3515

Introduction

Invagination of epithelial sheets is an important process duringgastrulation of many organisms and the formation of tissuessuch as the vertebrate lung, gut, insect trachea and the salivaryglands. During invagination, the cells specified to be particulartissues break down their cell adhesion with the rest of theepithelium, reorganize their cytoskeleton and apically constrictto ingress into the embryo (Schöck and Perrimon, 2002). Theprocess of invagination has been extensively studied duringventral furrow formation in Drosophila embryos. During thisprocess, cells close to the ventral midline constrict apically toproduce a shallow groove, which is deepened by cellshortening along the apicobasal axis (Leptin and Grunewald,1990). Prior to apical constriction, nonmuscle myosin II andβH spectrin are translocated to the apical side of the ventralfurrow cells. Disruption of this accumulation prevents ventralfurrow formation, suggesting that actin-myosin contractility isrequired for apical constriction (Nikolaidou and Barrett, 2004;Thomas and Kiehart, 1994; Young et al., 1991). In addition tothe cytoskeletal changes, ventral furrow formation requirescoordination between the cell cycle and invagination.Normally, ventral furrow cells delay mitosis until after they areinside the embryo. As shown by tribbles mutants, prematureentry into mitosis before invagination prevents ventral furrowformation (Grosshans and Wieschaus, 2000; Mata et al., 2000;Seher and Leptin, 2000). As invagination is a fundamentalprocess used repeatedly during organogenesis, we areinterested in understanding whether the process of invaginationand its genetic control are similar in other epithelial tissues.

The embryonic salivary glands of Drosophila provideanother good system to study epithelial invagination. The

salivary glands are derived from a disc of columnar, postmitoticepithelial cells in parasegment 2 of the Drosophila embryoknown as the salivary placodes (Fig. 1B,O). The salivaryplacodes are specified at stage 10 by three positive regulators– the homeotic gene Sex combs reduced, extradenticle andhomothorax (Henderson and Andrew, 2000; Panzer et al.,1992). The dorsal and ventral boundaries of the salivaryplacodes are determined by the decapentaplegic and Egfrsignaling pathways, respectively (Abrams et al., 2003; Panzeret al., 1992). Following their specification, the salivaryplacodes invaginate into the embryo to form the tubularsalivary glands. The process of invagination begins with a waveof apical constriction and basal movement of nuclei thatprogresses from the dorsal posterior cells to the dorsal anteriorcells 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 thedorsal anterior cells and then the ventral cells (Myat andAndrew, 2000a; Zhou et al., 2001). This sequentialinternalization of the cells results in a narrow tubular glandwhere the first invaginated cells form the distal tip of the glandand the last cells to ingress into the embryo form the proximalpart of the salivary gland.

We are just beginning to understand the genetic control ofsalivary invagination. Interestingly, at least one of the signalingpathways used during ventral furrow formation is also involvedin salivary invagination. Signaling by folded gastrulation (fog)activates RhoGEF2, a RhoGTPase exchange factor, in theventral furrow and the salivary placodes. Both fog andRhoGEF2 are necessary for the invagination of the ventralfurrow and the salivary glands (Hacker and Perrimon, 1998;

Epithelial invagination is necessary for formation of manytubular organs, one of which is the Drosophila embryonicsalivary gland. We show that actin reorganization andcontrol of endocycle entry are crucial for normalinvagination of the salivary placodes. Embryos mutant forTec29, the Drosophila Tec family tyrosine kinase, showeddelayed invagination of the salivary placodes. Thisinvagination delay was partly the result of an accumulationof G-actin in the salivary placodes, indicating that Tec29 isnecessary for maintaining the equilibrium between G- andF-actin during invagination of the salivary placodes.Furthermore, normal invagination of the salivary placodes

appears to require the proper timing of the endocycle inthese cells; Tec29 must delay DNA endoreplication in thesalivary placode cells until they have invaginated into theembryo. Taken together, these results show that Tec29regulates both the actin cytoskeleton and the cell cycle tofacilitate the morphogenesis of the embryonic salivaryglands. We suggest that apical constriction of the actincytoskeleton may provide a temporal cue ensuring thatendoreplication does not begin until the cells have finishedinvagination.

Key words: Tec kinase, Actin, Endocycle, Btk29A

Summary

Tec29 controls actin remodeling and endoreplication during

invagination of the Drosophila embryonic salivary glands

Vidya Chandrasekaran and Steven K. Beckendorf*

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA*Author for correspondence (e-mail: beckendo@berkeley.edu)

Accepted 2 June 2005

Development 132, 3515-3524

Published by The Company of Biologists 2005

doi:10.1242/dev.01926

Research article

Dev

elop

men

t

3516

Nikolaidou and Barrett, 2004). In the ventral furrow, RhoGEF2causes the apical myosin II localization that is necessary forinvagination of the ventral furrow (Nikolaidou and Barrett,2004). It is therefore possible that RhoGEF2 has a similarfunction in the salivary glands, thereby facilitating apicalconstriction of the placode cells. In addition to thefog–RhoGEF2 signaling pathway, the apical constriction of thesalivary placodes cells requires fork head, a winged helixtranscription factor; in its absence, the salivary primordium failsto 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 endoreplicationcycle or endocycle, in which the cells alternate between G andS phase without cell division, leading to an increase in ploidy.The salivary gland cells are the first cells to enter the endocyclein the embryo, and endoreplication in the gland reliablyprogresses from the distal tip of the gland to the proximal partduring stages 12-14 (Smith and Orr-Weaver, 1991). Thus, thewave of endoreplication in the invaginated glands follows thesame order as the preceding wave of apical constriction andinvagination. This endoreplication is first of many in thesalivary cells, leading to the giant polytene chromosomespresent in mature larvae.

The early events in salivary morphogenesis, including thelocalized initiation of invagination, the orderly progression ofinvagination to other placode cells and the beginning ofendoreplication, appear to be carefully coordinated. Thus far,however, the mechanisms coordinating these processes haveremained elusive.

Here we find that the Tec29 (Btk29A – FlyBase) tyrosinekinase is necessary to coordinate two essential processes: actincytoskeletal organization and regulation of the cell cycle. Tec29is a member of the Tec family of non receptor tyrosine kinases,which includes BTK, TEC, ITK, ETK and TXK (Gregory etal., 1987, Mano, 1999). Mutations in BTK are known to causeX-linked agammaglobulinemia in humans and X-linkedimmunodeficiency in mice. The human disorder results fromabsence of mature B lymphocytes, whereas mice with theimmunodeficiency have abnormal B cells (Maas and Hendriks,2001; Satterthwaite and Witte, 2000). Other Tec kinasesregulate many processes during development of lymphocytes,including cell cycle, cell death, cell adhesion and migration(Mano, 1999; Takesono et al., 2002). By contrast, theDrosophila Tec kinase Tec29 has only been linked to the actincytoskeleton 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 actinfilament reorganization and bundling of actin during earlycellularization and dorsal closure in embryos, as well as in thering canals of the ovary. Our data show that lack of Tec29caused a delay in invagination of the salivary glands becauseof a shift in the equilibrium between F-actin and G-actin, andbecause of premature endoreplication in the salivary placodecells. Thus, like ventral furrow formation, invagination of thesalivary placodes requires both the reorganization of the actin-myosin cytoskeleton and a cell cycle delay.

Materials and methodsDrosophila stocks

Tec29K00206/CyO and Tec29K05610/CyO are two P-element alleles of

Tec29 that lack any Tec29 expression in embryos and behave asembryonic null alleles (Roulier et al., 1998). Tec29e482/CyO has a stopcodon in the kinase domain and is predicted to lack all catalyticactivity (Guarnieri et al., 1998). Src64PI/TM3, a P-element insertionallele of Src64 was a gift from M. Simon (Dodson et al., 1998).Src42AE1/CyO, a null allele of Src42A, was from Adachi-Yamada(Tateno et al., 2000). UAS-CycE was a gift from P. O’Farrell. All theremaining stocks were obtained from the Bloomington Stock Center.w1118 flies were used as wild type controls for all experiments.

Immunocytochemistry

Embryo fixation and immunocytochemistry were performed asdescribed previously (Chandrasekaran and Beckendorf, 2003). Thefollowing primary antibodies were used in this study: rat anti-CREB(1:5000) (Andrew et al., 1997), rabbit anti-FKH (1:1000), rabbit anti-β-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), mouseanti-actin (1:100,), rat anti-DEADRINGER (1:15000, D. Andrew) andmouse anti-CYCLIN E (1:50, H. Richardson). Embryos were thenincubated with the appropriate secondary antibodies that were eitherbiotinylated (1:200, Vector Laboratories) or Alexa-conjugated (1:500,Molecular Probes). The biotinylated secondary antibodies weredetected using the Vectastain ABC kit (Vector Labs), followed byincubation with 0.5 mg/ml diaminobenzidine and 0.06% hydrogenperoxide. The embryos were then cleared with methyl salicylate andphotographed using Nomarski optics on the Leica DMRBmicroscope. The fluorescent embryos were cleared in 70% glycerolin PBS containing 2% n-propyl gallate (Sigma) and visualized usingthe Zeiss 510 confocal microscope.

In situ hybridization

Whole-mount in situ hybridization was performed as described byTautz and Pfeifle (Tautz and Pfeifle, 1989) with modifications(Harland, 1991) using antisense digoxigenin-labeled probes. Thesignal was visualized using nitro blue tetrazolium and BCIP assubstrates for alkaline phosphatase. Following in situ hybridization,the embryos were immunostained for β-galactosidase as describedabove in the immunocytochemistry protocol. The embryos wererinsed and cleared in 50% glycerol followed by 70% glycerol andphotographed using Nomarski optics on the Leica DMRBmicroscope.

BrdU labeling of embryos

BrdU labeling and detection were performed using the protocoldescribed by Shermoen (Shermoen, 2000). Briefly, embryos weredechorionated and incubated in n-octane (Sigma) for 5 minutes,followed by incubation in 1 mg/ml BrdU in PBS for 40 minutes. Thenthe embryos were fixed in a 1:1:2 mixture of PBS, 10% formaldehydeand heptane, followed by devitellenization with methanol. Theembryos were stored in methanol at –20°C. Embryos were rehydratedand immunostained with rabbit anti-FKH as well as rabbit-anti-β-galactosidase, followed by a fluorescent-conjugated secondaryantibody as described above. Following the immunostaining, embryoswere treated with 2.2 N HCl containing 0.1% triton twice for 15minutes each followed by neutralization in 0.1 M sodium borate. Theembryos 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 andimaged as described above.

Results

Tec29 is expressed in the Drosophila embryonicsalivary primordium

Although previous studies described Tec29 RNA and protein

Development 132 (15) Research article

Dev

elop

men

t

3517Tec29 is necessary for salivary invagination

expression in embryos (Katzen et al., 1990; Vincent et al.,1989; Wadsworth et al., 1990), the details of salivary glandexpression were not reported. We find that Tec29 RNAexpression begins at stage 11 (5.5-7.5 hours) in the dorsalposterior part of the salivary placodes, colocalizing withthe site of initial invagination (Fig. 1A). By stage 12, allsalivary placode cells express Tec29 (Fig. 1B). Tec29continues to be expressed in the invaginating salivary glandsat stage 12 (Fig. 1C) and later during embryogenesis (datanot shown). Then as the placodes invaginate, Tec29expression expands into the more ventral cells that willbecome the salivary ducts (Fig. 1D) and is weakly expressedin the salivary ducts of older embryos (data not shown). Asimilar expression pattern was observed for TEC29 protein(Fig. 1G,H). TEC29 is localized to the apical membrane inthe salivary gland cells (Fig. 1H).

Most Tec family kinases have an N-terminal pleckstrinhomology (PH) domain, followed by Tec homology (TH),SH2, SH3 and kinase domains. In Drosophila, there are twoisoforms of Tec29 RNA. The longer form (type 2) is similar tothe vertebrate Tec kinases and includes the PH domain,whereas the shorter type 1 isoform lacks the PH and part of theTH domain at its N terminus (Baba et al., 1999a). We observedthat the longer isoform of Tec29 is expressed only in thenervous system, whereas the shorter isoform is expressed inthe epidermis and at high levels in the salivary glands (Fig.1E,F). Thus, TEC29 function in the salivary glands apparentlydoes not require a PH domain.

Embryos mutant for Tec29 have long salivary glands

Expression in the salivary glands suggested that Tec29 mightbe important for salivary gland morphogenesis. Embryosmutant for a P-element allele of Tec29, Tec29K00206 hadabnormally long salivary glands that did not fully invaginateand were still connected to the surface (Fig. 1I,J). Thephenotype was fully penetrant, and identical phenotype wasobserved 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-typeplacodes, resulting in long glands. However, cell counts did notsupport this idea. Tec29 salivary placodes had 113±36 cellscompared with 117±39 in wild type. In addition, location ofthe placodes is normal. As shown by co-staining forENGRAILED, the placodes respect both the normal APboundaries at the edges of parasegment 2 (Fig. 1O,P) and theDV boundaries that separate them from the dorsal epidermisand the more ventral salivary duct cells (data not shown). Inaddition, staining for the mitotic marker phosphohistone H3produced no evidence of extra mitoses, either in the placodesor at later stages. Thus, the long glands did not result from therecruitment of extra cells into the primordium or from theproduction of extra cells during development.

The salivary ducts were also defective in Tec29 mutantembryos (compare Fig. 1M,N). As evidenced by staining forduct markers such as dead ringer, duct cell fate appeared to be

Fig. 1. Tec29 is expressed inembryonic salivary gland andhas a salivary glandphenotype. In all the figures,anterior is towards the left.(A-D) Tec29 mRNA isexpressed in the salivaryplacodes at stage 11 and in theinvaginating salivary glands(arrowhead). Tec29 is alsoexpressed in the salivary ductsfrom stage 12 onwards (D,arrow). (E,F) The shorterisoform of Tec29, type 1, isexpressed in the salivaryprimordium (E), while thelonger isoform, type 2, isexpressed only in the nervoussystem (F).(G,H) Immunostaining forTEC29 protein shows that it isexpressed in the salivaryplacodes and is apicallylocalized in the invaginatinggland (white arrowhead).(I-L) Immunostaining withanti-CREB antibody showedthat multiple alleles ofTec29, Tec29K00206,Tec29K00206/Tec29K05610 andTec29e482 have long salivaryglands compared with w1118 embryos (I). (M,N) The salivary ducts in Tec29K00206, as visualized by dead ringer (dri), do not go through normalmorphogenesis (arrows). (O,P) Double labeling for FKH and EN show that the salivary placodes at stage 11 are normal and respect their APboundary in Tec29K00206 embryos.

Dev

elop

men

t

3518

normally specified in these embryos, but the duct cells did notundergo normal morphogenesis (Fig. 1N). Staining for alumenal marker, CRUMBS, showed that tubular ducts were notformed in these embryos (data not shown).

Tec29 is necessary for the invagination of salivaryglands

As the long salivary glands in Tec29 mutants were not due toextra cells and there were uninvaginated cells in the salivaryglands of Tec29 embryos, we reasoned that the detailedanalysis of salivary placode invagination in Tec29 embryosmight explain the salivary gland phenotype. In wild-typeembryos, the progress of salivary invagination can be

monitored relative to germ band retraction, as both processesoccur during stage 12 of embryogenesis.

In wild-type embryos, there was an inverse relationshipbetween the area of the placodes left on the surface and thelength of the invaginated gland, such that by end of stage 12there were no placodes left on the surface (Fig. 2B). Thelength of the glands increased as the embryos progressthrough stage 12, but then decreased at later stages (Fig. 2A).As germ band retraction appeared to be normal in Tec29embryos, similar analyses of salivary glands were performedin these embryos. The salivary placodes began invagination onschedule and at early stage 12 looked similar to wild type (Fig.2A,B). During mid stage 12, when there was a linear increasein the length of the invaginated gland in wild type, Tec29embryos stalled; the glands did not increase in length and theplacode area did not decrease. By late stage 12, the length ofthe invaginated salivary glands in Tec29 embryos wascomparable with wild type, but by stage 15 the salivary glandsin Tec29 embryos were significantly longer than wild type(Fig. 2A). Despite this, there were still many placode cellsremaining on the surface at late stage 12 and even at stage 13-14 (Fig. 2B). Thus, in Tec29 embryos there was an arrest inthe salivary gland invagination process at mid stage 12,followed by a late increase in gland length. These resultsindicate that the delay in invagination precedes thelengthening of the gland in Tec29 mutants and is the maincause of the Tec29 salivary gland phenotype.

There is a paradox in understanding the Tec29 phenotype.Although fewer cells invaginate, the mutant glands eventuallybecome much longer than in wild-type embryos. As theincrease in the salivary gland length occurred late in Tec29embryos, we guessed that it might come from stretching themduring head involution in older embryos. To test thispossibility, Tec29 embryos were immunostained either forCRUMBS, which outlines the apical ends of epithelial cellsor for SCRIBBLE, a septate junction protein that is localizedto the lateral margins of epithelial cells, more basal toCRUMBS (Bilder and Perrimon, 2000; Schöck and Perrimon,2002; Tepass et al., 1990). Both markers highlight the salivarygland cells, producing nearly isodiametric outlines in wild-type embryos at stage 15 (Fig. 3A,B,E). In Tec29 embryosstained for either SCRIBBLE or CRUMBS, the distal regionof the salivary glands looked normal, but the cells in theproximal region of the glands were elongated in the APdirection (Fig. 3C,D,F). These results show that the arrest ofinvagination coupled with the anterior movement of theepidermis during head involution caused stretching of themutant salivary glands, leading to the observed phenotype oflong salivary glands.

Stretching of the glands in Tec29 mutants suggests that theywere tethered at both ends. The anterior tether probably resultsfrom the invagination defect. As the placodes do notcompletely invaginate in Tec29 embryos, the anterior part ofthe gland appears to remain anchored to the moving ectoderm.To permit stretching, the posterior part of the gland must alsobe tethered, probably by attachment to tissues close to the distaltip of the gland, maybe the anterior midgut. This posteriorattachment may be part of normal salivary morphogenesis asthe posterior part of the Tec29-mutant glands are locatednormally and have the same apical outlines as wild-typeglands.

Development 132 (15) Research article

(A)

(B)

0

500

1000

1500

2000

2500

Are

a o

f p

laco

des (

μ m2)

* *

Stages

80 70 60 50 40 30 20

Early 12 Mid 12 Late 12 13-14

Position of the germ band tip (%)

0

20

40

60

80

100

120

140

160

Le

ng

th o

f s

ali

va

ry g

lan

ds

(μm

)

80 70 60 50 40 30 20

Early 12 Mid 12 Late 12 13-14 15-16Stages

Position of the germ band tip (%)

* *

**wild type

Tec29

Fig. 2. Tec29 embryos show delays in invagination of the salivaryplacodes. (A) Changes in the length of the salivary gland (μm) as theplacode cells invaginate during stage 12 of embryogenesis and atstages 13 through 16. Progression through stage 12 was monitoredby the position of the tip of the germ band (as percent embryo lengthmeasured from the posterior end of the embryo). The bars show themean length of the salivary glands in embryos whose germ band tiplies in the indicated intervals. Seventy-five wild-type salivary glandsand 46 Tec29K00206 salivary glands from embryos at different stageswere measured. As the germ band has finished retraction by the endof stage 12, the later stages are indicated only by stage numbers, notposition. In w1118 embryos (blue bars), the length of the salivaryglands gradually increases during stage 12 and then decreasesslightly at stages 13-16. In Tec29K00206 embryos (red bars), there isno increase in length of gland observed at mid stage 12 (*); however,the length of the gland increases and exceeds w1118 at stage 15 (**).(B) This graph shows the area of the salivary placodes on the surfacemeasured in the same embryos used for measurement in A. Althoughthere is a decrease in the surface area of the placodes in w1118

embryos (blue bars), in Tec29K00206 embryos (red bars) the area ofthe placodes does not decrease such that at stage 13-14, one-third ofthe placode is still on the surface. Uninvaginated cells remain on thesurface of Tec29K00206 embryos at stage 15-16; however, these cellshave moved more anteriorly in the head and are hard to quantitate.

Dev

elop

men

t

3519Tec29 is necessary for salivary invagination

Apical actin cytoskeleton is disorganized in Tec29embryos

Previous studies have shown that Tec29 can reorganize theactin cytoskeleton in the ovary and during the cellularizationof Drosophila embryos (Djagaeva et al., 2005; Roulier et al.,1998; Thomas and Wieschaus, 2004). Therefore, we examinedwhether the actin distribution was normal in the placodes ofTec29 embryos. Staining with phalloidin to visualize F actin orwith an α-spectrin antibody did not reveal any grossabnormalities (Fig. 4A,B; data not shown). However, use of anactin monoclonal antibody that detects both F and G actinshowed that in Tec29 embryos, actin appeared to bedisorganized at the apical end of the placode cells (Fig. 4C,D),but looked similar to wild type on the basolateral surface (datanot shown). The disorganization of actin occurred early instage 12 in the ventral cells of the placodes and preceded thedelay in invagination observed in these cells. In addition, wefound that there were genetic interactions between Tec29 andthe actin-binding proteins, profilin and cofilin. The Drosophilaprofilin homolog, chickadee (chic), is important for promotingactin polymerization, thereby increasing F-actin in the ovary,embryo and imaginal discs, whereas the Drosophila cofilin

homolog twinstar (tsr), promotes depolymerization, thuslimiting actin filament growth and increasing G-actin (Cooleyet al., 1992; Gunsalus et al., 1995). Embryos mutant for eitherchic or tsr alone had normal salivary glands. However, Tec29chic double mutants showed an enhancement of the Tec29salivary gland phenotype. The salivary glands in 80% of Tec29chic double mutants showed more severe invagination defectswith large placodes left on the surface (Fig. 5B,E,G). Theremaining 20% of the embryos had salivary glands similar toTec29. By contrast, 30% of the Tec29 tsr double mutants hadsalivary 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 Tec29mutants there is shift from F-actin to G-actin on the apicalsurface of the salivary placode cells. They also suggest that theapparent disorganization seen with the actin antibody resultsfrom increases in G- relative to F-actin. The partial rescue ofthe Tec29 salivary gland phenotype by tsr indicates that theshift in the balance between G-actin and F-actin in the salivaryplacodes caused the invagination delay in Tec29 mutantsalivary placodes. Therefore, Tec29 was necessary to facilitatethe formation or maintenance of F-actin at the apical surfaceof the salivary placodes cells, and this localization of actin wascrucial for normal invagination.

Endoreplication is aberrant in Tec29 embryos

In at least two cases, the Drosophila ventral furrow and theparaxial mesoderm in Xenopus, the cell cycle must also beregulated to allow normal invagination, (Grosshans andWieschaus, 2000; Leise and Mueller, 2004; Mata et al., 2000;Seher and Leptin, 2000). This prompted us to examine the cellcycle control in the salivary placodes of Tec29 mutants.

Fig. 3. The salivary glands at stage 15 are stretched in Tec29 mutantembryos. (A-D) SCRIBBLE and FKH staining in the salivary glandsshow that the cells are isodiametric in w1118 but are stretched alongthe axis of the glands in Tec29K00206 embryos (arrowheads).(E,F) Staining with CRUMBS and FKH also shows that the proximalcells of the salivary glands are stretched in Tec29 embryos (arrows).Because the salivary gland cells are wedge shaped with their apicalsurface facing the lumen, CRUMBS, being more apical, produces asmaller outline of the cells than does SCRIBBLE.

Fig. 4. Actin is disorganized in the salivary placodes of Tec29K00206

embryos. All the panels show optical sections of embryos focused onthe apical surface of the salivary placodes. In all the panels, the circleoutlines the salivary placodes. (A,B) The apical staining for α-spectrin looks normal in Tec29K00206 embryos. (C,D) Actindisorganization on the apical surface of the cells in Tec29K00206

embryos is evident at early stage 12 when the embryos are stainedwith a monoclonal antibody against ACTIN, which detects both G-actin and F-actin.

Dev

elop

men

t

3520

Though the salivary placode cells are postmitotic, they doregulate a specialized cell cycle, the endoreplication cycle(endocycle), during their invagination. Therefore, we sought tounderstand if the control of the endocycle was necessary forsalivary invagination and whether the endocycle was normal inTec29 embryos. The salivary glands in Drosophila embryos arethe first cells in the embryo to leave the mitotic cycle and enterendoreplication (Smith and Orr-Weaver, 1991).Endoreplication begins in these cells immediately followingtheir invagination into the embryo at stage 12 (Fig. 6A) (Smithand Orr-Weaver, 1991). In wild-type salivary glands,endoreplication proceeds as a wave from the distal tip of thegland, progressing proximally until all the cells are included.It is rare for cells to enter endoreplication while still on thesurface of the embryo (Fig. 6E). Following one round ofendoreplication, the salivary gland cells enter G phase and donot resume replication until larval stages. During the early partsof invagination (early to mid stage 12), we found few BrdU-labeled, S-phase nuclei in wild-type glands (Fig. 6A).However, in Tec29 embryos, the coordinated progression ofsalivary gland endoreplication disappeared. Though thesalivary gland cells began endoreplication on schedule,endoreplicating cells were not confined to the distal tip.Isolated cells in the rest of the gland incorporated BrdU andeventually many cells that were still on the surface becamelabeled (Fig. 6B,F). Thus, in Tec29 embryos the synchronizedwave of endoreplication in the salivary glands is disrupted,resulting in uncoordinated endoreplication throughout theplacodes and invaginating glands.

Rescuing endoreplication can rescue the salivarygland phenotype of Tec29 embryos

To see whether premature endoreplication contributes to theinvagination defects in Tec29 embryos, we delayedendoreplication in Tec29 embryos. Previous studies haveshown that, although pulses of cyclin E are necessary forendoreplication, continuous expression can blockendoreplication (Knoblich et al., 1994; Weiss et al., 1998).

When we used paired-GAL4 to express UAS-cyclin E in thesalivary primordium, we found ectopic expression in thesalivary placodes beginning at stage 11 and continuing to beexpressed in the salivary glands in older embryos. Althoughexpression of prd-GAL4 in the gland is patchy, cells thatexpress cyclin E have a corresponding absence of BrdUlabeling, indicating that ectopic cyclin E efficiently blockedendoreplication (data not shown). Continuous expression ofcyclin E did not alter wild-type salivary gland invagination;however, continuous cyclin E caused a partial or completesuppression of the mutant phenotype in 69% of the Tec29homozygous embryos (Fig. 6I,J). These results indicate thatsynchronized endoreplication is necessary for invagination ofthe salivary glands and that Tec29 controls the progression ofthe endoreplication wave in the salivary glands.

The effects of Tec29 on endoreplication may be dueits effects on actin cytoskeleton

As Tec29 has effects on both actin cytoskeleton andendoreplication, we wanted to understand if Tec29independently affects these processes. Hence, we examinedendoreplication in Tec29 chic and Tec29 tsr double mutants.Though Tec29 tsr mutants show a suppression of the salivarygland phenotype, there was no clear decrease in the number orextent of the endoreplication defects in these embryos (Fig.6D,H). However, Tec29 chic double mutants showed a markedenhancement of the endoreplication defects observed in Tec29alone. There were more endoreplicating cells in embryos

Development 132 (15) Research article

Fig. 5. Tec29 phenotype in the salivary glands isenhanced by chickadee and suppressed bytwinstar. (A-C) More cells in the salivary glandsof Tec29K00206 chic221 double mutants (B) are onthe surface at stage 14 compared withTec29K00206 alone (A). However, Tec29K00206

tsrK05633 double mutants can rescue theTec29K00206 phenotype in the salivary glands(C). (D-F) The actin disorganization inTec29K00206 embryos is increased by chic221 andrescued by tsrK05633 (circle). (G) The graphicalrepresentation of the enhancement andsuppression by chic221 and tsrK05633,respectively. At stage 14, a third of the salivaryplacodes is left on the surface in Tec29K00206

embryos (759±36 μm2, n=30), whereas it isalmost doubled in Tec29K00206chic221 embryos(1304±45 μm2, n=30) as well as inTec29K00206Src42AE1 embryos (1278±43 μm2,n=30) and it is halved in Tec29K00206tsrK05633

(330±59 μm2, n=30).

Dev

elop

men

t

3521Tec29 is necessary for salivary invagination

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. Theseresults suggest that the defects in actin remodeling can affectthe endoreplication cycle in the salivary glands.

There is a genetic interaction between Tec29 andSrc family kinases in the salivary glands

Src family kinases have been shown to activate Tec kinases invertebrates (Takesono et al., 2002). In addition, the DrosophilaSrc-family kinases, Src42A and Src64, genetically interact withTec29 either in the embryo or in the ovary (Djagaeva et al.,2005; Guarnieri et al., 1998; Tateno et al., 2000; Thomas andWieschaus, 2004). We found that Tec and Src kinases alsointeracted genetically in the salivary glands. Though Src42A byitself did not show salivary placode invagination defect,mutations in Src42A enhanced the Tec29 salivary glandphenotype (Fig. 7B). In Tec29 Src42A double mutants, theinvagination defects are more severe than in Tec29 singlemutants. In 70% of the double mutant embryos fewer salivarygland cells invaginate, resulting in large placodes that remainon the surface (Fig. 7B and Fig. 5G). In addition, more salivarygland cells prematurely entered endoreplication in these doublemutants than in Tec29 alone (Fig. 7G,H). These Tec29 Src42Aembryos also showed disorganization of actin in the salivaryplacodes, but it is unclear if the disorganization is more severethan Tec29 alone (Fig. 7E,F).

There were also genetic interactions between Tec29 and theother Src kinase, Src64. Similar to Src42A mutants, Src64mutant embryos did not show any salivary gland abnormalities.However, many of the Tec29 Src64 double mutant embryosshowed gross abnormalities such as lack of head segments,making it difficult to evaluate the salivary gland phenotype. Ofthe Tec29 Src64 double mutant embryos that looked normal inoutline, 16% showed more severe salivary gland phenotypesthan Tec29 alone (Fig. 7C). The Tec29-Src64 interaction ismost clearly shown with heterozygous Tec29 embryos. Thesalivary glands are normal in Tec29/+ Src64/+ embryos.However, about one-third of Tec29/+ Src64/Src64 embryosshow Tec29-like invagination defects (Fig. 7D). These resultsshow that there is a strong genetic interaction between Src64and Tec29 in the salivary glands. The lack of salivary glandinvagination defects in embryos mutant for either Src kinaseand the enhancement of the null mutant Tec29 phenotype bySrc mutations suggest that Tec29 and the Src kinases mayfunction in a parallel pathways to promote salivary glandinvagination.

Discussion

Salivary placode cells must constrict apically and move theirnuclei basally in order to invaginate into the embryo (Myat andAndrew, 2000a; Myat and Andrew, 2000b). Therefore, theprocess of salivary gland invagination is expected to requireactin-myosin contractility. We show that Tec29, a non receptortyrosine kinase, is responsible for regulating the actinreorganization in the salivary placodes prior to invagination.Lack of Tec29 in the salivary placodes resulted in a shift ofactin in the salivary placodes from F-actin to more G-actin,leading to incomplete invagination of the salivary placodescells. This change in the balance between F-actin and G-actinwas observed only on the apical surfaces of these cells,suggesting that the delayed invagination was due to aberrant

Fig. 6. Tec29 is necessary to delay endoreplication in the salivaryglands. There are more BrdU-labeled nuclei in the invaginated glandlabeled with FKH antibody (green) at early stage 12 in Tec29K00206

compared with w1118 (A,B, arrowhead) and also ventrally in thesalivary placodes at mid stage 12 (arrowhead, compare E with F). Inaddition, there are more BrdU labeled nuclei in Tec29K00206chic221

embryos compared with Tec29K00206 alone at early stage 12 (C) andat mid stage 12 (G). BrdU labeling in Tec29K00206tsrK05633 is similarto Tec29K00206 embryos (D,H). (I,J) Delaying endoreplication bydriving UAS-CycE using prd-GAL4 in Tec29K00206 embryos rescuesthe long salivary gland phenotype of Tec29K00206 embryos (arrows).

Dev

elop

men

t

3522

apical constriction of the salivary placodes cells in Tec29embryos. Increasing actin depolymerization further in Tec29mutants by a mutation in chic increased the salivaryinvagination delay, whereas promoting actin polymerization inTec29 mutants by mutating tsr partially rescued theinvagination delay. Thus, our results provide the first evidencethat actin reorganization is necessary for salivary glandinvagination.

The shift in the equilibrium towards more G-actin in Tec29mutants might be due either to decreased polymerization ofactin or decreased stability of F-actin. Tec kinases affect bothactin polymerization and actin crosslinking in vertebrates andin Drosophila (Finkelstein and Schwartzberg, 2004). ITK-deficient mice show impaired actin polymerization in responseto T-cell activation (Labno et al., 2003). In addition, both BTKand ITK interact with WASP, a protein that can activate theArp2/3 complex and promote new actin filament formation(Baba et al., 1999b; Bunnell et al., 1996; Guinamarda et al.,1998). During early Drosophila oogenesis, accumulation of G-actin has been observed in the cortex of Tec29 mutants, leadingto defective fusome formation (Djagaeva et al., 2005). Inaddition, Tec29 has been suggested to affect actin crosslinkingby phosphorylating KELCH, an actin-bundling protein in thering canals of the ovary. The phosphorylation of KELCH isnecessary to decrease actin crosslinking, thereby allowingexpansion of the ring canals (Kelso et al., 2002). It is thereforepossible that in the salivary placodes, Tec29 might be neededfor new F-actin formation or bundling of the actin filaments topromote invagination.

Besides salivary gland invagination, Tec29 affects actinorganization during two other processes in Drosophilaembryogenesis: dorsal closure and cellularization. Tec29 inconjunction with Src42A is necessary for dorsal closure andlack of both these kinases result in a dorsal open phenotype.So, during dorsal closure as with salivary invagination, there isa positive interaction between Tec29 and Src42A. However,

Src42 is the main player during dorsal closure, whereas Tec29is more important for salivary invagination. Interestingly, thereis a reduction of F-actin observed at the leading edge of thedorsal epidermal cells in these double mutants, causing delayeddorsal closure (Tateno et al., 2000). Thus, the lack of thesetyrosine kinases causes changes in actin dynamics that resultin delayed migration of both the salivary placodes and duringdorsal closure. Similarly, the process of basal closure duringcellularization resembles apical constriction prior toinvagination and requires an actin-myosin based contraction atthe base of the cellularization front. In embryos arising fromTec29 germline clones, the actin microfilament ring does notcontract, resulting in membrane invagination of varying depthsand impairment of basal closure during late cellularization(Thomas and Wieschaus, 2004). It is possible that, similar tothe salivary invagination and dorsal closure, the absence of thecontractile ring during cellularization is due to decreased F-actin and/or increased G-actin. In general, Tec29 appears to beneeded for regulation of actin during periods of extensive andrapid reorganization of the actin cytoskeleton as observedduring migration and contraction of cells.

Although Tec kinases are known to alter the actincytoskeleton in many systems, we are the first to show arelationship between Tec29 and the endocycle. Our datasupport a previous observation that the salivary placodes inwild-type embryos enter endoreplication only after theyinvaginate into the embryo (Smith and Orr-Weaver, 1991). InTec29 mutants, however, the wave of endoreplication isdisrupted, such that the ventral cells in the placodes initiateendoreplication prior to invagination. As a result, these cellsfail to invaginate on schedule, resulting in the long salivaryglands. Therefore, delaying endoreplication appears to benecessary to allow invagination, and coordinating the twoevents is crucial for normal development of the salivary glands.Similar coordination between the cell cycle and morphogenesisis observed during ventral furrow formation in Drosophila

Development 132 (15) Research article

Fig. 7. Tec29 interacts with Src42 and Src64 in the salivary glands. (A-D) The invagination defects in Tec29K00206 embryos (A) are enhanced bySrc42AE1 (B). In addition, phenotypes of both homozygous and heterozygous Tec29K00206 embryos are enhanced by Src64PI (C,D, arrow).(E-H) Disorganization of actin observed in Tec29K00206 Src42AE1 double mutants is not obviously more severe than Tec29K00206 alone (E,F,circle); however, there are more cells labeled with BrdU in the Tec29K00206Src42AE1 compared with Tec29K00206 (arrowhead, compare H withG).

Dev

elop

men

t

3523Tec29 is necessary for salivary invagination

embryos and in the paraxial mesoderm in Xenopus embryos.In the ventral furrow and the paraxial mesoderm, the cell cycledelay is established by maintaining the Cdks in theirphosphorylated form. In the ventral furrow, this isaccomplished by tribbles inhibiting the Drosophila Cdc25homolog string, a protein that dephosphorylates and activatesCdks (Grosshans and Wieschaus, 2000; Mata et al., 2000;Seher and Leptin, 2000). In the paraxial mesoderm, thelocalized phosphorylation of Cdks by Wee2 is sufficient toprevent the cells from entering mitosis prior to invagination(Leise and Mueller, 2004). In both these cases, as in thesalivary gland, the coordination between cell cycle andinvagination is crucial for normal morphogenesis of theembryo. An important difference is that unlike the salivaryglands, it is the mitotic cycle that is regulated with invaginationof the ventral furrow and the paraxial mesoderm. Our studyprovides the first indication of a link between the endocycleand morphogenesis, and suggests that coordination ofendoreplication with invagination is crucial for normaldevelopment.

As shown in the ventral furrow and the paraxial mesoderm,mitosis and invagination use the same cytoskeletal componentsand require opposing levels of cell adhesion, making these twoprocesses incompatible with each other (Grosshans andWieschaus, 2000; Leise and Mueller, 2004; Mata et al., 2000;Seher and Leptin, 2000). However, a normal endocycle in fliesdoes not involve nuclear membrane breakdown or otherprocesses that might require the same cytoskeletal componentsas invagination (Edgar and Orr-Weaver, 2001). Thus, a normalendocycle might not interfere with invagination. But this maynot be a normal endocycle. Unlike the other endocycling cellsin the embryo, which enter the endocycle from G1, it has beensuggested that the salivary placodes may enter the endocyclefrom G2 (Smith and Orr-Weaver, 1991). It is therefore possiblethat the endocycle in the salivary placodes retains some aspectsof mitosis that would interfere with invagination. If so, thisendocycle would be similar to those in some endocyclingmammalian cells that enter the endoreplication cycle after G2during early M phase (Edgar and Orr-Weaver, 2001). Perhapsbecause this first salivary gland endocycle is unusual, it mustbe delayed until after the salivary cells are safely inside theembryo.

Our data indicate that Tec29 is necessary for actinremodeling during apical constriction in the salivary placodesand for endocycle progression as the glands invaginate. Inaddition, we found that manipulating the actin cytoskeleton inTec29 mutants can have effects on endoreplication in thesecells. When the actin defects were enhanced by eliminatingchic in Tec29 embryos, there was also an enhancement of theendoreplication defects of Tec29, suggesting that the actincytoskeleton and endoreplication cycle are linked. We proposethe following model to explain how these two events might becoordinated in the salivary placodes. The actin remodelingduring apical constriction and endoreplication follow eachother such that the first cells to apically constrict are the firstcells to invaginate and endoreplicate, and the remaining cellsfollow in sequence. These processes might be causally linked;the apical constriction wave might trigger the wave ofendoreplication. This coupling and a lag between the twoevents would ensure that endoreplication does not occurprematurely, while the cells are still on the surface of the

embryo. With this model, Tec29 would then regulate theendocycle indirectly, rather than independently regulating bothactin and endoreplication. Disruption of the apical constrictionwave in Tec29 mutants would lead to subsequent disruption ofthe endoreplication wave. An invagination delay due toinadequate actin polymerization would be compounded byplacode cells endoreplicating prior to invagination. This modelalso explains the ability of cyclin E to partially rescue the Tec29phenotype. Cyclin E overexpression would delayendoreplication and relieve some of the effects of inadequateactin polymerization. Studies in progress to identify TEC29targets are likely to provide further insight into its effects onendoreplication and invagination, and should enable us tobetter understand the link between these processes.

We are grateful to Deborah Andrew, David Bilder, Mike Simon, PatO’Farrell, T. Adachi-Yamada and the Bloomington Stock Center forproviding the fly stocks and the antibodies used in this study. Wewould like to thank Eileen Beall, Mark Stern, Tereza Kolesnikov andKatherine Harris for their advice and critical reading of themanuscript. This work was supported by NIH grant DE12519 toS.K.B.

ReferencesAbrams, E. W., Vining, M. S. and Andrew, D. J. (2003). Constructing an

organ: the Drosophila salivary gland as a model for tube formation. TrendsCell Biol. 13, 247-254.

Andrew, D. J., Baig, A., Bhanot, P., Smolik, S. M. and Henderson, K. D.(1997). The Drosophila dCREB-A gene is required for dorsal/ventralpatterning of the larval cuticle. Development 12, 181-193.

Baba, K., Takeshita, A., Majima, K., Ueda, R., Kondo, S., Juni, N. andYamamoto, D. (1999a). The Drosophila Bruton’s tyrosine kinase (Btk)homolog is required for adult survival and male genital formation. Mol. Cell.Biol. 19, 4405-4413.

Baba, Y., Nonoyama, S., Matsushita, M., Yamadori, T., Hashimoto, S.,Imai, K., Arai, S., Kunikata, T., Kurimoto, M., Kurosaki, T. et al.(1999b). Involvement of wiskott-aldrich syndrome protein in B-cellcytoplasmic tyrosine kinase pathway. Blood 93, 2003-2012.

Bilder, D. and Perrimon, N. (2000). Localization of apical epithelialdeterminants by the basolateral PDZ protein Scribble. Nature 403, 676-680.

Bunnell, S. C., Henry, P. A., Kolluri, R., Kirchhausen, T., Rickles, R. J.and Berg, L. J. (1996). Identification of Itk/Tsk Src homology 3 domainligands. J. Biol. Chem. 271, 25646-25656.

Chandrasekaran, V. and Beckendorf, S. K. (2003). senseless is necessaryfor the survival of embryonic salivary glands in Drosophila. Development130, 4719-4728.

Cooley, L., Verheyen, E. and Ayers, K. (1992). chickadee encodes a profilinrequired for intercellular cytoplasm transport during Drosophila oogenesis.Cell 69, 173-184.

Djagaeva, I., Doronkin, S. and Beckendorf, S. K. (2005). Src64 is involvedin fusome development, cytokinesis and in karyosome formation duringDrosophila oogenesis (in press).

Dodson, G. S., Guarnieri, D. J. and Simon, M. A. (1998). Src64 is requiredfor ovarian ring canal morphogenesis during Drosophila oogenesis.Development 125, 2883-2892.

Edgar, B. A. and Orr-Weaver, T. L. (2001). Endoreplication cell cycles: morefor less. Cell 105, 297-306.

Finkelstein, L. D. and Schwartzberg, P. L. (2004). Tec kinases: shaping T-cell activation through actin. Trends Cell Biol. 14, 443-451.

Gregory, R. J., Kammermeyer, K. L., Vincent, W. S., 3rd and Wadsworth,S. G. (1987). Primary sequence and developmental expression of a novelDrosophila melanogaster src gene. Mol. Cell. Biol. 7, 2119-2127.

Grosshans, J. and Wieschaus, E. (2000). A genetic link betweenmorphogenesis and cell division during formation of the ventral furrow inDrosophila. Cell 101, 523-531.

Guarnieri, D. J., Dodson, G. S. and Simon, M. A. (1998). SRC64 regulatesthe localization of a Tec-family kinase required for Drosophila ring canalgrowth. Mol. Cell. 1, 831-840.

Guinamarda, R., Aspenströmb, P., Fougereaua, M., Chavriera, P. and

Dev

elop

men

t

3524

Guillemota, J. (1998). Tyrosine phosphorylation of the Wiskott-AldrichSyndrome protein by Lyn and Btk is regulated by CDC42. FEBS Lett. 434,431-436.

Gunsalus, K. C., Bonaccorsi, S., Williams, E., Verni, F., Gatti, M. andGoldberg, M. L. (1995). Mutations in twinstar, a Drosophila gene encodinga cofilin/ADF homologue, result in defects in centrosome migration andcytokinesis. J. Cell Biol. 131, 1243-1259.

Hacker, U. and Perrimon, N. (1998). DRhoGEF2 encodes a member of theDbl family of oncogenes and controls cell shape chages during gastrulationin Drosophila. Genes Dev. 12, 274-284.

Harland, R. M. (1991). In situ hybridization: an improved whole-mountmethod for Xenopus embryos. Methods Cell Biol. 36, 685-695.

Henderson, K. D. and Andrew, D. J. (2000). Regulation and function of Scr,exd, and hth in the Drosophila salivary gland. Dev. Biol. 217, 362-374.

Katzen, A. L., Kornberg, T. and Bishop, J. M. (1990). Diverse expressionof dsrc29A, a gene related to src, during the life cycle of Drosophilamelanogaster. Development 110, 1169-1183.

Kelso, R. J., Hudson, A. M. and Cooley, L. (2002). Drosophila Kelchregulates actin organization via Src64-dependent tyrosine phosphorylation.J. Cell Biol. 156, 703-713.

Knoblich, J. A., Sauer, K., Jones, L., Richardson, H., Saint, R. and Lehner,C. F. (1994). Cyclin E controls S phase progression and its down-regulationduring Drosophila embryogenesis is required for the arrest of cellproliferation. Cell 77, 107-120.

Labno, C. M., Lewis, C. M., You, D., Leung, D. W., Takesono, A.,Kamberos, N., Seth, A., Finkelstein, L. D., Rosen, M. K., Schwartzberg,P. L. and Burkhardt, J. K. (2003). Itk functions to control actinpolymerization at the immune synapse through localized activation of Cdc42and WASP. Curr. Biol. 13, 1619-1624.

Leise, W. F., 3rd and Mueller, P. R. (2004). Inhibition of the cell cycle isrequired for convergent extension of the paraxial mesoderm during Xenopusneurulation. Development 131, 1703-1715.

Leptin, M. and Grunewald, B. (1990). Cell shape changes during gastrulationin Drosophila. Development 110, 73-84.

Maas, A. and Hendriks, R. W. (2001). Role of Bruton’s tyrosine kinase in Bcell development. Dev. Immunol. 8, 171-181.

Mano, H. (1999). Tec family of protein-tyrosine kinases: an overview of theirstructure and function. Cytokine Growth Factor Rev. 10, 267-280.

Mata, J., Curado, S., Ephrussi, A. and Rorth, P. (2000). Tribblescoordinates mitosis and morphogenesis in Drosophila by regulatingstring/CDC25 proteolysis. Cell 101, 511-522.

Myat, M. M. and Andrew, D. J. (2000a). Organ shape in the Drosophilasalivary gland is controlled by regulated, sequential internalization of theprimordia. Development 127, 679-691.

Myat, M. M. and Andrew, D. J. (2000b). Fork head prevents apoptosis andpromotes cell shape change during formation of the Drosophila salivaryglands. Development 127, 4217-4226.

Nikolaidou, K. K. and Barrett, K. (2004). A Rho GTPase signaling pathwayis used reiteratively in epithelial folding and potentially selects the outcomeof Rho activation. Curr. Biol. 14, 1822-1826.

Panzer, S., Weigel, D. and Beckendorf, S. K. (1992). Organogenesis inDrosophila melanogaster: embryonic salivary gland determination iscontrolled by homeotic and dorsoventral patterning genes. Development114, 49-57.

Roulier, E. M., Panzer, S. and Beckendorf, S. K. (1998). The Tec29 tyrosinekinase is required during Drosophila embryogenesis and interacts withSrc64 in ring canal development. Mol. Cell 1, 819-829.

Satterthwaite, A. B. and Witte, O. N. (2000). The role of Bruton’s tyrosinekinase in B-cell development and function: a genetic perspective. Immunol.Rev. 175, 120-127.

Schöck, F. and Perrimon, N. (2002). Molecular mechanisms of epithelialmorphogenesis. Annu. Rev. Cell Dev Biol. 18, 463-493.

Seher, T. C. and Leptin, M. (2000). Tribbles, a cell-cycle brake thatcoordinates proliferation and morphogenesis during Drosophilagastrulation. Curr. Biol. 10, 623-629.

Shermoen, A. W. (2000). BrdU labeling of chromosomes. In DrosophilaProtocols (ed. W. Sullivan, M. Ashburner and R. S. Hawley), pp. 57-65.New York: Cold Spring Harbor Press.

Smith, A. V. and Orr-Weaver, T. L. (1991). The regulation of the cell cycleduring Drosophila embryogenesis: the transition to polyteny. Development112, 997-1008.

Takesono, A., Finkelstein, L. D. and Schwartzberg, P. L. (2002). Beyondcalcium: new signaling pathways for Tec family kinases. J. Cell Sci. 115,3039-3048.

Tateno, M., Nishida, Y. and Adachi-Yamada, T. (2000). Regulation of JNKby Src during Drosophila development. Science 287, 324-327.

Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridizationmethod for the localization of specific RNAs in Drosophila embryos revealstranslational control of the segmentation gene hunchback. Chromosoma 98,81-85.

Tepass, U., Theres, C. and Knust, E. (1990). crumbs encodes an EGF-likeprotein expressed on apical membranes of Drosophila epithelial cells andrequired for organization of epithelia. Cell. 61, 787-799.

Thomas, G. H. and Kiehart, D. P. (1994). Beta heavy-spectrin has a restrictedtissue and subcellular distribution during Drosophila embryogenesis.Development 120, 2039-2050.

Thomas, J. H. and Wieschaus, E. (2004). Src64 and Tec29 are required formicrofilament contraction during Drosophila cellularization. Development131, 863-871.

Vincent, W. S., 3rd, Gregory, R. J. and Wadsworth, S. C. (1989).Embryonic expression of a Drosophila src gene: alternate forms of theprotein are expressed in segmental stripes and in the nervous system. GenesDev. 3, 334-347.

Wadsworth, S. C., Muckenthaler, F. A. and Vincent, W. S., 3rd (1990).Differential expression of alternate forms of a Drosophila src protein duringembryonic and larval tissue differentiation. Dev. Biol. 138, 296-312.

Weigel, D., Bellen, H., Jürgens, G. and Jäckle, H. (1989). Primordiumspecific requirement of the homeotic gene fork head in the developing gutof the Drosophila embryo. Roux’s Arch. Dev. Biol. 198, 201-210.

Weiss, A., Herzig, A., Jacobs, H. and Lehner, C. F. (1998). ContinuousCyclin E expression inhibits progression through endoreduplication cyclesin Drosophila. Curr. Biol. 8, 239-242.

Young, P. E., Pesacreta, T. C. and Kiehart, D. P. (1991). Dynamic changesin the distribution of cytoplasmic myosin during Drosophila embryogenesis.Development. 111, 1-14.

Zhou, B., Bagri, A. and Beckendorf, S. K. (2001). Salivary glanddetermination in Drosophila: a salivary-specific, fork head enhancerintegrates spatial pattern and allows fork head autoregulation. Dev. Biol. 237,54-67.

Development 132 (15) Research article

Dev

elop

men

t