Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
* Author for correspondence (e-mail: beckendo{at}berkeley.edu)
Accepted 2 June 2005
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SUMMARY |
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Key words: Tec kinase, Actin, Endocycle, Btk29A
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Introduction |
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The embryonic salivary glands of Drosophila provide another good
system to study epithelial invagination. The salivary glands are derived from
a disc of columnar, postmitotic epithelial cells in parasegment 2 of the
Drosophila embryo known as the salivary placodes
(Fig. 1B,O). The salivary
placodes are specified at stage 10 by three positive regulators the
homeotic gene Sex combs reduced, extradenticle and
homothorax (Henderson and Andrew,
2000; Panzer et al.,
1992
). The dorsal and ventral boundaries of the salivary placodes
are determined by the decapentaplegic and Egfr signaling
pathways, respectively (Abrams et al.,
2003
; Panzer et al.,
1992
). Following their specification, the salivary placodes
invaginate into the embryo to form the tubular salivary glands. The process of
invagination begins with a wave of apical constriction and basal movement of
nuclei that progresses from the dorsal posterior cells to the dorsal anterior
cells and finally to the ventral cells of the salivary placodes. The order of
invagination follows the apical constriction wave, beginning with the dorsal
posterior cells, followed by the dorsal anterior cells and then the ventral
cells (Myat and Andrew, 2000a
;
Zhou et al., 2001
). This
sequential internalization of the cells results in a narrow tubular gland
where the first invaginated cells form the distal tip of the gland and the
last cells to ingress into the embryo form the proximal part of the salivary
gland.
We are just beginning to understand the genetic control of salivary
invagination. Interestingly, at least one of the signaling pathways used
during ventral furrow formation is also involved in salivary invagination.
Signaling by folded gastrulation (fog) activates
RhoGEF2, a RhoGTPase exchange factor, in the ventral furrow and the
salivary placodes. Both fog and RhoGEF2 are necessary for
the invagination of the ventral furrow and the salivary glands
(Hacker and Perrimon, 1998;
Nikolaidou and Barrett, 2004
).
In the ventral furrow, RhoGEF2 causes the apical myosin II
localization that is necessary for invagination of the ventral furrow
(Nikolaidou and Barrett,
2004
). It is therefore possible that RhoGEF2 has a
similar function in the salivary glands, thereby facilitating apical
constriction of the placode cells. In addition to the
fogRhoGEF2 signaling pathway, the apical constriction of the
salivary placodes cells requires fork head, a winged helix
transcription factor; in its absence, the salivary primordium fails to
invaginate (Myat and Andrew,
2000b
; Weigel et al.,
1989
).
Once the salivary placode cells invaginate into the embryo, they enter a
modified cell cycle known as the endoreplication cycle or endocycle, in which
the cells alternate between G and S phase without cell division, leading to an
increase in ploidy. The salivary gland cells are the first cells to enter the
endocycle in the embryo, and endoreplication in the gland reliably progresses
from the distal tip of the gland to the proximal part during stages 12-14
(Smith and Orr-Weaver, 1991).
Thus, the wave of endoreplication in the invaginated glands follows the same
order as the preceding wave of apical constriction and invagination. This
endoreplication is first of many in the salivary cells, leading to the giant
polytene chromosomes present in mature larvae.
The early events in salivary morphogenesis, including the localized initiation of invagination, the orderly progression of invagination to other placode cells and the beginning of endoreplication, appear to be carefully coordinated. Thus far, however, the mechanisms coordinating these processes have remained elusive.
Here we find that the Tec29 (Btk29A FlyBase)
tyrosine kinase is necessary to coordinate two essential processes: actin
cytoskeletal organization and regulation of the cell cycle. Tec29 is
a member of the Tec family of non receptor tyrosine kinases, which includes
BTK, TEC, ITK, ETK and TXK (Gregory et
al., 1987, Mano,
1999
). Mutations in BTK are known to cause X-linked
agammaglobulinemia in humans and X-linked immunodeficiency in mice. The human
disorder results from absence of mature B lymphocytes, whereas mice with the
immunodeficiency have abnormal B cells
(Maas and Hendriks, 2001
;
Satterthwaite and Witte,
2000
). Other Tec kinases regulate many processes during
development of lymphocytes, including cell cycle, cell death, cell adhesion
and migration (Mano, 1999
;
Takesono et al., 2002
). By
contrast, the Drosophila Tec kinase Tec29 has only been
linked to the actin cytoskeleton during Drosophila embryogenesis and
oogenesis (Guarnieri et al.,
1998
; Roulier et al.,
1998
; Tateno et al.,
2000
; Thomas and Wieschaus,
2004
). Tec29 is needed for actin filament reorganization
and bundling of actin during early cellularization and dorsal closure in
embryos, as well as in the ring canals of the ovary. Our data show that lack
of Tec29 caused a delay in invagination of the salivary glands
because of a shift in the equilibrium between F-actin and G-actin, and because
of premature endoreplication in the salivary placode cells. Thus, like ventral
furrow formation, invagination of the salivary placodes requires both the
reorganization of the actinmyosin cytoskeleton and a cell cycle delay.
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Materials and methods |
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Immunocytochemistry
Embryo fixation and immunocytochemistry were performed as described
previously (Chandrasekaran and Beckendorf,
2003). The following primary antibodies were used in this study:
rat anti-CREB (1:5000) (Andrew et al.,
1997
), rabbit anti-FKH (1:1000), rabbit anti-ß-galactosidase
(1:1000, Vector Laboratories), mouse anti-TEC29 (1:10), mouse anti-CRUMBS
(1:100) and guinea pig anti-SCRIBBLE (1:1000, D. Bilder), mouse anti-ENGRAILED
(1:10, DSHB), mouse anti-actin (1:100,), rat anti-DEADRINGER (1:15000, D.
Andrew) and mouse anti-CYCLIN E (1:50, H. Richardson). Embryos were then
incubated with the appropriate secondary antibodies that were either
biotinylated (1:200, Vector Laboratories) or Alexa-conjugated (1:500,
Molecular Probes). The biotinylated secondary antibodies were detected using
the Vectastain ABC kit (Vector Labs), followed by incubation with 0.5 mg/ml
diaminobenzidine and 0.06% hydrogen peroxide. The embryos were then cleared
with methyl salicylate and photographed using Nomarski optics on the Leica
DMRB microscope. The fluorescent embryos were cleared in 70% glycerol in PBS
containing 2% n-propyl gallate (Sigma) and visualized using the Zeiss 510
confocal microscope.
In situ hybridization
Whole-mount in situ hybridization was performed as described by Tautz and
Pfeifle (Tautz and Pfeifle,
1989) with modifications
(Harland, 1991
) using
antisense digoxigenin-labeled probes. The signal was visualized using nitro
blue tetrazolium and BCIP as substrates for alkaline phosphatase. Following in
situ hybridization, the embryos were immunostained for ß-galactosidase as
described above in the immunocytochemistry protocol. The embryos were rinsed
and cleared in 50% glycerol followed by 70% glycerol and photographed using
Nomarski optics on the Leica DMRB microscope.
BrdU labeling of embryos
BrdU labeling and detection were performed using the protocol described by
Shermoen (Shermoen, 2000).
Briefly, embryos were dechorionated and incubated in n-octane (Sigma) for 5
minutes, followed by incubation in 1 mg/ml BrdU in PBS for 40 minutes. Then
the embryos were fixed in a 1:1:2 mixture of PBS, 10% formaldehyde and
heptane, followed by devitellenization with methanol. The embryos were stored
in methanol at 20°C. Embryos were rehydrated and immunostained with
rabbit anti-FKH as well as rabbit-anti-ß-galactosidase, followed by a
fluorescent-conjugated secondary antibody as described above. Following the
immunostaining, embryos were treated with 2.2 N HCl containing 0.1% triton
twice for 15 minutes each followed by neutralization in 0.1 M sodium borate.
The embryos were then incubated with mouse anti-BrdU overnight at 4°C. The
BrdU labeling in the nuclei was detected using a fluorescent-conjugated
secondary antibody and embryos were processed and imaged as described
above.
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Results |
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Embryos mutant for Tec29 have long salivary glands
Expression in the salivary glands suggested that Tec29 might be
important for salivary gland morphogenesis. Embryos mutant for a P-element
allele of Tec29, Tec29K00206 had abnormally long salivary
glands that did not fully invaginate and were still connected to the surface
(Fig. 1I,J). The phenotype was
fully penetrant, and identical phenotype was observed in embryos mutant for at
least two other Tec29 alleles, Tec29K05610 and
Tec29e482, as well as
Tec29K00206/Tec29K05610 heterozygotes
(Fig. 1K,L).
A possible explanation for this phenotype was that the mutant salivary placodes had more cells than wild-type placodes, resulting in long glands. However, cell counts did not support this idea. Tec29 salivary placodes had 113±36 cells compared with 117±39 in wild type. In addition, location of the placodes is normal. As shown by co-staining for ENGRAILED, the placodes respect both the normal AP boundaries at the edges of parasegment 2 (Fig. 1O,P) and the DV boundaries that separate them from the dorsal epidermis and the more ventral salivary duct cells (data not shown). In addition, staining for the mitotic marker phosphohistone H3 produced no evidence of extra mitoses, either in the placodes or at later stages. Thus, the long glands did not result from the recruitment of extra cells into the primordium or from the production of extra cells during development.
The salivary ducts were also defective in Tec29 mutant embryos (compare Fig. 1M,N). As evidenced by staining for duct markers such as dead ringer, duct cell fate appeared to be normally specified in these embryos, but the duct cells did not undergo normal morphogenesis (Fig. 1N). Staining for a lumenal marker, CRUMBS, showed that tubular ducts were not formed in these embryos (data not shown).
Tec29 is necessary for the invagination of salivary glands
As the long salivary glands in Tec29 mutants were not due to extra
cells and there were uninvaginated cells in the salivary glands of
Tec29 embryos, we reasoned that the detailed analysis of salivary
placode invagination in Tec29 embryos might explain the salivary
gland phenotype. In wild-type embryos, the progress of salivary invagination
can be monitored relative to germ band retraction, as both processes occur
during stage 12 of embryogenesis.
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There is a paradox in understanding the Tec29 phenotype. Although
fewer cells invaginate, the mutant glands eventually become much longer than
in wild-type embryos. As the increase in the salivary gland length occurred
late in Tec29 embryos, we guessed that it might come from stretching
them during head involution in older embryos. To test this possibility,
Tec29 embryos were immunostained either for CRUMBS, which outlines
the apical ends of epithelial cells or for SCRIBBLE, a septate junction
protein that is localized to the lateral margins of epithelial cells, more
basal to CRUMBS (Bilder and Perrimon,
2000; Schöck and
Perrimon, 2002
; Tepass et al.,
1990
). Both markers highlight the salivary gland cells, producing
nearly isodiametric outlines in wild-type embryos at stage 15
(Fig. 3A,B,E). In
Tec29 embryos stained for either SCRIBBLE or CRUMBS, the distal
region of the salivary glands looked normal, but the cells in the proximal
region of the glands were elongated in the AP direction
(Fig. 3C,D,F). These results
show that the arrest of invagination coupled with the anterior movement of the
epidermis during head involution caused stretching of the mutant salivary
glands, leading to the observed phenotype of long salivary glands.
Stretching of the glands in Tec29 mutants suggests that they were tethered at both ends. The anterior tether probably results from the invagination defect. As the placodes do not completely invaginate in Tec29 embryos, the anterior part of the gland appears to remain anchored to the moving ectoderm. To permit stretching, the posterior part of the gland must also be tethered, probably by attachment to tissues close to the distal tip of the gland, maybe the anterior midgut. This posterior attachment may be part of normal salivary morphogenesis as the posterior part of the Tec29-mutant glands are located normally and have the same apical outlines as wild-type glands.
Apical actin cytoskeleton is disorganized in Tec29 embryos
Previous studies have shown that Tec29 can reorganize the actin
cytoskeleton in the ovary and during the cellularization of
Drosophila embryos (Djagaeva et
al., 2005; Roulier et al.,
1998
; Thomas and Wieschaus,
2004
). Therefore, we examined whether the actin distribution was
normal in the placodes of Tec29 embryos. Staining with phalloidin to
visualize F actin or with an
-spectrin antibody did not reveal any
gross abnormalities (Fig. 4A,B;
data not shown). However, use of an actin monoclonal antibody that detects
both F and G actin showed that in Tec29 embryos, actin appeared to be
disorganized at the apical end of the placode cells
(Fig. 4C,D), but looked similar
to wild type on the basolateral surface (data not shown). The disorganization
of actin occurred early in stage 12 in the ventral cells of the placodes and
preceded the delay in invagination observed in these cells. In addition, we
found that there were genetic interactions between Tec29 and the
actin-binding proteins, profilin and cofilin. The Drosophila profilin
homolog, chickadee (chic), is important for promoting actin
polymerization, thereby increasing F-actin in the ovary, embryo and imaginal
discs, whereas the Drosophila cofilin homolog twinstar
(tsr), promotes depolymerization, thus limiting actin filament growth
and increasing G-actin (Cooley et al.,
1992
; Gunsalus et al.,
1995
). Embryos mutant for either chic or tsr
alone had normal salivary glands. However, Tec29 chic double mutants
showed an enhancement of the Tec29 salivary gland phenotype. The
salivary glands in 80% of Tec29 chic double mutants showed more
severe invagination defects with large placodes left on the surface
(Fig. 5B,E,G). The remaining
20% of the embryos had salivary glands similar to Tec29. By contrast,
30% of the Tec29 tsr double mutants had salivary glands that
invaginated normally or nearly so, indicating a partial suppression of the
Tec29 mutant phenotype (Fig.
5C,F,G). These genetic interactions show that in Tec29
mutants there is shift from F-actin to G-actin on the apical surface of the
salivary placode cells. They also suggest that the apparent disorganization
seen with the actin antibody results from increases in G-relative to F-actin.
The partial rescue of the Tec29 salivary gland phenotype by
tsr indicates that the shift in the balance between G-actin and
F-actin in the salivary placodes caused the invagination delay in
Tec29 mutant salivary placodes. Therefore, Tec29 was
necessary to facilitate the formation or maintenance of F-actin at the apical
surface of the salivary placodes cells, and this localization of actin was
crucial for normal invagination.
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The effects of Tec29 on endoreplication may be due its effects on actin cytoskeleton
As Tec29 has effects on both actin cytoskeleton and
endoreplication, we wanted to understand if Tec29 independently
affects these processes. Hence, we examined endoreplication in Tec29
chic and Tec29 tsr double mutants. Though Tec29 tsr
mutants show a suppression of the salivary gland phenotype, there was no clear
decrease in the number or extent of the endoreplication defects in these
embryos (Fig. 6D,H). However,
Tec29 chic double mutants showed a marked enhancement of the
endoreplication defects observed in Tec29 alone. There were more
endoreplicating cells in embryos mutant for both Tec29 and
chic than in Tec29 mutants alone
(Fig. 6C,G). This effect on
endoreplication was observed at early stage 12, prior to the onset of
invagination defects. These results suggest that the defects in actin
remodeling can affect the endoreplication cycle in the salivary glands.
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There were also genetic interactions between Tec29 and the other Src kinase, Src64. Similar to Src42A mutants, Src64 mutant embryos did not show any salivary gland abnormalities. However, many of the Tec29 Src64 double mutant embryos showed gross abnormalities such as lack of head segments, making it difficult to evaluate the salivary gland phenotype. Of the Tec29 Src64 double mutant embryos that looked normal in outline, 16% showed more severe salivary gland phenotypes than Tec29 alone (Fig. 7C). The Tec29-Src64 interaction is most clearly shown with heterozygous Tec29 embryos. The salivary glands are normal in Tec29/+ Src64/+ embryos. However, about one-third of Tec29/+ Src64/Src64 embryos show Tec29-like invagination defects (Fig. 7D). These results show that there is a strong genetic interaction between Src64 and Tec29 in the salivary glands. The lack of salivary gland invagination defects in embryos mutant for either Src kinase and the enhancement of the null mutant Tec29 phenotype by Src mutations suggest that Tec29 and the Src kinases may function in a parallel pathways to promote salivary gland invagination.
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Discussion |
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Besides salivary gland invagination, Tec29 affects actin
organization during two other processes in Drosophila embryogenesis:
dorsal closure and cellularization. Tec29 in conjunction with
Src42A is necessary for dorsal closure and lack of both these kinases
result in a dorsal open phenotype. So, during dorsal closure as with salivary
invagination, there is a positive interaction between Tec29 and
Src42A. However, Src42 is the main player during dorsal
closure, whereas Tec29 is more important for salivary invagination.
Interestingly, there is a reduction of F-actin observed at the leading edge of
the dorsal epidermal cells in these double mutants, causing delayed dorsal
closure (Tateno et al., 2000).
Thus, the lack of these tyrosine kinases causes changes in actin dynamics that
result in delayed migration of both the salivary placodes and during dorsal
closure. Similarly, the process of basal closure during cellularization
resembles apical constriction prior to invagination and requires an
actin-myosin based contraction at the base of the cellularization front. In
embryos arising from Tec29 germline clones, the actin microfilament
ring does not contract, resulting in membrane invagination of varying depths
and impairment of basal closure during late cellularization
(Thomas and Wieschaus, 2004
).
It is possible that, similar to the salivary invagination and dorsal closure,
the absence of the contractile ring during cellularization is due to decreased
F-actin and/or increased G-actin. In general, Tec29 appears to be
needed for regulation of actin during periods of extensive and rapid
reorganization of the actin cytoskeleton as observed during migration and
contraction of cells.
Although Tec kinases are known to alter the actin cytoskeleton in many
systems, we are the first to show a relationship between Tec29 and
the endocycle. Our data support a previous observation that the salivary
placodes in wild-type embryos enter endoreplication only after they invaginate
into the embryo (Smith and Orr-Weaver,
1991). In Tec29 mutants, however, the wave of
endoreplication is disrupted, such that the ventral cells in the placodes
initiate endoreplication prior to invagination. As a result, these cells fail
to invaginate on schedule, resulting in the long salivary glands. Therefore,
delaying endoreplication appears to be necessary to allow invagination, and
coordinating the two events is crucial for normal development of the salivary
glands. Similar coordination between the cell cycle and morphogenesis is
observed during ventral furrow formation in Drosophila embryos and in
the paraxial mesoderm in Xenopus embryos. In the ventral furrow and
the paraxial mesoderm, the cell cycle delay is established by maintaining the
Cdks in their phosphorylated form. In the ventral furrow, this is accomplished
by tribbles inhibiting the Drosophila Cdc25 homolog
string, a protein that dephosphorylates and activates Cdks
(Grosshans and Wieschaus,
2000
; Mata et al.,
2000
; Seher and Leptin,
2000
). In the paraxial mesoderm, the localized phosphorylation of
Cdks by Wee2 is sufficient to prevent the cells from entering mitosis prior to
invagination (Leise and Mueller,
2004
). In both these cases, as in the salivary gland, the
coordination between cell cycle and invagination is crucial for normal
morphogenesis of the embryo. An important difference is that unlike the
salivary glands, it is the mitotic cycle that is regulated with invagination
of the ventral furrow and the paraxial mesoderm. Our study provides the first
indication of a link between the endocycle and morphogenesis, and suggests
that coordination of endoreplication with invagination is crucial for normal
development.
As shown in the ventral furrow and the paraxial mesoderm, mitosis and
invagination use the same cytoskeletal components and require opposing levels
of cell adhesion, making these two processes incompatible with each other
(Grosshans and Wieschaus,
2000; Leise and Mueller,
2004
; Mata et al.,
2000
; Seher and Leptin,
2000
). However, a normal endocycle in flies does not involve
nuclear membrane breakdown or other processes that might require the same
cytoskeletal components as invagination
(Edgar and Orr-Weaver, 2001
).
Thus, a normal endocycle might not interfere with invagination. But this may
not be a normal endocycle. Unlike the other endocycling cells in the embryo,
which enter the endocycle from G1, it has been suggested that the salivary
placodes may enter the endocycle from G2
(Smith and Orr-Weaver, 1991
).
It is therefore possible that the endocycle in the salivary placodes retains
some aspects of mitosis that would interfere with invagination. If so, this
endocycle would be similar to those in some endocycling mammalian cells that
enter the endoreplication cycle after G2 during early M phase
(Edgar and Orr-Weaver, 2001
).
Perhaps because this first salivary gland endocycle is unusual, it must be
delayed until after the salivary cells are safely inside the embryo.
Our data indicate that Tec29 is necessary for actin remodeling during apical constriction in the salivary placodes and for endocycle progression as the glands invaginate. In addition, we found that manipulating the actin cytoskeleton in Tec29 mutants can have effects on endoreplication in these cells. When the actin defects were enhanced by eliminating chic in Tec29 embryos, there was also an enhancement of the endoreplication defects of Tec29, suggesting that the actin cytoskeleton and endoreplication cycle are linked. We propose the following model to explain how these two events might be coordinated in the salivary placodes. The actin remodeling during apical constriction and endoreplication follow each other such that the first cells to apically constrict are the first cells to invaginate and endoreplicate, and the remaining cells follow in sequence. These processes might be causally linked; the apical constriction wave might trigger the wave of endoreplication. This coupling and a lag between the two events would ensure that endoreplication does not occur prematurely, while the cells are still on the surface of the embryo. With this model, Tec29 would then regulate the endocycle indirectly, rather than independently regulating both actin and endoreplication. Disruption of the apical constriction wave in Tec29 mutants would lead to subsequent disruption of the endoreplication wave. An invagination delay due to inadequate actin polymerization would be compounded by placode cells endoreplicating prior to invagination. This model also explains the ability of cyclin E to partially rescue the Tec29 phenotype. Cyclin E overexpression would delay endoreplication and relieve some of the effects of inadequate actin polymerization. Studies in progress to identify TEC29 targets are likely to provide further insight into its effects on endoreplication and invagination, and should enable us to better understand the link between these processes.
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ACKNOWLEDGMENTS |
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