Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095-1606, USA
* Author for correspondence (e-mail: jlengyel{at}ucla.edu)
Accepted 14 October 2002
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SUMMARY |
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Key words: JAK/STAT pathway, Cell rearrangement, Tubulogenesis, Morphogenesis, Drosophila melanogaster
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INTRODUCTION |
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An unsolved problem of great interest is the source of the polarizing
information that causes rearranging cells to intercalate in one axis, and not
in another. Although convergent extension in both frogs and fish has been
shown to require the planar cell polarity pathway initiated by non-canonical
Wnt signaling, the nature of the positional cues that orient the rearranging
cells has not been defined (reviewed by
Wallingford et al., 2002).
Similarly, known genes required for oriented cell rearrangement in the C.
elegans dorsal epidermis and Drosophila posterior spiracles
encode putative transcription factors that are thought to confer morphogenetic
capacity, rather than regulate expression of cue-providing molecules
(Heid et al., 2001
;
Brown and Castelli-Gair Hombria,
2000
). As localized, secreted signals have been demonstrated to
guide various types of cell migration and epithelial outgrowth in both
Drosophila and mammals (Duchek et
al., 2001
) (reviewed by
Metzger and Krasnow, 1999
;
Hogan and Kolodziej, 2002
;
Moore, 2001
;
Rollins, 1997
), an appealing
hypothesis is that rearranging cells orient with respect to spatially
localized cell signaling molecules.
The conserved JAK/STAT (Janus Kinase/Signal Transducer and Activator of
Transcription) signaling pathway is widely used; it has been shown to play a
required role in a variety of processes including hematopoiesis, sex
determination, lymphocyte migration, and border cell migration (reviewed by
Ward et al., 2000;
Luo and Dearolf, 2001
;
Sefton et al., 2000
;
Moore, 2001
;
Vila-Coro et al., 1999
;
Silver and Montell, 2001
;
Beccari et al., 2002
). A
suggestion that JAK/STAT signaling might be involved in epithelial cell
rearrangement comes from the observation that the ligand for the
Drosophila JAK/STAT pathway, Unpaired (Upd; Os FlyBase) is
expressed in a highly localized position at the anterior of the embryonic
hindgut epithelium (Iwaki et al.,
2001
). Mutants that block the elongation of the hindgut, which
occurs largely by cell rearrangement, also alter the localized pattern of
upd expression (Iwaki et al.,
2001
).
Exploiting the genetic simplicity of Drosophila, we present
evidence that the JAK/STAT pathway orients cell rearrangement in the hindgut,
a simple epithelial tubule. In contrast to the situation in mammals where
there are four different JAKs and seven different STATs
(Imada and Leonard, 2000), the
Drosophila genome encodes only one known ligand (upd), one
receptor (the cytokine-like domeless, dome, also known as master
of marelle), one JAK (hopscotch, hop) and one STAT
(Stat92E, also known as marelle) (reviewed by
Castelli-Gair Hombria and Brown,
2002
). For examining cell movement during tubulogenesis, the
hindgut is a particularly useful model as it elongates by cell rearrangement
without either cell proliferation or apoptosis
(Iwaki et al., 2001
) (reviewed
by Lengyel and Iwaki, 2002
).
Analysis of loss-of-function mutants shows that the key components of the
Drosophila JAK/STAT pathway are required to achieve a fully elongated
hindgut; gain-of-function (overexpression) studies show that uniform high
level activation of the pathway is not sufficient, while localized production
of ligand is necessary to promote oriented cell rearrangement. Our results
support a model in which an anteroposterior gradient of ligand activates STAT
activity in a similar gradient, leading to orientation of cell rearrangement.
This is the first example of a required role for JAK/STAT signaling in
orienting cell rearrangement that drives elongation of an epithelium.
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MATERIALS AND METHODS |
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Generation of germline clones
Germline clones of hop and Stat92E were made using the
FLP-DFS technique (Chou et al.,
1993). For hop, larvae of the genotype
hopC111 FRT101/ovoD1
FRT101; hsFLP38 were heat shocked at 37°C for 2 hours on each of
days 4 and 5 after egg deposition. The eclosed females of this genotype were
then mated to FM7, act-lacZ males to obtain progeny, half of which
lacked both maternal and zygotic contributions of hop (referred to as
hopm-z-). For Stat92E, larvae of the
genotype hsFLP122;FRT82B ovoD1/FRT82B
Stat92EP1681 were similarly heat shocked and
mated to Stat92E06346/TM3 ftz-lacZ males
to obtain Stat92Em-z-progeny.
Histology
Whole-mount in situ hybridization was carried out as described
(Pignoni and Zipursky, 1997;
Tautz and Pfeifle, 1989
).
Digoxigenin-labeled RNA probes (Roche Molecular Biochemicals) were made from
cDNA templates of upd (Harrison
et al., 1998
), dome
(Brown et al., 2001
),
hop (Binari and Perrimon,
1994
), Stat92E (Hou
et al., 1996
; Yan et al.,
1996
), drm (Green et
al., 2002
), Ser
(Thomas et al., 1991
),
hh (Lee et al., 1992
)
and dri (Gregory et al.,
1996
). Antibody staining of embryos was performed using standard
techniques (Ashburner, 1989
).
Antibodies (and dilutions) used were
-Crb (1:100),
-Wg (1:100)
and
-En (1:5), all available from the Developmental Studies Hybridoma
Bank at the University of Iowa Department of Biological Sciences;
-Stat92E (Chen et al.,
2002
) (1:1000);
-phosphorylated histone H3 (Upstate
Biotechnology, 1:500);
-Con
(Meadows et al., 1994
) (1:30);
and
-ß-Galactosidase (Promega, 1:1000, Cappel, 1:500). For
transverse sections, embryos were fixed in either 2% glutaraldehyde plus 1%
osmium tetroxide, or in 4% formaldehyde, embedded in Epon and sectioned at 2
µm. Sections were stained with 0.025% Methylene Blue and 0.01% Toluidine
Blue in 0.025% sodium tetraborate buffer. Light microscopy was carried out
using a Zeiss Axiophot microscope, and images were captured with a Sony
DKC-5000 digital camera. Confocal microscopy was carried out using a Carl
Zeiss LSM 310 with a 40x objective and 1.5x digital zoom; images
were acquired and processed using Zeiss LSM software. Hindgut lengths were
measured as previously described (Iwaki et
al., 2001
) using images acquired with a Hamamatsu camera and
Axiovision software. Total hindgut cell number in embryos of different mutant
genotypes was determined by counting nuclei (identified by
anti-ß-Galactosidase staining) that were expressing lacZ due to
the presence of either bynapro or
bynGAL4,UAS-lacZ.nls, following previously published procedures
(Iwaki et al., 2001
). The
number of cells in the hindgut circumference was determined by counting cells
in eight to ten serial transverse sections through the large intestine of
three to four different embryos. Embryos were staged according to
Campos-Ortega and Hartenstein
(Campos-Ortega and Hartenstein,
1997
).
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RESULTS |
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wg is expressed throughout the hindgut primordium starting at stage 5; by late stage 10 it is expressed at the most anterior and most posterior of the hindgut (Fig. 1C). To assess the required role of wg expression at these different stages, we carried out temperature shift experiments with a temperature-sensitive wg mutant (see supplementary figure S1 at http://dev.biologists.org/supplemental/). While early lack of wg function results in a dramatically smaller hindgut, the elimination of wg function during stage 10 (prior to the period of major elongation of the hindgut) or later allows essentially normal hindgut elongation. Thus, localized expression of Ser, hh, spi, dpp, Dl and wg does not appear to play a required role in hindgut cell rearrangement.
Localized expression of upd, dome and Stat92E in
the developing hindgut
upd, encoding the ligand for the Drosophila JAK/STAT
pathway, is only expressed in the small intestine
(Fig. 1C,H) and is regulated by
genes controlling hindgut cell rearrangement. In drm and
bowl mutants, expression of upd is missing from the small
intestine (Fig. 1I,J), while in
lin mutants, upd expression is expanded throughout much of
the hindgut (Fig. 1K). These
results raise the possibility that localized Upd might provide an orienting
cue for rearranging hindgut cells.
If it plays a role in hindgut cell rearrangement, upd must be expressed before and during the period of major hindgut elongation, i.e. between stages 11 and 16 (Fig. 1A); genes encoding the other known components of the Drosophila JAK/STAT signaling pathway, summarized in Fig. 2A, should also be expressed at the same stages, both within and adjacent to upd-expressing cells. We used in situ hybridization to characterize the expression of upd, dome, hop and Stat92E during stages just prior to and during hindgut elongation; characterization of Stat92E protein expression is presented in a subsequent section.
|
Expression of upd in the hindgut is first detected at stage 9 in a narrow ring of cells that will become the small intestine (Fig. 2B). Expression in the prospective small intestine is maintained during stages 10 and 11 (Fig. 2C), where it can be seen just posterior to the everting renal tubules (note that in the hindgut at these germband-extended stages, `posterior' is toward the head). During stages 12-14, when the hindgut undergoes a major part of its elongation, upd expression is seen throughout the now distinct small intestine (Fig. 2D). Expression of upd is maintained throughout the small intestine during the remainder of embryogenesis.
In addition to upd, we examined expression in the hindgut of genes
encoding the JAK/STAT receptor (dome), JAK (hop) and STAT
(Stat92E). hop is expressed uniformly throughout the embryo,
including the hindgut as it elongates (data not shown)
(Binari and Perrimon, 1994).
Expression of both dome and Stat92E is detected weakly at
the anterior of the hindgut beginning at stage 9, becomes significantly
stronger by stage 11, and is maintained through stage 14
(Fig. 2E-J). For both the
receptor- and STAT-encoding genes, expression domains in the hindgut
epithelium overlap with and extend beyond the narrow domain of upd
expression (Fig. 2, brackets).
Most significantly, expression of dome and Stat92E extends
to a more posterior position in the hindgut epithelium than does expression of
upd (Fig. 2, compare
C,F,I, brackets). Thus, the mRNA expression of the ligand, receptor and STAT
components in the hindgut prior to and during its elongation is consistent
with a role for JAK/STAT signaling in hindgut cell rearrangement.
Required role of upd and JAK/STAT pathway components in
hindgut elongation
To assess the requirement for upd and JAK/STAT signaling in
hindgut elongation, we examined hindgut morphology, length and circumference
in wholemounts and transverse sections of embryos deficient for upd, dome,
hop or Stat92E. In embryos lacking zygotic upd
function, the hindgut reaches only about half its normal length and is
somewhat wider (Fig. 3B).
Hindguts are also incompletely elongated and wider in embryos lacking both
maternal and zygotic activity of either hop or Stat92E
(Fig. 3C,D). Embryos lacking
zygotic dome function have hindguts only slightly shorter than wild
type (data not shown), presumably because of the maternal contribution of
dome (Brown et al.,
2001). However, when a dominant negative form of Dome
(UAS-dome
CYT3.2) is expressed uniformly using
bynGAL4, hindguts are significantly shorter and wider
(Fig. 3I). Length measurements
reveal that upd-, hop- and Stat92E-deficient, as
well as DomeDN- expressing hindguts, while not as short and wide as
those of drm, bowl and lin embryos (compare
Fig. 1B-D), are nevertheless
40-50% shorter than those of wild-type (summarized in
Fig. 3Q). Consistent with their
wider appearance in whole-mount embryos, upd, dome, hop and
Stat92E-deficient hindguts have a greater number of cells in their
circumference (19-27) than do wild-type hindguts (12)
(Fig. 3F-H,M,Q). Overall, the
shorter and wider appearance of the hindgut is roughly similar among embryos
lacking the different components of the JAK/STAT pathway.
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The shorter hindgut length is not due to a deficiency of cells, nor is the
excess number of cells in the circumference due to overproliferation, as the
total number of hindgut epithelial cells is 96, 89 and 85% of wild-type for
upd, hop and Stat92E mutant embryos, respectively
(Fig. 3Q). Further, staining of
upd embryos with anti-phosphorylated histone H3, a marker of mitosis,
did not detect more cell division than observed in wild type (data not shown).
The number of cells in the wild-type hindgut at stage 11 is 50; by stage
16, this number has been reduced dramatically (to
12) by cell
rearrangement (Iwaki et al.,
2001
). Because by stage 16 the number of cells in the
circumference of upd, hop and Stat92E embryos is reduced
only partially (to roughly 20-30), while total cell number is essentially the
same as seen in wild-type (Fig.
3Q), it must be concluded that, in the JAK/STAT loss-of-function
mutants tested, there is a defect in hindgut cell rearrangement.
Requirement for localized JAK/STAT signaling
If localized JAK/STAT signaling provides an orienting cue for cell
rearrangement, then ectopic JAK/STAT signaling throughout the hindgut would be
expected to disrupt this process. To test this, we used bynGAL4 to
drive various UAS constructs uniformly in the hindgut epithelium. Uniform
expression of upd results in hindguts that elongate to only about 65%
of the wild-type length and have about 50% more than the normal number of
cells in their circumference (Fig.
3J,N,Q). In the testis, eye and hemocytes, ectopic expression of
upd or activated JAK causes increased cell proliferation
(Kiger et al., 2001;
Tulina and Matunis, 2001
;
Chen et al., 2002
;
Luo et al., 1997
). However,
total cell number in hindguts ectopically expressing upd is only 76%
of normal (Fig. 3Q); consistent
with this, staining with anti-phosphorylated histone H3 did not detect excess
cell proliferation (data not shown). As uniform expression of upd in
the hindgut does not result in an increase (but rather a reduction) in hindgut
cell number, the excess number of circumferential cells seen in upd
overexpressing hindguts must arise from a defect in cell rearrangement.
To ask whether localized activation of other components of the JAK/STAT pathway is required for hindgut elongation, we expressed uniformly an activated form of the Drosophila JAK (UAS-hopTML). Similar to what was seen for uniform expression of upd, total cell number did not differ significantly from wild type, but the resulting hindguts were shorter and had more circumferential cells (Fig. 3K,O,Q). When both UAS-hopTML and UAS-Stat92E are driven by bynGAL4 (Fig. 3L,P), the hindgut elongation defect is more severe than UAS-hopTML alone.
The ectopic expression studies presented here demonstrate that, while components of JAK/STAT signaling are required, activation of the pathway at uniformly high levels throughout the hindgut is not compatible with normal cell rearrangement. Experiments to be presented in a subsequent section further support the idea that spatially restricted JAK/STAT signaling is necessary for hindgut cell rearrangement.
Disruption of JAK/STAT signaling does not affect hindgut
patterning
As proper cell rearrangement is correlated with correct hindgut patterning
(Iwaki et al., 2001), it could
be argued that, rather than affecting the orientation of rearranging cells
directly, upd and JAK/STAT signaling control cell rearrangement by
affecting patterning. To test this idea, we assessed gene expression
characteristic of the different hindgut regions
(Fig. 4A) in upd
mutants and in embryos ectopically expressing upd throughout the
hindgut. Examination of all markers tested (except wg, see below)
supports the conclusion that all three domains small intestine, large
intestine, and rectum as well as boundary cell rows and rings are
present and correctly patterned in upd mutant hindguts
(Fig. 4B;
updos1A row). Similarly, all three hindgut
domains and boundary cells are present when upd is uniformly
expressed in the hindgut (Fig.
4B; bynGAL4; UAS-upd row). The only domain missing from
hindguts lacking upd is a small, wg-expressing region at the
extreme anterior of the small intestine that is established during stages
10/11 (Fig. 4A,B). As our
temperature-shift experiments showed that activity of wg is not
required for hindgut elongation after stage 10, this defect in wg
expression cannot be the basis for the effect of upd loss-of-function
on hindgut elongation.
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In summary, all domains of the hindgut, with the exception of a small number of cells at its anteriormost tip, are correctly patterned in hindguts either lacking or uniformly expressing upd. We therefore conclude that, rather than affecting patterning primarily and morphogenesis secondarily, upd and JAK/STAT signaling directly affect the cell rearrangement that elongates the hindgut.
Spatially restricted upd is required for hindgut cell
rearrangement
If localized expression of upd at the anterior of the hindgut
epithelium is required for hindgut cell rearrangement, then expression of
upd in this domain should rescue the elongation defect in embryos
lacking upd. We therefore used drmGAL4 to drive expression
of UAS-upd only at the anterior of the hindgut in embryos lacking
upd function. The resulting hindguts appeared morphologically normal,
with an average length 93% that of wild type
(Fig. 5C,D,I). Thus, anteriorly
localized (in the small intestine) expression of upd in an
upd mutant background is sufficient for elongation.
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Is restriction of upd expression only to the hindgut anterior
necessary for the cell rearrangement that drives elongation? To answer this,
we used the uniformly expressed bynGAL4 to drive upd in an
upd mutant background. Rather than rescuing (like the
drmGAL4 driver), this resulted in severely defective, short and wide
hindguts (Fig. 5A,B). It could
be argued that the early and high level of expression driven by
bynGAL4 causes a level of activation of JAK/STAT pathway that
inhibits cell rearrangement. We therefore performed the same experiment with
fkhGAL4 and 455.2GAL4, which (respectively) drive expression
uniformly in the hindgut at progressively lower levels and later times
(Iwaki and Lengyel, 2002;
Fuss et al., 2000
;
San Martin and Bate, 2001
) (D.
D. I., unpublished). We observed neither rescue of the upd hindgut
phenotype nor a phenotype more defective than that of upd alone (in
contrast to the result with bynGAL4; data not shown). Thus,
anteriorly localized upd expression appears to be both necessary and
sufficient for cell rearrangement in the large intestine.
An important issue is whether upd plays a permissive role in the hindgut (giving cells the ability to rearrange), or an instructive role (orienting cells as they rearrange). As none of three different levels of uniform upd expression rescued the upd loss-of-function phenotype, it seems unlikely that the function of JAK/STAT signaling in the hindgut is simply to promote the capacity of cells to rearrange. Rather, the data support the notion that the required role of localized expression of upd is to provide an instructive cue that orients cell rearrangement.
upd signaling mediates drm function in hindgut
Although upd cannot entirely mediate the effect of drm on
hindgut cell rearrangement (as drm hindguts are shorter and wider
than those of upd; Fig.
5E,F), we asked whether, and to what extent, expression of
UAS-upd under control of drmGAL4 could rescue the
drm loss-of-function phenotype. Strikingly, expression of
upd at the anterior of the drm mutant hindgut is sufficient
to bring about significant rescue of the drm hindgut phenotype, as
assessed in both wholemounts and transverse sections
(Fig. 5G,H). Compared with
drm hindguts, the partially rescued hindguts are 45% longer, and have
35% fewer cells in their circumference
(Fig. 5I). The rescue of the
drm hindgut phenotype by anteriorly expressed upd thus
demonstrates that upd is a key mediator of drm function in
the hindgut.
Upd signal is received by cells of the large intestine
Given the small number of cells in its circumference by stage 16
(Fig. 3E)
(Iwaki et al., 2001), the
large intestine must undergo significant cell rearrangement as it elongates.
Yet upd, which we have shown controls cell rearrangement throughout
much of the hindgut, is expressed only in the small intestine, and not in the
large intestine. Because action of Upd over a distance has been described in
the eye disc (Zeidler et al.,
1999
), it seemed possible that Upd produced in the small intestine
might control cell rearrangement by activating JAK/STAT signaling in the large
intestine (Fig. 1C,H). During
stages 11 and 12, as cell rearrangement is elongating the hindgut, the
prospective large intestine is 50 to 60 µm in length
(Fig. 6A,B); this is the
distance over which Upd would have to diffuse to affect the entire large
intestine.
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In addition to activating phosphorylation of Stat92E
(Luo and Dearolf, 2001),
expression of upd has also been shown to upregulate Stat92E protein
levels during embryogenesis (Chen et al.,
2002
). We therefore investigated expression of Stat92E
mRNA and protein as reporters for receipt of the Upd signal. Antibody staining
shows that, in the stage 11 hindgut, Stat92E protein is greatly reduced in the
absence of upd function, and dramatically upregulated when
upd is uniformly expressed (Fig.
6C,D). This regulation appears to occur at the transcriptional
level, as Stat92E mRNA is similarly reduced in the absence of
upd function, and upregulated when upd is uniformly
expressed in the hindgut (Fig.
6E,F). We conclude that staining with anti-Stat92E identifies
cells receiving Upd signal.
During stages 11 to 12, Stat92E protein is detected in a domain that extends more posteriorly than the upd expression domain at the hindgut anterior (compare Fig. 2C and Fig. 6G,I); this can be seen most clearly when both upd mRNA and Stat92E protein are labeled (Fig. 6K). Sagittal sections reveal that Stat92E is present in the nuclei of the hindgut epithelium at stages 11 and 12, (Fig. 6H,J); therefore, during these stages, at least some of the large intestine epithelial cells are receiving Upd signal. Significantly, Stat92E appears to be present in the hindgut as a gradient, with the most anterior cells (at the point of renal tubule evagination) most strongly labeled; during stages 11 to 12, this gradient extends to at least 20% to 45% of the length of the large intestine primordium (Fig. 6G-J). Because of limits in dynamic range of the anti-Stat92E assay, we cannot presently determine whether, at some point, this gradient extends over the entire large intestine.
Stat92E is present not only in the nuclei of the hindgut epithelium, but
also in the nuclei of the hindgut visceral mesoderm
(Fig. 6J; see supplementary
figure S2 at
http://dev.biologists.org/supplemental/).
However, as determined by staining with anti-Connectin, the hindgut visceral
mesoderm is normal in upd mutant embryos (see supplementary figure
S2); furthermore, the hindguts of twi mutants, which lack all
mesoderm, are able to undergo a significant amount of elongation (see
supplementary figure S2) (San Martin and
Bate, 2001). Thus, even though cells of the visceral mesoderm
receive the Upd signal, they do not appear to play a significant role in the
rearrangement of cells within the hindgut epithelium.
The expression of Stat92E in the hindgut epithelium, while suggestive, does not reveal whether receipt of Upd signal by prospective large intestine cells is required for their rearrangement. Because in an upd mutant embryo expression of upd at the anterior of the hindgut rescues elongation (Fig. 5C), we asked whether expression of dominant active Hop (hopTML) might also rescue. As a control, we expressed hopTML uniformly in the hindgut; similar to what we observed for upd, this results in a dramatic upregulation of Stat92E (Fig. 6, compare D with L). A key finding is that, in contrast to the essentially complete rescue of the upd hindgut phenotype when upd is expressed anteriorly (Fig. 6M), anterior expression of hopTML in an upd background does not rescue hindgut elongation (Fig. 6N). These results demonstrate that activation of the JAK/STAT pathway only in cells of the prospective small intestine (i.e. the domain that expresses upd) is not sufficient for normal hindgut cell rearrangement. Thus, upd is required in a non-cell autonomous fashion for hindgut cell rearrangement.
The observed upregulation of Stat92E in cells of the hindgut posterior to
the small intestine is consistent with the cell non-autonomous function of
upd in the hindgut. The fact that cell rearrangement is severely
abnormal when uniform levels of Stat92E and HopTML are driven
together (Fig. 3L,P) further
supports the idea that Stat92E must be distributed non-uniformly, i.e. as a
gradient, in order for cells to rearrange. The observed gradient of Stat92E,
reaching to at least 20% of the length of the large intestine during stage 11,
and to at least 45% during stage 12 (Fig.
6H,J), suggests that there is a corresponding gradient of the
activating ligand Upd in the hindgut. Both the distance (roughly 20 to 40
µm, see Fig. 6A,B) and time
(stages 9 through 12, roughly 4-6 hours) over which this postulated Upd
gradient is established are consistent with parameters described for formation
of gradients of Upd, Dpp and Wg in imaginal disc tissues
(Zeidler et al., 1999;
Entchev et al., 2000
;
Teleman and Cohen, 2000
;
Strigini and Cohen, 2000
), as
described below.
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DISCUSSION |
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Upd is required non-autonomously for hindgut cell rearrangement
The rescue of the upd phenotype by anteriorly localized expression
in the hindgut of upd, but not of activated JAK (Hopscotch),
demonstrates that there is a requirement for upd function that is not
cell autonomous. In other words, upd is required in cells (those of
the large intestine that undergo the greatest rearrangement) different from
cells that produce it (those of the small intestine). A number of examples
have been described in which localized expression of a signaling molecule
(including Upd) is required non-autonomously for cell rearrangement,
morphogenesis or motility. In the Drosophila eye imaginal disc,
expression of Upd at the midline is required to establish a dorsoventral
polarity that orients ommatidial rotation
(Zeidler et al., 1999). In
both Drosophila tracheae and the vertebrate lung, branching
morphogenesis of the epithelium depends on localized expression of FGF in
adjacent mesenchyme (reviewed by Metzger
and Krasnow, 1999
; Hogan and
Kolodziej, 2002
).
Localized activation of JAK/STAT signaling has been shown to play a role in
cell motility in a number of contexts. In Drosophila, localized
expression of Upd in the anterior polar cells of the egg chamber acts to
coordinate the migration of the adjacent border cells
(Silver and Montell, 2001;
Beccari et al., 2002
). In
mammals, cytokines expressed in target tissues act to attract both migrating
lymphocytes and tumor (reviewed by Moore,
2001
; Muller et al.,
2001
; Murphy,
2001
; Vila-Coro et al.,
1999
). Our finding that localized (only in the small intestine)
expression of upd is both necessary and sufficient for rearrangement
of cells in the large intestine indicates that Upd must have an
organizational, action-at-a-distance function in controlling cell
rearrangement during tubular morphogenesis.
A predicted gradient of Upd in the hindgut
The rescue experiments discussed above establish that there is a cell
non-autonomous requirement for upd in hindgut elongation. Consistent
with this, there is evidence that Upd is present and required in an
anteroposterior gradient in the hindgut. Prior to and during hindgut
elongation, both Stat92E mRNA and Stat92E protein are detected not
only in the small intestine epithelium (and the visceral mesoderm surrounding
the small intestine), but also in the epithelium posterior to the small
intestine; this expression of Stat92E appears to be in a gradient. In the
Drosophila eye imaginal disc, a gradient of Upd is required to orient
the rotation of ommatidial cell clusters
(Zeidler et al., 1999); in
addition, there is evidence for a gradient of Upd and Stat92E in patterning of
the follicular epithelium of the Drosophila egg chamber (R. Xi, J. R.
McGregor and D. A. Harrison, unpublished). As expression of Stat92E depends on
upd (this work) (Chen et al.,
2002
), it is likely that Upd protein is present in the hindgut
epithelium as an anteroposterior gradient, with its highest level in the
upd-expressing cells of the small intestine, and lowest level in
posterior, upd non-expressing cells of the large intestine.
Expression of SOCS36E (suppressor of cytokine signaling at
36E), which is regulated by upd, overlaps with and extends
significantly beyond the domain of upd expression
(Karsten et al., 2002
),
further supporting the idea that there is a gradient of Upd in the hindgut. A
model that summarizes the observed localized expression of upd mRNA,
a gradient of Stat92E protein, and cell rearrangement leading to elongation is
shown in Fig. 7.
|
In the Drosophila eye imaginal disc, anti-Upd staining and the
behavior of clones of mutant cells that have lost components of the JAK/STAT
pathway indicate that Upd is present in a gradient that extends at least 50
µm beyond its midline mRNA expression domain
(Zeidler et al., 1999). In the
Drosophila hindgut, we have shown that Stat92E is a reliable reporter
for the presence of Upd. Two to four hours after upd is first
expressed at the anterior of the hindgut (stage 9), Stat92E can be detected at
least 30-40 µm from the site of upd expression (stages 11 and 12).
These time and distance parameters are similar to those observed during
generation of the Upd gradient in the eye, and the Dpp and Wg gradients in
wing imaginal discs, which form over distances of roughly 40-80 µm in 1-8
hours (Zeidler et al., 1999
;
Entchev et al., 2000
;
Teleman and Cohen, 2000
;
Strigini and Cohen, 2000
).
Thus, it is reasonable to imagine that a gradient of Upd is established in the
developing hindgut in a short enough time frame to affect cell
rearrangement.
The essential consequence of JAK/STAT signaling is activation of the STAT
protein, which leads to altered transcriptional programs
(Darnell, 1997;
Horvath, 2000
). STAT has been
shown in a number of contexts to be required for cell motility
(Sano et al., 1999
;
Yamashita et al., 2002
;
Silver and Montell, 2001
;
Beccari et al., 2002
), and
therefore probably regulates expression of genes controlling cytoskeletal
assembly and cell adhesion. In these contexts, however, activation of STAT
does not appear to be required to orient cell movement, but rather to
facilitate or promote it. As Stat92E is required for hindgut elongation, and
its protein product appears to be present in a gradient along the
anteroposterior axis, this raises the intriguing question of how a gradient of
a transcription factor might orient cell rearrangement.
Concluding remarks
Our results demonstrate a new role for Upd and the JAK/STAT pathway, namely
the control of cell rearrangement that elongates a tubular epithelium. Given
the widespread occurrence of JAK/STAT signaling, further analysis of the
mechanism by which JAK/STAT signaling controls hindgut cell rearrangement in
Drosophila is likely to provide insights into the control of cell
motility during many processes, including organogenesis, wound healing and
cancer metastasis.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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---|
Ashburner, M. (1989). Drosophila: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Beccari, S., Teixeira, L. and Rorth, P. (2002). The JAK/STAT pathway is required for border cell migration during Drosophila oogenesis. Mech. Dev. 111,115 -123.[CrossRef][Medline]
Binari, R. and Perrimon, N. (1994). Stripe-specific regulation of pair-rule genes by hopscotch, a putative Jak family tyrosine kinase in Drosophila. Genes Dev. 8,300 -312.[Abstract]
Brown, S. and Castelli-Gair Hombria, J. (2000).
Drosophila grain encodes a GATA transcription factor required for
cell rearrangement during morphogenesis. Development
127,4867
-4876.
Brown, S., Hu, N. and Hombria, J. C. (2001). Identification of the first invertebrate interleukin JAK/STAT receptor, the Drosophila gene domeless. Curr. Biol. 11,1700 -1705.[CrossRef][Medline]
Campos-Ortega, J. A. and Hartenstein, V. (1997). The Embryonic Development of Drosophila melanogaster. Berlin: Springer-Verlag.
Castelli-Gair Hombria, J. and Brown, S. (2002). The fertile field of Drosophila JAK/STAT signalling. Curr. Biol. 12,R569 -R575.[CrossRef][Medline]
Chen, H. W., Chen, X., Oh, S. W., Marinissen, M. J., Gutkind, J.
S. and Hou, S. X. (2002). mom identifies a receptor
for the Drosophila JAK/STAT signal transduction pathway and encodes a
protein distantly related to the mammalian cytokine receptor family.
Genes Dev. 16,388
-398.
Chou, T. B., Noll, E. and Perrimon, N. (1993).
Autosomal P[ovoD1] dominant female-sterile insertions in
Drosophila and their use in generating germ-line chimeras.
Development 119,1359
-1369.
Darnell, J. E., Jr (1997). STATs and gene
regulation. Science 277,1630
-1635.
Duchek, P., Somogyi, K., Jekely, G., Beccari, S. and Rorth, P. (2001). Guidance of cell migration by the Drosophila PDGF/VEGF receptor. Cell 107, 17-26.[Medline]
Entchev, E. V., Schwabedissen, A. and Gonzalez-Gaitan, M. (2000). Gradient formation of the TGF-beta homolog Dpp. Cell 103,981 -991.[Medline]
Ettensohn, C. A. (1985). Gastrulation in the sea urchin embryo is accompanied by the rearrangement of invaginating epithelial cells. Dev. Biol. 112,383 -390.[Medline]
Ferrus, A., Llamazares, S., de la Pompa, J. L., Tanouye, M. A.
and Pongs, O. (1990). Genetic analysis of the Shaker
gene complex of Drosophila melanogaster. Genetics
125,383
-398.
Fuss, B. and Hoch, M. (2002). Notch signaling controls cell fate specification along the dorsoventral axis of the Drosophila gut. Curr. Biol. 12,171 -179.[CrossRef][Medline]
Fuss, B., Meissner, T., Bauer, R., Lehmann, C., Eckardt, F. and Hoch, M. (2000). Control of endoreplication domains in the Drosophila gut by the knirps and knirps-related genes. Mech. Dev. 100,15 -23.[CrossRef]
Green, R. B., Hatini, V., Johansen, K. A., Liu, X. J. and
Lengyel, J. A. (2002). Drumstick is a zinc finger protein
that antagonizes Lines to control patterning and morphogenesis of the
Drosophila hindgut. Development
129,3645
-3656.
Gregory, S. L., Kortschak, R. D., Kalionis, B. and Saint, R. (1996). Characterization of the dead ringer gene identifies a novel, highly conserved family of sequence-specific DNA-binding proteins. Mol. Cell. Biol. 16,792 -799.[Abstract]
Harrison, D. A., Binari, R., Nahreini, T. S., Gilman, M. and Perrimon, N. (1995). Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and developmental defects. EMBO J. 14,2857 -2865.[Abstract]
Harrison, D. A., McCoon, P. E., Binari, R., Gilman, M. and
Perrimon, N. (1998). Drosophila unpaired encodes a
secreted protein that activates the JAK signaling pathway. Genes
Dev. 12,3252
-3263.
Hatini, V., Bokor, P., Goto-Mandeville, R. and DiNardo, S.
(2000). Tissue-and stage-specific modulation of Wingless
signaling by the segment polarity gene lines. Genes
Dev. 14,1364
-1376.
Heid, P. J., Raich, W. B., Smith, R., Mohler, W. A., Simokat, K., Gendreau, S. B., Rothman, J. H. and Hardin, J. (2001). The zinc finger protein DIE-1 is required for late events during epithelial cell rearrangement in C. elegans. Dev. Biol. 236,165 -180.[CrossRef][Medline]
Hoch, M. and Pankratz, M. J. (1996). Control of gut development by forkhead and cell signaling molecules in Drosophila. Mech. Dev. 58,3 -14.[CrossRef][Medline]
Hogan, B. L. and Kolodziej, P. A. (2002). Organogenesis: molecular mechanisms of tubulogenesis. Nat. Rev. Genet. 3,513 -523.[CrossRef][Medline]
Horvath, C. M. (2000). STAT proteins and transcriptional responses to extracellular signals. Trends Biochem. Sci. 25,496 -502.[CrossRef][Medline]
Hou, X. S., Melnick, M. B. and Perrimon, N. (1996). Marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the mammalian STATs. Cell 84,411 -419.[Medline]
Imada, K. and Leonard, W. J. (2000). The Jak-STAT pathway. Mol. Immunol. 37, 1-11.[CrossRef][Medline]
Irvine, K. D. and Wieschaus, E. (1994). Cell
intercalation during Drosophila germband extension and its regulation
by pair-rule segmentation genes. Development
120,827
-841.
Iwaki, D. D., Johansen, K. A., Singer, J. B. and Lengyel, J. A. (2001). drumstick, bowl, and lines are required for patterning and cell rearrangement in the Drosophila embryonic hindgut. Dev. Biol. 240,611 -626.[CrossRef][Medline]
Iwaki, D. D. and Lengyel, J. A. (2002). A Delta-Notch signaling border regulated by Engrailed/Invected repression specifies boundary cells in the Drosophila hindgut. Mech. Dev. 114,71 -84.[CrossRef][Medline]
Karsten P., Elend, S. and Zeidler, M. P. (2002). Cloning and expression of Drosophila SOCS36E and its potential regulation by the JAK/STAT pathway. Mech. Dev. 117,343 -346.[CrossRef][Medline]
Keller, R., Davidson, L., Edlund, A., Elul, T., Ezin, M., Shook, D. and Skoglund, P. (2000). Mechanisms of convergence and extension by cell intercalation. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355,897 -922.[CrossRef][Medline]
Kiger, A. A., Jones, D. L., Schulz, C., Rogers, M. B. and
Fuller, M. T. (2001). Stem cell self-renewal specified by
JAK-STAT activation in response to a support cell cue.
Science 294,2542
-2545.
Lee, J. J., von Kessler, D. P., Parks, S. and Beachy, P. A. (1992). Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog.Cell 71,33 -50.[Medline]
Lengyel, J. A. and Iwaki, D. D. (2002). It takes guts: the Drosophila hindgut as a model system for organogenesis. Dev. Biol. 243, 1-19.[CrossRef][Medline]
Leung, B., Hermann, G. J. and Priess, J. R. (1999). Organogenesis of the Caenorhabditis elegans intestine. Dev. Biol. 216,114 -134.[CrossRef][Medline]
Luo, H. and Dearolf, C. R. (2001). The JAK/STAT pathway and Drosophila development. BioEssays 23,1138 -1147.[CrossRef][Medline]
Luo, H., Rose, P., Barber, D., Hanratty, W. P., Lee, S., Roberts, T. M., D'Andrea, A. D. and Dearolf, C. R. (1997). Mutation in the Jak kinase JH2 domain hyperactivates Drosophila and mammalian Jak-Stat pathways. Mol. Cell. Biol. 17,1562 -1571.[Abstract]
Meadows, L. A., Gell, D., Broadie, K., Gould, A. P. and White,
R. A. (1994). The cell adhesion molecule, connectin, and the
development of the Drosophila neuromuscular system. J.
Cell Sci. 107,321
-328.
Metzger, R. J. and Krasnow, M. A. (1999).
Genetic control of branching morphogenesis. Science
284,1635
-1639.
Moore, M. A. (2001). The role of chemoattraction in cancer metastases. BioEssays 23,674 -676.[CrossRef][Medline]
Muller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M. E., McClanahan, T., Murphy, E., Yuan, W., Wagner, S. N. et al. (2001). Involvement of chemokine receptors in breast cancer metastasis. Nature 410,50 -56.[CrossRef][Medline]
Murakami, R., Shigenaga, A., Kawakita, M., Takimoto, K., Yamaoka, I., Akasaka, K. and Shimada, H. (1995). aproctous, a locus that is necessary for the development of the proctodeum in Drosophila embryos, encodes a homolog of the vertebrate Brachyury gene. Roux's Arch. Dev. Biol. 205, 89-96.
Murphy, P. M. (2001). Chemokines and the
molecular basis of cancer metastasis. New Engl. J.
Med. 345,833
-835.
Nusslein-Volhard, C., Wieschaus, E. and Kluding, H. (1984). Mutations affecting the pattern of the larval cuticle of Drosophila melanogaster. I.Zygotic loci on the second chromosome. Roux's Arch. Dev. Biol. 192,267 -282.
Perrimon, N. and Mahowald, A. P. (1986). l(1)hopscotch, A larval-pupal zygotic lethal with a specific maternal effect on segmentation in Drosophila. Dev. Biol. 118, 28-41.[Medline]
Pignoni, F. and Zipursky, S. L. (1997).
Induction of Drosophila eye development by decapentaplegic.Development 124,271
-278.
Rollins, B. J. (1997). Chemokines.
Blood 90,909
-928.
San Martin, B. and Bate, M. (2001). Hindgut
visceral mesoderm requires an ectodermal template for normal development in
Drosophila. Development
128,233
-242.
Sano, S., Itami, S., Takeda, K., Tarutani, M., Yamaguchi, Y.,
Miura, H., Yoshikawa, K., Akira, S. and Takeda, J. (1999).
Keratinocyte-specific ablation of Stat3 exhibits impaired skin remodeling, but
does not affect skin morphogenesis. EMBO J.
18,4657
-4668.
Sefton, L., Timmer, J. R., Zhang, Y., Beranger, F. and Cline, T. W. (2000). An extracellular activator of the Drosophila JAK/STAT pathway is a sex-determination signal element. Nature 405,970 -973.[CrossRef][Medline]
Silver, D. L. and Montell, D. J. (2001). Paracrine signaling through the JAK/STAT pathway activates invasive behavior of ovarian epithelial cells in Drosophila. Cell 107,831 -841.[Medline]
Skaer, H. (1993). The alimentary canal. InThe Development of Drosophila melanogaster (ed. M. Bate and A. Martinez Arias), pp. 941-1012. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Strigini, M. and Cohen, S. M. (2000). Wingless gradient formation in the Drosophila wing. Curr. Biol. 10,293 -300.[CrossRef][Medline]
Takashima, S. and Murakami, R. (2001). Regulation of pattern formation in the Drosophila hindgut by wg, hh, dpp, and en. Mech. Dev. 101, 79-90.[CrossRef][Medline]
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[Medline]
Tearle, R. and Nusslein-Volhard, C. (1987). Tubingen mutants and stock list. Dros. Inf. Serv. 66,209 -269.
Teleman, A. A. and Cohen, S. M. (2000). Dpp gradient formation in the Drosophila wing imaginal disc. Cell 103,971 -980.[Medline]
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.[Abstract]
Tulina, N. and Matunis, E. (2001). Control of
stem cell self-renewal in Drosophila spermatogenesis by JAK- STAT
signaling. Science 294,2546
-2549.
Vila-Coro, A. J., Rodriguez-Frade, J. M., Martin De Ana, A.,
Moreno-Ortiz, M. C., Martinez, A. C. and Mellado, M. (1999).
The chemokine SDF-1alpha triggers CXCR4 receptor dimerization and activates
the JAK/STAT pathway. FASEB J.
13,1699
-1710.
Wallingford, J. B., Fraser, S. E. and Harland, R. M. (2002). Convergent extension: the molecular control of polarized cell movement during embryonic development. Dev. Cell 2, 695-706.[Medline]
Wang, L. and Coulter, D. E. (1996). bowel, an odd-skipped homolog, functions in the terminal pathway during Drosophila embryogenesis. EMBO J. 15,3182 -3196.[Abstract]
Ward, A. C., Touw, I. and Yoshimura A. (2000).
The Jak-Stat pathway in normal and perturbed hematopoiesis.
Blood 95,19
-29.
Yamashita, S., Miyagi, C., Carmany-Rampey, A., Shimizu, T., Fujii, R., Schier, A. F. and Hirano, T. (2002). Stat3 controls cell movements during zebrafish gastrulation. Dev. Cell 2,363 -375.[Medline]
Yan, R., Small, S., Desplan, C., Dearolf, C. R. and Darnell, J. E., Jr (1996). Identification of a Stat gene that functions in Drosophila development. Cell 84,421 -430.[Medline]
Zeidler, M. P., Perrimon, N. and Strutt, D. I.
(1999). Polarity determination in the Drosophila eye: a
novel role for unpaired and JAK/STAT signaling. Genes
Dev. 13,1342
-1353.