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Cell Biology International (2010) 34, 1113–1118 (Printed in Great Britain)
Rab11 regulates JNK and Raf/MAPK-ERK signalling pathways during Drosophila wing development
Tanmay Bhuin and Jagat K Roy1
Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221 005, India


Developmental signalling pathways are regulated by intracellular vesicle trafficking in multicellular organisms. In our earlier communication, we have shown that mutation in Rab11 (a subfamily of the Ypt/Rab gene family) results in the activation of JNK signalling pathways in Drosophila eye. Here, we report that Rab11 regulates JNK and Raf/MAPK-ERK signalling pathways during Drosophila wing development. Using immunofluorescence and immunohistochemical analyses, we show that overexpression of Rab11 in mutant wing imaginal disc cells triggers the induction of apoptosis and activation of JNK and ERK. Further, using a genetic approach it has been shown that Rab11 interacts with the components of these pathways during Drosophila wing development. In addition to this, in Rab11 mutant wing imaginal discs JNK activity was monitored using pucE69, a P-lacZ enhancer-trap line inserted in puckered (puc). A strong induction of puc in Rab11 mutant wing imaginal disc cells provided a strong support that Rab11 regulates the JNK signalling pathway during Drosophila wing development.


Key words: activation, βPS integrin, Drosophila, JNK, mutation, Rab11, Raf/MAPK-ERK, vesicle trafficking

Abbreviations: DAB, 3,3′-diaminobenzidine, EGFR, epidermal growth factor receptor, MAPK, mitogen activated protein kinase, RE, recycling endosome

1To whom correspondence should be addressed (email jkroy@bhu.ac.in).


1. Introduction

During development of multicellular organisms, growth of tissues and organs is accompanied by a careful co-ordination in the rates of cell proliferation, cell death and cell differentiation (Neufeld et al., 1998; Conlon and Raff, 1999). A general principle, during development of tissues and organs, involves an initial generation of excess cells prior to the cell fate determination, after which surplus cells are eliminated by an evolutionary conserved mechanism of physiological cell death or cell suicide known as apoptosis. After completion of a multicellular organism development, survival of the organism mainly depends upon the maintenance and renewal of these cell types. Various extracellular and intracellular signals regulate apoptosis. A constant supply of growth factors from the extracellular matrix and neighbouring cells has been implicated for cell survival in most of the tissues (Barres et al., 1992; Raff, 1992; Raff et al., 1993).

The JNKs, evolutionarily conserved protein kinases, belong to the subfamily of MAPKs (mitogen activated protein kinases), which are also known as SAPKs (stress activated protein kinases). The JNK pathway is evolutionarily conserved (Noselli, 1998), and this pathway is well known as a regulator of stress defence, cell and tissue morphogenesis and immune cell function, proliferation, differentiation and apoptosis (Stronach and Perrimon, 1999; Davis, 2000; Kockel et al., 2001; Xia and Karin, 2004). Genetically amenable organism Drosophila melanogaster has a single JNK encoding gene, basket (bsk), which regulates several developmental processes viz., cell morphogenesis, planner cell polarity, dorsal closure, thorax closure, wound healing (Martín-Blanco et al., 1998; Zeitlinger and Bohmann, 1999; Suzanne et al., 2001; Rämet et al., 2002) and apoptosis (Adachi-Yamada et al., 1999). The JNK pathway acts as an effector of apoptotic cell death during imaginal disc development in Drosophila (Adachi-Yamada et al., 1999; Moreno et al., 2002).

Mammalian cell line studies have implicated the Ras/Raf/MAPK-ERK pathway in growth factor mediated survival signalling (Xia et al., 1995). In the Raf pathway, Ras-GTP activates a cascade of kinases: Raf (MAPKKK), MEK (MAPKK) and ERK (MAPK), constituting the Ras/Raf/MAPK effector pathway (Xia et al., 1995; Gardner and Johnson, 1996). The Drosophila homologue of MAPK is encoded by the gene rolled (rl) (Biggs et al., 1994). The role of Ras/MAPK signalling in regulating cell proliferation and cell differentiation has been well established genetically in Drosophila (Wassarman and Therrien, 1997). The Raf-MEK-ERK signalling pathway regulates cell fate specification, proliferation and cell survival downstream of EGFR (epidermal growth factor receptor) in the imaginal discs of Drosophila (Diaz-Benjumea and Hafen, 1994; Prober and Edgar, 2000). However, a direct mechanistic link between the Raf/MAPK-ERK survival pathway and the cell death machinery has not been demonstrated so far.

Trafficking of activated EGFR through endosomes controls the spatial and temporal regulation of MAPK signalling (Taub et al., 2007). Rab11, a subfamily of Rab GTPases, is a well-known marker for REs (recycling endosomes) and plays a key role in regulating the transport of vesicles/proteins from REs and EEs (early endosomes) to the TGN (trans-Golgi network) and plasma membrane through recycling pathways (Ullrich et al., 1996; Ren et al., 1998; Wilcke et al., 2000). Developmental signalling pathways are regulated by the endocytic and exocytic trafficking of receptors and their ligands (Gonzalez-Gaitan 2003; Piddini and Vincent 2003).

Earlier, we have shown that mutation in Rab11 results in the activation of JNK signalling pathways in Drosophila eye (Tiwari and Roy, 2009). In the present study, we show that Rab11 mutation induces apoptosis in wing imaginal disc cells and trigger the activation of JNK and ERK. Further, it is shown that Rab11 genetically interacts with the components of these pathways during Drosophila wing development. Using pucE69, a P-lacZ enhancer-trap line inserted in puckered (puc), we also show modulation in JNK activity in Rab11 mutant wing imaginal disc cells providing a strong support in Rab11 regulation with the JNK signalling pathway during Drosophila wing development.

2. Materials and methods

2.1. Fly stocks and genetics

The following stocks were used: UAS-Rab11N124I (Satoh et al., 2005; gift from D. Ready), UAS-Rab11QL (Emery et al., 2005; a gift of M. Gonzalez-Gaitan), UAS-RafDN24.1 (Martín-Blanco et al., 1999; a gift of Martín-Blanco), UAS-rlSem (gain-of-function allele of ERK, a gift of D. Bohmann), UAS-bskDN/TM6B (Adachi-Yamada et al., 1999; a gift of Adachi-Yamada), pucE69 (Ring and Martinez-Arias, 1993; a gift of Martinez-Arias). Other stocks were from the Bloomington Stock Centre.

2.2. Immunohistochemistry and microscopy

Wing imaginal discs from third-instar larvae were dissected in PBS and fixed in 4% paraformaldehyde/PBS for 20 min and antibody staining was performed by standard procedures. The primary antibodies were used at the following concentrations: rabbit polyclonal cleaved caspase-3,1:500 (Sigma); rabbit polyclonal anti-phosphospecific-JNK antibody, 1:100 (Promega); mouse anti-ERK antibody, 1:100 (Promega); and rabbit anti-β-galactosidase, 1:5000 (Molecular Probes). Secondary antibodies used were as follows: Alexa-488-coupled secondary antibodies (Molecular Probes, 1:200), biotinylated goat anti-rabbit IgG (Jackson Immunoresearch, 1:300), alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma, 1:1000). Biotinylated secondary antibodies were used in combination with Vector Elite ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. For improved signal intensity and colour contrast, the staining was developed by using 0.5 mg/ml DAB (3,3′-diaminobenzidine) and 0.02% hydrogen peroxide; 8% nickel chloride was used for further enhancement of regular DAB procedure. Fluorescent samples were mounted in 70% glycerol in PBS supplemented with 15% DABCO (1,4-diazabicyclo[2.2.2]octane) and images were taken on a BioRad MRC laser scanning confocal microscope while HRP and alkaline phosphatase stained samples were mounted in 70% glycerol in PBS, then examined and photographed in bright-field optics. Captured images were processed using BioRad software and/or Adobe Photoshop.

2.3. Wing preparations

Wings of adult flies and from dead pupae were dissected and mounted in 70% ethanol, and then photographed using a phase contrast microscope.

3. Results and discussion

3.1. Overexpression of Rab11 induces apoptosis along with activation of the JNK and ERK in Drosophila wing imaginal discs

In our unpublished observation (Bhuin and Roy), it has been shown that Rab11 mediates transcytic and exocytic trafficking of βPS integrin and it was noted that alteration of Rab11 activity results in mislocalization/abnormal accumulation of βPS integrin in cytoplasm instead of being transported to its proper adhesion site. The phenotypic consequence of expressing dominant-negative (Rab11N124I) or constitutively active (Rab11QL) or Rab11-dsRNA in wing pouch using MS1096-GAL4 was blisters in adult wings with a reduction in wing size. The presence of blisters suggests a loss of integrin function, while reduced wing size was due to apoptosis as detected by anti-cleaved caspase 3 staining (Figure 1b–c) as compared with wild type (1a). Thus, we examined whether the induction in apoptosis can trigger the activation of JNK and ERK in mutant Rab11 wing imaginal disc cells. As expected, JNK phosphorylation was dramatically increased in the wing imaginal discs expressing dominant-negative or constitutively active Rab11 (Figure 2b–c) as compared with the wild type (Figure 2a), which directly demonstrated an increase in JNK activity by altered Rab11 activity. Similarly, as expected, ERK activation was also detected throughout the whole wing pouch (Figure 2e–f) as compared with the wild type, where ERK expression is detected in wing margin and wing vein (Figure 2d). The activation of JNK and ERK in Rab11 mutant wing imaginal disc cells indicates that Rab11 might regulate JNK and ERK signalling pathways during wing development in Drosophila.

3.2. Rab11 genetically interacts with the candidates of JNK and Raf/MAPK-ERK signalling pathways

To confirm the histochemical and immunofluorescence data of activation of JNK and ERK in overexpressed mutant Rab11 wing imaginal discs, a genetic interaction study of Rab11 with the candidates of JNK and Raf/MAPK-ERK pathways was carried out during wing development. When constitutively active or dominant-negative Rab11 alleles were driven by MS1096-GAL4, pupal lethality ranged from 11% to 12%; whereas after driving a dominant-negative allele of DJNK, UAS-bskDN or a dominant-negative allele of Raf, UAS-RafDN or a gain-of-function allele of Drosophila ERK, UAS-rlSem by MS1096-GAL4, pupal lethality ranged between 2% to 3%. But when dominant-negative or constitutively active Rab11 mutation was brought with a candidate of JNK and Raf/ERK mutant, pupal lethality was greatly enhanced. Rab11 mutation in bsk mutant background showed 100% pupal lethality; in Raf, mutant background it showed pupal lethality ranging from 75% to 76%, and in ERK mutant background the pupal lethality ranged from 88% to 89% (Figure 3). The enhancement of pupal lethality in double mutant backgrounds indicated that Rab11 genetically interacts with the candidates of JNK and Raf/ERK signalling pathways during wing development. Further, the wing morphology of dissected lethal pupae and the emerged flies from double mutants of Rab11 and bsk, Raf or ERK showed reduced wing size (Figure 4d, e, g, h, j and k) as compared with Rab11 (Figure 4a, b) or bsk (Figure 4c) or Raf (Figure 4f) or ERK (Figure 4i) mutant alone. To check whether the 100% pupal lethality in the Rab11 and bsk double mutant and reduction of wing size in Rab11 and Raf or Rab11 and ERK double mutants were due to enhanced apoptosis, anti-cleaved caspase 3 staining was carried out in double mutants. All the double mutant combinations showed enhanced number of apoptotic cells (Figure 5d, e, g, h, j and k) as compared with no apoptotic cells detected in mutants of bsk and Raf/ERK (Figure 5c, f and i) and relatively fewer apoptotic cells in Rab11 mutant (Figure 5a, b). Therefore, an aggravation of Rab11-induced apoptosis in bsk and Raf/ERK mutant backgrounds further suggest that the apoptosis might be a result of the activation of JNK and ERK signalling pathways.

As the induction of apoptosis mediated by JNK signalling pathway is well established in the wing of Drosophila (Adachi-Yamada et al., 1999; Goberdhan and Wilson, 1988), and the Ras-Raf-MEK-ERK signalling pathway is known to regulate cell fate specification, proliferation and cell survival downstream of EGFR in the imaginal discs of Drosophila (Diaz-Benjumea and Hafen, 1994; Prober and Edgar, 2000), the enhanced apoptotic cells as well as enhanced lethality detected in double mutants indicate that Rab11 is involved in the regulation of JNK and Raf/MAPK-ERK signalling pathways during wing development in Drosophila. Moreover, the overexpression of mutant Rab11 inducing the activation of JNK and ERK suggests that Rab11 regulates apoptosis via regulation of the JNK pathway and Raf/ERK signalling pathway which is downstream of EGFR rather than via direct regulation of the transcription of apoptotic factors. Our results show that alterations of Rab11 function through these different genetic procedures exhibit the same range of phenotypes. Therefore, it has been suggested that an optimum function of Rab11 is essential for a normal wing to develop, and any up-regulation or down-regulation of Rab11 function disturbs the balance, resulting in anomalies in wing development, thus interfering with different signalling pathways.

Further, activity of the JNK pathway was monitored in the wing discs by transcriptional activation of puckered (puc), which encodes a JNK phosphatase and negatively regulates the activity of the JNK pathway (Martín-Blanco et al., 1998). The transcriptional activity of a puc-lacZ reporter gene is very low or absent in wing imaginal disc, but when apoptotic stimuli are received, puc-lacZ is induced in a JNK-dependent manner (Adachi-Yamada et al., 1999). Therefore, in Rab11 mutant wing imaginal discs, JNK activity was monitored using pucE69, a P-lacZ enhancer-trap line inserted in puckered (puc) (Ring and Martinez-Arias, 1993). In the wing pouch where mutant Rab11 was driven by MS1096-GAL4, a strong induction of puc expression was seen (Figure 6b, c) as compared with the control, where mutant Rab11 has not been driven (Figure 6a). This result once again supports the notion that Rab11 is linked to the JNK signalling pathway during Drosophila wing development.

Several independent pathways are involved which activate JNK, and it has been suggested that induction of apoptosis mediated by JNK activation is a controlling mechanism which eliminates excess/abnormal cells during wing development in Drosophila (Adachi-Yamada and O'Connor, 2004). Although the other possibilities governing the activation of JNK mediating apoptosis cannot be ruled out, Rab11-mediated JNK-dependent apoptosis might be one of the ways to eliminate abnormal cells during wing development. Therefore, it is concluded that Rab11 regulates JNK and Raf/MAPK-ERK signalling pathways during Drosophila wing development.

Author Contribution

Planning, execution of work and manuscript preparation were done by Tanmay Bhuin under the guidance of Jagat Roy.

Acknowledgements

We thank M. Gonzales-Gaitan, D. Ready, Martín-Blanco, D. Bohmann, Adachi-Yamada, Martinez-Arias and the Bloomington Stock Centre for the fly stocks. The use of the national scanning confocal microscopy facility from DST is also gratefully acknowledged.

Funding

This work was supported by grants from DST to JKR and SRF from ICMR, New Delhi, to TB.

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Received 11 March 2010/15 June 2010; accepted 19 July 2010

Published as Cell Biology International Immediate Publication 19 July 2010, doi:10.1042/CBI20100155


© The Author(s) Journal compilation © 2010 Portland Press Limited


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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