© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 654-664 doi:10.1242/dev.114959
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Generation of enteroendocrine cell diversity in midgut stem cell
lineages
ABSTRACT
The endocrine system mediates long-range peptide hormone
signaling to broadcast changes in metabolic status to distant target
tissues via the circulatory system. In many animals, the diffuse
endocrine system of the gut is the largest endocrine tissue, with the
full spectrum of endocrine cell subtypes not yet fully characterized.
Here, we combine molecular mapping, lineage tracing and genetic
analysis in the adult fruit fly to gain new insight into the cellular and
molecular mechanisms governing enteroendocrine cell diversity.
Neuropeptide hormone distribution was used as a basis to generate a
high-resolution cellular map of the diffuse endocrine system. Our
studies show that cell diversity is seen at two distinct levels: regional
and local. We find that class I and class II enteroendocrine cells can
be distinguished locally by combinatorial expression of secreted
neuropeptide hormones. Cell lineage tracing studies demonstrate
that class I and class II cells arise from a common stem cell lineage
and that peptide profiles are a stable feature of enteroendocrine cell
identity during homeostasis and following challenge with the enteric
pathogen Pseudomonas entomophila. Genetic analysis shows that
Notch signaling controls the establishment of class II cells in the
lineage, but is insufficient to reprogram extant class I cells into class II
enteroendocrine cells. Thus, one mechanism by which secretory cell
diversity is achieved in the diffuse endocrine system is through cellcell signaling interactions within individual adult stem cell lineages.
KEY WORDS: Drosophila, Diffuse endocrine system, Neuropeptide,
Stem cell, Notch
INTRODUCTION
On 16 January 1902, Ernest Starling observed a striking increase in
pancreatic secretion when he prepared lysate from a segment of
freshly excised intestinal mucosa rubbed with sand and weak HCl,
filtered it through cotton wool and injected the extract into the
jugular vein of an anesthetized dog (Bayliss and Starling, 1902).
Documentation of this novel ‘reflex’ marked the inception of
endocrinology as a field and implicated the intestinal epithelium as
a potent source of peptides capable of acting over a great distance via
the circulatory system to profoundly alter physiology. We now know
that the gut epithelium is a rich source of secreted hormones that
originate from specialized enteroendocrine cells and comprise a
diffuse endocrine system distinct from the well-studied glandular
endocrine system (Engelstoft et al., 2013; Schonhoff et al., 2004). Gut
endocrine cells are embedded in the barrier epithelium and mediate
luminal chemosensitivity (Furness et al., 2013; Reimann et al., 2012).
In response to environmental stimuli, peptides secreted from the gut
Washington University School of Medicine, Department of Developmental Biology,
660 South Euclid Avenue, St Louis, MO 63110, USA.
*Author for correspondence ([email protected])
Received 1 July 2014; Accepted 15 December 2014
654
epithelium act broadly on endocrine, nervous and immune systems to
encode adaptive physiologic responses. Such peptides (also called
neuropeptides) belong to distinct families, such as allatostatins,
diuretic hormones and tachykinins (Bendena et al., 1999; Gäde, 2004;
Nässel and Wegener, 2011; Van Loy et al., 2010). Knowledge differs
widely with respect to each peptide family member and their
experimentally defined roles within a given species. However,
neuropeptide hormones have been implicated in processes such as
growth control, muscle activity, feeding behavior, osmotic balance,
reproduction and epithelial secretion. The prodigious number of
enteroendocrine cells and their varied neuropeptide expression
combine to make the gut the largest endocrine organ in the body.
And yet, the mechanisms underlying endocrine diversity in this
central endocrine tissue have not been fully understood.
The invertebrate gastrointestinal (GI) tract, like its mammalian
counterpart, contains a diffuse endocrine system providing a
tractable experimental model to investigate the basis of enteroendocrine diversity. This population of cells can be easily detected
along the entire anterior-posterior (A/P) axis of the adult midgut
with the pan-enteroendocrine marker Prospero (Pros). Adult
enteroendocrine cells are continuously maintained by stem cell
divisions that occur in physiologically and functionally distinct
regions of the midgut (Micchelli and Perrimon, 2006; Ohlstein and
Spradling, 2006; Strand and Micchelli, 2011). Genomic analysis at
the organ level has shown that a wide variety of neuropeptides and
their cognate receptors, many of which are conserved, are expressed
both within the gut and in the periphery (Marianes and Spradling,
2013; Buchon et al., 2013; Veenstra et al., 2008; Veenstra, 2009;
Veenstra and Ida, 2014). It is also now possible to specifically
isolate enteroendocrine cells and to obtain expression profiles at the
cellular level (Dutta et al., 2013). Moreover, experimental means to
challenge the barrier epithelium with pathogenic or nutritional
insults are now available and combine readily with existing
molecular genetic tools (Vodovar et al., 2005; Lee and Micchelli,
2013; Piper et al., 2014). Thus, the GI tract of Drosophila provides
an unsurpassed experimental model to investigate aspects of diffuse
endocrine system biology.
Studies of adult Drosophila enteroendocrine cells have focused
on the molecular events that determine whether a newly formed
intestinal progenitor, called enteroblast, will adopt the secretory
(endocrine) or absorptive (enterocyte) cell fate (Micchelli and
Perrimon, 2006; Ohlstein and Spradling, 2006). The idea that Notch
signaling might control this decision was first suggested following
the observation that high levels of a transcriptional reporter for
Notch could be detected in the enteroblast (Micchelli and Perrimon,
2006). Subsequent functional studies showed that the Notch ligand
Delta can be detected at high levels in the neighboring intestinal
stem cell and that Delta is both necessary and sufficient
to distinguish the endocrine and enterocyte fate (Beebe et al.,
2010; Ohlstein and Spradling, 2007). Together, these observations
led to a simple model in which stem cells signal to enteroblasts to
DEVELOPMENT
Ryan Beehler-Evans and Craig A. Micchelli*
RESEARCH ARTICLE
Development (2015) 142, 654-664 doi:10.1242/dev.114959
influence cell fate: low levels of Notch transduction in the
enteroblast promote the enteroendocrine differentiation and high
levels of Notch transduction in the enteroblast promote the
enterocyte differentiation. This asymmetry between stem cell and
daughter cell is reinforced by segregation of Sara endosomes into
the newly formed enteroblast (Montagne and Gonzalez-Gaitan,
2014). However, precisely when the specifying Notch signaling
event occurs remains an open question (Perdigoto et al., 2011).
Transcription factors of the Enhancer of split and achaete-scute
complexes have both been shown to control endocrine
differentiation (Bardin et al., 2010). More recently, Slit/Robo
signaling has been implicated in enteroendocrine specification
(Biteau and Jasper, 2014). Possible interactions between Notch and
Robo signaling pathways have not yet been fully explored.
The goal of the current study is to characterize the extent of
cellular diversity in the diffuse endocrine system of adult
Drosophila and to gain insight into the molecular mechanisms
underlying this process. Here, we focus on the posterior midgut and
use differential neuropeptide expression as a criterion to map
enteroendocrine cell types in situ with great spatial resolution. We
show that even immediately adjacent endocrine cells along
the midgut express different combinations of neuropeptides,
dividing the diffuse endocrine system into class I and class II
endocrine cells. Directed cell lineage-tracing studies show that, once
an enteroendocrine cell subtype is established, it is stably
maintained in the adult, even following environmental challenge.
Thus, diversity of endocrine cells is seen at two levels: regional and
local. We demonstrate that individual gut stem cells generate pairs
of class I and class II enteroendocrine cells displaying distinct
neuropeptide profiles. Moreover, the precise combination of
neuropeptide expression depends on stem cell position within the
midgut. Finally, our analysis shows that Notch signaling is initially
required to generate class II enteroendocrine cell diversity in
posterior midgut stem cell lineages, but is dispensable for both
underlying regional identity and neuropeptide expression in extant
endocrine cells. We conclude that local endocrine diversity is
generated in the diffuse endocrine system during adult homeostasis
through cell-cell signaling interactions within individual stem cell
lineages.
The adult Drosophila midgut epithelium is a rich source of
secretory neuropeptide hormones, showing elevated levels
of Allatostatin A (AstA), Allatostatin B (AstB), Allatostatin
C (AstC), Neuropeptide F (Npf ), Diuretic hormone 31 (DH31)
and Tachykinin (Tk) (Veenstra et al., 2008; Reiher et al., 2011;
Veenstra and Ida, 2014). Neuropeptide expression subdivides the
gut into broad regions along the A/P axis, which are evident with
regards to both anatomical and molecular markers (supplementary
material Fig. S1A-N; Table S1) (Veenstra et al., 2008; Veenstra and
Ida, 2014). For example, the posterior midgut is divided into two
primary neuropeptide-expressing regions that we refer to as region
1 and region 2, respectively (Fig. 1A; supplementary material
Fig. S1A,B, Fig. S2A-F; Table S1). A panel of transgenic
molecular markers have been used to subdivide the posterior
midgut into regions (Marianes and Spradling, 2013; Buchon et al.,
2013). However, careful comparison of relevant marker strains
with neuropeptide antisera revealed segmental boundaries that
were distinct (supplementary material Fig. S2A-F). Therefore,
for the purpose of this study, which focuses primarily on the
Fig. 1. Diversity of adult enteroendocrine cells is both regional and local.
(A) Regional differences in peptide hormone expression distinguish segments
of the adult posterior midgut (shading). (B) Within each region, local
differences in peptide hormones are detected among endocrine cell pairs.
Class I and II enteroendocrine cells are distinguished by peptide antisera and
Gal4 lines. Yellow indicates Notch-dependent cell fates. DH, DH31.
(C-H) Peptides are differentially detected in Pros+ cell pairs. (C) Anti-AstA, red;
anti-Pros, red. (D) Anti-AstB, red; anti-Pros, green. (E) Anti-AstC, red; antiPros, green. (F) Anti-DH31, red; anti-Pros, green. (G) Anti-Npf, red; anti-Pros,
green. (H) Anti-Tk, red; anti-Pros, green. (I) Pairs of endocrine cells express
different peptides. Anti-AstA, red; anti-Tk, green. (J) At the transition between
region 1 and 2, AstA increases posteriorly, whereas AstB decreases
posteriorly. Anti-AstA and anti-AstB, white; Tk>GFP, green. Scale bars: 10 µm.
peptide-rich posterior midgut, we use the ‘region 1’ and ‘region 2’
designations to refer directly to native domains of peptide
expression in the adult midgut.
655
DEVELOPMENT
RESULTS
Neuropeptide expression distinguishes adult
enteroendocrine cells in the posterior midgut
RESEARCH ARTICLE
Development (2015) 142, 654-664 doi:10.1242/dev.114959
Adult stem cells maintain the differentiated epithelial lining of
the midgut (Micchelli and Perrimon, 2006; Ohlstein and
Spradling, 2006). This monolayer consists primarily of large
absorptive enterocyes, but also includes ∼1000 (∼10%) secretory
enteroendocrine cells that can be identified by the pan-endocrine
marker Pros. However, it has been unclear whether neuropeptide
expression in the midgut is restricted only to endocrine cells, or
the extent to which individual cells might express specific
combinations of particular neuropeptides. To determine whether
secretory neuropeptides are expressed in Pros+ cells, we
conducted a series of double-labeling experiments on five-dayold adult midguts. Our studies show that distinct subsets of Pros+
cells also stain positive for AstA, AstB, AstC, DH31, Npf and
Tk when assayed in a pairwise manner (Fig. 1C-H). Initial
characterization indicated that, with the exception of AstC in
region 1, only one of the two adjacent Pros+ cells stained positive
for the peptide assayed. This impression was confirmed by scoring
the percentage of Pros+ cells within a given region that also
expressed a particular neuropeptide (Table 1). Values ranged from
48 to 52%, or approximately half of the Pros+ cells. Neuropeptide
expression was specific to endocrine cells, with no obvious
expression detected above background levels elsewhere in the gut
epithelium. These results were independently confirmed using
specific neuropeptide Gal4 driver lines, which we found to
be expressed in the adult midgut (Materials and Methods;
supplementary material Fig. S1F,J,N and Fig. S3; Table S1).
Thus, neuropeptide expression in the gut is limited to endocrine
cells, and in all but one case, labels half of the Pros+ cells in a
particular midgut region.
Adjacent Pros+ enteroendocrine cells can be unambiguously
distinguished by unique neuropeptide expression profiles (Fig. 1B,I;
supplementary material Fig. S1A-N and Fig. S3A-N; Table S1). A
comprehensive series of double- and triple-staining studies,
employing both antibodies and Gal4 lines, was performed. This
analysis showed that in region 1, for example, class I enteroendocrine
cells are AstB+, AstC+, whereas class II enteroendocrine cells are
AstC+ (supplementary material Fig. S3E,F). In region 2, class
I enteroendocrine cells are AstA+, AstC+ (supplementary material
Fig. S3C,D,G,H), whereas class II enteroendocrine cells are DH31+,
Tk+ (supplementary material Fig. S3I,J,M,N). To determine
which fraction of Pros+ cells express either class I or class II
neuropeptide markers we simultaneously labeled AstC+, Tk+ and
Pros+ cells (Table 1). Using combinations of either antisera or
Gal4 lines revealed that the vast majority (>95%) of Pros+ cells
also express neuropeptides. A transition zone between region 1
and region 2 was identified, where neuropeptide protein is
detected in a graded manner within class I endocrine cells (Fig. 1J;
supplementary material Table S1). We note that pairs of class I
and class II enteroendocrine cells were also detected in the
anterior and middle regions of the adult midgut (Fig. 1G;
supplementary material Fig. S1; Table S1). Thus, precise
combinations of secretory neuropeptides can be used to
distinguish each cell within an enteroendocrine pair 5 days after
eclosion.
Neuropeptide expression is a fixed characteristic of
enteroendocrine cell subtypes
Neuropeptides actively modulate organismal physiology. It is therefore
possible that established cellular profiles of peptide expression
associated with a class of enteroendocrine cells can change during
adulthood. Alternatively, peptide expression might be a fixed
characteristic of endocrine cell subtypes. To test these possibilities,
we conducted directed cell lineage-tracing experiments using specific
neuropeptide Gal4 driver lines (Fig. 2). Class I enteroendocrine cells in
region 2 of the posterior midgut were genetically labeled by crossing
Antigen
Neuropeptide+/
Neuropeptide+ Pros+ Pros+ ratio (%) Region
n (guts)
AstA
AstB
AstC
DH31
Npf
Tk
AstC/Tk
GFP*
106
42
261
196
245
252
749
1487
6
3
6
6
6
6
6
8
206
88
269
383
500
525
792
1557
51
48
97
51
49
48
95
96
2
1
1
2
Anterior+CC
2
1+2
1+2
*AstC-Gal4/Tk-Gal4×UAS-GFP; CC, Copper cell region.
656
Fig. 2. Peptide expression stably distinguishes class I and II
enteroendocrine cells. (A-H) Directed lineage tracing of class I and II
enteroendocrine cells using actin5C>lacZ; UAS-flp. Anti-β-Gal, red; anti-AstA/
anti-DH31, green. (A,C,E,G) AstA-Gal4. (B,D,F,H) Tk-Gal4. (A,B) Gal4 lines
are not detected in newly eclosed adult midguts. (C,D) 4 days following
eclosion Gal4 drivers mark endocrine subtypes. Marked cells are distinguished
from their neighbors with neuropeptide antisera. (E,F) 10 days following
eclosion. (G,H) Challenge with Pe. Scale bar: 10 µm.
DEVELOPMENT
Table 1. Distribution of neuropeptides in Prospero+ cells
RESEARCH ARTICLE
Development (2015) 142, 654-664 doi:10.1242/dev.114959
Table 2. Directed cell lineage tracing of enteroendocrine cell subtypes
AstA>Trace
Tk>Trace
Timepoint (days)
DH31+β-Gal+
β-Gal+
DH31+β-Gal+/β-Gal+ ratio (%)
n (guts)
0
4
10
8*
0
0
1
0
0
54
197
161
0
0
<1
0
5
4
7
6
Timepoint (days)
AstA+β-Gal+
β-Gal+
AstA+β-Gal+/β-Gal+ ratio (%)
n (guts)
0
4
10
8*
0
0
0
1
0
146
244
161
0
0
0
<1
6
7
8
7
* 24 h Pe challenge.
Class I and class II enteroendocrine cells are lineally related
The presence of enteroendocrine cells with distinct neuropeptide
expression profiles raised the question whether such cells might
arise from a common cell lineage or whether they are lineally
distinct (Fig. 3A). To distinguish these possibilities, we
conducted cell lineage-tracing studies. Wild-type lineages were
labeled using the MARCM system (Lee and Luo, 1999) and
stained to identify enteroendocrine subtypes. We focused first on
region 2 of the posterior midgut, where simultaneous detection of
class I and II enteroendocrine cells was possible. Here, marked
lineages were found to span both class I and II endocrine cell
subtypes (Fig. 3B-E). To further characterize the distribution of
endocrine cell subtypes, the number of AstA+ and Tk+ cells per
clone was scored for each marked lineage recovered (Table 3).
The total number of AstA+ and Tk+ cells per clone ranged from 0
to 4. In every case in which a marked lineage contained two or
more endocrine cells, we scored the presence of both AstA+ and
Tk+ enteroendocrine cells. We conclude that in region 2 of the
posterior midgut, class I and II enteroendocrine cell pairs arise
from a common cell lineage.
Characterization of wild-type lineages was also performed in
region 1 of the posterior midgut, yielding similar results. However,
analysis of the region 1 was limited by the lack of markers to
simultaneously distinguish class I and class II enteroendocrine
cells (e.g. Fig. 1B and Fig. 3F,G; supplementary material
Table S1). Thus, we scored the fraction of Pros+ cells that were
also AstB+ (Fig. 3H,I; Table 4). We predicted that half of the Pros+
enteroendocrine cells present in each clone would be AstB+. We
found that marked lineages recovered in region 1 contained
between 0 and 3 Pros+ cells. However, among the clones that
contained two Pros+ cells, 48% fit this criterion (Table 4). In
summary, nearly half of the wild-type clones observed in region 1
were consistent with a model in which class I and II cells arise
from a common lineage, as shown for region 2. It is likely that the
method of clone scoring used for analysis in region 1 enriched for
a set of early clones containing less differentiated cells (i.e. Pros+
only). By contrast, the analysis in region 2 was biased toward late
clones identified by the presence of highly differentiated
endocrine cells (i.e. AstA+, Tk+). Taken together, analysis of
wild-type cell lineages supports a general model in which class I
and class II endocrine cell pairs of the posterior midgut arise from
a common stem cell lineage.
Class II enteroendocrine cells arise from a Su(H)-Gal4
expressing progenitor
The choice of cell fate between absorptive enterocytes and secretory
endocrine cells in the midgut is controlled by the Notch signaling
pathway (Bardin et al., 2010; Beebe et al., 2010; Micchelli and
Perrimon, 2006; Ohlstein and Spradling, 2006, 2007; Perdigoto
et al., 2011). Activation of Notch signaling in stem cell daughters,
called enteroblasts, promotes enterocyte formation, whereas reduced
Notch signaling leads to the production of ectopic enteroendocrine
cells. This model predicts that directed cell lineage-tracing of
progenitors transducing high levels of Notch signaling should label
only enterocytes. We tested this prediction using a pulse/chase
experiment and the conditional Su(H)-Gal4, tub-Gal80TS strain to
label a fraction of adult cell lineages with actin5C FRT stop FRT
lacZnuc; UAS-flp under both baseline conditions and following
environmental challenge (Table 5; Fig. 4A-D). As predicted,
enterocytes were readily labeled by this procedure when cell
marking was initiated, following a shift to the non-permissive
temperature (Fig. 4A-D). However, quite unexpectedly, we also
recovered labeled Tk+ class II endocrine cells at a frequency of
0.65% under baseline conditions (Table 5; Fig. 4A). Similarly, we
observed labeled DH31+ cells, a second marker correlated with class
II endocrine fate, following environmental challenge (Table 5;
Fig. 4C). By contrast, Tk+ class II cells were never labeled in
unshifted control experiments; nor were AstA+ class I cells labeled at
657
DEVELOPMENT
the AstA-Gal4 to the actin5C FRT stop FRT lacZnuc; UAS-flp lineage
tracer (AstA>Trace; Fig. 2A,C,E,G). The AstA-Gal4 driver line was
first detected in the adult midgut 4 days following eclosion (Fig. 2A,C).
Ten days following eclosion, midguts were stained to identify DH31+
cells, a marker of class II enteroendocrine cells in region 2 of the
posterior midgut. We observed that cells labeled by AstA-Gal4 were
DH31−, although DH31 staining was readily detected in adjacent
endocrine cells (Fig. 2E). In a complementary experiment, we labeled
class II enteroendocrine cells in region 2 using Tk-Gal4 (Fig. 2B,D,F,
H). The Tk-Gal4 driver line was also first detected in the adult midgut
4 days following eclosion (Fig. 2B,D). Ten days following eclosion,
we stained for AstA+, a neuropeptide that marks class I enteroendocrine
cells in region 2. We observed that cells labeled by Tk-Gal4 were
AstA−, although AstA+ cells were readily detected in adjacent
endocrine cells (Fig. 2F). To further document these observations we
counted the number of lacZ+, DH31+ and lacZ+, AstA+ cells present in
adult midguts at 0, 4 and 10 days after eclosion, and again 24 h
following challenge with the Gram-negative enteric pathogen
Pseudomonas entomophila (Pe) (Fig. 2G,H; Table 2). This analysis
shows that AstA-Gal4 and Tk-Gal4 driver lines mark endocrine cell
subtypes that remain DH31− or AstA− during adult homeostasis, as
well as following pathogenic challenge. Taken together, these
experiments suggest that the class I and class II neuropeptide profiles
remain stable in enteroendocrine cells during adult gastrointestinal
homeostasis as well as following pathogenic challenge. Thus,
neuropeptide expression provides a reliable means of distinguishing
distinct enteroendocrine cell subtypes in the adult midgut.
RESEARCH ARTICLE
Development (2015) 142, 654-664 doi:10.1242/dev.114959
Table 3. Analysis of neuropeptides in region 2 cell lineages
WT
0 AstA+
1 AstA+
2 AstA+
≥3 AstA+
0 Tk+
1 Tk+
2 Tk+
≥3 Tk+
31
5
0
0
8
10
2
0
0
1
1
0
0
0
0
0
N55e11
0 AstA+
1 AstA+
2 AstA+
≥3 AstA+
89
0
0
0
0
0
0
0
0
0
0
0
29
0
0
0
+
0 Tk
1 Tk+
2 Tk+
≥3 Tk+
signaling in the generation of regional diversity among midgut
endocrine cell subtypes.
Class II enteroendocrine cells are established by Notch/
Delta signaling
Table 4. Analysis of neuropeptides in region 1 cell lineages
0 Pros+
1 Pros+
2 Pros+
3 Pros+
0 AstB
1 AstB+
2 AstB+
3 AstB+
92
–
–
–
15
9
–
–
27
28
3
–
2
2
3
0
Genotype
AstB+Pros+
Pros+
AstB+Pros+/
Pros+ ratio (%)
n (guts)
WT
N55e11
51
149
161
249
32
60
18
7
WT
+
the non-permissive experimental temperature (Table 5). This
suggested that Notch signaling might be active specifically in the
class II enteroendocrine cell lineage. Consistently, Notch pathway
reporters were also detectable in Tk+ cells (Fig. 4E-H). From this, we
conclude that DH31+ and Tk+ class II enteroendocrine cells arise
from a Su(H)-Gal4-expressing progenitor. These findings prompted
a series of experiments to directly test the function of Notch
658
DEVELOPMENT
Fig. 3. Class I and II enteroendocrine cells arise from a common lineage.
(A) Wild-type class I ( pink) and class II (red) endocrine cells are present in
pairs distributed among enterocytes (gray). Alternate lineage models are
distinguished by cell-labeling studies (green). (B-I) Wild-type MARCM lineages
5 days after induction, dashed line. (B-E) Lineages spanning class I and II
entoendocrine cells, region 2. Anti-AstA, red; anti-Tk, green. Distinct endocrine
subtypes arise from a common lineage. (F-I) Marked lineages, region 1. AntiPros, white. (G) Anti-AstC, red. (I) Anti-AstB, red. Scale bar: 10 µm.
Notch loss in the intestinal stem cell lineage leads to the production
of ectopic Pros+ cells, suggesting the formation of enteroendocrine
tumors (Micchelli and Perrimon, 2006; Ohlstein and Spradling,
2006). Yet, the full extent of endocrine cell differentiation in these
tumors is unclear. Therefore, we first confirmed and extended
previous studies by generating null Notch clones and monitoring the
endocrine fate using an expanded panel of endocrine cell markers,
including the pan-endocrine marker Pros; the ELKS family protein
Bruchpilot (Brp), which supports calcium-dependent secretory
vesicle release; and cell morphology. In addition, we examined the
levels of Peptidylglycine α-hydroxylating monoxygenase (Phm).
Phm is an enzyme that catalyzes the rate-limiting step in C-terminal
α-amidation, a necessary step for the production of active
neurosecretory peptides from pro-forms of the peptides. We found
that Notch loss led to ectopic cells displaying the characteristic
morphology of mature enteroendocrine cells with polarized cellular
processes projecting toward the gut lumen (Fig. 5A). In addition,
these cells were Pros+, Brp+ and Phm+ (Fig. 5B-D). Thus, we
conclude that the ectopic cells that arise in Notch mutant lineages
exhibit multiple features of well-differentiated endocrine tumors.
By contrast, Notch loss was found to differentially affect the
distribution of specific secretory neuropeptides. We examined
distinguishing markers for both class I and class II enteroendocrine
cells in region 2 of the posterior midgut (Fig. 5E-T). Notch clones in
region 2 led to loss of both DH31+ and Tk+, whereas AstA+ cells
remained present in the lineage (Fig. 5E-L). We quantified this
phenotype and found that, in every case in which two or more
endocrine cells were present, clones retained AstA+ cells but lost
Tk+ cells (Table 3). Similar results were observed using additional
class I and class II markers in the anterior midgut, copper cell region
and region 1 of the posterior midgut (Fig. 5M-T; supplementary
material Fig. S4; Table S4). Importantly, these findings were
RESEARCH ARTICLE
Development (2015) 142, 654-664 doi:10.1242/dev.114959
Table 5. Analysis of Su(H)>Trace
+
Peptide
Peptide β-Gal
Tk
Tk
AstA
Pros
DH31
DH31
0
11
0
5
8
19
+
β-Gal
9
1690
1141
731
1214
2693
+
Peptide+β-Gal+/
β-Gal+ ratio (%)
‘Chase’
(days)
n
(guts)
0
0.65
0
0.68
0.71
0.71
10*
10-12‡
10-12‡
10§
14§
15¶
6
5
5
5
10
10
*Unshifted, unchallenged; ‡shifted, unchallenged; § shifted, challenged (heat
stress); ¶shifted, challenged (Pe).
independently confirmed using conditional Notch knockdown
(supplementary material Fig. S5A-J). We note that, whereas Notch
loss affected local neuropeptide profiles among endocrine pairs, it
was not associated with changes in underlying regional identity in
neuropeptide expression (supplementary material Fig. S5K). Thus,
Notch signaling is necessary to specify the class II endocrine cell
subtype in the adult midgut, without affecting neuropeptide
processing enzymes or the cellular machinery implicated in
secretory vesicle release.
The Notch pathway can be activated by Delta and Serrate ligands.
As in the case of wild type, marked lineages lacking Serrate
produced both AstA+ and Tk+ endocrine cells (Fig. 6A,B).
However, loss of Delta was necessary for the production of Tk+
enteroendocrine cells (Fig. 6C-F). We note that Delta clones
exhibited greater heterogeneity in the progenitor marker esg-lacZ
than Notch, which correlated with heterogeneity of the neuropeptide
phenotype (Fig. 6G,H; supplementary material Fig. S6). Thus,
Delta, but not Serrate, is necessary for establishing class II
enteroendocrine cells during adult midgut homeostasis.
Notch function is dispensable for endocrine cell
maintenance
Notch activation is sufficient to establish class II
enteroendocrine cells
We tested whether Notch signaling is also sufficient to establish
local differences in endocrine cell subtype. In a first test, we
examined the consequences of global Notch activation on
endocrine cell identity by monitoring DH31+ cells at 6 and 24 h
following induction of an hs-Notchintra transgene (supplementary
material Fig. S8). To distinguish newly formed cells from
previously differentiated cells, we performed the experiment in
an esg>GFP background. As previously reported, we readily
observed that Notch pathway activation promoted differentiation
of esg>GFP + into large enterocytes 24 h following induction
(supplementary material Fig. S8A,C). We also observed a low
frequency of DH31+ doublets under these conditions, which are
not typically seen in wild-type cell lineages (supplementary
Fig. 4. Class II endocrine cells arise from a Su(H)-Gal4 progenitor.
(A-D) Conditional lineage tracing of Su(H)-Gal4TS cells using actin5C>lacZ;
UAS-flp, 12 days after induction. (A,B) Cellular products of Su(H)-Gal4TS
labeling, red/white; anti-Tk, green. Arrows indicate Tk+ cells derived from Su
(H)-Gal4TS cells. Asterisks show unlabeled Tk+ cells. (B) Single channel.
(C,D) Cellular products of Su(H)-Gal4TS, 7 days following exposure to Pe, red/
white; anti-DH31, green. Arrows indicate DH31+ cells derived from Su(H)Gal4TS cells. Asterisks show unlabeled DH31+ cells. (D) Single channel.
(E-H) Markers of Notch signaling in Tk+ cells, red. Arrows indicate Tk+ cells.
Asterisks show unlabeled Tk+ cells. (E,F) Su(H)GBE-lacZ reporter. Note
lower levels of the reporter in the Tk+ cells. (E) esg>GFP, green; anti-Tk, red.
(F) Su(H)GBE-lacZ, white. (G,H) Dl-lacZ labeling, red/white; anti-Tk, green.
(H) Single channel. Scale bars: 10 µm.
material Fig. S8C,D; Table 3). This observation is consistent with
the idea that Notch activation is sufficient to promote class II
endocrine cell fate. The low frequency phenotype is predicted
because endocrine progenitors are an order of magnitude rarer
than epithelial progenitors and because, under baseline
conditions, proliferation of all progenitors is infrequent. We
reasoned that, if progenitor pools are first increased, using genetic
means, and then Notch is ectopically activated, the phenotype
should be detected with greater frequency. Therefore, in a second
test of Notch sufficiency, conditional Presenillin knockdown with
esg-Gal4; UAS-GFP, tub-Gal80TS was followed by induction of
659
DEVELOPMENT
Notch might function only in the establishment of endocrine
diversity. Alternatively, Notch might function in both the
establishment and maintenance of endocrine diversity. To
distinguish these possibilities, we pursued a conditional, cell typespecific genetic loss-of-function approach. Conditional Notch
knockdown was achieved in extant class II enteroendocrine cells
using the UAS-GFP, tub-Gal80TS; Tk-Gal4 strain. However, despite
strong Notch knockdown with this construct (e.g. supplementary
material Fig. S5), no change in either DH31+ or AstA+ cells was
detected (supplementary material Fig. S7A-D). This suggests that
Notch signaling is dispensable for maintenance of neuropeptide
expression in enteroendocrine cells.
RESEARCH ARTICLE
Development (2015) 142, 654-664 doi:10.1242/dev.114959
the hs-Notchintra transgene. Under these conditions we observed a
rapid, robust and significant increase in the number of Pros+,
DH31+ cells (Fig. 7A-F). Finally, we tested whether Notch
activation is sufficient to reprogram fully differentiated
enteroendocrine cell fate from class I to class II. However,
conditional activation of Notchintra in AstA+ cells was insufficient
to induce DH31 peptide in these cells (Fig. 7G,H). Altogether,
these results suggest that Notch signaling is both necessary and
sufficient to specify the class II endocrine cell subtype during
660
adult midgut homeostasis. However, once a class I endocrine cell
is established, ectopic Notch activation is insufficient to
reprogram subtype identity.
DISCUSSION
The barrier epithelium of the gut is a unique biological interface. It
is here that a complex stream of continuously changing luminal
stimuli are presented to the organism and must be converted into
appropriate physiologic and neuromodulatory states. The cells of
DEVELOPMENT
Fig. 5. Notch is necessary to establish class II endocrine cells. (A-T) Mosaic analysis of Notch using MARCM; representative clones are highlighted by dotted
lines. (A-D) General endocrine markers, 7 days after induction. (A) Anti-GFP, green; phalloidin, red, staining F-actin (muscle). (B) Anti-Pros, red. (C) Anti-Brp, red.
(D) Anti-Phm, red. (E-T) Distribution of neuropeptides in Notch clones, 5 days after induction. Clones on left in paired micrographs, green. Asterisks, internal
controls. (E-L) Region 2. (F,H) Anti-AstA, red; anti-Tk, green. Endocrine cell morphology is evident in cross-section. (I-M,O,Q,S) Anti-Pros, red. (J,L) Anti-DH31,
green. Note the absence of DH31+ cells within the clone. (M-T) Region 1. (N,P) Anti-AstC, red. (R,T) Anti-AstB, red. Scale bars: 10 µm.
RESEARCH ARTICLE
Development (2015) 142, 654-664 doi:10.1242/dev.114959
indicates that >95% of Pros+ cells express one or more distinct
neuropeptides. We predict that the remaining ∼5% of Pros+ cells
correspond either to newly formed endocrine cells in the process of
differentiating, dividing progenitors (see Biteau and Jasper, 2014;
Zielke et al., 2014) or differentiated endocrine cells expressing
peptides other than those tested.
The mapping data presented here confirm and extend a rapidly
growing body of work, which has provided essential insight into the
distribution of regulatory neuropeptides in Drosophila (Buchon
et al., 2013; Chintapalli et al., 2007; Marianes and Spradling, 2013;
Reiher et al., 2011; Veenstra et al., 2008; Veenstra and Ida, 2014).
Overall, these reports are in general agreement, despite significant
differences in approach, methodology and scope.
Our systematic, combinatorial mapping strategy provides high
cellular and temporal resolution of neuropeptide distribution in vivo
under baseline conditions. A pairwise comparison of available
antisera with Gal4 driver lines suggests that patterns of neuropeptide
expression and protein distribution are similar, if not identical, in the
adult midgut. Importantly, these validated reagents provide
powerful and specific new tools for manipulating the endocrine
system, as we demonstrate. Finally, directed cell lineage analysis
shows that neuropeptide profiles are stable features of endocrine
cells during both normal homeostasis and following challenge with
the enteric Gram-negative pathogen Pe.
In mammals, the classical view of the diffuse endocrine system
is the ‘one cell-one hormone’ dogma (Engelstoft et al., 2013;
Schonhoff et al., 2004). However, recent studies in mouse and
human have challenged this perspective (Egerod et al., 2012; Habib
et al., 2012; Sei et al., 2011). It is now clear that the mammalian GI
tract exhibits not only regional diversity but also local diversity in
the combinatorial expression of neuropeptides in single endocrine
cells of the diffuse endocrine system. For example, in the murine
intestine, endocrine cells in the crypt express multiple endocrine
hormones, but endocrine cells on the villus do not (Sei et al., 2011).
Interestingly, in mammals, peptides that are coexpressed also appear
to have overlapping or coordinated functions. Thus, broad
coexpression of secretory neuropeptides is a conserved feature of
the diffuse endocrine system among metazoans.
the diffuse endocrine system represent an early, if not the primary,
level at which these changes are sensed and broadcast. We have used
Drosophila as a model system to address the molecular mechanisms
by which cellular diversity in the diffuse endocrine system is
homeostatically maintained.
Organization of the adult enteroendocrine system
Endocrine cells can be characterized by the neuropeptides they
secrete. From this perspective, the adult diffuse endocrine system
exhibits both regional and local organization. We show that
neuropeptide expression in the adult midgut is restricted to cells
positive for Pros, a pan-endocrine marker defining the extent of the
diffuse endocrine system. Among Pros+ cells, distinct segmental
regions of neuropeptide expression along the A/P axis are evident.
Within each region, local differences in combinatorial neuropeptide
distribution distinguish class I and II endocrine cells. Our analysis
Class I and class II endocrine subtypes arise from a common
stem cell lineage
The diffuse endocrine system differs from the glandular endocrine
system in that it must be continuously renewed. The combinatorial
differences among endocrine neuropeptides described here provide
an experimental basis to address how diversity is maintained in the
diffuse endocrine system. We distinguish two possible models and
directly demonstrate that endodermal stem cell lineages give rise to
both class I and class II endocrine cells from a single lineage. Thus,
local diversity within the diffuse endocrine system is controlled by
individual stem cell lineages situated at distinct positions along the
length of the GI tract. Developmental studies have characterized
the stepwise process of endocrine cell development (Micchelli et al.,
2011; Takashima et al., 2011). Cell divisions occurring during late
pupal stages initially establish pairs of gut enteroendocrine cells.
Moreover, recent studies have implicated the gut neurosecretory
peptide Bursicon as a key peptide in the complex behavioral process
of pupal eclosion (Scopelliti et al., 2014). The fact that cellular
diversity in the diffuse endocrine system is actively maintained by
adult stem cell lineages suggests that endocrine cells have broad
functional significance in maintaining many aspects of metabolic
homeostasis throughout the course of adulthood. These crucial
functions remain to be fully characterized.
661
DEVELOPMENT
Fig. 6. Delta is necessary to establish class II endocrine cells.
(A-H) Analysis of Delta and Serrate lineages using MARCM, dotted line.
(A,B) Serrate clone, 5 days after induction. (B) Anti-AstA, red; anti-Tk, green.
(C,D) Delta clone, 5 days after induction. (D) Anti-AstA, red; anti-Tk, green.
Asterisk shows Tk+ cell outside clone. (E,F) Delta clone, 10 days after
induction. (F) Anti-AstA, red. (G,H) Delta clone, 10 days after induction.
(H) Anti-AstA, red; esg-lacZ, white. Scale bars: 10 µm.
RESEARCH ARTICLE
Development (2015) 142, 654-664 doi:10.1242/dev.114959
Controlling cell type in the endocrine lineage
Simultaneous detection of class I and II endocrine cells in marked
endodermal lineages suggested that endocrine cell fate is controlled
by signaling events within individual stem cell lineages. Su(H)-Gal4
662
progenitors were found to give rise to class II, but not class I,
endocrine cells, under a wide variety of experimental conditions that
included baseline homeostasis and different environmental
challenges, suggesting a key role for the Notch signaling pathway
in generating enteroendocrine diversity. We note that this finding
differs significantly from another report, which concluded that
endocrine cells do not arise from a Su(H)Gal4+ progenitor (Biteau
and Jasper, 2014). For example, in one experiment, we detected five
Pros+ cells among a total of 731 labeled cells (10 days following
induction, five posterior midguts), whereas Biteau and Jasper report
no Pros+ cells among 418 (4 days following induction, eight guts).
This disparity is therefore most likely explicable by the short chase
period used and the relative rarity of endocrine progenitors in the gut.
Our subsequent functional characterization clearly showed that
Notch signaling is both necessary and sufficient to control cell
diversity in the diffuse endocrine system. Although Notch loss leads
to well-differentiated endocrine tumors, they exhibit specific changes
in the type of endocrine cells produced. By contrast, Notch function
was dispensable in controlling underlying regional identity along the
length of the gut. Gaining insight into the process underlying regional
specification represents an interesting line of future investigation.
Although our analysis focused on the posterior midgut, where peptide
diversity is greatest, our studies also indicate that similar requirements
for Notch in class II endocrine cells exist along the entire length of the
GI tract. Finally, targeted manipulation of Notch signaling in
differentiated endocrine cells failed to alter endocrine identity,
consistent with a model in which Notch signaling functions
specifically in endocrine progenitors. Thus, Notch signaling plays
at least two separable roles in controlling cell fate within the gut stem
cell lineages: a previously known requirement in specifying
absorptive enterocytes, and the newly described role in controlling
the diversity of secretory enteroendocrine cells along the length of the
adult midgut demonstrated here (Fig. 7I, yellow).
Non-autonomous consequences of endocrine tumors are implicit
by the very nature of the peptides these cells secrete. Indeed,
carcinoid tumors are neuroendocrine malignancies that secret
hormones, which cause debilitating symptoms in patients. In
addition, loss of endocrine cell subtypes has been associated with
metabolic disruptions, including lipid absorption and glucose
homeostasis (Mellitzer et al., 2010). Our study suggests that these
DEVELOPMENT
Fig. 7. Notch activation is sufficient to establish class II enteroendocrine
cells. (A-F) Analysis of Notch pathway activation on endocrine cell fate,
8 days following conditional Presenilin knockdown using esgTS. Adults were
shifted to the non-permissive temperature for 7 days to induce Presenilin
knockdown and then stained against DH31+ cells 24 h later, in the presence
or absence of global Notch pathway activation. (A,C,E) Anti-Pros, red.
(A-D) Anti-DH31, green. (A,B) Presenilin knockdown leads to endocrine
hyperplasia and a reduction in DH31+ cells. (C,D) Presenilin knockdown
leads to endocrine hyperplasia and a rapid increase in DH31+ cells following
global Notch activation. Inset, DH31+ cells exhibit endocrine morphology.
(E) Notch activation is sufficient to promote differentiation of esg+ cells to
large enterocytes, green. (F) Quantitation. The average number of DH31+
cells per gut scored for each genotype (n=7-12 guts per genotype, error bars
represent s.e.m.; ***P<0.0001). (G-H) Analysis of Notch pathway activation in
fully differentiated class I endocrine cells using AstA-Gal4TS. Adults were
shifted to the non-permissive temperature for 7 days. AstA>GFP, green;
Anti-DH31, red. (G) Controls shifted to non-permissive temperature show
conditional expression of GPF. (H) Notch overexpression in AstA+ cells does
not cause expression of the class II marker DH31. (I) Schematic cell lineage
diagram. Undifferentiated stem cell daughters (enteroblasts) give rise to
either the secretory (enteroendocrine) or absorptive (enterocyte) cell fate.
Branch points indicate Notch-dependent cell fate decisions. Progenitors,
green; differentiated cells, pink, red and blue; Notch-dependent cell fates,
yellow. ISC, intestinal stem cell; EB, enteroblast. Scale bars: 20 µm.
RESEARCH ARTICLE
MATERIALS AND METHODS
Fly stocks
The following fly stocks were used: from Bloomington Stock Center: w1118
(#39352; wild-type control); Canton-S (#1; wild-type control); w1118;
GMR65D06-Gal4 (#39352; AstA-Gal4, this study); w1118; GMR61H08Gal4 (#39283; AstC-Gal4, this study); w1118; GMR57F07-Gal4 (#46389;
DH31-Gal4, this study); w1118; GMR61H07-Gal4 (#39282; called Tk-Gal4,
this study); w1118; GMR50A12-Gal4/TM3 (#47618); w1118; GMR42C06Gal4 (#50150); w1118; GMR46B08-Gal4 (#47361); pros10419, ry506(#11744;
pros-lacZ); y,w; UAS-GFP.nls (#4775); w; FRT82B, hsπM (#2004; wild type);
UAS-dicer 2 (#24648); tub-Gal80TS(#7108); tub-Gal80TS (#7017); ry506
P{PZ} Dl05151/TM3 (#11651; Dl-lacZ); w, hsflp, tub-Gal80, FRT19A (#5133);
y,w; esgk606 (#10359); UAS-NRNAi (#7078); UAS-psnRNAi (#27681); UASNintra (#52008). w1118; npf-Gal4 (Wu et al., 2003); Fer1-GFP (Flytrap
#G00188); w; actin5C FRT stop FRT lacZnuc; UAS-flp (actin5C>lacZ; Struhl
and Basler, 1993); y,w, UAS-GFP, hsflp; tub-Gal4, FRT82B, tub-Gal80/TM6B;
Su(H)GBE-Gal4 (Zeng et al., 2010); Su(H)GBE-lacZ (Furriols and Bray,
2001); esg-Gal4, UAS-GFP (Micchelli and Perrimon, 2006); N55e11 FRT19A/
FM7 (Kidd et al., 1983); w, hsflp, tub-Gal80, FRT19A; UAS-GFP, UAS-lacZ;
tub-Gal4; w; esg-Gal4, UAS-GFP, tub-Gal80TS (Micchelli and Perrimon,
2006; esgTS); Dlrev10 FRT82B (Haenelin et al., 1990); Serrx106 FRT82B (Thomas
et al., 1991); y,w, UAS-GFP, hsflp; esgk606; tub-Gal4, FRT82B, tub-Gal80/
TM6B; hs-Nintra (Struhl et al., 1993). For additional information, see
http://flybase.org.
Characterization of neuropeptide distribution
Characterization of adult midgut neuropeptide distribution was carried
out in wild-type flies (w1118 or Canton-S). We present the results of
neuropeptide distribution in newly eclosed flies selected from low-density
cultures, aged for 5 days at 25°C and dissected directly in fixative.
Directed cell lineage tracing
Directed tracing of adult endocrine cell lineages was performed by crossing
specific Gal4 driver lines to the w; actin5C FRT stop FRT lacZ; UAS-flp
lineage tracer and culturing at 25°C. Neuropeptide Gal4 lineage tracing
experiments were analyzed as newly eclosed virgins (day 0) and then 4 and
10 days after eclosion under baseline conditions; they were also analyzed
8 days after eclosion following a 24 h Pe (OD5) infection. Conditional lineage
tracing was performed by combining either neuropeptide Gal4 or Su(H)-Gal4
lines with tub-Gal80TS. The resultant flies were then crossed to the w; actin5C
FRT stop FRT lacZnuc; UAS-flp lineage tracer. Crosses were established and
cultured at 18°C until adulthood. F1 female progeny were aged 3-5 days at 18°
C and were then shifted to 29°C for defined periods. Su(H)-Gal4TS lineagetracing experiments were analyzed 10-12 days following the shift to 29°C and
compared with unshifted controls grown at 18°C for 10 days. Su(H)-Gal4TS
lineage-tracing experiments were also analyzed following challenge by heat
stress or ad libitum exposure to Pe. For heat challenge, adults were grown at
29°C for 5 days, heat-shocked (37°C water bath, 45 min in the morning, plus
45 min at night) and cultured for an additional 5 days (10 day ‘chase’). An
additional heat challenge protocol was also tested. Adults were grown at 29°C
for 7 days, heat-shocked (37°C water bath, 45 min in the morning, plus 45 min
at night) and cultured for an additional 7 days (14 day ‘chase’). For bacterial
challenge, adults were grown at 29°C 7 days, exposed to Pe (24 h on Pe OD20laced food vials at 29°C) and cultured for an additional 7 days (15 day
‘chase’).
Mosaic analysis
Positively marked cell lineages were generated in female midguts using
the MARCM system. Fly crosses were cultured on standard media
supplemented with yeast paste at 25°C. Newly eclosed females of the
appropriate genotype were aged 3-5 days prior to clone induction. Clones
were induced by placing vials in a 37°C water bath for 30-40 min. Flies were
heat-shocked 2-3 times within a 24 h period.
Pe infection
Flies were infected ad libitum with Pe. Infected flies were fed on food
supplemented with 0.5 ml of Pe at OD5-OD20 in 5% sucrose. Mock-infected
flies were placed on food supplemented with 0.5 ml 5% sucrose. Flies were
infected with Pe for 24 h and subsequently maintained at 29°C throughout
the course of the experiment.
Conditional manipulation of Notch pathway
To conditionally target progenitor cells or differentiated endocrine cell
populations in the adult midgut, esg-Gal4, Tk-Gal4 or AstA-Gal4 was first
combined with tub-Gal80TS and then crossed to UAS-NotchRNAi, UASPresenilinRNAi or UAS-Nintra. Crosses were established and cultured at 18°C
until adulthood. F1 female progeny of the appropriate genotype were aged
3-5 days at 18°C and then shifted to 29°C for 7 days. Global activation of the
Notch pathway using the hs-Nintra transgene was accomplished by providing
two 30-min heat shocks at 37°C, one in the morning and the second at night.
Histology
Generation of whole-mount samples for immunostaining was performed as
previously described (Micchelli, 2014). Briefly, adult females were in fixed
0.5× PBS and 4% EM-grade formaldehyde (Polysciences) overnight,
washed in 1× PBS, 0.1% Triton X-100 (PBST) for a minimum of 2 h, and
incubated in primary antisera (see below) overnight. Samples were washed
and incubated in secondary antibodies for 3 h, washed again in PBST and
mounted in Vectashield+DAPI. All steps were completed at 4°C.
Antisera
Chicken anti-GFP (Abcam, #ab13970; 1:10,000); mouse anti-β-Gal (DSHB,
#40-1a; 1:100); mouse anti-Pros (DSHB, #MR1A; 1:100); mouse anti-Brp
(DSHB, #nc82; 1:10); mouse anti-Cut (DSHB, #2B10; 1:100); mouse antiAstA (DSHB, #5F10; 1:20); rabbit anti-β-Gal (Cappel, #08559761; 1:4000);
rabbit anti-Tk (1:500, a generous gift from Dr Dick Nässel, Stockholm
University, Sweden) rabbit anti-Npf (1:750, a generous gift from Dr Paul
Taghert, Washington University, USA); rabbit anti-Phm (1:250, a generous
gift from Dr Paul Taghert); rabbit anti-AstB (1:500, a generous gift from Drs
Paul Taghert and Jan Veenstra, Université de Bordeaux, France); rabbit antiAstC (1:500, a generous gift from Dr Jan Veenstra); rabbit anti-DH31 (1:500,
a generous gift from Dr Jan Veenstra). Alexa Fluor-conjugated secondary
antibodies (Molecular Probes, #A-11039, #A-11029, #A-11031, #A-21236,
#A-11034, #A-11036, #A-21245; 1:2000).
Microscopy and imaging
Samples were analyzed on a Leica DM5000 or Leica TCS SP5 confocal
microscope. Images were processed for brightness and contrast in
Photoshop CS (Adobe).
Acknowledgements
We wish to acknowledge the collegiality of members of the fly community who freely
shared reagents. We are indebted to Drs D. Nä ssel, P. Taghert and J. Veenstra for
generous gifts of very limited antibodies, without which this study would simply not
have been possible. We also acknowledge Joseph Burclaff for initial screening
and characterization of neuropeptide Gal4 lines. Finally, we thank members of the
Micchelli lab for constructive discussions and comments on the manuscript.
Competing interests
The authors declare no competing financial interests.
Author contributions
R.B.-E. performed the experiments. R.B.-E. and C.A.M. wrote the manuscript.
Funding
This work was supported by grants from the American Cancer Society and the
National Institutes of Health [NIH RO1 DK095871 to C.A.M.]. Deposited in PMC for
release after 12 months.
663
DEVELOPMENT
effects might result from either the loss of specific peptides or
overabundance of others in the diffuse endocrine system. Thus,
further insight into how endocrine peptides are produced and how
secretion is controlled might have important consequences for
understanding tumor pathophysiology and for developing targeted
treatments for metabolic disease.
Development (2015) 142, 654-664 doi:10.1242/dev.114959
RESEARCH ARTICLE
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.114959/-/DC1
References
Montagne, C. and Gonzalez-Gaitan, M. (2014). Sara endosomes and the
asymmetric division of intestinal stem cells Development 141, 2014-2023.
Nä ssel, D. R. and Wegener, C. (2011). A comparative review of short and long
neuropeptide F signaling in invertebrates: any similarities to vertebrate
neuropeptide Y signaling? Peptides 32, 1335-1355.
Ohlstein, B. and Spradling, A. (2006). The adult Drosophila posterior midgut is
maintained by pluripotent stem cells. Nature 439, 470-474.
Ohlstein, B. and Spradling, A. (2007). Multipotent Drosophila intestinal stem cells
specify daughter cell fates by differential notch signaling. Science 315, 988-992.
Perdigoto, C. N., Schweisguth, F. and Bardin, A. J. (2011). Distinct levels of Notch
activity for commitment and terminal differentiation of stem cells in the adult fly
intestine. Development 138, 4585-4595.
Piper, M. D. W., Blanc, E., Leitao-Gonçalves,
R., Yang, M., He, X., Linford, N. J.,
̃
Hoddinott, M. P., Hopfen, C., Soultoukis, G. A., Niemeyer, C. et al. (2014).
A holidic medium for Drosophila melanogaster. Nat. Methods 11, 100-105.
Reiher, W., Shirras, C., Kahnt, J., Baumeister, S., Isaac, R. E. and Wegener, C.
(2011). Peptidomics and peptide hormone processing in the Drosophila midgut.
J. Proteome Res. 10, 1881-1892.
Reimann, F., Tolhurst, G. and Gribble, F. M. (2012). G-protein-coupled receptors
in intestinal chemosensation. Cell Metabol. 15, 421-431.
Schonhoff, S. E., Giel-Moloney, M. and Leiter, A. B. (2004). Minireview:
development and differentiation of gut endocrine cells. Endocrinology 145,
2639-2644.
Scopelliti, A., Cordero, J. B., Diao, F., Strathdee, K., White, B. H., Sanson, O. J.
and Vidal, M. (2014). Local control of intestinal stem cell homeostasis by
enteroendocrine cells in the adult Drosophila midgut. Curr. Biol. 24, 1199-1211.
Sei, Y., Lu, X., Liou, A., Zhao, X. and Wank, S. A. (2011). A stem cell markerexpressing subset of enteroendocrine cells resides at the crypt base in the small
intestine. Am. J. Physiol. Gastrointest. Liver Physiol. 300, G345-G356.
Strand, M. and Micchelli, C. A. (2011). Quiescent gastric stem cells maintain the
adult Drosophila stomach. Proc. Natl. Acad. Sci. USA 108, 17696-17701.
Struhl, G. and Basler, K. (1993). Organizing activity of wingless protein in
Drosophila. Cell 72, 527-540.
Struhl, G., Fitzgerald, K. and Greenwald, I. (1993). Intrinsic activity of the Lin-12
and Notch intracellular domains in vivo. Cell 74, 331-345.
Takashima, S., Adams, K. L., Ortiz, P. A., Ying, C. T., Moridzadeh, R., YounossiHartenstein, A. and Hartenstein, V. (2011). Development of the Drosophila
entero-endocrine lineage and its specification by the Notch signaling pathway.
Dev. Biol. 353, 161-172.
Thomas, U., Speicher, S. A. and Knust, E. (1991). The Drosophila gene Serrate
encodes an EGF-like transmembrane protein with a complex expression pattern in
embryos and wing discs. Development 111, 749-761.
Van Loy, T., Vandersmissen, H. P., Poels, J., Van Hiel, M. B., Verlinden, H. and
Broeck, J. V. (2010). Tachykinin-related peptides and their receptors in
invertebrates: a current view. Peptides 31, 520-524.
Veenstra, J. A. (2009). Peptidergic paracrine and endocrine cells in the midgut of
the fruit fly maggot. Cell Tissue Res. 336, 309-323.
Veenstra, J. A. and Ida, T. (2014). More Drosophila enteroendocrine peptides:
Orcokinin B and the CCHamides 1 and 2. Cell Tissue Res. 357, 607-621.
Veenstra, J. A., Agricola, H.-J. and Sellami, A. (2008). Regulatory peptides in fruit
fly midgut. Cell Tissue Res. 334, 499-516.
Vodovar, N., Vinals, M., Liehl, P., Basset, A., Degrouard, J., Spellman, P.,
Boccard, F. and Lemaitre, B. (2005). Drosophila host defense after oral infection
by an entomopathogenic Pseudomonas species. Proc. Natl. Acad. Sci. USA 102,
11414-11419.
Wu, Q., Wen, T., Lee, G., Park, J. H., Cai, H. N. and Shen, P. (2003).
Developmental control of foraging and social behavior by the Drosophila
neuropeptide Y-like system. Neuron 39, 147-161.
Zeng, X., Chauhan, C. and Hou, S. X. (2010). Characterization of midgut stem celland enteroblast-specific Gal4 lines in Drosophila. Genesis 48, 607-611.
Zielke, N., Korzelius, J., van Straaten, M., Bender, K., Schuhknecht, G. F. P.,
Dutta, D., Xiany, J. and Edgar, B. (2014). Fly-FUCCI: a versatile tool for studying
cell proliferation in complex tissues. Cell Rep. 7, 588-598.
DEVELOPMENT
Bardin, A. J., Perdigoto, C. N., Southall, T. D., Brand, A. H. and Schweisguth, F.
(2010). Transcriptional control of stem cell maintenance in the Drosophila
intestine. Development 137, 705-714.
Bayliss, W. M. and Starling, E. H. (1902). The mechanism of pancreatic secretion.
J. Physiol. 28, 325-353.
Beebe, K., Lee, W.-C. and Micchelli, C. A. (2010). JAK/STAT signaling coordinates
stem cell proliferation and multilineage differentiation in the Drosophila intestinal
stem cell lineage. Dev. Biol. 338, 28-37.
Bendena, W. G., Donly, B. C. and Tobe, S. S. (1999). Allatostatins: a growing family
of neuropeptides with structural and functional diversity. Ann. N. Y. Acad. Sci. 897,
311-329.
Biteau, B. and Jasper, H. (2014). Slit/Robo signaling regulates cell fate decisions in
the intestinal stem cell lineage of Drosophila. Cell Rep. 7, 1867-1875.
Buchon, N., Osman, D., David, F. P. A., Fang, H. Y., Boquete, J.-P., Deplancke,
B. and Lemaitre, B. (2013). Morphological and molecular characterization of
adult midgut compartmentalization in Drosophila. Cell Rep. 3, 1725-1738.
Chintapalli, V. R., Wang, J. and Dow, J. A. T. (2007). Using FlyAtlas to identify
better Drosophila melanogaster models of human disease. Nat. Genet. 39,
715-720.
Dutta, D., Xiang, J. and Edgar, B. A. (2013). RNA expression profiling from FACSisolated cells of the Drosophila intestine. Curr. Protocol Stem Cell Biol. 27,
2F.2.1-2F.2.12.
Egerod, K. L., Engelstoft, M. S., Grunddal, K. V., Nøhr, M. K., Secher, A., Sakata,
I., Pedersen, J., Windeløv, J. A., Fü chtbauer, E.-M., Olsen, J. et al. (2012).
A major lineage of enteroendocrine cells coexpress CCK, secretin, GIP, GLP-1,
PYY, and neurotensin but not somatostatin. Endocrinology 153, 5782-5795.
Engelstoft, M. S., Egerod, K. L., Lund, M. L. and Schwartz, T. W. (2013).
Enteroendocrine cell types revisited. Curr. Opin. Pharmacol. 13, 912-921.
Furness, J. B., Rivera, L. R., Cho, H.-J., Bravo, D. M. and Callaghan, B. (2013).
The gut as a sensory organ. Nat. Rev. Gastroenterol. Hepatol. 1010, 729-740.
Furriols, M. and Bray, S. (2001). A model Notch response element detects
Suppressor of Hairless-dependent molecular switch. Curr. Biol. 11, 60-64.
Gä de, G. (2004). Regulation of intermediary metabolism and water balance of
insects by neuropeptides. Annu. Rev. Entomol. 49, 93-113.
Habib, A. M., Richards, P., Cairns, L. S., Rogers, G. J., Bannon, C. A. M., Parker,
H. E., Morley, T. C. E., Yeo, G. S. H., Reimann, F. and Gribble, F. M. (2012).
Overlap of endocrine hormone expression in the mouse intestine revealed by
transcriptional profiling and flow cytometry. Endocrinology 153, 3054-3065.
Haenelin, M., Kramatschek, B. and Campos-Ortega, J. A. (1990). The pattern of
transcription of the neurogenic gene Delta of Drosophila melanogaster.
Development 110, 905-914.
Kidd, S., Lockett, T. J. and Young, M. W. (1983). The Notch locus of Drosophila
melanogaster. Cell 34, 421-433.
Lee, T. and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies
of gene function in neuronal morphogenesis. Neuron 22, 451-461.
Lee, W.-C. and Micchelli, C. A. (2013). Development and characterization of a
chemically defined food for Drosophila. PLoS ONE 8, e67308.
Marianes, A. and Spradling, A. C. (2013). Physiological and stem cell
compartmentalization within the Drosophila midgut. Elife 2, e00886.
Mellitzer, G., Beucher, A., Lobstein, V., Michel, P., Robine, S., Kedinger, M. and
Gradwohl, G. (2010). Loss of enteroendocrine cells in mice alters lipid absorption
and glucose homeostasis and impairs postnatal survival. J. Clin. Invest. 120,
1708-1721.
Micchelli, C. A. (2014). Whole-mount immunostaining of the adult Drosophila
gastrointestinal tract. Methods 68, 273-279.
Micchelli, C. A. and Perrimon, N. (2006). Evidence that stem cells reside in the
adult Drosophila midgut epithelium. Nature 439, 475-479.
Micchelli, C. A., Sudmeier, L., Perrimon, N., Tang, S. and Beehler-Evans, R.
(2011). Identification of adult midgut precursors in Drosophila. Gene Expr.
Patterns 11, 12-21.
Development (2015) 142, 654-664 doi:10.1242/dev.114959
664