© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 644-653 doi:10.1242/dev.113357
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Enteroendocrine cells are generated from stem cells through a
distinct progenitor in the adult Drosophila posterior midgut
Xiankun Zeng* and Steven X. Hou*
Functional mature cells are continually replenished by stem cells to
maintain tissue homoeostasis. In the adult Drosophila posterior midgut,
both terminally differentiated enterocyte (EC) and enteroendocrine
(EE) cells are generated from an intestinal stem cell (ISC). However, it
is not clear how the two differentiated cells are generated from the ISC.
In this study, we found that only ECs are generated through the
Su(H)GBE+ immature progenitor enteroblasts (EBs), whereas EEs are
generated from ISCs through a distinct progenitor pre-EE by a novel
lineage-tracing system. EEs can be generated from ISCs in three ways:
an ISC becoming an EE, an ISC becoming a new ISC and an EE
through asymmetric division, or an ISC becoming two EEs through
symmetric division. We further identified that the transcriptional factor
Prospero (Pros) regulates ISC commitment to EEs. Our data provide
direct evidence that different differentiated cells are generated by
different modes of stem cell lineage specification within the same
tissues.
KEY WORDS: Intestinal stem cell, Secretory enteroendocrine cell,
Pre-enteroendocrine cell, Prospero, Drosophila
INTRODUCTION
Adult stem cells maintain tissue homeostasis by continuously
replenishing damaged, aged and dead cells in any organism
(Weissman, 2000). As expected, adult stem cell self-renewal and
differentiation are tightly regulated processes, and an imbalance in
adult stem cell homeostasis can result in complications such as
tumor formation and degenerative diseases. Moreover, adult stem
cell behavior must be precisely regulated to ensure a prompt
response to tissue damage and stress.
The Drosophila midgut is one of its largest organs, and it serves
as the entry site not only for nutrients like food and water, but also
for pathogens, such as harmful bacteria, viruses and toxins (Hakim
et al., 2010). As a result, the midgut epithelium is constantly
exposed to environmental assault and undergoes rapid turnover.
The integrity of the epithelium is maintained by replenishing lost
cells with intestinal stem cells (ISCs) (Micchelli and Perrimon,
2006; Ohlstein and Spradling, 2006). These ISCs lie adjacent to
the basement membrane and divide approximately once a day
to give rise to either absorptive enterocytes (ECs) or secretory
enteroendocrine (EE) cells. Because of the simple cell lineage, ease
of performing genetic analysis and availability of abundant mutants,
the Drosophila midgut has served as a powerful model system
for studying adult stem cell-mediated tissue homeostasis and
regeneration.
Basic Research Laboratory, Center for Cancer Research, National Cancer Institute,
Frederick, MD 21701, USA.
*Authors for correspondence ([email protected]; [email protected])
Received 25 May 2014; Accepted 9 December 2014
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Previous studies suggest that an ISC divides asymmetrically to
produce one new ISC (self-renewal) and one immature daughter
cell, an enteroblast (EB), which further differentiates into an EC or
an EE cell (Micchelli and Perrimon, 2006; Ohlstein and Spradling,
2006). Notch (N) signaling plays a major role in regulating ISC selfrenewal and differentiation (Micchelli and Perrimon, 2006; Ohlstein
and Spradling, 2006, 2007). The ligand of the N pathway, Delta
(Dl), is specifically expressed on an ISC and unidirectionally
switches on the N-signaling pathway in the neighboring EB to
promote the differentiation of an EB to an EC and to inhibit the
differentiation of an EB to an EE (Bardin et al., 2010; Micchelli and
Perrimon, 2006; Ohlstein and Spradling, 2006, 2007). In addition,
commitment and terminal differentiation of ISCs require distinct
levels of N activity; commitment requires high activity and terminal
differentiation requires low activity (Perdigoto et al., 2011).
Furthermore, the overexpression of Dl in ISCs does not affect ISC
proliferation, but promotes EC fate at the expense of EE cells,
suggesting that N signaling is not only necessary, but also
sufficient, for specifying EC cell fate (Beebe et al., 2010; Jiang
et al., 2009).
The differentiated ECs can also regulate ISCs through a feedback
mechanism to maintain tissue homeostasis (Biteau et al., 2011;
Jiang and Edgar, 2011). Cells in the intestine are constantly exposed
to numerous insults, from tissue damage to bacterial infection.
It was reported that these events initially affect differentiated ECs,
causing either ablation or activated JNK-mediated stress signaling
in the ECs (Jiang and Edgar, 2011). The affected ECs would
upregulate unpaired (Upd) ligands (Upd2 and Upd3) of the
JAK-STAT signal transduction pathway, which would activate the
JAK-STAT signal transduction pathway in ISCs. This activation
would induce ISC division and differentiation to generate new ECs
that would replenish the damaged epithelium.
Compared with the extent of knowledge on EC specification and
regulation, relatively little is known about EE cell fate specification
and regulation. In this study, we performed lineage-tracing
experiments using a newly developed tracing system and found
that EE was generated from ISCs through a distinct progenitor. We
further found that Prospero (Pros) functions downstream of or
parallel to the achaete-scute complex (AS-C) members to determine
ISC commitment to EE.
RESULTS
Su(H)GBE+ EBs are progenitors of ECs but not EEs
Different cell types in the adult Drosophila posterior midgut can be
identified morphologically as well as by their expression of different
markers. ISCs are diploid, have a small nucleus, and express Dl and
Sanpodo (Spdo). EBs are diploid and have a small nucleus;
Su(H)GBE-lacZ, a transcriptional reporter of N signaling, was found
to be highly expressed in the daughter cell (EB). ECs are polyploid,
have a large nucleus and express the transcriptional factor Pdm-1.
EEs are diploid, have a small nucleus, and express the transcription
DEVELOPMENT
ABSTRACT
factor Pros and synaptic protein Bruchpilot (Brp/nc82) (Jiang et al.,
2009; Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006,
2007; Perdigoto et al., 2011; Zeng et al., 2013). However, it is not
clear whether all ISCs are Dl positive (Dl+), whether all EBs are
Su(H)GBE+, whether all ECs are Pdm-1+ and whether all EEs
are Pros+. In this study, we only examined Dl+ ISCs, Su(H)GBE+
EBs, Pdm-1+ ECs and Pros+ EEs.
Both ISCs and EBs in the midgut express the transcriptional
factor escargot (Esg) (Micchelli and Perrimon, 2006). Esg-Gal4 is
widely used to manipulate gene expression in ISCs and EBs of the
posterior midgut. During a routine staining of esg-Gal4, UAS-GFP
midguts with the Pros antibody, we unexpectedly found that 3.3%
of Esg+ diploid cells express the EE marker Pros (n=45 guts;
Fig. 1A,B). Interestingly, these Esg+ Pros+ cells do not express the
EB marker Su(H)GBE-lacZ (n=45 guts; Fig. 1A′,B). These Esg+ Su
(H)GBE− Pros+ cells may be EE progenitors (or pre-EEs). We
reasoned that some of the newly generated ECs and EEs might
inherit weak green fluorescent protein (GFP) from Su(H)GBE-Gal4,
UAS-mCD8-GFP-labeled EBs if they were developed from EBs.
Indeed, we found that, among all GFP+ cells, 7.6% (32/472, n=12;
Fig. 1C-D) of the cells are ECs that express weak GFP inherited
from Su(H)GBE+ EBs; but surprisingly, we did not find any EEs
that have inherited GFP from Su(H)GBE+ EBs (0/472, n=12;
Fig. 1C-D). We further examined cell fate identification using
the mosaic analysis with a repressible cell marker (MARCM)
Development (2015) 142, 644-653 doi:10.1242/dev.113357
technique (Lee and Luo, 1999). In wild-type MARCM clones with
Su(H)GBE-lacZ, we found that none of the Pros+ EEs inherited the
β-gal from Su(H)GBE+ EB among 75 clones (Fig. 1E-E″), whereas
23 out of 112 clones had at least one Pdm-1+ EC-expressing β-gal
(Fig. 1F-F″). Additionally, we generated MARCM clones using Su
(H)GBE-Gal4 instead of Tub-Gal4. Consistently, we found that
some Pdm1+ ECs inherited weak GFP from Su(H)GBE+ EB (14
Pdm1+ cells expressing GFP among 153 GFP+ cells; supplementary
material Fig. S1A-A″), but none of the EEs inherited GFP from Su
(H)GBE+ EBs (0 Pros+ cells expressed GFP among 153 GFP+ cells;
supplementary material Fig. S1A-A″). These data again indicate
that ECs, but not EEs, are developed from Su(H)GBE+ EBs. The
above data suggest that EEs may not be developed from Su(H)GBE+
EBs, but may be generated from ISCs through another mechanism.
To further test this possibility, we developed a novel lineage-tracing
system to trace cell lineage in the posterior midgut.
Developing a new lineage-tracing system
The lineage-tracing system that combines a tamoxifen-inducible Cre
knock-in allele with the Rosa26-lacZ reporter strain has been
successfully used to identify cell ( particularly stem cell) lineages in
adult mice (Barker et al., 2007; Takeda et al., 2011). In Drosophila
melanogaster, FLP/FRT, an alternative site-specific recombination
system originally identified in yeast, has been predominantly used,
with great success, to generate a genetic mosaic for clonal analysis
Fig. 1. Su(H)GBE+ EBs are not EE progenitors. (A,A′) Esg+ (green) Pros+ (red) cells (arrows) do not express the EB marker Su(H)GBE-lacZ (arrowheads,
purple in A′). (B) The quantification of Esg+ Pros+ Su(H)GBE− and Esg+ Pros+ Su(H)GBE+ cells among all Esg+ cells. Data are represented as mean±s.e.m.
(C,C′) Some of the ECs (arrowheads) inherited weak GFP from Su(H)GBE+ EBs; none of the EEs (arrow) inherited GFP from Su(H)GBE+ EBs, suggesting
that ECs, but not EEs, are developed from Su(H)GBE+ EBs. (D) The quantification of GFP+ ECs and EEs among all GFP+ cells in Su(H)GBE>GFP posterior
midguts. Data are represented as mean±s.e.m. (E-E″) Wild-type MARCM clones with Su(H)GBE-lacZ. Some polyploid ECs (asterisks) express β-Gal inherited
from Su(H)GBE-lacZ+ EBs (arrows). However, none of the EEs (arrowhead) expresses β-Gal, suggesting that ECs, but not EEs, are developed from Su(H)GBE+
EBs. (F-F″) Wild-type MARCM clones with Su(H)GBE-lacZ. The arrows indicate a β-Gal+ Pdm-1− EB. The asterisks indicate a β-Gal+ Pdm-1+ EC. The
arrowheads indicate a β-Gal− Pdm-1+ EC. Scale bars: 10 μm.
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DEVELOPMENT
RESEARCH ARTICLE
(Chou and Perrimon, 1996; Golic and Lindquist, 1989; Lee and
Luo, 1999; Xu and Rubin, 1993). These experiments depend on
inducing recombination in a few isolated progenitor cells, followed
by clonal expansion of these recombined cells (Decotto and
Spradling, 2005; Micchelli and Perrimon, 2006; Ohlstein and
Spradling, 2006; Singh et al., 2007, 2011; Strand and Micchelli,
2011; Tulina and Matunis, 2001; Xie and Spradling, 1998). The
mosaic clonal systems are effective for identifying a marked cell
lineage, but they are not ideal for accurately identifying the origin of
individual marked cells. A Gal4 technique for real-time and clonal
expression (G-TRACE) was developed to identify more precisely
the origin of individual cells (Evans et al., 2009). The TARGET
system, in which Gal4 is combined with the temperature-sensitive
GAL80 protein (Gal80ts), can be used for inducible control of Gal4
activity (McGuire et al., 2003). Theoretically, a combination of the
G-TRACE and TARGET systems, which is akin to the mouseinducible Cre/Rosa26-lacZ reporter system, would enable a precise
lineage-tracing experiment to be performed. However, in our
experience, the TARGET system always had a low level of leakage
that complicated the interpretation of the lineage-tracing results. The
Gal4s used in these experiments were all expressed in various cell
types in embryos or during other earlier developmental stages. The
marked progeny cells might come from other labeled cells during
the earlier developmental stages instead of coming from the adult
stem cells, if the leakage occurred during the earlier developmental
stages. To overcome these complications, we developed a new
(to our knowledge) lineage-tracing system (Fig. 2A,B), which
combines the main features of the TARGET, G-TRACE and
inducible Cre/loxP (Heidmann and Lehner, 2001) systems. We call
Development (2015) 142, 644-653 doi:10.1242/dev.113357
this new system the TARGET technique for real-time and clonal
expression system, or, for simplicity, the T-TRACE system. To
prevent possible leakage before the adult stage, we used the doubleleakage proofs: the first is a temperature-sensitive Gal4 inhibitor,
Gal80ts (McGuire et al., 2003), which suppresses Gal4 activity at the
permissive temperature (18°C); the second is an estrogen-inducible
Cre whose activity depends on the presence of estrogen. To initiate
cell lineage tracing (Heidmann and Lehner, 2001), the flies have to
be cultured at a non-permissive temperature (29°C) and fed with
food containing 150 μg/ml of estrogen. The stem cell/progenitorspecific Gal4 mediates expression of the Cre recombinase, which in
turn removes a loxP-flanked transcriptional termination cassette
inserted between a ubiquitin-p63E (Ubi-p63E) promoter fragment
and the GFP open reading frame. Therefore, the Ubi-p63E promoter
will drive GFP expression in all subsequent daughter cells derived
from the initial Gal4-expressing progenitor cells.
We tested the T-TRACE system by using esg-Gal4 (in both stem
cell ISCs and EBs) and Su(H)GBE-Gal4 [in Su(H)GBE+ EBs only]
in the adult Drosophila posterior midgut (Micchelli and Perrimon,
2006; Zeng et al., 2010). When cultured on food without estrogen at
18°C, the flies of both Esg-Gal4/T-TRACE (supplementary material
Fig. S2D) and Su(H)GBE-Gal4/T-TRACE (supplementary material
Fig. S2H) grew to adulthood with no obvious phenotype, and they
did not express GFP in their midguts; when cultured on food without
estrogen at 29°C, or on food with estrogen at 18°C, the flies of both
Esg-Gal4/T-TRACE (supplementary material Fig. S2B,C) and
Su(H)GBE-Gal4/T-TRACE (supplementary material Fig. S2F,G)
expressed GFP in a few cells in their midguts, indicating that
either condition is causing partial leakage; when cultured on food
Fig. 2. The T-TRACE analysis system. (A,B) The molecular mechanisms of the T-TRACE system. In this system, TARGET mediates the expression of an
estrogen-inducible Cre, which in turn removes a loxP-flanked transcriptional termination cassette inserted between a ubiquitin-p63E (Ubi-p63E) promoter
fragment and the GFP open reading frame. Therefore, the Ubi-p63E promoter will drive GFP expression in all subsequent daughter cells derived from the initial
Gal4-expressing progenitor cells. (C,C′) Fluorescence images showing T-TRACE analysis of the esg-Gal4 line at 29°C on food with estrogen (C). A schematic
shows the result of lineage tracing using esg-Gal4 (C′). (D,D′) Fluorescence images showing T-TRACE analysis of the Dl-Gal4 line at 29°C on food with estrogen
(D). A schematic diagram shows the result of lineage tracing using Dl-Gal4 (D′). In C and D, the adult fly posterior midguts were stained with anti-GFP (green),
anti-Dl (cytoplasmic, red), anti-Pros (nuclear, red) and DAPI (blue). Arrows, GFP+ Pros+ EEs; arrowheads, GFP+ polyploid ECs. Scale bars: 10 μm.
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RESEARCH ARTICLE
with estrogen at 29°C, the flies of both Esg-Gal4/T-TRACE
(supplementary material Fig. S2A) and Su(H)GBE-Gal4/T-TRACE
(supplementary material Fig. S2E) expressed GFP in the cells of the
respective Gal4 driver and daughter cells. These results suggest that
the T-TRACE system is a reliable and leakage-proof system.
ECs, but not EEs, develop from Su(H)GBE+ EBs
To clarify cell lineage in the posterior midgut, we performed
lineage-tracing experiments with the T-TRACE system on
esg-Gal4, Dl-Gal4 (in ISCs only) and Su(H)GBE-Gal4 in the
adult Drosophila posterior midgut (Micchelli and Perrimon, 2006;
Zeng et al., 2010). We cultured the adult flies on food with estrogen
at a restrictive temperature (29°C). After 1 week, the flies were
dissected, stained and examined for expression of GFP, Dl (ISC
marker) and Pros (EE cell marker) under a confocal microscope. We
found that EE cells represent 7.5% (80/1067) of the GFP-positive
cells from the Esg-Gal4/T-TRACE (Figs 2C,C′ and 3B) and 8.6%
(80/925) of the GFP-positive cells from the Dl-Gal4/T-TRACE
(Figs 2D,D′ and 3B). Surprisingly, Pros-positive EE cells are totally
absent in GFP-positive cells from the Su(H)GBE-Gal4/T-TRACE
(Fig. 3A-B). These data suggest that EE cells may be generated from
ISCs through another cell type instead of Su(H)GBE+ EBs.
Consistently, a recent study has also found that Su(H)GBE+ EBs
do not have the capacity to generate EEs, but are rather
EC-committed progenitors (Biteau and Jasper, 2014).
Su(H)GBE-lacZ was found highly expressed in the daughter cell
(EB) of ISCs with high Dl levels (Ohlstein and Spradling, 2007);
thus, EEs may develop from some Su(H)GBElow EBs. To rule out
the possibility that some Su(H)GBElow EBs may be the progenitors
of EEs, we performed further lineage-tracing experiments using a
steroid (RU486)-inducible ‘GeneSwitch’ Gal4 (5966GS) (Mathur
et al., 2010; Nicholson et al., 2008) in combination with an
Development (2015) 142, 644-653 doi:10.1242/dev.113357
Act<y+<EGFP reporter line. The 5966GS is expressed in EBs and
ECs, but is excluded from ISCs and EEs (Fig. 3C,C′) (Guo et al.,
2014; Hur et al., 2013; Kapuria et al., 2012; Mathur et al., 2010). We
cultured the adult flies on food with RU486 at room temperature
(∼22°C). Again, we found that none of the 243 Pros-positive EE
cells were GFP positive from the UAS-flp/5866GS/Act<y+<EGFP
flies (Fig. 3E-E″), indicating that EEs are not generated from
5966GS+ EBs and ECs. No GFP-labeled cells were found when
flies were not fed with RU486 (Fig. 3D). These data together
suggest that Su(H)GBE+ and 5966GS+ EBs are the progenitors of
only ECs, and EEs are likely generated from ISCs through another
cell type.
EEs are generated from ISCs through a distinct progenitor
The data above suggest that EEs may not be developed from
Su(H)GBE+ and 5966GS+ EBs, but are developed from ISCs
through another way. This information suggests that there may be
some transit cells expressing both the ISC marker Dl and EE marker
Pros that exist during EE maturation from ISCs in the posterior
midgut. To test this, we stained posterior midguts with the ISC
marker Dl and the EE marker Pros and looked for some transit cells
expressing both Dl and Pros. Indeed, about 1.9% of ISCs (11/566,
n=11) expressed both Dl and Pros (Fig. 4A-A″) in wild-type
posterior midguts. We renamed the Dl+ Pros+ cells ‘pre-EEs (or EE
progenitor)’ and the Dl− Pros+ cells ‘mature EEs’ or just ‘EEs’.
We unexpectedly found that Pros was expressed in some of the
dividing phospho-Histone H3 ( pH3)-positive ISC-like cells in
N DN (esg ts>N DN) midguts (supplementary material Fig. S3A-A″)
when we were checking the mitotic index of N DN (esg ts>N DN)
midguts. This encouraged us to further examine the expression of
Pros in dividing pH3+ ISCs in the wild-type posterior midgut.
Strikingly, we found that 6.7% (42/628) of pH3+ ISCs express
Fig. 3. EEs are not developed from Su(H)GBE+ and
5966GS+ EBs. (A) A fluorescence image showing
T-TRACE analysis of the Su(H)GBE-Gal4 line at 29°C on
food with estrogen. Arrowheads, GFP+ polyploid ECs.
(A′) A schematic diagram of the result of lineage tracing
using Su(H)GBE-Gal4. (B) The quantitative percentages of
Pros+ cells among GFP+ cells from T-TRACE analysis of
esg-Gal4, Dl-Gal4 and Su(H)GBE-Gal4. Data are mean±
s.e.m. (C,C′) 5966GS (PswitchPC)-Gal4/UAS-mCD8-GFP
expression. GFP is expressed in EBs (white arrows) and
ECs (asterisks), but is not expressed in ISCs (red arrows)
and EEs (white arrowheads). The inset shows an enlarged
view. (D-E″) Fluorescence images showing UAS-flp/
5966GS/Act<y+<EGFP analysis at 22°C on food without
RU486 (D), or at 22°C on food with RU486 (E-E″).
Arrowheads, Pros+ EEs; arrows, Dl+ ISCs. The adult fly
posterior midguts were stained with anti-GFP (green), antiDl (cytoplasmic, red), anti-Pros (nuclear, red) and DAPI
(blue). Scale bars: 10 μm.
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RESEARCH ARTICLE
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Development (2015) 142, 644-653 doi:10.1242/dev.113357
division behaviors of individual ISCs, and it is technically difficult
to precisely track cell lineages in the multiple-cell ISC clones.
Consistent with symmetric and asymmetric division of ISCs to
generate EEs, in some of the two-cell ISC lineages, both cells were
Pros+ EE cells (n=12 from seven guts; Fig. 5A,A′); in others, one
cell is a Pros+ EE and the other is a Dl+ ISC (n=5 from seven guts;
Fig. 5B,B′). We also found one-cell ISC lineages in which the single
cell is a Pros+ EE (n=32 from seven guts; Fig. 5C,C′), indicating that
an ISC may directly convert to an EE through a post-mitotic EE
progenitor.
The data above suggest that EE cells can be generated from ISCs
in one of three ways: an ISC directly becoming one EE cell
(32/49=65%; Fig. 5C-D); an ISC becoming two EE cells through
symmetric division (12/49=24%; Fig. 5A,A′,D); or an ISC
becoming one new ISC and one EE cell through asymmetric
division (5/49=10%; Fig. 5B,B′,D). An EE cell occurs in either twocell pairs (Ohlstein and Spradling, 2006) or in a single cell in the
wild-type Drosophila midgut (supplementary material Fig. S5);
the ratio of an EE cell pair to a single cell is 1:4.5. These EE cell
pairs may be generated through symmetric ISC division, whereas
the single EE cells may be generated through either asymmetric ISC
division or direct conversion of an ISC into an EE cell.
Fig. 4. EE generation during ISC division. (A-A″) A representative image
showing a Dl+ Pros+ pre-EE cell (arrows) and Dl− Pros+ EE cell (arrowheads).
(B-B‴) Representative images of a pH3+ Pros+ Dl+ cell in the posterior midgut
of wild-type flies. (C-C‴) Representative images showing an ISC becoming
two pre-EEs through symmetric division. α-Tubulin was used to label the
spindle of the dividing cell. (D-D″) Representative images showing an ISC
becoming a new ISC and a pre-EE through asymmetric division. In all images,
the 1-week-old wild-type flies were stained using the antibodies indicated.
Scale bars: 2 μm. (E) Schematic showing the quantitation of EE generation
from ISCs through symmetric and asymmetric division in the posterior midguts
of wild-type flies.
Pros (Fig. 4B-D″; supplementary material Fig. S4) in the wildtype posterior midguts. Consistently, a recent study shows that a
few of Pros+ EEs can undergo S-phase and mitotic division
(Zielke et al., 2014). We further found that these Pros+ dividing
ISCs still also express the ISC marker Dl ( pH3+ Dl+ Pros+ cells;
Fig. 4B-B‴; supplementary material Fig. S4). Among the pH3+
Dl+ Pros+ cells, some (18/42) went through symmetric division to
generate two pre-EEs from one ISC (Fig. 4C-C‴,E; supplementary
material Fig. S4B-C‴), and some (7/42) went through asymmetric
division to generate one new ISC and one pre-EE (Fig. 4D-E;
supplementary material Fig. S4D-D‴) in the wild-type posterior
midguts.
EEs are generated from ISCs in one of three ways
The data above indicate that an ISC can generate two EEs after
symmetric division and one ISC and one EE after asymmetric
division. To further verify the division pattern, we carefully
examined the division patterns of ISCs during lineage tracing in
the Dl-Gal4/T-TRACE flies (Fig. 5) at 5 days on food with estrogen
at 29°C. In addition to the previously reported multiple-cell ISC
lineages that contain ISCs, EBs, ECs and EEs (72% of ISC lineages
contained at least three cells; Micchelli and Perrimon, 2006;
Ohlstein and Spradling, 2006), we also detected that 11% of ISC
lineages contained two cells and that 17% of ISC clones were
one-cell ISC lineages. To understand EE generation in detail, we
focused our analysis on the two-cell and one-cell ISC lineages
containing EE cell(s) because they more likely represent the early
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The expression of Pros in pre-EEs suggests that Pros may play an
important role in the conversion of ISCs into EEs. To understand the
function of pros in EE formation, we generated GFP-marked ISC
clones that were homozygous for the loss-of-function allele pros 17
(Cook et al., 2003), using the MARCM technique (Lee and Luo, 1999).
Seven days after clone induction, we found that GFP-marked clones of
pros 17 were completely devoid of nc82+ EEs (Fig. 6B,D,D′) compared
with their wild-type counterparts (Fig. 6B-C′), whereas the ISCs in the
GFP-marked clones showed normal proliferation (Fig. 6A).
Consistently, the EE population dramatically decreased when we
knocked down pros in both ISCs and EBs by expressing UAS-pros RNAi
in these cells using esg ts driver (Fig. 6E). To rule out the possibility that
Pros directly regulates the expression of the EE cell marker Brp (the
antigen of nc82) in contexts other than in EE cell formation, we
identified another EE cell-specific marker, Dachshund, which is a
transcriptional regulator required for eye and leg development (Mardon
et al., 1994). The anti-Dac antibody Mabdac1-1 specifically labeled
Pros+ EEs in wild-type midguts (supplementary material Fig. S6A-A″),
as well as excessive EEs in UAS-NDN- (supplementary material Fig.
S6B) and UAS-ase- (supplementary material Fig. S6C) overexpressing
midguts. Consistently, we did not detect Dac+ EEs in GFP-marked
pros 17 clones (supplementary material Fig. S6D,F,F′) compared with
their wild-type counterparts (supplementary material Fig. S6D-E′).
Together, these data suggest that Pros regulates EE cell fate
determination.
To further clarify whether Pros functions in ISCs or Su(H)GBE+
EBs to regulate EE cell fate determination, we generated an ISCspecific Gal4 system, in which Su(H)GBE-Gal80 blocks the
activation by Gal4 of Esg-Gal4 in Su(H)GBE+ EBs, but keeps it
active in ISCs only (this study; Wang et al., 2014). We named this
Gal4 ‘ISC-Gal4’ and found that it is much stronger than the
previously published Dl-Gal4 (Zeng et al., 2010). Knockdown of
pros in ISCs using the ISC-Gal4 system combined with tub-Gal80ts
(esg-Gal4, UAS-GFP; Su(H)-Gal80, tub-Gal80 ts; referred to as
ISC ts) resulted in fewer nc82+ EEs (Fig. 6E), whereas knockdown of
pros in Su(H)GBE+ EBs using Su(H)GBE-Gal4.UAS-mCD8-GFP;
tub-Gal80 ts (referred to as Su(H)GBE ts) generated a phenotype that
was no different from that of the wild-type control (Fig. 6E).
DEVELOPMENT
Pros determines ISCs commitment to EEs
RESEARCH ARTICLE
Development (2015) 142, 644-653 doi:10.1242/dev.113357
Fig. 5. EEs are generated by symmetric and asymmetric
division of ISCs and are directly converted from ISCs.
Fluorescence images showing T-TRACE analysis of the
Dl-Gal4 line at 29°C on food with estrogen. The adult fly
posterior midguts were stained with anti-GFP (green), anti-Dl
(cytoplasmic, red), anti-Pros (nuclear, red) and DAPI (blue).
Scale bars: 5 μm. (A,A′) An ISC becomes two EEs (arrows)
through symmetric division. (B,B′) An ISC becomes a new
ISC (arrowhead) and an EE (arrow) through asymmetric
division. (C,C′) An ISC directly becomes an EE (arrow).
(D) Schematic showing the quantitation of EE generation
from ISCs. The GFP-marked cells are illustrated in the insets
of A′,B′,C′.
Pros functions downstream of or parallel to the achaetescute complex to determine EE fate in ISCs
The members of the achaete-scute complex (AS-C), Scute (Sc)
and Asense (Ase), have been shown to play a major role in EE
cell fate determination and to be upregulated in the midgut that
expressed a dominant-negative form of N (NDN) (Bardin et al.,
2010). The overexpression of Sc or Ase in both ISCs and EBs
by Esg-Gal4 resulted in a dramatic increase in EEs (Bardin
et al., 2010). To further clarify whether Sc and Ase function in
ISCs or EBs to regulate EE cell fate determination, we expressed
UAS-sc specifically in ISCs or Su(H)GBE+ EBs using ISC ts or
Su(H)GBE ts. Compared with the wild-type control (supplementary
material Fig. S7A,D,I), the expression of UAS-sc in ISCs generated
an excess number of EE cells (supplementary material Fig. S7B,I),
which were indistinguishable from those generated previously by
the expression of UAS-sc in both ISCs and EBs using Esg-Gal4
(Bardin et al., 2010); however, the expression of UAS-sc in Su(H)
GBE+ EBs (supplementary material Fig. S7E,I) generated a
Fig. 6. Pros regulates EE cell fate determination in
ISCs. (A) The quantitative cell numbers per clone in
C-D′. (B) The quantitative percentages of nc82+ EEs in
GFP+ clones in C-D′. Data are represented as mean±
s.e.m. (C-D′) MARCM clones of wild-type control (C,C′)
and pros17 (D,D′). Seven days after clone induction,
GFP-marked clones of pros17 (D,D′) were completely
devoid of nc82+ EEs (arrow) compared with their wildtype counterparts (C,C′), whereas the clone sizes are
similar (A). (E) The numbers of nc82+ EE cells in the
posterior midguts of prosRNAi driven by indicated Gal4,
compared with wild-type controls. Data are represented
as mean± s.e.m. *P<0.01. (F,F′) The overexpression
of sc in ISCs and EBs (esg ts>sc). (G,G′) The
overexpression of sc and pros RNAi in ISCs and EBs
(esg ts>sc+pros RNAi). The overexpression of pros RNAi
suppressed the excess EE phenotype associated with
sc overexpression, indicating that Pros functions either
downstream of or parallel to Sc. The adult fly posterior
midguts were stained with anti-GFP (green), anti-nc82
(red) and DAPI (blue). Scale bars: 10 μm.
649
DEVELOPMENT
Surprisingly, however, the overexpression of pros in ISCs and EBs
did not increase EEs in comparison with the wild-type control in the
posterior midgut epithelium (data not shown).
These data suggest that Pros is necessary, but not sufficient, for
EE cell fate determination in ISCs.
phenotype that was no different from the wild-type control
(supplementary material Fig. S7D,I). Likewise, the overexpression
of the other three members of AS-C, including ase, l(1)sc and
achaete (ac), in ISCs but not in Su(H)GBE+ EBs, resulted in a
dramatic increase in EEs (data not shown). Conversely, we knocked
down sc in ISCs or EBs by expressing UAS-scRNAi in these cells, and
we found that the knockdown of sc in ISCs resulted in reduced EEs
(supplementary material Fig. S7C,I), whereas the knockdown of
sc in Su(H)GBE+ EBs did not change the EE cell population
(supplementary material Fig. S8F,I).
Similarly, we expressed UAS-NDN in ISCs using cell typespecific Gal4 and found that the expression of UAS-NDNin ISCs
caused both ISC and EE cell expansion phenotype, which is
indistinguishable from those generated previously by the expression
of UAS-NDN in both ISCs and EBs using Esg-Gal4 (supplementary
material Fig. S8A,B; Bardin et al., 2010; Ohlstein and Spradling,
2007; Zeng et al., 2013). To explicitly evaluate the function of N in
Su(H)GBE+ EBs, we expressed UAS-NDN in Su(H)GBE+ EBs using
Su(H)GBE-Gal4 along with the T-TRACE system. Interestingly,
compared with the wild-type control (supplementary material
Fig. S8C), the expression of UAS-NDN in Su(H)GBE+ EBs
completely blocked the differentiation of Su(H)GBE+ EBs into
ECs (supplementary material Fig. S8D), whereas the EE cell density
was not changed at all (supplementary material Fig. S8E). These
data, plus previously published data, suggest that N regulates EE
cells through Sc and Ase only in ISCs.
To further determine an epistatic relationship between AS-C and
Pros, we expressed UAS-sc and UAS-prosRNAi in ISCs at the same
time using esg ts. Interestingly, compared with a dramatic increase in
nc82+ EEs in the sc overexpression (esg ts>sc) midgut, the
expression of pros RNAi in the sc overexpression midgut (esg ts>sc+
pros RNAi; Fig. 6G,G′) suppressed the excess EE cell phenotypes
associated with overexpressing sc (esg ts>sc; Fig. 6F,F′). Likewise,
the expression of prosRNAi in the ase midgut (esg ts>ase+pros RNAi;
supplementary material Fig. S7H) also suppressed the excess EE
cell phenotypes associated with overexpressing ase (esg ts>ase;
supplementary material Fig. S7G). Together, Pros functions either
downstream of or parallel to AS-C to regulate EE cell fate in ISCs.
DISCUSSION
In the adult Drosophila posterior midgut, both terminally
differentiated EC and EE cells are generated from the ISC.
However, it is not clear how the differentiated cells are generated
from the ISC. In this study, we found that only ECs are generated
through the Su(H)GBE+ immature progenitor EBs, whereas EEs are
generated from ISCs through a distinct type of progenitors by an ISC
converting to an EE, an ISC becoming a new ISC and an EE through
asymmetric division, or an ISC becoming two EEs through
symmetric division. The transcriptional factor Pros functions
specifically in ISCs to guide the commitment of ISCs into EEs.
The anatomy and cell types in the Drosophila midgut are similar
to those in the mammalian small intestine: both systems are a tubular
epithelium composed of absorptive and secretory cells, which are
constantly replenished by ISCs (Wang and Hou, 2010). We
demonstrated that EEs are generated from ISCs through a distinct
type of progenitor in the Drosophila, based on the following
evidence. First, we found that some Esg+ cells express the EE
marker Pros, but none of the Su(H)GBE+ cells expresses Pros.
Second, we identified that Su(H)GBE+ EBs are the progenitors of
ECs, instead of EEs, by using a novel lineage-tracing system:
T-TRACE. To rule out the possibility that some Su(H)GBElow EBs
may be the progenitors of EEs, we performed a lineage-tracing
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Development (2015) 142, 644-653 doi:10.1242/dev.113357
experiment using another EB Gal4 driver, 5966GS (Guo et al.,
2014; Hur et al., 2013; Kapuria et al., 2012; Mathur et al., 2010),
and further confirmed that 5966GS+ EBs are also not the
progenitors of EE cells. Third, we identified a transit cell type
called pre-EE (or EE progenitor), which expresses both the ISC
marker Dl and the EE marker Pros. Finally, we demonstrated that
ISCs can convert into EE cells without cell division, by which 65%
of EE cells are generated. In addition to direct conversion of an ISC
into an EE cell, ISCs can undergo mitotic division to generate two
EE cells by symmetric division, and an EE cell and a new ISC by
asymmetric division, by which 35% of EEs are generated.
Consistently, quiescent ISCs in mice have been recently identified
as direct precursors of secretory Paneth cells and EE cells (Buczacki
et al., 2013). These quiescent ISCs can differentiate into secretory
Paneth cells and EE cells with minimal cell division (Buczacki et al.,
2013). Recent studies in ISCs of mice also suggest that stem cell
pools are maintained through population asymmetry. Some stem
cells are lost due to differentiation or damage and their positions are
filled by other stem cells through symmetric division (Snippert
et al., 2010; Simons and Clevers, 2011). Further insight into the
mechanism of mouse ISC regulation has been provided by a
combination of in vivo and in vitro assays (Sato et al., 2011). It was
found that the Lgr5+ stem cells are closely associated with the nichesupporting Paneth cells. Lgr5hi cells undergo neutral competition
for contact with Paneth cell surface after symmetrical division. The
detached stem cells are unable to maintain stem cell competence
without access to short-range signals and progressively
differentiate. In Drosophila posterior midgut, the predominance of
ISC symmetric division is one of the main mechanisms for
increasing the gut cell number during adaptive growth (O’Brien
et al., 2011). The method of EE generation by ISCs may also change
during environmental and dietary stresses to meet physiological
need. For example, symmetric division of an ISC into two EEs may
predominate when more secreting EEs are needed.
Based on the results of this study and previously published
information (Bardin et al., 2010; Beebe et al., 2010; Ohlstein and
Spradling, 2007; Perdigoto et al., 2011), we proposed a model of
ISC lineage specification (Fig. 7). An ISC can generate an
Su(H)GBE+ EB for EC lineage differentiation or a pre-EE for EE
cell lineage differentiation. The N signaling promotes EC lineage
differentiation and inhibits EE lineage differentiation through
promoting ISC to Su(H)GBE+ EB commitment and blocking ISC
to pre-EE commitment by repressing expression of sc and ase in
ISCs, whereas N signaling in Su(H)GBE+ EBs is required for
promoting Su(H)GBE+ EB to EC differentiation. The transcriptional
Fig. 7. Model of intestinal stem cell lineage generation. A model of intestinal
stem cell lineage development in the adult Drosophila posterior midgut. More
details are provided in the text.
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
The T-TRACE lineage-tracing method
In mouse, the combination of a tamoxifen-inducible Cre knock-in
allele with the Rosa26-lacZ reporter strain has been successfully used
to identify stem cell lineages in adult mice (Barker et al., 2007; Takeda
et al., 2011). In Drosophila, the FLP/FRT-based site-specific
recombination system and its derivatives (including MARCM
system) have been successfully used to identify cell (particularly
stem cell) lineages. The FLP/FRT-based mosaic clonal systems are
very effective for identifying a marked cell lineage, but they are not
ideal for accurately identifying the origin of individual marked cells.
In this study, we developed a new lineage-tracing system: T-TRACE.
Similar to the mouse lineage-tracing system, the T-TRACE lineagetracing method is a gene-based system and allows the accurate
identification of all cells derived from one marked cell. However, the
T-TRACE method does not require chromosome segregation at
mitosis to label cells and may have limitation in some applications.
Additionally, as the T-TRACE system is estrogen inducible, the
variable efficiency of estrogen delivery to different tissue may obscure
the tracing result. Thus, the optimal concentration of estrogen has to
be determined for T-TRACE for different tissue. The combination of
the FLP/FRT-based site-specific recombination system and the
T-TRACE system, such as first using the FLP/FRT-based sitespecific recombination system to identify general cell (particularly
stem cell) lineages and then using the T-TRACE system to accurately
identify the origin of individually marked cells, will give more precise
and accurate information.
MATERIALS AND METHODS
Fly stocks
The following fly strains were used: esg-Gal4 line (from Shigeo Hayashi,
RIKEN, Japan); Dl-Gal4 and Su(H)GBE-Gal4 were generated in our
laboratory and have been described previously (Zeng et al., 2010); UASN DN (from Mark Fortini, Thomas Jefferson University, Philadelphia, PA,
USA); UAS-ase (from Yuh-Nung Jan, UCSF, CA, USA); UAS-Cre-EBD304
(from Christian Lehner, University of Zurich, Switzerland); FRT 82B-pros 17
and UAS-prosRNAi (from Tiffany Cook, University of Cincinnati, OH,
USA); 5966 GS (from Haig Keshishian, Yale University, New Haven, CT,
USA); and Su(H)GBE-lacZ (from Sarah Bray, University of Cambridge,
UK). The following strains were obtained from the Bloomington
Drosophila Stock Center: UAS-sc; tub-Gal80ts; UAS-pros RNAi-1
(BL26745); and UAS-pros and fly lines used for MARCM clones,
including FRT 82B-piM; SM6,hs-flp; MKRS,hs-flp, and FRT 82B tub-Gal80.
UAS-scRNAi (v105951) and UAS-pros RNAi-2 (v101477) were obtained from
the Vienna Drosophila RNAi Center (VDRC).
Flies were raised on standard fly food at 25°C and 65% relative humidity,
unless otherwise indicated.
Generation of Ubi-loxP-Stop-loxP-GFP (Ubi-p63E<Stop<GFP)
transgenic flies
A 1.96 kb promoter of Ubi-p63E (CG11624) (Evans et al., 2009) was
amplified from Drosophila genomic DNA and cloned into vector pStinger at
the EcoRI and KpnI sites, using the following primers to produce the
Ubi-GFP construct: 5′-GATGAATTCTGTCCGTATCCTTTAGGC-3′ and
5′-TGAGGTACCTTGGATTATTCTGCGGGA-3′.
Then the stop cassette with flanking loxP sites was amplified from
the UAS-dBrainbow construct (Hampel et al., 2011) using the following
primers: 5′- AGTGGTACCATAACTTCGTATAAAGTATCC-3′ and 5′-AGTGGTACCATAACTTCGTATAGGATACTT-3′.
The PCR product was then cloned into Ubi-pStiner at the KpnI sites to
produce the Ubi-loxP-Stop-loxP-GFP. The above construct was sequenced,
purified, and microinjected into embryos using the standard method.
Lineage tracing
T-TRACE
The lineage-tracing system is doubly controlled by both temperature shift and
estrogen induction. To perform lineage-tracing experiments, we used the
following flies: Ubi-p63E<Stop<EGFP,esg-Gal4; tub-Gal80ts, UAS-creEBD304; Ubi-p63E<Stop<EGFP; Dl-Gal4/tub-Gal80ts, UAS-cre-EBD304;
and Ubi-p63E<Stop<EGFP, Su(H)GBE-Gal4; tub-Gal80ts, UAS-cre-EBD304.
These flies were cultured on food without estrogen at 18°C. To initiate the
lineage-tracing experiment, 3- to 5-day-old flies were transferred to new
food containing 150 μg/ml of estrogen at 29°C for 7 days before dissection.
Pswitch tracing
UAS-flp/5966GS (PswitchPC)-Gal4/Act<y+<EGFP flies were cultured on
food containing RU-486 (10 µg/ml) (Sigma) at 29°C for 7 days before
dissection.
MARCM clonal analysis
To induce MARCM clones of FRT 82B-piM (as a wild-type control) and
FRT 82B-pros 17, we generated the following flies: act>y +>Gal4, UAS-GFP/
SM6, hs-flp; FRT 82B tub-Gal80/-piM (or -pros 17). Three- or four-day-old
adult female flies were heat-shocked for 45 min twice, at an interval of
8-12 h, at 37°C. The flies were transferred to fresh food daily after the final
heat shock, and their posterior midguts were processed for staining at the
indicated times.
RNAi-mediated gene depletion
Male UAS-RNAi transgene flies were crossed with female virgins of esgGal4, UAS-GFP; tub-Gal80 ts, esg-Gal4,UAS-GFP; Su(H)GBE-Gal80, tubGal80 ts (for ISC-specific expression) or of Su(H)GBE-Gal4,UAS-GFP;
tub-Gal80 ts [for Su(H)GBE+ EB-specific expression]. The flies were
cultured at 18°C. Three- to five-day-old adult flies with the appropriate
genotype were transferred to new vials at 29°C for 7 days before dissection.
Fly genotypes
Fig. 1A: Su(H)GBE-lacZ; esg-Gal4, UAS-GFP.
Fig. 1E-F‴: Su(H)GBE-lacZ/hs-flp, tub-Gal4, UAS-GFP; FRT82b, tubGal80/FRT82b, piM.
Fig. 2C: Ubi-p63E<Stop<EGFP, esg-Gal4; tub-Gal80ts, UAS-cre-EBD304.
Fig. 2D: Ubi-p63E<Stop<EGFP; Dl-Gal4/tub-Gal80ts, UAS-cre-EBD304.
Fig. 3A: Ubi-p63E<Stop<EGFP, Su(H)GBE-Gal4; tub-Gal80ts, UAScre-EBD304.
Fig. 3D-E″: UAS-flp; 5966GS/Act<y+<EGFP.
Fig. 5: Ubi-p63E<Stop<EGFP; Dl-Gal4/tub-Gal80ts, UAS-cre-EBD304.
Fig. 6C: act>y+>Gal4, UAS-GFP/SM6, hs-flp; FRT82B tub-Gal80/
FRT82B, piM.
Fig. 6D: act>y +>Gal4, UAS-GFP/SM6, hs-flp; FRT82B tub-Gal80/
FRT82B, pros17.
Fig. 6F-F′: esg-Gal4, UAS-GFP/UAS-scute; Tub-Gal80ts.
Fig. 6G-G′: esg-Gal4, UAS-GFP/UAS-scute; Tub-Gal80ts/UAS-ProsRNAi.
Histology and image capture
The fly intestines were dissected in PBS and fixed in PBS containing 4%
formaldehyde for 20 min. After three 5-min rinses with PBT (PBS+0.1%
Triton X-100), the samples were blocked with PBT containing 5% normal goat
serum and kept overnight at 4°C. Then the samples were incubated with the
primary antibody at room temperature for 2 h and incubated with the
fluorescence-conjugated secondary antibody for 1 h at room temperature.
Samples were mounted in the Vectashield mounting medium with DAPI
(Vector Laboratories). We used the following antibodies: mouse anti-β-Gal
651
DEVELOPMENT
factor Pros functions specifically in ISCs to determine commitment
of ISCs into EEs. Both Pre-EEs and Su(H)GBE+ EBs are
intermediate diploid cells, but they are different from each other.
First, Su(H)GBE+ EBs do not express the ISC marker Dl, but preEEs do. Second, Su(H)GBE+ EBs are post-mitotic cells (Ohlstein
and Spradling, 2007), but some pre-EEs are able to undergo mitotic
division. Our understanding of the flexibility of stem cell
commitment is of the utmost importance for not only basic stem
cell biology, but also targeted tumor therapy.
Development (2015) 142, 644-653 doi:10.1242/dev.113357
(Promega, Z3781; 1:200); mouse anti-Dl (DSHB, C594.9B; 1:50); rabbit antiPdm-1 (a gift from Xiaohang Yang, Zhejiang University, Hangzhou, China;
1:1000); mouse anti-Pros (DSHB, MR1A; 1:50); nc82 (DSHB, nc82; 1:20);
guinea pig anti-Pros (a gift from Tiffany Cook, Cincinnati Children’s Hospital
Medical Center, Cincinnati, OH, USA; 1:3000); and chicken anti-GFP
(Abcam, ab101863; 1:3000). Secondary antibodies used were goat antimouse, anti-chicken, and goat anti-rabbit IgG conjugated to Alexa 488, Alexa
568 or Alexa 647 (Molecular Probes, A-11039, A-11031, A-11029, A-11034,
A-11036, A-21244, A-21236; all 1:400). Images were captured with the Zeiss
LSM 510 confocal system and processed with the LSM Image Browser and
Adobe Photoshop. Different subregions of the midgut have distinct molecular
signatures and stem cells (Buchon et al., 2013; Marianes and Spradling, 2013).
To avoid confusion, we focused only on the posterior midgut in this study.
Quantification and statistical analysis
To quantify the percentage of Pros+ or nc82+ EEs, the Pros+ or nc82+ EEs
and total cells were counted in a 1×105 µm2 area of a single confocal plane.
All the data were analyzed using Student’s t-test; sample size (n) is shown in
the text or figure.
Acknowledgements
We thank Jiangsha Zhao, Chhavi Chauhan and Jennifer Taylor for critical reading
of the manuscript; Shigeo Hayashi, Mark Fortini, Yuh Nung Jan, Tiffany Cook,
Christian Lehner, Haig Keshishian, Sarah Bray, VDRC, the Bloomington Stock
Centers and TRiP at Harvard Medical School for fly stocks; Julie Simpson for the
UAS-dBrainbow DNA construct; Utpal Banerjee for the Ubi-p63E DNA construct;
Tiffany Cook, Xiaohang Yang and the Developmental Studies Hybridoma Bank for
antibodies; and S. Lockett for help with the confocal microscope.
Competing interests
The authors declare no competing or financial interests.
Author contributions
X.Z. and S.X.H. conceived the project, designed and performed the experiments
and wrote the manuscript.
Funding
This research was supported by the Intramural Research Program of the National
Cancer Institute, National Institutes of Health. Deposited in PMC for release after 12
months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.113357/-/DC1
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