking any hormone expression. In
addition to being expressed in developing endocrine cells throughout
the gut, Insm1 is also expressed in the developing central nervous
system, where it contributes to the formation and expansion of
intermediate (basal) neural progenitors from early apical progenitor
cells (Farkas et al., 2008), in the peripheral neural system and in the
olfactory epithelium, where it is involved in regulating the
differentiation of neurogenic progenitor cells (Wildner et al., 2008;
Rosenbaum et al., 2011).
The acquisition of robust quantitative global gene transcription
datasets, which are necessary for understanding the gene regulatory
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DEVELOPMENT
Anna B. Osipovich1,2, Qiaoming Long3, Elisabetta Manduchi4, Rama Gangula1, Susan B. Hipkens1,
Judsen Schneider1,2, Tadashi Okubo5, Christian J. Stoeckert, Jr4, Shinji Takada6 and Mark A. Magnuson1,2, *
RESEARCH ARTICLE
network that dictates the formation and function of endocrine
cells, requires the combined use of fluorescent reporter alleles,
fluorescence-activated cell sorting (FACS) and next-generation
sequencing technology. To this end, we have derived mice
containing an Insm1GFPCre reporter allele that enabled us to isolate
highly purified populations of Insm1-expressing and -deficient
pre-endocrine cells, and to characterize these cell populations by
RNA-Seq. In addition, we performed multi-channel FACS to isolate
and differentially characterize Insm1-positive cell populations that
express either high or low levels of Pdx1. In doing so, we identified
many genes that are likely to contribute to both the formation and
function of pancreatic endocrine cell types. In addition, the function
of Ripply3 and the alternative RNA processing of Neurog3 mRNA
were examined. Together, these studies provide multiple new
insights into the gene regulatory network controlling pancreatic
endocrine cell formation and function.
RESULTS
Generation of Insm1GFPCre reporter mice
A two-step strategy utilizing both gene targeting and recombinasemediated cassette exchange (RMCE) was used to derive mice that
express a green fluorescent protein-Cre fusion protein (GFPCre)
under control of the endogenous Insm1 gene locus (Fig. 1A;
supplementary material Fig. S1A-F). Insertion of GFPCre
sequences into the Insm1 gene locus disrupted Insm1 protein
expression, as confirmed by western blot analysis of homozygous
null embryos (supplementary material Fig. S1F). Mice heterozygous
for this allele (hereafter termed Insm1+/−) appeared normal, whereas
animals that were homozygous for Insm1GFPCre (hereafter termed
Insm1−/−) died between E15.5 and E18.5 due to defects in
catecholamine biosynthesis and secretion (Wildner et al., 2008).
Green fluorescence was observed in pancreatic pre-endocrine cells
(Fig. 1C) and GFPCre was readily detectable by immunofluorescence
in islet cells after birth [at postnatal day (P) 1, P7 and P60; data not
shown] and in the developing central nervous system between E9.5
and E18.5 (Fig. 1B). GFPCre expression was also detected in the
peripheral nervous system and gut endocrine cells (data not shown).
Development (2014) 141, 2939-2949 doi:10.1242/dev.104810
Co-staining with anti-GFP and anti-Insm1 antibodies at E15.5-18.5 in
Insm1+/− pancreata showed that the majority of Insm1-positive cells
co-expressed both proteins (supplementary material Fig. S2).
Functionality of the Cre portion of the GFPCre fusion was also
confirmed (data not shown).
Insm1 knockout mice have altered pancreatic hormone cell
differentiation, replication, size and migration
To investigate the role of Insm1 in pancreas development we
quantified the percentage of different pancreatic hormone-positive
cells among GFPCre-expressing endocrine cells in Insm1
heterozygous and knockout animals at E18.5 (supplementary
material Fig. S3). Consistent with the results of Gierl et al. (2006),
54% of endocrine cells expressed insulin in heterozygous animals,
whereas only 8% of GFPCre-expressing cells were insulin positive
(a 6-fold decrease) in Insm1−/− embryos. There were also less
pronounced but significant decreases in cells expressing glucagon
(from 24% to 11%), somatostatin (from 11% to 7%) and ghrelin (from
8% to 5%) in the null embryos. Also, the number of pancreatic
polypeptide-positive cells increased from 7% to 12% in the knockout
animals, as is also consistent with the findings of Gierl et al. (2006).
Since it has been suggested that Insm1 inhibits the progression of
endocrine cells through the cell cycle (Zhang et al., 2009), we
quantified the proliferation rates of GFPCre-positive endocrine
cells by immunostaining for Ki67, a marker of cell proliferation
(Fig. 2A). At E18.5 we observed up to a 7-fold decrease (from
11.6% to 1.5%) in the number of Ki67/GFPCre double-positive
cells in heterozygous versus knockout pancreata (Fig. 2B).
Concurrently, the size of the Insm1−/− endocrine progenitor cells
is increased (Fig. 2C,D) and their shape becomes irregular. No
differences in the number of apoptotic cells were observed between
heterozygous and homozygous null animals by TUNEL assay (data
not shown). To assess whether the proliferation defect is visible at
an earlier stage, we also quantified proliferation rates at E15.5
(supplementary material Fig. S4). Although the majority of
endocrine cells are postmitotic at E15.5, we were still able to
detect a slight decrease in proliferation from 1.4% in Insm1+/− cells
to 0.9% in Insm1−/− cells.
In addition to a reduced proliferation rate of Insm1−/− cells, we
also observed that the GFPCre-positive endocrine cells remained
closely associated with pancreatic ductal cells. We quantified the
apparent cell migration defect by calculating the percentage of
GFPCre-expressing cells that were in contact with ductal cells
(DBA-positive cells) and found an increase (from 24% to 55%) in
the knockout animals compared with heterozygous animals
(Fig. 2E,F). This finding suggests that Insm1, in addition to
its other roles, is necessary for the migration of the differentiating
pre-endocrine cell away from pancreatic ductal regions.
Fig. 1. The Insm1GFPCre allele. (A) Schematic of the Insm1GFPCre allele. Insm1
coding sequences were replaced with those encoding GFPCre using
combined gene targeting/recombinase-mediated cassette exchange (RMCE)
as described in supplementary material Fig. S1. The triangles represent
heterotypic loxP sites and the circle a remnant FLP recognition target (FRT)
site. (B) Green fluorescence in a whole mouse embryo at E11.5 broadly marks
the neural system. (C) Green fluorescence in a pancreas at E15.5 marks
pre-endocrine cells. Fluorescence images were overlaid with images taken
with white light.
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To quantitatively assess the gene expression changes brought about by
the absence of Insm1, we performed RNA-Seq using FACS-purified
progenitor cells isolated at E15.5. On average, we obtained 13,456
(±2886) cells per Insm1+/− embryo and 8505 (±1602) cells per
Insm1−/− embryo at E15.5, consistent with a proliferation defect.
RNA-Seq resulted in the detection of 13,865 individual mRNAs with
an RPKM >0.1. Pairwise comparison of the transcriptional profiles
from Insm1+/− and Insm1−/− mice revealed that the expression levels
for the majority of genes were highly correlated, but with a subset of
the genes being differentially expressed (Fig. 3B; supplementary
material Table S1). After counting only those transcripts altered in
DEVELOPMENT
Identification of Insm1-dependent genes in developing
endocrine cells
RESEARCH ARTICLE
Development (2014) 141, 2939-2949 doi:10.1242/dev.104810
Fig. 2. Impaired proliferation, cell size and migration of
pre-endocrine cells in Insm1 knockout mice.
(A) Immunofluorescence labeling of pancreatic tissues from
Insm1+/− and Insm1−/− mice at E18.5 with anti-GFP antibodies
(green) and antibodies against cell proliferation marker Ki67 (red).
Arrows indicate cells co-expressing GFP and Ki67. (B) Percentage
of Ki67-positive cells among GFP-positive cells demonstrates a
proliferation defect in Insm1−/− pancreas. (C) Immunofluorescence
labeling of pancreatic tissues from Insm1+/− and Insm1−/− mice at
E18.5 with anti-GFP antibodies (green) and antibodies against cell
surface marker E-cadherin (red) enabled the measurement of
endocrine cell area. Representative cells for each phenotype are
outlined. (D) Quantification of cell area shows increased cell size in
Insm1−/− endocrine cells. (E) Immunofluorescence labeling of
pancreatic tissues from Insm1+/− and Insm1−/− mice at E18.5 with
anti-GFP antibodies (green) and the ductal marker DBA (red).
(F) Quantification of the percentage of GFP-positive cells in contact
with DBA-positive cells shows a defect in the migration of Insm1−/−
endocrine cells away from the ducts. Error bars indicate s.e.m.
(n≥6); P-values were determined by Student’s t-test. Scale bars:
50 μm.
expression of transcription factors that play important roles in the
maintenance of progenitor cells (Bhlhe22, Rest, Nr5a2, Hes1, Acsl1,
Foxp2, Sox6). Markers of EMT (Zeb2, Mmp14, Cldn11, Cdh2, Vim,
Cdh11, Fn) and components of Notch signaling pathway (Hes1, Hes5,
Hey1, Dll1, Notch2, Notch1) were also increased in the Insm1−/−
progenitor cells. Similarly, we observed an upregulation in genes
involved in other signaling pathways controlling cell progenitor states,
such as TGF/BMP (Smad3, Bmpr1b, Tgfb2, Bmp1), FGF (Fgfr1,
Fgfr4) and IGF (Igfbpl1, Igfbp5). Several genes involved in cell
proliferation (Cdkn1c, Bcl2, Mertk, Anxa2, Ccnd1) were increased in
Insm1−/− cells, whereas others (Cdkn1b, Manf and Akt3) were more
highly expressed in the Insm1+/− cells. Insm1+/− cells also showed
greater expression of genes involved in cytoskeleton reorganization
and cell migration (Gng12, Pak3, Filip1), vascularization-promoting
growth factors (Pgf, Vegfa) and a β-cell-specific MPK8 scaffold
protein (Mapk8ip2).
We performed chromatin immunoprecipitation (ChIP) analysis
of the Insm1-regulated genes Rest, Cdkn1c, Cdkn1b and Ripply3,
all of which contain putative Insm1 binding sites within their
promoters based on the JASPAR database (Mathelier et al., 2014)
(supplementary material Table S2). All genes showed enrichment
that was similar or greater than that of the Insm1 promoter itself,
which was previously shown to be downregulated by Insm1
(Breslin et al., 2002) (supplementary material Fig. S6).
Differential expression of alternatively spliced transcripts of
Neurog3
Since alternative RNA splicing plays an important role in the
differentiation of embryonic stem cells (ESCs) and the
specification of many cell lineages (Fu et al., 2009; Grabowski,
2011; Llorian and Smith, 2011), and our results showed a strong
Insm1-dependent increase in the expression of many RNAbinding splicing factors, we examined the datasets for splice
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DEVELOPMENT
expression by more than 1.5-fold (with confidence values ≥70%), we
identified 228 genes that were increased in expression and another
1115 genes that were decreased in expression in Insm1+/− versus
Insm1−/− samples. Gene ontology analysis revealed that the majority
of the more highly expressed transcripts in the Insm1+/− cells are
involved in secretory cell functions, including vesicular traffic, protein
processing and degradation. By contrast, the transcripts that were more
highly expressed in the knockout cells were grouped into broader
categories, including cytoskeleton remodeling, transcriptional
regulation and components of developmental, differentiation and
morphogenesis pathways (Fig. 3C; supplementary material Fig. S5).
Genes most dependent on Insm1 for expression include those
encoding pancreatic hormones, especially Ins1 and Ins2, as well as
components of the hormone secretory machinery such as Chgb, Scg3,
Snap25, Nnat and Vatl1. Consistent with our immunohistochemical
findings, Ppy was also more highly expressed in the Insm1−/− cells, as
was Cartpt, which encodes a neuropeptide. In the absence of Insm1
there was also a reduction in the expression of several transmembrane
channels and transporters (Kctd12b, Slc2a2, Slc30a8, Cacna1a), gap
junction proteins (Gjd2), a calcium-sensing protein (Hpca), several
extracellular matrix proteins (Cryba2, Muc4, Spock2), and other less
well characterized transmembrane proteins (Tmem215, Lrp11,
Amigo2). Insm1−/− cells showed an increase in the expression of
receptors previously associated with different progenitor cell types
(Tspan8, Prom1, Itgam, Cd59a, Lgr4) and neural progenitor cells
(Nrp2, Pmp22, Fat4, Gfra1, Lamc1). Interestingly, among the nucleic
acid-binding proteins in Insm1+/− cells, there was a marked increase in
the expression of genes encoding RNA-processing proteins such as
Elavl4, Celf3 and Rod1, all of which have been shown to be involved
in alternative RNA splicing (Chapple et al., 2007; Spellman et al.,
2007; Ince-Dunn et al., 2012). Genes for DNA-binding proteins, such
as Fev, St18, Ripply3, Lmo3 and Jazf1, were upregulated to a lesser
extent. Conversely, Insm1−/− cells exhibited a marked increase in the
RESEARCH ARTICLE
Development (2014) 141, 2939-2949 doi:10.1242/dev.104810
junctions for a number of pro-endocrine genes. Interestingly,
Neurog3 mRNA was found to be alternatively spliced in endocrine
progenitors at E15.5, with two splice variants present – a long
coding and a short non-coding, with the shorter variant having
about twice as many sequence tags in Insm1−/− than in Insm1+/−
cells. To validate the presence of alternatively spliced Neurog3
transcripts, we designed primers to simultaneously amplify both
splicing variants or just the long or short variant (Fig. 4A), and
performed semi-quantitative RT-PCR. This experiment, as well as
RT-qPCR, confirmed the expression of both Neurog3 mRNA
isoforms (Fig. 4B,C; supplementary material Fig. S7), with the
ratio of long to short variant expression being 2-fold higher
in Insm1+/− than in Insm1−/− cells. Immunostaining of E15.5
embryos also showed a decrease in the number of GFP/Neurog3
co-expressing cells (19% in Insm1 +/− compared with 12% in
Insm1 −/− tissues; Fig. 4D,E), as might be expected if there was a
reduction in Neurog3 protein levels.
Validation of RNA-Seq results by RT-qPCR and further
characterization of novel genes
To further validate and quantify the differential expression of
selected genes, we performed RT-qPCR using 96-well TaqMan
2942
array cards that were designed to detect genes showing higher
expression in Insm1+/− versus Insm1−/− cells as well as some known
pancreatic endocrine genes as a reference (supplementary material
Table S3). Cells were isolated at E15.5 and E18.5, with the later time
point serving to better detect changes occurring as the endocrine
progenitor cells became more differentiated. This analysis confirmed
the upregulation or downregulation of all these genes at E15.5
(supplementary material Fig. S8A) and allowed us to distinguish
between those required for later stages of endocrine differentiation
and maturation (upregulated in Insm1+/− versus Insm1−/− samples at
E18.5, e.g. Mnx1, Mafa, Ripply3 and Fev) and those active at earlier
stages of endocrine specification (downregulated in Insm1+/− versus
Insm1−/− samples at E18.5, e.g. Neurod1, Runx1t1, Pax6 and Pax4).
The expression of some of these genes in pancreatic endocrine cells
was confirmed by examining in situ RNA hybridization images in
the EUREXPRESS database obtained from E14.5 mouse embryos
(Diez-Roux et al., 2011) (supplementary material Fig. S8B). In
addition, we performed staining with antibodies against surface
proteins, such as cadherin Celsr3, adhesion molecule Amigo2, and
low-density lipoprotein receptor-related protein Lrp11 (supplementary
material Fig. S9), and found preferential staining in each case for
pre-endocrine cells.
DEVELOPMENT
Fig. 3. RNA-Seq analysis of Insm1+/− and Insm1−/− endocrine progenitor cell populations at E15.5. (A) Representative FACS profiles of GFPCre-positive
Insm1+/− and Insm1−/− endocrine cell populations isolated at E15.5 and used for RNA isolation and RNA-Seq. (B) Analysis of RNA-Seq results demonstrates
differential gene expression between Insm1+/− and Insm1−/− cells. Correlation plot of RPKM (reads per kilobase per million mapped reads, log10) values for
identified genes in Insm1+/− and Insm1−/− cell populations shows that although the majority of reads fall along the line representing equal expression, a number of
transcripts are upregulated in Insm1+/− or Insm1−/− cells. (C) Functional groupings of genes upregulated in Insm1+/− and Insm1−/− cell populations. Enrichment
scores for functional gene ontology groups represent the geometric mean (in −log scale) of P-values in the corresponding annotation cluster.
RESEARCH ARTICLE
Development (2014) 141, 2939-2949 doi:10.1242/dev.104810
Temporal expression profiling of endocrine populations
expressing high and low levels of Pdx1
Beginning at ∼E14.5 during pancreas development, Pdx1 is expressed
at higher levels in β-cells than in non-β-cells (Ohlsson et al., 1993).
Thus, to identify Insm1-regulated genes that might be specifically
important for β-cell development and function we used a Pdx1CFP
allele (Potter et al., 2011) to isolate populations of Insm1-positive
endocrine cells that express either high (HI) or low (LO) levels of Pdx1
at time points from E15.5 to E18.5. Confocal imaging of direct
fluorescence confirmed that Insm1GFPCre/+;Pdx1CFP/+ mice expressed
both GFP and CFP in endocrine cells, with different levels of CFP
expression marking endocrine subpopulations (Fig. 5A), which we
were able to separate by FACS (Fig. 5B). This allowed us to assess
changes in the relative proportions of different cell types during
pancreas development over a 4 day interval (Fig. 5C). Insm1/Pdx1
double-positive endocrine cells comprised ∼20-30% of total sorted
cells, with the greatest increase in cell numbers occurring between
E16.5 and E17.5. As expected, the proportion of Pdx1-HI cells in the
double-positive cell populations increased between E15.5 and E18.5,
mirroring the increase in β-cells during pancreas development.
To explore the differences between the Insm1/Pdx1-HI and
Insm1/Pdx1-LO pre-endocrine cell populations as a function of
time, we performed both RT-qPCR and RNA-Seq analysis.
Pairwise comparison of RNA-Seq results between these cell
populations at E15.5 and E18.5 revealed an increase in the
expression of a number of genes important for β-cell specification
(supplementary material Tables S4 and S5). Gene ontology analysis
showed that most of the Insm1/Pdx1-HI enriched genes are involved
in hormone secretion as well as ion transport and glucose
metabolism (supplementary material Fig. S10A,B). Ninety-three
genes were commonly upregulated in Insm1/Pdx1-HI versus Insm1/
Pdx1-LO cells at E15.5 and E18.5. Correlation analysis showed that
many of these genes have similar differential expression levels and
many are known to be important for β-cell function (supplementary
material Fig. S10C).
RT-qPCR analysis of pre-endocrine cell populations from E15.5
to E18.5 allowed hierarchical clustering based on relative levels of
expression of the genes in the Insm1/Pdx1-HI versus Insm1/Pdx1LO cells (supplementary material Fig. S11). Genes expressed at
higher levels in the Insm1/Pdx1-LO cells included the α-cell
transcription factor Arx, the hormones Glc and Ghrl, as well as
many transcription factors known to be involved in early endocrine
specification, such as Neurod1, Neurog3, Isl1 and Pax4. These
results confirmed our prediction that the Insm1/Pdx1-LO population
consists mostly of non-β-cells and non-committed pre-endocrine
cells. Interestingly, somatostatin and Ppy were expressed at a higher
level in the Insm1/Pdx1-HI population, especially at E15.5. Other
genes that were more highly expressed in the Insm1/Pdx1-HI cells
included known β-cell genes and transcription factors such as Ins2,
Glut2, Mafa, Mnx1 and Nkx6.1. This population also contained the
transcriptional regulators Mafg and Ripply3 and the signaling
molecule Igfbp7.
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DEVELOPMENT
Fig. 4. Neurog3 is alternatively spliced in endocrine progenitor cells. (A) Neurog3 gene and mRNA splicing isoforms. Primer pair N1+N3 is designed to
amplify both isoforms, N1+N2 the long coding isoform, and N1+N4 the short non-coding isoform. (B) Semi-quantitative PCR analysis for Neurog3 mRNA
isoforms. The proportion of the short non-coding isoform is higher in Insm1−/− than in Insm1+/− cells. (C) Quantification of relative expression of the Neurog3 long
versus short isoforms by real-time PCR. (D) Immunofluorescence labeling of pancreatic tissues from Insm1+/− and Insm1−/− mice at E15.5 with anti-GFP
antibodies (green) and anti-Neurog3 antibodies (red). (E) The percentage of Neurog3-positive cells among GFP-positive cells is decreased in Insm1−/− pancreas,
implying changes in Neurog3 expression on a protein level. Error bars indicate s.e.m. (n≥6); P-values were determined by Student’s t-test. Scale bar: 50 μm.
RESEARCH ARTICLE
Development (2014) 141, 2939-2949 doi:10.1242/dev.104810
β-cell and α-cell areas were increased (Fig. 6C,D; supplementary
material Fig. S12C). In addition, there was a notable (although not
statistically significant) increase in somatostatin-positive and Ppypositive cell area (data not shown). Co-staining of insulinexpressing and glucagon-expressing cells for Ki67 indicated that
the proliferation of β-cells was increased in the Ripply3 knockout
pancreata (Fig. 6E,F). The increase in proliferation was not
accompanied by the loss of differentiated β-cell phenotype since
knockout tissues expressed levels of the maturation markers Glut2,
Mafa, Mafa and Nkx6.1 similar to those of the heterozygote
(supplementary material Fig. S12D). TUNEL assay showed no
increase in apoptosis of β-cells in Ripply3 null animals at E18.5
(data not shown).
Fig. 5. Isolation of Insm1-positive endocrine cell populations expressing
high (Insm1/Pdx1-HI) and low (Insm1/Pdx1-LO) levels of Pdx1.
(A) Confocal image of direct fluorescence in a pancreas from animals that were
doubly heterozygous for both the Insm1GFPCre and Pdx1CFP alleles. GFP and
CFP expression marks pre-endocrine cells, with different levels of CFP
marking heterogeneous endocrine populations expressing Pdx1 at high and
low levels. Based on the known expression patterns of Insm1 and Pdx1 (Guz
et al., 1995; Gu et al., 2002; Mellitzer et al., 2006), the perdurance of CFP for
46 h, and immunohistochemical staining of pancreatic tissues with anti-Pdx1
and anti-hormone antibodies (data not shown), these populations are thought
to represent the following cells: Pdx1-LO, predominantly acinar cells with a
small proportion of ductal cells; Insm1/Pdx1-HI, endocrine cells including
mostly β-cells but also with some δ-cells; Insm1/Pdx1-LO, endocrine cells
including uncommitted endocrine cells and other non-β-cells; and Insm1positive neural cells of non-pancreatic origin. (B) FACS profile of pancreatic
populations sorted from Insm1GFPCre/+;Pdx1CFP/+ animals. Four distinct
populations are sorted: GFP positive, CFP positive and two GFP/CFP doublepositive populations expressing high (Pdx1-HI) and low (Pdx1-LO) levels of
CFP. (C) Temporal changes in the relative proportions of the different sorted
pancreatic cell populations. Scale bar: 50 μm.
Ripply3 is expressed in pancreatic β-cells and contributes to
their proliferation
Since the function of Ripply3, a gene we found to be both upregulated
by Insm1 and enriched in Insm1/Pdx1-HI cell populations, had not
been previously explored in pancreas, we analyzed mouse pancreatic
tissues from Ripply3β-Gal mice, which express β-galactosidase instead
of Ripply3 (Okubo et al., 2011). Co-staining for β-galactosidase,
chromogranin A and islet hormones confirmed that Ripply3 is
expressed in nascent endocrine cells, with higher levels detected in
β-cells at E18.5 (Fig. 6A; supplementary material Fig. S12A).
RT-qPCR on embryonic and adult tissues showed that Ripply3 is
strongly expressed in heart and lung, and to a lesser degree in liver and
kidney. In the adult pancreas the expression of Ripply3 was highest in
islets (Fig. 6B). Based on RNA-Seq profiling of distinct pancreatic
developmental populations, Ripply3 is expressed in early endoderm at
E8.0, with lower levels detected in pancreatic endoderm and MPCs,
and increased expression in Neurog3-positive endocrine progenitor
cells at E15.5 and further in nascent β-cells at E16.5 (supplementary
material Fig. S12B). Although pancreatic endocrine cell types were
present at E18.5 in Rippy3β-Gal heterozygous and knockout mice, both
2944
In agreement with previously published data (Gierl et al., 2006),
mice without Insm1 have a reduced number of endocrine cells and
hormone expression in these cells is either lacking or imbalanced in
terms of specific endocrine cell types. Given that Insm1 has long
been known to contain a SNAG repressor domain (Chiang and
Ayyanathan, 2013), our finding that approximately twice as many
genes in Insm1−/− than in Insm1+/− mice were increased in
expression is consistent with the major role of Insm1 being as a
repressor. In the absence of Insm1, pancreatic pre-endocrine cells
display many features of earlier stage progenitor cells, including an
increase in the expression of transcription factors and cell surface
receptors associated with the progenitor state, EMT markers, and
components of morphogenic signaling pathways (e.g. Notch, Tgf/
BMP and Wnt). Although EMT markers increased in Insm1 null
cells, they failed to migrate away from the ducts. This might be due
to incomplete EMT, since some the genes upregulated in Insm1+/−
cells are involved in actin remodeling and cell migration. Moreover,
signaling molecules and extracellular matrix proteins that are
expressed upon Insm1-dependent endocrine differentiation might be
necessary for the migration and aggregation of newly differentiated
endocrine cells into islets. Insm1 null cells also have decreased rates
of proliferation that starts at E15.5 and is well pronounced at E18.5.
This decrease was not accompanied by an increase in apoptosis, and
the knockout cells appeared larger in size, a characteristic that often
accompanies cell cycle arrest at G1/G2 phase (Bjorklund et al.,
2006). Consistent with cell cycle arrest without apoptosis, Insm1
null cells express more of the cell cycle inhibitor Cdkn1c and the
pro-survival gene Bcl2. Interestingly, Insm1-expressing cells make
more of the cell cycle inhibitor Cdkn1b ( p27Kip1), which is involved
in controlling the proliferation of newly differentiated β-cells
(Georgia and Bhushan, 2006). This suggests that Insm1 promotes a
switch in the mode of cell cycle regulation from being Cdkn1c based
to being Cdkn1b regulated, with both genes being directly regulated
by Insm1. A similar developmental switch in cell cycle regulators
has been observed during pituitary development, in which noncycling precursors express Cdkn1c and more differentiated cells
express Cdkn1b (Bilodeau et al., 2009). Although the differentiation
of endocrine cells in the presence of Insm1 is associated with an
increase in proliferation, cell proliferation itself is under the control
of multiple genes, including cell cycle inhibitors such as Cdkn1b
and Ripply3. The complex regulation of endocrine progenitor cell
proliferation is not surprising given that overexpression of Insm1 in
the developing neural cortex has been shown to decrease the
proliferation of less differentiated apical progenitor cells but to
increase the proliferation of more differentiated intermediate basal
progenitor cells (Farkas et al., 2008).
DEVELOPMENT
DISCUSSION
Role of Insm1 in pancreatic pre-endocrine cell differentiation
RESEARCH ARTICLE
Development (2014) 141, 2939-2949 doi:10.1242/dev.104810
Fig. 6. Ripply3 is involved in the regulation of endocrine cell replication. (A) Ripply3 is expressed in pancreatic endocrine cells at E18.5. Immunofluorescence
staining of pancreata from Ripply3β-Gal heterozygous mice with anti-β-galactosidase and antibodies against the pancreatic hormones insulin (Ins), glucagon
(Gcg), pancreatic polypeptide (Ppy) and somatostatin (Sst). (B) Ripply3 is expressed in adult pancreatic islets. RT-qPCR analysis of Ripply3 relative expression in
different tissues and during embryonic development (normalized to expression in ESCs). (C) Immunofluorescence staining for insulin and glucagon for
representative sections of Ripply3β-Gal heterozygous and homozygous mice. The hormone-positive endocrine area appeared to be expanded in Ripply3 knockout
animals. (D) Quantification of total insulin-positive and glucagon-positive areas in Ripply3β-Gal heterozygous and knockout animals. Both areas are significantly
increased in knockout animals. (E) Immunofluorescence staining for insulin, glucagon and Ki67 for representative sections of Ripply3β-Gal heterozygous and
homozygous mice. (F) The percentage of Ki67-positive cells among total insulin-positive or glucagon-positive cells shows an increase in the proliferation of
endocrine cells in Ripply3 knockout animals. Error bars indicate s.e.m. (n≥6); P-values were determined by Student’s t-test. Scale bars: 50 μm.
Identification of novel endocrine surface markers
This study revealed a number of surface proteins that mark early
endocrine cells. For example, mRNAs for Amigo2, Celsr3 and Lrp11
were strongly upregulated in Insm1-positive cells, and immunostaining
for the encoded proteins confirmed their localization along the
membrane of endocrine cells. Amigo2 showed the strongest endocrinespecific staining pattern and may function in cell adhesion and
migration (Rabenau et al., 2004). Celsr3, the cadherin associated with
planar polarity, displayed localized membrane staining and was
recently shown to promote endocrine cell differentiation (Tissir and
Goffinet, 2006; Cortijo et al., 2012). Another early endocrine cell
marker validated by our study is Lrp11, as was previously suggested by
Hald et al. (2012). Interestingly, although Lrp11 is present in all
endocrine cells, it seems to be most highly expressed in δ-cells.
Neurog3 is alternatively spliced in endocrine progenitor cells
RNA-Seq revealed that ∼30% of the differentially expressed genes
had two or more differentially expressed RNA variants (data not
shown). This suggests that the Insm1-dependent differentiation
program might also function at a post-transcriptional level by
promoting endocrine lineage-specific alternative splicing. Although
the mechanisms involved in this regulation are unclear, this could
occur through the activation of genes encoding RNA-binding
2945
DEVELOPMENT
This study also brings to light several genes that might play
important but previously unrecognized roles in the formation of islet
cells. Indeed, although a key function of Insm1 might be to repress
genes (e.g. Rest) that maintain pancreatic ductal epithelial cells in a
progenitor-like state, which would be expected to promote endocrine
cell differentiation, many genes were also found to be upregulated in
Insm1-expressing cells. These include genes necessary for hormone
secretion, membrane transporters and channels, extracellular matrix
proteins, RNA-processing proteins and cell migration proteins. For
example, Insm1-expressing progenitor cells are characterized by
higher levels of transcription factors that are found in differentiating
β-cells (Mafa, Mafb, Mnx1, Vdr, Hmgn3, Fev, Lmx1b), as well as
novel genes that have not been studied in the pancreas (Lmo3,
Ripply3, Jazf1, Nhlh1). Among the Myt1 family, St18, Myt1 and
Myt1l1 were all upregulated, suggesting that all three of these genes
are expressed in differentiating endocrine cells, contrary to a
previous report (Gu et al., 2004). Considering that many of the
Insm1-regulated transcription factors have also been shown to play
key roles during neural development, and that neural and islet cells
share many phenotypic properties, these genes probably also
contribute to endocrine development. Interestingly, one such gene,
the transcription factor Jazf1, is associated with the development of
type 2 diabetes (Zeggini et al., 2008).
RESEARCH ARTICLE
Development (2014) 141, 2939-2949 doi:10.1242/dev.104810
proteins, such as Elavl4, Celf3 and Rod1. All of these transcripts were
strongly upregulated in Insm1-positve cells and so could be important
in regulating splicing changes during endocrine differentiation. In
particular, we found that Neurog3 mRNA is alternatively spliced in
endocrine progenitor cells, with a long coding and short non-coding
isoform present. The latter is formed through splicing to the alternative
splice acceptor site located in the second exon of Neurog3
(supplementary material Fig. S7). Interestingly, the ratio of the
Neurog3 coding to non-coding isoform was higher in Insm1-positive
cells, suggesting that alternative splicing of the Neurog3 pre-mRNA in
an Insm1-dependent manner provides a positive-feedback mechanism
for maintaining Neurog3 expression during development.
Transcriptional profiling of subpopulations of pre-endocrine
cells
Ripply3 as a downstream modulator of endocrine cell
proliferation
In this study we discovered that Ripply3 is also expressed in
endocrine progenitor cell populations and that Insm1 binds to the
Ripply3 promoter. Ripply3 belongs to a family of related proteins,
the other two members of which, Ripply1 and Ripply2, are
necessary for the proper formation of somite boundaries and
function by antagonizing T-box transcription factor activity during
development (Kawamura et al., 2005; Hitachi et al., 2009;
Takahashi et al., 2010). Ripply3 has also been shown to regulate
pharyngeal apparatus development through suppression of Tbx1
activity (Okubo et al., 2011). The importance of other Ripply gene
family members prompted us to explore the role of Ripply3 in
pancreas development. Although our analysis did not reveal any
defects in endocrine cell differentiation in Ripply3 null pancreatic
tissues, we observed that both β-cell and α-cell areas and
proliferation were increased.
The increase in proliferation in Ripply3 null tissues suggests that,
similar to Cdkn1b, Ripply3 might control the proliferation of newly
committed β-cells in order to allow them to properly differentiate.
2946
Fig. 7. Model for the role of Insm1 in endocrine progenitor cells. (A) The
Insm1 transcriptional program regulates a progression of pre-endocrine cells
from Neurog3-positive endocrine progenitors residing in a duct to islet-bound
committed endocrine cells. (B) Endocrine progenitor cells are characterized by
Notch signaling and other stemness-controlling pathways. As endocrine cell
differentiation occurs, the proliferation of these cells slows as they exit the cell
cycle and delaminate from the duct through the activation of the EMT program.
Insm1 promotes these changes by repressing progenitor cell functions while
simultaneously activating genes involved in hormone secretion, formation of
extracellular matrix, cell migration, and promotion of alternative splicing and
vascularization. The Insm1-dependent endocrine differentiation program is
accompanied by an increase in the coding isoform of Neurog3 and expression
of Ripply3, a gene we have shown to regulate β-cell proliferation.
The mechanism by which Ripply3 functions in pre-endocrine cells
is unclear. As mentioned above, members of this class of proteins
have been shown to bind and suppress the activity of T-box
transcription factors. Previous reports (Begum and Papaioannou,
2011; Oropez and Horb, 2012) have shown that Tbx2 is expressed
in pre-endocrine cells, and our current findings indicate that it is
upregulated in the absence of Insm1. Thus, given that Tbx2 was
previously shown to promote cell proliferation (Vance et al., 2005),
Ripply3 might function by antagonizing Tbx2 activity in
differentiating endocrine cells. In any case, the functional
interactions between Ripply3 and Tbx2, and how each gene might
contribute to the growing network of genes that promote and/or
regulate the proliferation of pancreatic endocrine progenitor cells,
merit additional study.
Conclusions
These studies provide valuable new insights into the role that Insm1
plays in developing pancreatic endocrine cells (as summarized in
the model shown in Fig. 7). We expect that the RNA-Seq datasets,
besides identifying many genes that are affected in expression by
Insm1, will serve as a platform for further discoveries, especially as
more comprehensive models of the gene regulatory network in
endocrine cells are built.
MATERIALS AND METHODS
Antibodies and primers
Antibodies employed in immunohistochemistry and ChIP and primers used
for genotyping, qPCR and ChIP analysis are described in the Methods in the
supplementary material.
DEVELOPMENT
Combinatorial FACS using two or more fluorescent reporter alleles
is a powerful means of isolating specific progenitor cell
subpopulations. For this reason, we also made use of a recently
derived Pdx1CFP fluorescent reporter allele to explore the
differences in gene expression between developing endocrine
progenitor cells that express either high or low levels of Pdx1. It
is well known that Pdx1 marks all endocrine cells (Herrera, 2000;
Gu et al., 2002) and that developing β-cells, as well as a small
fraction of δ-cells, express high levels of Pdx1 (Leonard et al., 1993;
Ohlsson et al., 1993; Guz et al., 1995). As expected, Insm1/Pdx1-HI
cells express more β-cell-specific genes, such as Ins2, Slc2a2 and
Mafa. However, we were surprised to find that this cell population
(at E15.5) also contains many somatostatin and Ppy transcripts. This
suggests that the Insm1/Pdx1-HI cell population might contain a
common β/δ-progenitor, as well as newly formed δ-cells, the
existence of which has been predicted by Pax4 and Arx knockout
studies (Collombat et al., 2003, 2005). Our data are also consistent
with previous lineage-tracing studies that have shown that a Ppy-Cre
transgene marks both β-cells and Ppy-producing cells (PP-cells)
(Herrera et al., 1994; Herrera, 2000), a finding that suggests that PPcells and a common β/δ-cell might share common a progenitor, or
that Ppy is expressed in the common β/δ-cell progenitor. Analysis of
differential expression between Insm1/Pdx1-HI and Insm1/Pdx1LO endocrine subpopulations allowed us to define Insm1-dependent
genes that are enriched in Pdx1-HI populations and therefore
potentially important for specific functions of β-cells and/or δ-cells.
RESEARCH ARTICLE
The Insm1GFPCre allele was made using a sequential gene targeting and
RMCE strategy using methods and vectors that have been described
previously (Chen et al., 2011). Insm1GFPCre and Pdx1CFP mice (Potter et al.,
2011) were maintained on a CD1 outbred background (90% or higher) to
extend the survival of the embryos, as previously described (Gierl et al.,
2006). Approximately 10% of the Insm1 homozygous null pups survived to
E18.5, consistent with the results of Gierl et al. (Gierl et al., 2006), with all
other embryos dying between E15.5 and E18.5. Ripply3β-Gal mice were of
mixed C57BL6/129SV background. All animal procedures and husbandry
protocols were approved by the Vanderbilt University Institutional Animal
Care and Use Committee.
Embryo isolation and imaging, tissue dissociation and FACS
Timed matings were carried out by designating noon on the day vaginal plugs
were identified as E0.5. Embryos at different stages were obtained by dissection
and imaged by direct fluorescence visualization using a Leica MZ 16 FA
stereoscope equipped with a QImaging RETIGA 4000R camera. For FACS,
embryos were isolated at E15.5-18.5 and initially genotyped by direct
fluorescence. Pancreata were dissected in cold PBS and dissociated with
Accumax solution (Sigma-Aldrich) containing 5 μg/ml DNase I (SigmaAldrich) at 37°C for 1 h with periodic resuspension. Quenching solution [L15
medium, 10 mM Hepes (pH 7.0), 1 mg/ml bovine serum albumin (Life
Technologies), 5 μg/ml DNase I] was added to stop the reaction. The cell
suspension was filtered through a 35 μm filter into FACS tubes, washed with
quenching solution and resuspended in 0.5 ml quenching solution containing
7AAD viability dye (1:1000; Life Technologies). Fluorescent cells were sorted
directly into Trizol LS reagent (Life Technologies) containing 40 μg/ml mussel
glycogen (Sigma-Aldrich) using a FACSAria I cell sorter (BD Biosciences).
RNA isolation, library construction and RNA-Seq
Three independent RNA isolates from each genotype were used for cDNA
amplification and sequencing. Cells from multiple embryos were pooled to
obtain a sufficient amount of RNA for analysis. The methods used to isolate
RNA, validate integrity and purity, amplify cDNA and make libraries have
been described previously (Choi et al., 2012). Single-end sequencing
(110 bp) was performed on an Illumina HiSeq2000 genome analyzer. Read
alignment to the mouse genome (mm9) was performed using RNA-Seq
Unified Mapper (RUM) (Grant et al., 2011). Expression level was
quantified as reads per kilobase of gene model per million mapped reads
(RPKM). Genome alignment of sequencing data yielded total mapped reads
in a range from 30 to 47 million per sample, from which 66-70% of reads
were uniquely mapped. RPKM values of the different samples were highly
correlated between replicates (R2=0.99). Differential expression analysis
was performed using Patterns of Gene Expression (PaGE) software (Grant
et al., 2005). Summaries of the differential expression analyses are provided
in supplementary material Tables S1, S3 and S4. Gene ontology analysis
was performed using DAVID Bioinformatics Resources v.6.7 (Huang et al.,
2009). RNA-Seq data are available at http://genomics.betacell.org (study
numbers 4294 and 4674) and are also deposited at ArrayExpress (accession
number E-MTAB-1929).
RT-qPCR analysis
Ninety-six gene custom TaqMan Low-Density Array (TLDA; Life
Technologies) cards were used for validation of gene expression. The
TaqMan probe and primer sets were selected from predesigned TaqMan
gene expression assays (Life Technologies). Three independent RNA
isolates for each genotype or developmental time point were assayed. cDNA
was prepared from 30 μg total RNA using a high-capacity cDNA Archive
Kit (Life Technologies) and divided between two ports of the TLDA cards.
PCR was performed with an ABI 7900HT real-time PCR system (Life
Technologies) and amplification data were analyzed using Sequence
Detection System version 2.1 (Life Technologies) and Excel software
(Microsoft). 18S ribosomal RNA and Gapdh were used as endogenous
housekeeping controls for normalization, and the comparative Ct method
was used to calculate relative fold expression by 2−ΔΔCt (supplementary
material Table S2). Hierarchical clustering analysis of the data was
performed using GenePattern software (Reich et al., 2006). The clustering
was performed on relative expression levels (log10) using Euclidean distance
and pairwise average linkage. The Hierarchical Clustering Viewer from
GenePattern was used for graphical representation, with coloring relative for
each gene row. For analysis of Neurog3 alternative splicing variants, 1 ng
amplified cDNA (used for RNA-Seq) was amplified using either real-time
PCR with Power SYBR Green PCR Master Mix (Life Technologies) or
semi-quantitative PCR using Phusion Taq (New England Biolabs).
Immunohistochemistry
Immunofluorescence staining of frozen sections was essentially as
previously described (Burlison et al., 2008). Ductal cells were stained
with biotinylated Dolichos biflorus agglutinin (DBA; Vector Laboratories)
followed by streptavidin-Cy5 conjugate (Vector Laboratories). Coverslips
were mounted using ProLong Gold antifade reagent with DAPI (Life
Technologies). Fluorescence images were acquired using an Axioplan2
microscope (Zeiss) with 20× and 40× objectives and QImaging RETIGA
EXi camera. Confocal microscopy images were acquired using an LSM 510
Meta microscope and imaging software (Zeiss). Images taken with different
color channels were merged using Photoshop (Adobe Systems).
Morphometric analysis
The fractions of GFP-positive or β-galactosidase-positive endocrine cells
co-expressing other proteins were calculated by manual cell counting on
step sections (50 μm apart) of pancreatic tissues from at least six different
animals of the same age and genotype. At least 500 cells were counted for
each phenotype in each experiment. The percentage of GFP-positive cells
adjacent to ducts was calculated based on the number of these cells in
contact with DBA-positive cells. The cell area of GFP-positive endocrine
cells was measured using ImageJ (NIH) software to outline E-cadherinpositive cell borders using a total of 1000 cells from three different animals
of each phenotype. The insulin-positive and glucagon-positive areas of
Ripply3 heterozygous and mutant animals were measured using every fifth
section from three different animals of the same phenotype.
ChIP
Pancreata were dissected from E15.5 wild-type embryos, cross-linked with
1% formaldehyde in PBS for 8 min, quenched with 125 mM glycine and
washed with PBS. Tissues were lysed in 100 μl lysis buffer [50 mM TrisHCl ( pH 8.0), 10 mM EDTA, 1% SDS, 1× protease inhibitor mix (Sigma),
1 mM PMSF], sonicated (Bioraptor, Diagenode) and lysates diluted to 1 ml
with RIPA buffer [10 mM Tris-HCl ( pH 7.5), 140 mM NaCl, 1 mM EDTA,
0.5 mM EGTA, 1% Triton X-100, 0.1% (w/v) SDS, 1× protease inhibitor
mix, PMSF]. 20 μg sonicated chromatin was incubated with 8 μg antibody
coupled to IgG magnetic beads (Cell Signaling Technology) overnight at
4°C. The beads were washed five times with RIPA buffer and once with TE
buffer [10 mM Tris ( pH 8.0), 1 mM EDTA]. After washing, bound DNA
was eluted at 65°C in elution buffer [20 mM Tris-HCl ( pH 7.5), 5 mM
EDTA, 50 mM NaCl, 1% SDS, 50 μg/ml proteinase K] for 2 h. After crosslinking reversal, the immunoprecipitated DNA was purified by phenolchloroform extraction and ethanol precipitation and resuspended in 30 μl TE
buffer. Enrichment at target promoters was determined by real-time PCR
with Power SYBR Green PCR Master Mix. Relative fold enrichment at
different sites was calculated by the 2−ΔΔCT method from five different ChIP
experiments.
Acknowledgements
We thank Guoqiang Gu, Jennifer Stancill and Hannah Worchel for critically
reviewing the manuscript; David K. Flaherty and Brittany Matlock for providing FACS
expertise and technical assistance; Jennifer Skelton for help with deriving the mice;
and Travis Clark for assistance with the next-generation sequencing.
Competing interests
The authors declare no competing financial interests.
Author contributions
A.B.O., Q.L., J.S., C.J.S. and M.A.M. were involved in the conception and design of
the experiments and co-wrote the manuscript. A.B.O. performed most of the
experiments and analyzed the data; Q.L. designed the gene targeting vector;
R.G. performed embryo dissections and stereoscopy; S.B.H. performed the gene
2947
DEVELOPMENT
Mouse lines
Development (2014) 141, 2939-2949 doi:10.1242/dev.104810
targeting and RMCE procedures; E.M. and C.J.S. performed initial RNA-Seq data
analysis; T.O. and S.T. provided tissues from Ripply3 knockout mice. All authors
were provided with the opportunity to comment on the manuscript.
Funding
These studies were supported by National Institutes of Health (NIH) grants
[DK72473 and DK89523] to M.A.M. Use of shared resources at Vanderbilt University
School of Medicine was supported by NIH grants [CA68485, DK58404 and
DK20593]. Deposited in PMC for immediate release.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.104810/-/DC1
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