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Regulation of JNK by Src During Drosophila Development

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Science  14 Jan 2000:
Vol. 287, Issue 5451, pp. 324-327
DOI: 10.1126/science.287.5451.324

Abstract

In Drosophila, the Jun amino-terminal kinase (JNK) homolog Basket (Bsk) is required for epidermal closure. Mutants for Src42A, a Drosophila c-srcprotooncogene homolog, are described. Src42A functions in epidermal closure during both embryogenesis and metamorphosis. The severity of the epidermal closure defect in the Src42A mutant depended on the amount of Bsk activity, and the amount of Bsk activity depended on the amount of Src42A. Thus, activation of the Bsk pathway is required downstream of Src42A in epidermal closure. This work confirms mammalian studies that demonstrated a physiological link between Src and JNK.

Genes that regulate cell shape changes in Drosophila are required for dorsal closure of the embryonic epidermis and thorax closure of the pupal epidermis (1). Mutations in genes such as hemipterous(hep) and basket (bsk, also known asDJNK) result in abnormal embryos with a dorsal hole or abnormal adults with a dorsal midline cleft (1,2). Hep and Bsk are homologous to the mammalian MKK7 (MAPK kinase 7) and JNK, and they are components of a MAPK (mitogen-activated protein kinase) cascade (3). Although the role of the Hep-Bsk cascade during dorsal closure has been extensively studied, the upstream trigger of this cascade is poorly understood. To identify the trigger, we screened mutants showing the dorsal midline cleft phenotype, like a mild hep mutant (Fig. 1Ab). We found that the mutant forSrc42A showed this phenotype and that Src42A regulates Bsk during Drosophila development.

Figure 1

Adult and embryonic phenotypes of a zygotic mutant of Src42A and genetic interaction with mutants for other Src-family kinases and Hep-Bsk pathway. (A) Dorsal midline cleft in hep and Src42A mutants. (a) Wild-type (Canton S). (b) hep1 /Y (1). (c to e) Src42AJp45 adults exhibited the cleft with variable severity. (c) Class I, nearly normal. (d) Class II, midline devoid of bristles. (e) Class III, cleft notum. (B) Severity of dorsal midline cleft inSrc42A mutant varied depending on Bsk activity. Classes I to III correspond to clefts shown in (Ac) to (Ae), respectively. Triangle on right represents the strength of the Bsk signal. (C) Synergism of Src42A and Tec29 in dorsal closure and the phenotypic rescue by activated DJun. (a) Wild-type. (b)bsk2 . Arrowheads in (b), (e), and (f) mark the posterior edge of the dorsal hole in the cuticles. Percentage of embyros in which the posterior edge of the dorsal hole extended posterior to the 50% length of the embryo is indicated. (c) ASrc42Amyri homozygote. A similar mouth part defect was observed in Src42AE1 . (d)Tec29206 /Tec295610 trans-heterozygote. (e) Tec29206Src42Amyri /Tec295610Src42Amyri . (f) Tec29206Src42AmyrisevE(hs)-DJunAct /Tec295610Src42Amyri . A larger proportion of the embryos of this genotype had a small anterior hole, as shown. (g)Src42Amyri ;Src64P1 .

From our mutant collection of the P-element–inserted semilethal lines, we identified one line, Jp45, that survived to adulthood but showed various degrees of the dorsal midline cleft phenotype (Fig. 1A, c to e). Excision of the P-element eliminated the semilethality and restored the cleft phenotype. The P-element was inserted in the 5′ untranslated region (UTR) of the Src42Agene, which encodes a Src-family nonreceptor tyrosine kinase (4–6). We used ethyl methanesulfonate (EMS) mutant screening to isolate two strong alleles of Src42A,Src42AE1 andSrc42Amyristylation (myri) (7). InSrc42AE1 , a stop codon at codon 483 eliminated the COOH-terminal part of the kinase domain of Src42A (8,9). Src42Amyri has a point mutation in codon 2, which causes an amino acid substitution from Gly2to Asp. Gly2 is conserved in all members of the Src family and must be myristylated for localization of Src to the cellular membrane in mammals (10). About 50% of theSrc42Amyri homozygotes died before they hatched (Table 1), and most of the remainder died during the first-instar larval stage. Therefore, Gly2 is required for development.

Table 1

Synergism of Src42A and Tec29found in embryonic lethality.

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Because adult Src42AJp45 phenotypes resembled that of hep, we suspected that Src42A was involved in Hep and Bsk function. A mutation in hep or bskdominantly enhanced the lethality and the phenotypic severity ofSrc42AJp45 homozygotes (Table 2 and Fig. 1B). Conversely, reducing the gene dosage of puckered (puc), a gene encoding a phosphatase that inactivates Bsk (11), suppressed the lethality and the severity of the cleft phenotype ofSrc42AJp45 . Thus, Src42A may function in the Bsk pathway during metamorphosis.

Table 2

Effect of Tec29, hep,bsk, and puc mutations on Src42Amutant lethality. All crosses were performed at 18°C because the viability of Src42AJp45 was reduced at 25°C, the standard temperature.

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Dorsal closure is the process in which a pair of epidermal layers elongates dorsally and fuses at the dorsal midline of the embryo (1) (Fig. 1Ca). This process is not completed inhep and bsk mutants, yielding a dorsal open phenotype (Fig. 1Cb). Strong Src42A mutants did not show the dorsal open phenotype but displayed the malformed mouth parts (Fig. 1Cc). This defect is similar to the defect in the embryo of theTec29 mutant (Fig. 1Cd) (12). Tec29 is a Src-related nonreceptor tyrosine kinase and is regulated by SRC64, another Drosophila Src homolog, during oogenesis (13). Thus, Tec29 may be involved in the function of Src42A. A mutation in Tec29 dominantly enhanced the lethality ofSrc42AJp45 (Table 2).

Furthermore, the Tec29 Src42A double mutant showed complete embryonic lethality (Table 2), and a certain part of the dead embryos showed the dorsal open phenotype (Fig. 1Ce) (2). Activated DJun, a transcription factor downstream of Bsk (14, 15), partially rescued the dorsal open phenotype in the Tec29 Src42A double mutant (Fig. 1Cf). Thus, Src42A appears to regulate Bsk in the fusion of epithelial sheets during embryogenesis and metamorphosis, and Tec29 is involved in this regulation. We further observed the double mutant forSrc64 and Src42A. It manifested a mild but clear dorsal open phenotype (Fig. 1Cg), which suggests a functional redundancy between Src64 and Src42A.

Expression of puc is known to be induced by the Bsk signal (2, 11). In the wing disc of the wild-type third-instar larva, puc is expressed in the dorsal midline of the adult notum (Fig. 2Aa) (16). In the wing disc of theSrc42AJp45 mutant, puc expression was reduced (Fig. 2Ab). In contrast, larvae with a constitutively activated form of Src42A (Src42ACA) (17) showed ectopic expression of puc (18, 19) (Fig. 2Ac). Further, introduction of a hep null mutation reduced the amount of ectopic puc expression (Fig. 2Ad). It is known that Bsk induces expression of puc anddecapentaplegic (dpp) during embryonic dorsal closure (Fig. 2B, a and c) (2, 11,14, 15). The embryos of the Tec29 Src42Adouble mutant did not show any puc or dppexpression in the leading edge cells (Fig. 2B, b and d) (20). These results indicate that Src42A, Tec29, Hep, and Bsk regulate dpp and puc expression during embryonic dorsal closure.

Figure 2

Regulation of the Bsk pathway and cell shape change by Src42A. (A) puc expression in wing discs depended on the strength of the Src42A signal. (a) Control. Normal puc expression (arrow). (b)Src42AJp45 . (c)UAS-Src42ACA /+; 71B-GAL4/+. Ectopic puc expression (arrowhead) (20). (d)hepr75 /Y;UAS-Src42ACA /+; 71B-GAL4/+. (e) Region where 71B-GAL4 (19) is expressed is visualized by UAS-lacZ. (B) Levels ofpuc and dpp expression depended on Src42A and Tec29 during dorsal closure. Expression of puc (a, b) (dorsal view) and dpp (c, d) (lateral view) in stage 14 embryos is shown. (a and c) Control. (b and d)Tec29206 Src42Amyri /Tec295610 Src42Amyri . Leading edge cells (arrows). puc and dppexpression was detected by X-gal staining of thepuc-lacZ reporter (pucE69 ) and in situ hybridization todpp mRNA, respectively. (C) Phosphorylation of Bsk by the Src42A signal. Antibodies to JNK1 and P-JNK crossreact with total Bsk protein (2) and phosphorylated Bsk protein (21), respectively. Lanes: 1 to 3, controls; 4, in the presence of the Src42ACA protein. (D) Cell shape and F-actin (a and b) and P-Tyr (c and d) accumulations in the leading edge cells (arrowheads) of the lateral epidermis of stage 14 embryos. Cell shape was observed by phalloidin-FITC staining and P-Tyr antibody staining. (a and c) Control. Leading edge cells elongated dorsally, and F-actin and P-Tyr accumulations were seen in the cellular membranes. (b and d) Tec29206Src42Amyri / Tec295610Src42Amyri .

To investigate the ability of Src42ACA to activate Bsk, we directly assessed the amount of phosphorylated Bsk by immunoblot analysis (21). Forced expression of Src42ACA did not affect the quantity of total Bsk protein (Fig. 2C, upper lanes) but induced more phosphorylated Bsk (Fig. 2C, lane 4) than the controls. Thus, Src42A appears to regulate the phosphorylation level of Bsk.

During embryonic dorsal closure, the Hep-Bsk signal is required for elongation of the leading edge cells (1). In the absence of the Bsk signal, these cells do not fully elongate (2). The accumulation of F-actin and phosphotyrosine (P-Tyr) in leading edge cells is associated with the elongation of these cells (Fig. 2D, a and c). Accumulation of these substances is disturbed in theDJun and the puc mutants (11,15). In the double mutant for Tec29 andSrc42A, the leading edge cells contained reduced quantities of F-actin and P-Tyr, and these cells were only partially elongated (Fig. 2D, b and d). Thus, the defect in embryonic dorsal closure in theTec29 Src42A double mutant is caused by this failure in cell shape change, as is the case in the DJun mutant.

We propose a model in which Src42A, upon receiving an unidentified signal, activates the Hep-Bsk pathway to regulate cell shape change and epidermal layer movement. This is consistent with the observation in mammals that c-Src regulates the cell morphogenetic and migratory processes and is known to activate JNK (22). As inDrosophila, c-Src definitely affects F-actin organization and P-Tyr localization during cell morphogenesis (23). Therefore, Src regulation of JNK activity toward a change in cell shape may be conserved.

It can be also interpreted that Src42A acts upstream of DFos (24), a dimerization partner of DJun. Although theSrc42A, Tec29, and Src64 single mutants do not show a dorsal open phenotype (12,25, 26), the DFos mutant clearly exhibits it. This relationship is also analogous to that in mammals. Both c-src and c-fos knockout mice had a similar defect, osteopetrosis caused by reduced osteoclast function. But the phenotypic severity was milder in c-src than in c-fos knockouts (27), which can be explained by the functional overlap in multiple Src-family tyrosine kinases (28). Accordingly, in both Drosophila and mammals, multiple nonreceptor tyrosine kinases may cooperate to regulate the function of the Jun/Fos complex.

  • * To whom correspondence should be addressed. E-mail: adachi{at}bio.nagoya-u.ac.jp

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