Drs. Zahedi and Shen contributed equally to this work.
Leading edge-secreted Dpp cooperates with ACK-dependent signaling from the amnioserosa to regulate myosin levels during dorsal closure
Article first published online: 24 SEP 2008
Copyright © 2008 Wiley-Liss, Inc.
Special Issue: Special Focus on the Extracellular Matrix, in Memory of Dr. Elizabeth D. Hay
Volume 237, Issue 10, pages 2936–2946, October 2008
How to Cite
Zahedi, B., Shen, W., Xu, X., Chen, X., Mahey, M. and Harden, N. (2008), Leading edge-secreted Dpp cooperates with ACK-dependent signaling from the amnioserosa to regulate myosin levels during dorsal closure. Dev. Dyn., 237: 2936–2946. doi: 10.1002/dvdy.21722
- Issue published online: 24 SEP 2008
- Article first published online: 24 SEP 2008
- Manuscript Accepted: 30 JUL 2008
- Canadian Institutes of Health Research. Grant Number: MOP-74513
- dorsal closure;
Dorsal closure of the Drosophila embryo is an epithelial fusion in which the epidermal flanks migrate to close a hole in the epidermis occupied by the amnioserosa, a process driven in part by myosin-dependent cell shape change. Dpp signaling is required for the morphogenesis of both tissues, where it promotes transcription of myosin from the zipper (zip) gene. Drosophila has two members of the activated Cdc42-associated kinase (ACK) family: DACK and PR2. Overexpression of DACK in embryos deficient in Dpp signaling can restore zip expression and suppress dorsal closure defects, while reducing the levels of DACK and PR2 simultaneously using mutations or amnioserosa-specific knock down by RNAi results in loss of zip expression. ACK function in the amnioserosa may generate a signal cooperating with Dpp secreted from the epidermis in driving zip expression in these two tissues, ensuring that cell shape changes in dorsal closure occur in a coordinated manner. Developmental Dynamics 237:2936–2946, 2008. © 2008 Wiley-Liss, Inc.
There are striking parallels between wound healing and developmental events that involve epithelial fusions, and insight gained from the study of any one of these processes may be broadly applicable (Martin and Wood,2002; Martin and Parkhurst,2004). Dorsal closure of the Drosophila embryo is a developmental epithelial fusion event that seals a hole in the dorsal epidermis occupied by the amnioserosa (reviewed in Harden,2002). Key regulatory events in dorsal closure occur in the first row of epidermal cells flanking the amnioserosa, known as the leading edge or dorsalmost epidermal (DME) cells. The amnioserosa is also a critical player in dorsal closure, undergoing a dramatic change in morphology that aids movement of the epidermal flanks and is involved in bidirectional communication with the epidermis (Glise and Noselli,1997; Lamka and Lipshitz,1999; Kiehart et al.,2000; Jacinto and Martin,2001; Reed et al.,2001; Stronach and Perrimon,2001; Harden et al.,2002; Conder et al.,2004; Franke et al.,2005; Scuderi and Letsou,2005; Fernandez et al.,2007; Wada et al.,2007). Dpp is a member of the transforming growth factor-beta (TGF-β) superfamily of cytokines expressed in the DME cells that acts through a receptor complex comprised of Thickveins (Tkv) and Punt to regulate gene expression through the R-Smad Mothers against dpp (Mad) and the Co-Smad Medea (Harden,2002). Among the genes regulated by Dpp signaling in the DME cells is zipper (zip), encoding the non-muscle myosin II heavy chain (Young et al.,1993; Arquier et al.,2001). One mechanical force required for dorsal closure is created by accumulation of F-actin and nonmuscle myosin II at the leading edge, forming a cable that constricts the DME cells to elongate in a dorsalward direction (Young et al.,1993; Franke et al.,2005), and it is likely that loss of zip expression makes a significant contribution to dorsal closure failure in Dpp pathway mutants.
We previously showed that expression of a constitutively active version of the small GTPase Cdc42 could suppress the dorsal closure defects associated with a tkv loss-of-function allele, tkv7 (Ricos et al.,1999). A likely route by which Cdc42 drives this suppression of the tkv phenotype would be through activation of a downstream effector protein. DACK and PR2 are the two Drosophila members of the activated Cdc42-associated kinase (ACK) family of nonreceptor tyrosine kinases (Manser et al.,1993; Burbelo et al.,1995; Clemens et al.,2000; Sem et al.,2002; Worby et al.,2002). Throughout this study the general term ACK is used to refer to any or all members of the ACK family. When a point is made about a particular ACK protein, its specific name is used. DACK overexpression can suppress the dorsal closure defects caused by expression of a dominant negative version of Cdc42, and expression of constitutively active Cdc42 causes elevation of DACK transcript levels in the amnioserosa during dorsal closure (Sem et al.,2002). These results make the ACK proteins promising candidates for Cdc42-effectors impacting Dpp signaling, and here we show that ACK-dependent signaling from the amnioserosa cooperates with Dpp secreted from the leading edge to drive zip expression in these two tissues. As the zip product is required in both tissues for cell shape change (Franke et al.,2005), this transcriptional regulation may provide a means for coordinating morphogenetic events during dorsal closure.
Dpp Signaling Is Necessary but Not Sufficient for zip Expression in the DME Cells and Amnioserosa
In addition to controlling morphogenesis of the epidermis, the Dpp pathway is required for apical cell constriction in the amnioserosa, but the route by which Dpp regulates this cell shape change is not clear (Fernandez et al.,2007). To determine whether the amnioserosa cells are exposed to Dpp ligand produced in the DME cells, we expressed green fluorescent protein (GFP) -tagged, biologically active Dpp (Teleman and Cohen,2000) using the LE-Gal4 driver, which is expressed in a subset of DME cells (Glise and Noselli,1997; Fig. 1A), and stained embryos with anti-GFP antibodies to reveal Dpp-GFP over the amnioserosa (Fig. 1B). Dpp secreted from the DME cells signals back into these cells to promote their morphogenesis, at least in part by driving zip expression, and we wondered if Dpp migrating over the amnioserosa might similarly drive zip expression in the amnioserosa cells to promote apical constriction (Arquier et al.,2001; Franke et al.,2005). One way to ensure coordinated morphogenesis of neighboring tissues would be to have a signal from one tissue transcriptionally regulating a required component of morphogenesis in another, and we looked for Dpp-dependent zip expression in the amnioserosa. Using high sensitivity fluorescent RNA in situ hybridization (FISH) with tyramide signal amplification, we detected zip transcription in the amnioserosa that was highest at the beginning of germband retraction and had disappeared by the beginning of dorsal closure (Fig. 1C–E). In contrast, DME expression of zip was weak during germband retraction but high during dorsal closure. To assess the requirements for Dpp signaling in the regulation of zip expression, we performed zip FISH on tkv7 mutant embryos and saw a reduction in zip levels in the amnioserosa during germband retraction and a reduction in zip levels in the DME cells during dorsal closure (Fig. 1F,G). The latter finding confirms previous results using digoxigenin zip in situ hybridization (Arquier et al.,2001). The Dpp pathway can be ectopically activated in the embryo through expression of a constitutively active version of the Tkv receptor, TkvQ199D, or a Dpp transgene (Hoodless et al.,1996; Dorfman and Shilo,2001). Ectopic activation of Dpp signaling by expression of TkvQ199D with prd-Gal4 did not result in zip transcript elevation in prd stripes (data not shown), and we conclude that Dpp signaling is necessary but not sufficient for zip transcription. This result demonstrates a requirement for an additional input or inputs for transcriptional regulation of zip.
DACK Cooperates With Dpp Signaling in Regulating zip Expression
Given our previous finding that overexpression of DACK could suppress the dorsal closure defects caused by impaired Cdc42 signaling, and that activation of Cdc42 signaling could suppress the dorsal closure defects of tkv7 embryos, we tested to see if DACK overexpression would suppress the effects of loss of Dpp signaling (Ricos et al.,1999; Sem et al.,2002). Ubiquitous overexpression of DACK suppressed the dorsal defects of tkv7 embryos by around 40% (Table 1). The dorsal closure defects of tkv mutant embryos can be significantly suppressed by amnioserosa-specific expression of TkvQ199D and we wondered if similarly overexpressing DACK in this tissue alone would suppress Dpp pathway defects, particularly considering that global activation of Cdc42 signaling causes an up-regulation of DACK expression specifically in the amnioserosa (Sem et al.,2002; Fernandez et al.,2007). Expression of DACK using the amnioserosa-specific driver Gal4c381 (Manseau et al.,1997) suppressed the defects of tkv7 embryos by roughly 30% (Table 1). To see if DACK function had any bearing on zip transcription in the amnioserosa, we expressed wild-type and kinase-dead (KD) DACK transgenes (Sem et al.,2002) using Gal4c381 and performed FISH. Surprisingly, both forms of DACK caused a dramatic increase in zip expression not only in the amnioserosa but also in the dorsal epidermis (Fig. 1I–L). When these transgenes were expressed with prd-Gal4 (the striped expression of which extends into the amnioserosa, see Fig. 2I), elevated zip expression was seen throughout the amnioserosa and dorsal epidermis, but not in prd stripes in the epidermis (Fig. 1M,N). We found that with either Gal4 driver, KD-DACK had a stronger effect on zip levels than wild-type DACK, and that both forms of DACK were capable of promoting zip expression in the amnioserosa during germband retraction (Fig. 1I,L and data not shown) and during dorsal closure (Fig. 1M,N and data not shown), when the amnioserosa normally shows no zip expression (compare Fig. 1M with Fig. 1E). The two Gal4 drivers, the expression patterns of which overlap only in the amnioserosa, caused essentially the same pattern of zip elevation, and our interpretation of these results is that DACK functions in the amnioserosa in a kinase-independent and cell nonautonomous manner to promote zip transcription on the dorsal side of the embryo. Based on these results, DACK overexpression could suppress the dorsal closure defects of tkv mutant embryos by restoring some zip expression in cooperation with residual dorsal Tkv activity, and DME and amnioserosa zip transcription in tkv7 mutant embryos overexpressing DACK was restored to near wild-type levels (Fig. 1H and data not shown).
|Genotypea||% Dorsal defects in progeny of cross used to create indicated genotypeb||n|
|(A)||tkv7, hs-Gal4/tkv7||16.7 (66.8)||927|
|(B)||tkv7, hs-Gal4/tkv7,UAS-DACK||9.9 (39.6)||707|
|(E)||tkv7/tkv7,UAS-DACK (29°C)||25.3 (100)||572|
|(F)||tkv7/tkv7,UAS-DACK;Gal4c381/+ (29°C)||16.7 (66.8)||534|
Our data suggest that DACK could act in parallel to Dpp signaling rather than functioning as either a component or a target of the Dpp pathway, and we did several experiments to address this further. First, we evaluated DACK for its ability to activate the Dpp pathway at either the receptor level or at the level of transcription of a Dpp-responsive gene other than zip. Activation of the Dpp pathway at the level of the receptor complex can be visualized by staining embryos with an antibody (anti-pMad) that recognizes the receptor-phosphorylation of Mad (Persson et al.,1998)(Fig. 2A,B). As a likely Dpp-responsive gene during dorsal closure, we chose Daughters against dpp (Dad), a Dpp target in the wing that is expressed in the amnioserosa and dorsal epidermis (Tsuneizumi et al.,1997; Wada et al.,2007)(Fig. 2D,E). We determined that the Dpp pathway is necessary and sufficient for Dad expression in embryos during dorsal closure. tkv7 embryos lost Dad expression, whereas expression of Dpp using prd-Gal4 caused an elevation of Dad levels in prd stripes (Fig. 2G,H). Overexpression of a wild-type DACK transgene failed to induce either pMad or Dad accumulation in prd stripes (Fig. 2C,I′). Similarly, DACK could not drive ectopic expression of spalt major (salm), another target of Dpp signaling that shows a similar expression pattern to Dad during dorsal closure (Lecuit et al.,1996; Nellen et al.,1996)(Fig. 2F and data not shown). We then checked to see if DACK was a target of the Dpp pathway, at the level of regulation of the quantity or activity of DACK protein. prd-Gal4 expression of either Dpp or TkvQ199D did not result in an increase DACK protein levels (Fig. 3A′ and data not shown). Increased DACK tyrosine kinase activity due to overexpression of a DACK transgene in a tissue-specific manner can be visualized as an increase in phosphotyrosine levels in that tissue (Sem et al.,2002; Fig. 3B′). This assay did not detect increased DACK tyrosine kinase activity in embryos expressing Dpp in prd stripes (Fig. 3C′).
To characterize further the interaction of DACK with Dpp signaling, we generated loss-of-function alleles of DACK. Through excision of a P element, KG00869 (Bellen et al.,2004), inserted 5 base pairs upstream of the start point of transcription of the longer of the two DACK transcripts, we created DACK alleles that were homozygous viable and showed no detectable DACK transcripts on Northern blots of adult flies but retained expression of flanking genes (Fig. 4A,B). Cuticle preparations revealed that DACK mutant embryos had a low frequency of dorsal defects (Table 1), and we tested to see if reducing Dpp signaling at either the ligand or receptor level would have an effect on this phenotype. When DACK mutant embryos were made heterozygous for either of two embryonic lethal dpp alleles, dpphr27 and dpphr92 (St Johnston et al.,1990; Wharton et al.,1993), or tkv7, the frequency of dorsal defects increased significantly (Table 1).
zip expression levels appeared near normal in DACK mutant embryos (data not shown), and we wondered if this was due to overlapping function with the other ACK, PR2. There are no null alleles available in PR2, but we determined that a piggyBac transposon insertion in the first intron of PR2 (Thibault et al.,2004) led to decreased PR2 transcript levels but did not affect transcription of neighboring genes (Fig. 4C,D), and found that PR2 DACK double mutant embryos showed a loss of zip transcription in the dorsal embryo (Fig. 1O). Amnioserosa-specific expression of RNAi transgenes targeting DACK and PR2 to knock down the two ACKs in the amnioserosa also resulted in loss of dorsal zip expression (Fig. 1P). Expression of either RNAi transgene alone did not cause a detectable decrease in zip levels (data not shown) and we conclude that DACK and PR2 cooperate in regulating zip expression. We could not quantify the effects of reducing ACK function on dorsal closure, as double mutant embryos, as well as embryos with ACK knocked down by RNAi, either in the amnioserosa or globally by heat shock using an Hs-Gal4 driver, showed a high frequency of failures to secrete cuticle. However, many ACK-deficient embryos that secreted cuticle but did not hatch into larvae showed defects in the dorsal surface including holes in the cuticle and germband retraction failures (Fig. 5C–F). Failure to secrete cuticle, dorsal holes and germband retraction defects are also seen with DACK overexpression in the embryo (Sem et al.,2002). An interesting distinction was noted between the dorsal defects of embryos bearing RNAi knockdowns of both ACKs compared with embryos in which PR2 function was knocked down with RNAi and DACK function impaired with a DACK allele, in that the former tended to have large dorsal holes extending to the anterior end (indicative of defects in both dorsal closure and head involution), whereas the latter tended to have small anterior holes (indicative of head involution defect only; compare Fig. 5D,E with Fig, 5C,F). DACK RNAi could be eliminating maternal DACK transcripts that would be retained in a zygotic DACK mutant, and the result suggests that head involution may be more sensitive to loss of ACK than dorsal closure.
Effects of Excessive Dpp Signaling in the Embryo and Wing Are Suppressed in a DACK Mutant Background
We have demonstrated that ACK function positively affects the output of Dpp signaling on the dorsal side of the embryo and we checked to see if reducing ACK function could suppress the effects of excessive Dpp signaling in this region. Overexpression of Dpp with prd-Gal4 caused dorsal anterior holes in the cuticle and germband retraction defects, indicating effects on morphogenetic events that are also sensitive to changes in ACK signaling (Fig. 5B). These phenotypic effects were completely blocked when Dpp-overexpressing embryos were made homozygous for the DACK10b allele (data not shown).
Does ACK impact Dpp signaling elsewhere during development? Dpp signaling has been extensively studied in wing development, during which expression of TkvQ199D in the enhancer piracy line TAJ3 causes ectopic vein formation between L3 and L4 (Hoodless et al.,1996)(Fig. 6B). When TAJ3 flies were made homozygous for the DACK10b allele, the ectopic vein phenotype was significantly suppressed, again indicating a positive input of ACK on Dpp signaling (Fig. 6C).
ACK Mediates Communication Between the Amnioserosa and the Epidermis in the Regulation of zip Expression
Our results on the regulation of zip expression by Dpp are consistent with the model of Fernandez and colleagues in which two rounds of dpp expression in the DME cells regulate dorsal closure (Fernandez et al.,2007). In the first round of dpp expression, before completion of germband retraction, Dpp signals from the DME cells to the amnioserosa. This signaling can be visualized by pMad staining in the amnioserosa, which is obvious during germband retraction but fades away by the beginning of dorsal closure, a pattern paralleled by zip expression. In the second round of signaling, occurring during dorsal closure, Dpp signals to the dorsal epidermis, as demonstrated by robust pMad in this tissue (see Fig. 2A) and high zip levels in the DME cells. We have demonstrated that Dpp signaling and ACK function are both necessary but not sufficient for zip expression in the embryo during dorsal closure. Activation of either Dpp signaling or ACK function in prd stripes does not lead to ectopic zip expression, indicating that in each case additional inputs are required. DACK is able to elevate zip expression only on the dorsal side of the embryo in regions where zip is normally expressed, indicating that required additional inputs are present; we propose that one such input is Dpp signaling. When DACK is overexpressed in the amnioserosa, either in prd stripes or throughout the whole tissue, its effects on zip expression are non–cell-autonomous, leading to up-regulation of zip expression throughout the dorsal side of the embryo. With regard to the zip expression pattern we see with prd-Gal4-driven DACK transgenes, a diffusible signal emitted from prd stripes in the amnioserosa could attain a fairly uniform distribution over the dorsal side of the embryo. We propose that Dpp secreted from the DME cells cooperates with a diffusible signal from the amnioserosa (regulated by ACK in a kinase-independent manner) to drive coordinated zip expression in these two tissues (Fig. 7). ACK appears to make the larger input into zip expression as DACK overexpression results in a clear elevation in zip levels on the dorsal side of the embryo, but this is not seen with excessive Dpp signaling. Simultaneously activating Dpp signaling and overexpressing DACK with prd-Gal4 is not sufficient to promote ectopic zip expression (for example in the ventral epidermis; W.S., unpublished observations), indicating that other components are required for zip expression, consistent with DACK operating through downstream signaling events.
It is well established that communication between the amnioserosa and the epidermis is critical for embryonic morphogenesis (Glise and Noselli,1997; Lamka and Lipshitz,1999; Reed et al.,2001; Stronach and Perrimon,2001; Conder et al.,2004; Scuderi and Letsou,2005; Fernandez et al.,2007; Wada et al.,2007) and we have identified the zip locus as one target of such crosstalk, with zip transcription in both tissues dependent on signals secreted by both tissues. A diffusible signal from the amnioserosa to the epidermis has been proposed in the regulation of germband retraction by Hindsight, a transcription factor that is member of the U-shaped group of genes expressed in the amnioserosa, and our preliminary results indicate that the U-shaped group is involved in the regulation of zip expression in both the amnioserosa and the epidermis (W.S., unpublished observations; Frank and Rushlow,1996; Lamka and Lipshitz,1999). How could ACK tie into transcriptional regulation of a diffusible signal from the amnioserosa to the epidermis? There is evidence that ACK functions in clathrin-mediated receptor endocytosis in a kinase-independent manner (Hopper et al.,2000; Teo et al.,2001; Yang et al.,2001; Yeow-Fong et al.,2005), and is possible that ACK regulates by receptor endocytosis a pathway in the amnioserosa that leads to a transcriptional response. Our data suggest that the kinase activity of DACK may actually impair its ability to drive zip expression, as KD-DACK promoted higher zip levels than wild-type DACK. Interestingly the binding of mammalian ACK1 to SNX9/SH3PX1, a member of the sorting nexin family of proteins involved in the sorting of proteins in the endosomal pathway, is inhibited by ACK1 kinase activity (Yeow-Fong et al.,2005). The interaction of ACK with a sorting nexin is conserved in flies, where Drosophila SH3PX1 binds to DACK (Worby et al.,2002).
In addition to the transcription of myosin in the DME cells being dependent on input from the amnioserosa, assembly of the actomyosin contractile apparatus in the DME cells requires an expression border for the adhesion molecule Echinoid between the amnioserosa and the epidermis (Laplante and Nilson,2006). The juxtaposition in epithelia of cells expressing Ed with those not expressing Ed triggers actomyosin cable assembly. Ed is expressed in the epidermis but not the amnioserosa and this provides a means, in addition to ACK-mediated signaling, by which the amnioserosa “communicates” with the epidermis in regulating the actomyosin contractile apparatus.
We had previously found that global activation of Cdc42 signaling in tkv mutant embryos could suppress the dorsal closure defects caused by a reduction in Dpp signaling, and our present results indicate a major route of action for this suppression is ACK (Ricos et al.,1999). The activation of Cdc42 throughout the embryo leads to increased expression of DACK specifically in the amnioserosa, and we have shown here that overexpressing DACK in this tissue can suppress tkv dorsal closure defects (Sem et al.,2002). Our results indicate a tissue-specific regulation of DACK levels by Cdc42 that may be part of a sophisticated signaling network enabling the coordinated morphogenesis of tissues in the embryo. DACK does not bind Cdc42 but PR2 does and Cdc42 may also regulate ACK function during dorsal closure through direct interaction with PR2 (Burbelo et al.,1995; Sem et al.,2002). The serine/threonine kinase dPak, an effector for Rac/Cdc42, may also be a component of Cdc42-mediated communication between the amnioserosa and the epidermis during dorsal closure. We previously showed that dPak expression in the amnioserosa is regulated by Cdcd42, but in the opposite direction from DACK in that impairment of Cdc42 signaling leads to elevated dpak transcription in this tissue (Sem et al.,2002). Impairing dPak kinase activity through amnioserosa-specific expression of the dPak autoinhibitory domain leads to defects in head involution and dorsal closure (Conder et al.,2004).
Breadth of ACK Interaction With Dpp Signaling
We see head involution defects and germband retraction failures in ACK-deficient embryos and loss of DACK can suppress the head involution defects and germband retraction failure caused by overexpression of Dpp. These results suggest that Dpp signaling and ACK cooperate in the regulation of these morphogenetic events in addition to dorsal closure. A recent review has highlighted the parallels between dorsal closure and head involution in terms of morphogenetic events and the genes required, with both involving epithelial sheet migration, zip expression and Dpp signaling (VanHook and Letsou,2008). DACK overexpression leads to excessive zip levels in the head and it is likely that ACK and Dpp signaling work together to provide myosin for head involution. That signaling from the amnioserosa is involved in regulating head involution is supported by our earlier finding that impairing dPak function in the amnioserosa causes failures in this process (Conder et al.,2004).
Does ACK impact Dpp signaling other than at the level of zip transcription? Homozygosity for a DACK allele suppresses the ectopic wing vein phenotype caused by excessive Dpp signaling. It is likely that this phenotype is caused by something other than misregulation of zip expression, and ACK could be regulating the expression of a subset of Dpp target genes (other than dad or salm) or may be interacting with the Dpp pathway at another level.
TGF-β family signaling is a central regulator of dorsal closure and other epithelial fusions, but how Dpp controls dorsal closure has not been well-defined. We have shown that regulation of zip expression in cooperation with the Drosophila ACKs constitutes a major route of action of Dpp during dorsal closure. These findings may be relevant to vertebrate wound healing, in which closure of the wound involves both epithelial movement and TGF-β–dependent contraction of connective tissue in the wound (Grose and Martin,1999).
Unless otherwise stated, all crosses were raised at 25°C. w1118 was used as a wild-type control strain. DACK mutations were generated by imprecise excisions of the P element, KG00869, provided by H. Bellen. LE-Gal4 was from S. Noselli, UAS-tkvQ199D (TAJ3) was from M. O'Connor, dpphr27 from K. Wharton, UAS-dpp-GFP from S. Cohen, and PR2pBC02472 from S. Artavanis-Tsakonas. UAS-DACK and UAS-KD-DACK transgenes were described previously (Sem et al.,2002). UAS transgenic lines expressing dsRNA targeting DACK and PR2 were obtained from Ryu Ueda at National Institute of Genetics, Japan. All other fly strains were obtained from the Bloomington Drosophila Stock Center. RNAi lines were tested for their ability to specifically knock down DACK and PR2 transcripts in embryos (data not shown). Crosses involving RNAi transgenes were raised at 29°C to maximize expression of the Gal4 driver. For crosses in which RNAi transgenes were expressed by heat shock using Hs-Gal4, embryos were aged 6 to 12 hr after egg laying and heat shocked in a 37°C water bath for 1 hr.
For the experiments in which tkv7 defects were suppressed by heat shock expression of DACK, embryos were aged 7–15 hr after egg laying and heat shocked in a 37°C water bath for 30 min. After heat-shock, embryos were aged for 7 hr at 21°C and fixed for RNA in situ hybridization or aged 48 hr at 21°C before cuticle preparation. In some experiments, tkv7 mutant embryos were selected by fluorescence dissecting microscope before cuticle preparation based on lack of a twist-GFP balancer chromosome.
Cuticle Preparations, Immunohistochemistry, and RNA In Situ Hybridization
Cuticle preparations, fixing, and antibody staining of embryos have been described (Harden et al.,1996). pMad antibody (1:100) was a gift from P. ten Dijke. Mutant embryos were distinguished by lack of balancer chromosomes bearing GFP reporters. FISH was performed on embryos as described (Lecuyer et al.,2007).
We thank Esther Verheyen for comments on the manuscript; Dan Jover for technical assistance; S. Artavanis-Tsakonas, H. Bellen, S. Cohen, S. Noselli, M. O'Connor, R. Ueda, and K. Wharton for fly strains; and P. ten Dijke for anti-pMad antibody. N.H. received funding from the Canadian Institutes of Health Research. W.S. was supported by a scholarship from the Michael Smith Foundation for Health Research.
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