Downregulation of Jun kinase signaling in the ... - Semantic Scholar

1098 Research Paper

Downregulation of Jun kinase signaling in the amnioserosa is essential for dorsal closure of the Drosophila embryo Bruce H. Reed*, Ronit Wilk*† and Howard D. Lipshitz*† Background: During Drosophila embryogenesis, Jun kinase (JNK) signaling has been shown to play a key role in regulating the morphogenetic process of dorsal closure, which also serves as a model for epithelial sheet fusion during wound repair. During dorsal closure the JNK signaling cascade in the dorsal-most (leading edge) cells of the epidermis activates the AP-1 transcription factor comprised of DJUN and DFOS that, in turn, upregulates the expression of the dpp gene. DPP is a secreted morphogen that signals lateral epidermal cells to elongate along the dorsoventral axis. The leading edge cells contact the peripheral cells of a monolayer extraembryonic epithelium, the amnioserosa, which lies on the dorsal side of the embryo. Focal complexes are present at the dorsal-most membrane of the leading edge cells, where they contact the amnioserosa.

Addresses: * Program in Developmental Biology, Research Institute, The Hospital for Sick Children and † Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1X8, Canada.

Results: We show that the JNK signaling cascade is initially active in both the amnioserosa and the leading edge of the epidermis. JNK signaling is downregulated in the amnioserosa, but not in the leading edge, prior to dorsal closure. The subcellular localization of DFOS and DJUN is responsive to JNK signaling in the amnioserosa: JNK activation results in nuclear localization of DFOS and DJUN; the downregulation of JNK signaling results in the relocalization of DFOS and DJUN to the cytoplasm. The HINDSIGHT (HNT) Zn-finger protein and the PUCKERED (PUC) JNK phosphatase are essential for downregulation of the JNK cascade in the amnioserosa. Persistent JNK activity in the amnioserosa leads to defective focal complexes in the adjacent leading edge cells and to the failure of dorsal closure.

Current Biology 2001, 11:1098–1108

Correspondence: Dr. Howard D. Lipshitz E-mail: [email protected] Received: 30 April 2001 Revised: 7 June 2001 Accepted: 7 June 2001 Published: 24 July 2001

0960-9822/01/$ – see front matter  2001 Elsevier Science Ltd. All rights reserved.

Conclusions: Focal complexes are assembled at the boundary between high and low JNK activity. In the absence of focal complexes, miscommunication between the amnioserosa and the leading edge may lead to a premature “stop” signal that halts dorsalward migration of the leading edge. Spatial and temporal regulation of the JNK signaling cascade may be a general mechanism that controls tissue remodeling during morphogenesis and wound healing.

Background One of the most striking morphogenetic events during Drosophila embryogenesis is dorsal closure, a process by which two sheets of epidermal cells, one on each side of the extraembryonic amnioserosa, stretch, meet, and fuse at the dorsal midline. The majority of dorsal closure studies have focused on the dorsal-most cells of the lateral epidermis. These cells, collectively known as the leading edge, abut the amnioserosa and form an orderly row of columnar cells. As dorsal closure initiates, the leading edge cells are the first to elongate along the dorsoventral axis of the embryo, after which the lateral epidermal cells also elongate. During dorsal closure, the dorsal-most membrane of the leading edge conspicuously accumulates F-actin, non-

muscle myosin, and tyrosine-phosphorylated proteins that are thought to be components of a RAC-dependent focal complex [1–6]. In this study, we use the term “focal complex” in reference to regions of the plasma membrane that are enriched for proteins associated with the transduction of extracellular signals into dynamic changes in the actin cytoskeleton. Focal complexes are induced by CDC42 and RAC and are morphologically distinct from focal adhesions that are induced by RHO [7]. Focal complexes and their associated cellular projections have been implicated as sites of cell-cell or cell-extracellular matrix communication. In mammalian fibroblasts, for example, focal complexes are found in association with cellular projections called filopodia and lamellipodia [7]. During dorsal closure, the leading edge extends filopodia and lamellipodia that contact the amnioserosa as well as each other [8, 9]. These structures are thought to generate contractile

Research Paper JNK signaling and Drosophila dorsal closure Reed et al. 1099

Table 1 Analysis of cuticle preparations of weak hnt alleles with respect to germ band retraction (GBR) and dorsal closure (DC).

GBR defecta DC defectb Wild-type

hnt308 (n ⫽ 128)

hnt704a (n ⫽ 278)

hntXO01 (n ⫽ 213)

hnt308; 3x dpp⫹ (n ⫽ 172)

19.6% 65.6% 14.8%

65.8% 20.5% 13.7%

84.4% 12.3% 3.3%

40.5% 50.2% 9.3%

The data are from crosses to Oregon R, with the exception of hnt308; 3x dpp⫹, for which the cross was to Dp(2;2)DTD48/CyO, P{w ⫹ mC ⫽ dpp ⫺ P23}RP1. The proportions of lethal embryos displaying germ band retraction or dorsal closure defects are expressed as percentages of the total number of cuticle preparations examined (n).

force and to act as sensors of positional information during cell migration and the “zippering up” of the epidermis at the end of dorsal closure [8, 9]. Dorsal closure has become a model system for the study of Jun kinase (JNK) signaling pathways and their involvement in regulating morphogenesis and wound repair [8, 10–13]. The accumulation of F-actin, nonmuscle myosin, and tyrosine-phosphorylated proteins in the leading edge is disrupted by the inactivation of JNK signaling, consistent with RAC-dependent JNK signaling playing a crucial role in the assembly or maintenance of these focal complexes [1–3, 5]. Two known target genes that are activated in response to JNK signaling in the leading edge cells during dorsal closure are decapentaplegic (dpp), which encodes a secreted morphogen belonging to the TGF-␤ family, and puckered (puc), which encodes a JNK phosphatase that acts as a component of a negative feedback loop to limit JNK signaling [10–16]. Throughout most of dorsal closure, the lateral epidermis and the amnioserosa are in intimate contact. As the lateral epidermis extends dorsally, it maintains contacts with the peripheral cells of the amnioserosa: the dorsal-most one or two rows of cells of the lateral epidermis overlie the most peripheral one or two rows of cells of the amnioserosa [17]. Several lines of evidence, in addition to the discovery of contacts between the leading edge filopodia and the amnioserosa [8], suggest that the amnioserosa is not passive during dorsal closure and that there is communication between the leading edge and the amnioserosa. Kiehart et al. [17] used live imaging and laser ablation to show that the amnioserosa is under tension during dorsal closure. Their data suggest that both the amnioserosa and the leading edge contribute to dorsal closure. The involvement of the amnioserosa in this process can also be inferred from observations of mutations in the segmental patterning gene armadillo (arm), which encodes the Drosophila homolog of ␤-catenin. Strong hypomorphic or null alleles of armadillo are defective in dorsal closure; the defects include the separation or ripping of the amnioserosa from the leading edge [18]. Where separation occurs,

a The GBR class includes embryos that were defective in GBR and DC. b The DC class includes only DC-defective cuticles, ranging from a small dorsal hole to cuticles that were devoid of any dorsal cuticle (dorsal open phenotype).

the leading edge cells remain cuboidal, but elongation proceeds where contact with the amnioserosa is maintained [18], suggesting an induction of leading edge elongation or differentiation through contact with the amnioserosa. We previously reported that expression of hindsight (hnt) in the amnioserosa, particularly in those cells that contact the lateral epidermis, is required for germ band retraction, and we presented evidence suggesting communication between the amnioserosa and the lateral epidermis [19, 20]. Here, we demonstrate that hnt is also required for dorsal closure. Using genetic and molecular assays, we show that HNT and PUC downregulate JNK activity in the amnioserosa. We show that the localization of DJUN and DFOS in the nuclei of amnioserosal cells requires active JNK signaling. Without active signaling, DJUN and DFOS become cytoplasmic. If JNK activity is not downregulated in the amnioserosa, as in hnt or puc mutants, DFOS and DJUN remain nuclear. Furthermore, in these mutants, focal complexes fail to accumulate in the leading edge epidermal cells that abut the amnioserosa. We present evidence suggesting that focal complexes form at the boundary between the leading edge and the amnioserosa only if this is also a high/low JNK signaling boundary. In the absence of these focal complexes, signals necessary for dorsal closure either cannot be transduced or are transduced inappropriately.

Results hnt mutants fail in embryonic dorsal closure

We previously studied the genetic control of germ band retraction and showed that expression of the HNT Znfinger transcription factor in the amnioserosa, particularly in those cells that abut the leading edge of the epidermis, is essential for this morphogenetic process [19, 20]. Here, we show, through analyses of hypomorphic hnt alleles, that HNT function is necessary for dorsal closure. A subset of hnt mutant embryos carrying the embryonic lethal alleles hnt704a and hntXO01 successfully complete germ band retraction but do not hatch [19]. Analyses of cuticle preparations revealed that 60% of hnt704a and 79% of hntXO01

1100 Current Biology Vol 11 No 14

Figure 1 The hnt308 mutation results in reduced HNT expression and causes the failure of dorsal closure. (a,b) Cuticle preparations of hnt308 mutants showing the (a) dorsal-hole or (b) dorsal-open phenotype. (c,d) Stage 10, (e,f) stage 12, and (g,h) stage 13 embryos collected from a hnt308/FM7Z stock immunostained with anti-HNT (purple) and anti-␤-galactosidase (brown). (c,e,g) hnt308/ FM7Z embryos (FM7Z-carrying embryos express ␤-galactosidase) show strong HNT expression in the amnioserosa. (d,f,h) hnt308/Y embryos, identified by the lack of anti-␤galactosidase staining, have reduced antiHNT staining (the amnioserosa is indicated with arrows). In general, control sibs were overstained by the time HNT staining was detected in mutants; the control embryos pictured here were removed from the developing solution prior to mutants, and the actual differences in HNT expression are therefore greater than presented.

embryos that complete retraction exhibit an anterior-open or dorsal-hole phenotype characteristic of the failure of dorsal closure (Table 1). A role for HNT in directing dorsal closure was confirmed through analysis of a P elementinduced allele, hnt308, in which there is 41% embryonic lethality. Of the dead embryos, 80% complete germ band retraction; of these, 82% exhibit the anterior-open and/or dorsal-hole phenotype (Table 1; Figure 1a,b). Both hnt704a and hntXO01 have previously been shown to be antibody null alleles, with the HNTXO01 protein truncated after the third of 14 C2H2-type Zn-fingers [19]. We mapped the P element insertion site in hnt308 to 509 bp upstream of the 5⬘-end of the NB701 hnt cDNA (for information on NB701, see [19]). Stage 10 hnt308 embryos, which are fully germ band extended, showed HNT expression in the amnioserosa, albeit at lower levels than wild-type (compare Figure 1d with 1c). We infer that this early amnioserosal expression must be sufficient for germ band retraction. Stage 12 and 13 hnt308 mutant embryos, undergoing or having completed germ band retraction, respectively, showed a general reduction in HNT protein levels when compared with wild-type siblings (compare

Figure 1f,h with 1e,g). The dorsal closure defect in hnt308, thus, may be caused by the reduction in HNT protein levels at and after stage 12. hnt and JNK signaling pathway mutants interact genetically

Several studies have shown that the JNK signaling pathway plays a key role in the regulation of dorsal closure [10–15]. We therefore tested for genetic interactions between hnt and JNK pathway mutants. As most of the embryonic lethality in hnt308 mutants is a consequence of dorsal closure failure, we tested for interactions by comparing embryonic lethality in controls versus genetic backgrounds in which the gene dose of JNK pathway components was altered. hnt308 single mutants exhibited 41% embryonic lethality (Table 2). When the dose of the JNK-encoding gene, basket (bsk), was reduced in a hnt308 mutant background, embryonic lethality was suppressed approximately 2-fold (to 29% by the bsk1 allele, and to 18% by a chromosomal deficiency for bsk; Table 2). These results suggest that, in hnt mutants, JNK signaling is upregulated (i.e., that the function of HNT in dorsal closure is to downregulate JNK signaling). Thus, the re-


Table 2 Embryonic lethality of weak hnt alleles and genetic interaction assays.

Maternal Genotype

hnt308/FM7

hntXO01/FM7 hnt704a/FM7

Paternal Genotype

Lethal Embryos/Total

Embryonic Lethality (%)a

Embryonic Lethality (Corrected %)b

Oregon R dpp10638/CyO 4x dpp⫹ bsk1/CyO Df(2L)J27/CyO Oregon R Oregon R

127/1244 65/1136 221/1054 46/531 44/594 92/326 94/316

41% 23% 80% 35% 30% ⵑ100% ⵑ100%

NA 5% NA 29% 18% — —

a

Embryonic lethality is expressed as a percentage of the expected maximum of 25% (see Materials and methods). b For hnt308, corrected values represent the embryonic lethality of the

nonbalancer progeny (see Materials and methods). This calculation was not applicable (NA) in crosses to Oregon R or in the case of 4x dpp⫹, as all progeny of this last cross are 3x dpp⫹.

duction of JNK function is able to partially suppress the dorsal closure defect in hnt308 mutants.

to germ band retraction (Figure 2a,c; also see the legend to Figure 4 in [16]).

To further test the role of HNT in regulating JNK signaling, we assayed genetic interactions between hnt308 and dpp mutants. DPP acts downstream of JNK signaling in dorsal closure [10–15]. A 50% reduction of dpp gene dose led to an 8-fold reduction in hnt308 embryonic lethality (5% versus 41%; Table 2). Conversely, increasing the dose of the wild-type dpp gene from two to three copies led to a 2-fold increase in embryonic lethality (80% versus 41%; Table 2). Examination of the hnt308 embryos carrying three doses of dpp revealed that the frequency of embryos with germ band retraction defects had doubled (41% as compared to 20%; Table 1). This last result provides the first evidence that HNT may regulate both germ band retraction and dorsal closure through the JNK/DPP signaling pathways. The direction of the genetic interaction between hnt308 and dpp is consistent with the hypothesized role of HNT to downregulate JNK signaling.

Toward the end of germ band retraction, JNK activity, as assayed by puclacZ and dpplacZ, decreases in the interior of the amnioserosa but persists in the amnioserosa perimeter cells that abut the leading edge (data not shown). By the onset of dorsal closure, when JNK activity becomes elevated in the leading edge, the amnioserosa perimeter cells lose JNK activity, and there is reduced dpplacZ or puclacZ expression throughout the amnioserosa (Figure 2b,d). It should be noted that perdurance of ␤-galactosidase protein in the amnioserosa means that our analyses of the timing of loss of puclacZ and dpplacZ expression define the latest point in development at which JNK signaling is downregulated, not when such downregulation initiates.

During normal development, JNK activity is downregulated in the amnioserosa prior to dorsal closure

We conclude that JNK signaling occurs in the amnioserosa prior to and during germ band retraction but is downregulated at or before the initiation of dorsal closure. DJUN and DFOS subcellular localization in the amnioserosa change in response to JNK signaling activity

Previous analyses of embryonic dorsal closure have focused on JNK functions in the leading edge of the epidermis [10–15]. However, we have shown that HNT is expressed in the amnioserosa, but not in the epidermis of the embryo [19, 20]. Given the genetic interactions between hnt and JNK pathway mutants, we therefore asked whether JNK signaling occurs in the amnioserosa during normal embryogenesis. We show here that JNK signaling is initially present in the amnioserosa but is downregulated prior to dorsal closure.

DJUN is activated through phosphorylation by JNK [12, 13, 21–23], and although it is capable of forming transcriptional activation complexes through homodimerization, it also forms heterodimers with DFOS [24, 25]. JUN/FOS heterodimers belong to the AP-1 class of transcription factor complexes, are more stable than JUN homodimers [26], and are thought to be the biologically relevant form [27, 28]. In Drosophila, DJUN and DFOS are encoded by the Djun and kayak (kay) genes, respectively; mutations in Djun and kay result in embryos with defective dorsal closure [12, 13, 15, 23].

A readout of JNK signaling activity in the leading edge is the transcriptional activation of the genes dpp and puc [10–15]. We therefore made use of enhancer trap lines, dpplacZ and puclacZ, as reporters for the activation state of the JNK pathway in the amnioserosa. We found that these enhancer trap lines are expressed in the amnioserosa prior

To further investigate JNK signaling in the amnioserosa during dorsal closure, we examined the expression of DJUN and DFOS. In wild-type embryos, DJUN and DFOS accumulate at high levels in the amnioserosa prior to dorsal closure (Figure 2e,g) [15, 23]. During dorsal closure, DJUN and DFOS levels are highest in the leading


Figure 2

edge but persist in the amnioserosa (Figure 2f,h) [15, 23]. In the amnioserosa, DJUN and DFOS are strictly nuclear prior to germ band retraction (Figure 2e,g). Strikingly, we found that both proteins begin to accumulate in the cytoplasm as germ band retraction is completed. While DFOS becomes nearly exclusively cytoplasmic (Figure 2h), DJUN can be detected in both the cytoplasm and the nuclei during dorsal closure (DJUN is present in a punctate pattern in the cytoplasm; Figure 2f). To determine whether nuclear restriction of DJUN and DFOS is dependent on JNK signaling, we examined DJUN and DFOS expression and subcellular localization in genetic backgrounds that were either reduced or elevated with respect to JNK signaling. In bsk2 embryos, which are deficient in JNK activity, we found that the amnioserosal cells show strong cytoplasmic localization of DJUN and DFOS (Figure 3b,e). The cytoplasmic localization was also clearly enhanced in bsk2/⫹ embryos, relative to wild-type (Figure 3a,d), suggesting that nuclear versus cytoplasmic localization of DJUN and DFOS is particularly sensitive to reduction in JNK signaling levels. To test the effect of increasing JNK activity in the amnioserosa, we immunostained puc mutant embryos. In this background, JNK activity is upregulated, and both DJUN and DFOS were restricted to the nuclei of the amnioserosal cells throughout embryogenesis (Figure 3c,f). This is the first report of nucleo-cytoplasmic regulation of DJUN and DFOS localization in Drosophila in response to JNK signaling.

JNK signaling and intracellular localization of DJUN and DFOS in the amnioserosa. (a,c,e,g) Lateral views of stage 11 embryos. (b,d,f,h) Views of the lateral epidermis, leading edge, and amnioserosa in stage 14 embryos ([h] is a dorsal view). (a,b) Anti-␤-galactosidase, visualizing dpplacZ expression in dpplacZ/⫹ embryos. The expression is present in both the leading edge and the amnioserosa (arrowheads) at stage 11 (it is weaker in the amnioserosa than in the leading edge). Stage 14 embryos show no amnioserosal expression, prominent leading edge expression, and graded expression through the lateral epidermis. (c) Anti-␤-galactosidase in puclacZ/⫹ (using the pucB48 enhancer trap) shows strong expression in the amnioserosa and the leading edge during stage 11. (d) Anti-␤-galactosidase in puclacZ/⫹ (using the pucB48 enhancer trap) shows that puc expression is limited to the leading edge during stage 14. (e,f) Anti-DJUN in wild-type, showing strong nuclear expression in the amnioserosa at stages 11 and 14 as well as strong leading edge expression during stage 14. (e,f) Wildtype embryos at stages 11 and 14, respectively, double labeled with anti-DJUN (green) and anti-HNT (red). Black and white enlargements show amnioserosal cells from the upper panels separated into (eⴕ,fⴕ) anti-HNT and (eⴕⴕ,fⴕⴕ) anti-DJUN channels. (e) During stage 11, DJUN is strongly expressed throughout the amnioserosa and shows nuclear localization. HNT and DJUN colocalize in the amnioserosal nuclei, while DJUN (but not HNT) is expressed in the leading edge and lateral epidermis. (f) Stage 14 embryos show punctate staining in the cytoplasm and reduced nuclear DJUN staining in the amnioserosa. (g,h) Wild-type embryos at stages 11 and 14, respectively, double labeled with anti-DFOS (green) and anti-HNT (red). Black and white enlargements show amnioserosal cells from the upper panels separated into (gⴕ,hⴕ) anti-HNT and (gⴕⴕ,hⴕⴕ) anti-

We have shown that DJUN and DFOS nuclear localization as well as dpplacZ and puclacZ expression support the conclusion that JNK signaling occurs in the amnioserosa prior to dorsal closure. Reciprocally, the reduction of dpplacZ and puclacZ expression and the movement of DJUN and DFOS from the nucleus into the cytoplasm of amnioserosal cells are indicative of downregulation of JNK signaling in this tissue prior to and during dorsal closure. Abnormal persistence of JNK signaling in the amnioserosa correlates with failure of dorsal closure

Given the phenotypic similarities between hnt and JNK signaling mutants, the genetic interactions between hnt and the JNK pathway mutants and our observations that JNK signaling is normally downregulated in the amnioserosa prior to dorsal closure, we next asked whether we

DFOS channels. (g) During stage 11, DFOS is strongly expressed throughout the amnioserosa and shows nuclear localization. HNT and DFOS colocalize in the amnioserosal nuclei, while DFOS (but not HNT) is expressed in the leading edge and lateral epidermis. (h) Stage 14 embryos show strong cytoplasmic, and only weak nuclear DFOS, staining in the amnioserosa. Cytoplasmic DFOS is particularly pronounced in the cells in the central region of the amnioserosa.


Figure 3 DJUN and DFOS subcellular localization depends on JNK activity. The amnioserosa of stage 13–14 embryos with altered JNK signaling activity immunostained with (a,b,c) anti-DJUN (red), (d,e,f) anti-DFOS (red), and (all panels) anti-phosphotyrosine (green). (a,d) bsk2/⫹ heterozygous embryos and (b,e) bsk2 homozygous embryos are associated with decreased JNK activity and enhance the cytoplasmic localization of (a,b) DJUN and (d,e) DFOS. bsk2 homozygous mutants were recognized by the failure of leading edge elongation and show increased cytoplasmic DJUN and DFOS throughout the amnioserosa relative to the heterozygotes. (c,f) puc homozygous embryos, recognized by their aberrant leading edge morphology, are associated with increased amnioserosal JNK activity and show persistent nuclear (c) DJUN and (f) DFOS localization throughout the amnioserosa. (c) A pucH246 and (f) a puclacZ embryo (using the pucA251 enhancer trap).

could provide molecular confirmation for HNT as a negative regulator of JNK signaling. HNT is not required for DJUN and DFOS expression, since these proteins are present in the amnioserosa of hnt mutant embryos at levels roughly comparable to wildtype (compare Figure 4a,b with Figure 2f,h). Strikingly, in contrast to wild-type embryos, hnt mutant embryos (hnt308 and hntXO01) showed persistent nuclear localization of DJUN and DFOS (compare Figure 4a,b with 2f,h). We also assayed dpplacZ and puclacZ expression in hnt308, hntXO01, and hnt1142 mutants. Both dpplacZ and puclacZ expression inappropriately persisted throughout the amnioserosa during stages 14 and 15 (compare Figure 4c,d with 2b,d). These results are consistent with the postulated role of HNT as a negative regulator of JNK signaling in the amnioserosa. As further confirmation of a role for HNT in downregulating JNK signaling, we asked whether ectopic expression of HNT could downregulate JNK signaling in the leading edge of the epidermis. We used an enhancer-promoter (EP) insertion line EP55 [29] with an EP element inserted 585 bp upstream of the 5⬘-end of the NB701 cDNA (for mapping of the insertion site, see Materials and methods). Ectopic HNT protein was expressed in the lateral epidermis using either daGAL4 or pnrGAL4 drivers (for pnrGAL4, compare Figure 4f with 4e) [30]. Consistent with a role for HNT in the downregulation of JNK signaling, dpplacZ expression was reduced in the leading edge and the lateral epidermis (compare Figure 4h with 4g). Together, our data demonstrate that HNT function is necessary for downregulation of the JNK cascade in the amnioserosa at or before the initiation of dorsal closure

and that ectopic expression of HNT in the leading edge of the epidermis is sufficient to cause downregulation of JNK signaling in these cells. Persistent nuclear localization of DJUN and DFOS is seen, not only in the amnioserosa of hnt mutants, but also in the amnioserosa of puc mutants in which dorsal closure also fails (Figure 3c,f). Thus hnt and puc mutants provide independent lines of evidence that downregulation of JNK signaling in the amnioserosa is essential for dorsal closure. Formation or maintenance of focal complexes in the leading edge of the epidermis is disrupted by persistent JNK signaling in the amnioserosa

The leading edge of the epidermis has been shown to play a crucial role in regulating dorsal closure [10–13]. We therefore asked whether persistent JNK signaling in the amnioserosa disrupts the morphology and behavior of the leading edge. In wild-type embryos, phosphotyrosine and F-actin accumulate conspicuously along the dorsal-most leading edge cell membranes that abut the amnioserosa, representing focal complexes (Figure 5a,d, and see [1, 2, 5]). It has previously been shown that focal complexes fail to accumulate in leading edge cells of puc mutants [16]. Similarly, in hnt mutants, phosphotyrosine and F-actin fail to accumulate at the dorsal-most membrane of the leading edge cells (Figure 5b,e). Thus, HNT function in the amnioserosa is necessary for the adjacent leading edge cells to assemble or maintain focal complexes at their dorsal-most membranes.


Figure 4

[16], and hnt mutants (compare Figure 4c,d with 2b,d). Consistent with this result, the cells of the lateral epidermis undergo dorsal-ventral elongation in puc [16] and hnt mutants (see, e.g., Figure 5e), a process dependent on JNK-induced signals from the leading edge [11–13, 23]. The simplest interpretation of these data is that assembly or maintenance of focal complexes in the leading edge occurs only if there is a boundary between high and low JNK signaling at the junction of the leading edge (high) and the amnioserosa (low). In hnt and puc mutants, since JNK signaling persists in the amnioserosa, such a high/ low JNK activity boundary never forms, and therefore focal complexes are either not assembled or are not maintained at the dorsal membrane of the leading edge. In the absence of focal complexes, the leading edge is unable to move over the amnioserosa.

The loss of hnt causes persistent JNK signaling in the amnioserosa, while ectopic HNT expression can downregulate JNK signaling in the leading edge. (a) An hnt308/Y mutant stained with anti-DJUN, showing persistent nuclear DJUN in the amnioserosa and normal accumulation of DJUN in the leading edge (compare to Figure 2f). hntXO01 shows similar persistent nuclear DFOS (data not shown). (b) An hntXO01/Y mutant stained with anti-DFOS, showing strong persistent nuclear DFOS localization (compare to Figure 2h). hnt308 shows similar persistent nuclear DFOS (data not shown). (c) An hnt308/Y; dpplacZ/⫹ mutant stained with anti-␤-galactosidase, showing dpplacZ expression in the amnioserosa (arrowheads) (compare to Figure 2b). (d) An hnt308/Y; puclacZ/⫹ mutant, showing persistent puclacZ (using the pucB48 enhancer trap) expression in the amnioserosa (arrowheads) (compare to Figure 2d). Note that puclacZ, dpplacZ, and DFOS are all expressed in the leading edge of hnt mutants; while their expression patterns in the leading edge and lateral epidermis are normal, the levels of expression in these regions are slightly reduced relative to control sibs. (e) Wild-type and (f) EP55; pnrGAL4/⫹ embryos labeled with anti-HNT. As evidenced by the strong anti-HNT staining in the dorsal region of the lateral epidermis in (f), EP55 results in GAL4-inducible HNT expression. In (f), gain was reduced because of the very strong ectopic HNT expression in the epidermis; thus, HNT appears weaker in the amnioserosa compared with (e). (g,h) Anti-␤-galactosidase staining, showing dpplacZ expression in (g) wild-type and (h) EP55; pnrGAL4/⫹ backgrounds. Ectopic HNT causes dpplacZ expression to be greatly reduced in the leading edge and lateral epidermis during dorsal closure.

The failure of focal complex assembly in the leading edge cells of hnt and puc mutants is not a secondary consequence of the failure of JNK signaling in these cells. This conclusion derives from the fact that dpplacZ and puclacZ are expressed in the leading edge of wild-type [11, 16, 18], puc

If this logic is correct, then focal complexes might fail to form if JNK signaling is downregulated in both the amnioserosa and the leading edge, since, again, no high/ low JNK signaling boundary would be established. Because we had already shown that ectopic expression of HNT using EP55 results in downregulation of JNK activity in the leading edge (Figure 4h), we decided to extend our analyses to possible effects of ectopic HNT on focal complexes by analyzing phosphotyrosine and F-actin in the leading edge. The resultant phenotype was very similar to that seen in loss-of-function hnt mutants: focal complexes failed to accumulate in the leading edge cells (compare Figure 5c,f with 5b,e). Furthermore, as in hnt mutants, dorsal closure failed (data not shown). Despite defects in the leading edge, elongation of lateral epidermal cells occurs (see, e.g., Figure 5f). Thus, the failure of dorsal closure is independent of elongation of the lateral epidermis and correlates with the failure to assemble focal complexes in the leading edge cells. The hypothesis that focal complexes form only when there is a high/low JNK signaling boundary at the juxtaposition of the leading edge and the amnioserosa predicts that conversion of a high/high back to a high/low condition will lead to the restoration of focal complexes. We therefore decided to downregulate JNK activity in the amnioserosa of hnt mutants during the stages at which JNK signaling would abnormally persist. This was accomplished by expressing PUC or dominant-negative JNK using an amnioserosa-specific GAL4 driver, GAL4332.3 [31]. As described above, in hnt mutants (the high/high situation), focal complexes are absent from the leading edge, and the morphology of the leading edge cells is highly abnormal (Figure 5b,e). Consistent with our hypothesis, when either PUC or dominant-negative JNK is expressed in the amnioserosa of hnt mutants, focal complexes are restored to the dorsal-most membrane of the leading edge, and


Figure 5 A high/low JNK signaling boundary between the leading edge and the amnioserosa is required for the accumulation of focal complexes in leading edge cells. Two markers of focal complexes are shown: (a–c, g,h) anti-phosphotyrosine and (d–f) F-actin. (a,d) Wild-type, (b,e) hnt308 mutant, (c,f) EP55; daGAL4/⫹ embryos, and (g,h) an hnt308 embryo carrying GAL4332.3, which (g) drives UAS-bskDN expression, or (h) drives UAS-puc expression, in the amnioserosa. Phosphotyrosine and F-actin fail to accumulate along the dorsal-most membrane of the leading edge in (b,e) hnt mutants as well as (c,f) when HNT is misexpressed in the leading edge. Despite the absence of focal complexes, the cells of the leading edge and lateral epidermis elongate dorsoventrally. (g,h) Phosphotyrosine accumulation is restored to the dorsal-most membrane of the leading edge of a hnt308 mutant embryo in which dominantnegative (g) JNK or (h) PUC is misexpressed in the amnioserosa.

the morphology of the leading edge is shifted toward wildtype (Figure 5g,h). In summary, our analyses show that focal complexes fail to accumulate in the leading edge when there is no JNK signaling boundary between the leading edge and the amnioserosa. The restoration of a high/low situation by the expression of either PUC or dominant-negative JNK in a hnt mutant results in the restoration of focal complexes in the leading edge.

Discussion JUN and FOS subcellular localization change during development in response to JNK signaling

In mammals, active AP-1 transcription factors comprise heterodimers of JUN with FOS or ATF-family proteins (reviewed in [32, 33]). Levels of JUN and FOS proteins are regulated both transcriptionally and posttranscriptionally, the latter largely through ubiquitin-mediated proteolysis [34, 35]. FOS is particularly labile, with an estimated half-life of 10 min, considerably shorter than the estimated 60 min half-life of JUN [32, 35]. Phosphorylation is a major mechanism that regulates the activity of JUN and FOS: JNK is the major kinase involved in JUN phosphorylation, while FOS is a target of ERKs (reviewed in [33]). The phosphorylation status of JUN and FOS also affects their stability (reviewed in [33]). Intracellular localization of JUN and FOS has been less extensively studied than the control of protein activity and turnover [36, 37]. Nuclear localization of C-FOS, for example, is thought to depend both upon heterodimerization with JUN as well as upon extracellular signals whose transduction is JNK-independent [36, 37]. In contrast,

V-FOS (the oncogenic form of FOS) is localized constitutively to the nucleus [36, 37]. Given FOS’s short halflife, subcellular localization of FOS has been suggested as a mechanism of regulating AP-1 activity [37]. In this study, we have shown that, in Drosophila, amnioserosal DJUN and DFOS are strictly nuclear at early embryonic stages but relocalize to the cytoplasm during dorsal closure. To our knowledge, this is the first report of regulated intracellular localization of these proteins during development. Restriction of DJUN and DFOS to the nucleus correlates with high JNK activity. In contrast, the presence of these proteins in the cytoplasm correlates with low levels of JNK signaling; in this case, DFOS becomes largely cytoplasmic, while DJUN is present in both the nucleus and the cytoplasm. The persistence of nuclear DJUN may in part be a consequence of the fact that it has a longer half-life than DFOS. In the cytoplasm, DJUN shows a punctate distribution. While the nature of these DJUN-containing structures is unknown, we speculate that they might represent the 26S proteasome [35]. JNK signaling boundaries may be crucial for regulation of focal complex assembly and tissue interaction during morphogenesis

Focal complexes include signaling molecules and components of the actin-based contractile apparatus of the cell [7, 38]. They are thought to play a role in signal transduction events between cells, or between cells and the extracellular matrix, and in linking these signals to changes in cell shape and behavior [7, 38]. For example, filopodia form in a focal complex-dependent fashion and are thought to function both to generate contractile force and to sense signals and positional information through cell-


cell contacts [9]. Examples of epithelial sheet fusion events associated with filopodia include ventral enclosure in Caenorhabditis elegans [39] and imaginal disc fusion during metamorphosis in Drosophila [40]. Recently, it has been shown that filopodia and lamellipodia extend from the leading edge during dorsal closure and contact the amnioserosa [8]. Here, we have shown that the failure to form a boundary between high and low JNK signaling correlates with the failure to assemble or maintain focal complexes at the junction between the leading edge and the amnioserosa. The restoration of a high/low JNK activity boundary rescues focal complexes in the leading edge. The absence of focal complexes in the leading edge may result in defects in signaling between the leading edge and the amnioserosa and/or an inability to form filopodia. It has been suggested that the meeting of the two epidermal sheets at the dorsal midline is associated with a signal to “stop”, with consequent disassembly of the cell motility machinery [8, 9]. If such a stop signal were produced by cells with high JNK activity, then the signal would not normally be encountered until dorsal closure was close to complete and contact was made between the opposing leading edges. The failure to achieve dorsal closure in hnt and puc mutants, according to this model, would be due to the leading edge encountering a precocious stop signal generated by persistent JNK signaling in the amnioserosa.

Conclusions We have shown that spatial regulation of JNK signaling is essential for the tissue remodeling that drives dorsal closure. Focal complexes are assembled at the boundary between high and low JNK signaling. These complexes are likely to direct the formation of F-actin-based cellular protrusions that allow the epidermis to move dorsally and then to fuse at the dorsal midline [8]. We have shown that nuclear versus cytoplasmic localization of the AP-1 transcription factor is developmentally regulated and is a readout of the activity of the JNK cascade. Spatial and temporal regulation of this cascade may serve a conserved function in controlling epithelial remodeling during morphogenesis and wound repair.

Materials and methods Drosophila mutants and lines Mutants and lines were as follows: hnt308 (from S.L. Zipursky, UCLA), hntXO01, hnt704a, hntXE81 (described in [19]), hnt1142 (from J. Duffy, Indiana University; described in [41]), EP55 (from P. Rorth, EMBL, Heidelberg; described in [29]), pnrGAL4 (also known as pnrMD237; from P. Simpson, CNRS, Strasbourg; described in [30]), Dp(2;2)DTD48/CyO, P{w ⫹ mC ⫽ dpp ⫺ P23}RP1 (⫽ 4 x dpp⫹; from W. Gelbart, Harvard University), GAL4daG32 (also known as P{Gal4-da.G32}) and GAL4332.3 (also known as P{GawB.332.3}) (both described in [31]), dpplacZ (also known as dpp10638) and two puclacZ enhancer traps (pucA251 and pucB48) (described, respectively, in [16, 42]), UAS-bskDN (referred to here as dominantnegative JNK; described in [43]) and UAS-puc (described in [16]) (both from M. Mlodzik, Mt. Sinai School of Medicine, New York), Df(2R)J27

(a deletion that removes bsk), bsk1, bsk2, and pucH246 (Bloomington Drosophila Stock Center).

Embryonic lethality assays and cuticle preparations Embryos (0–18-hr-old) were washed from grape juice-agar plates in PBS-TX (0.12% Triton X-100) and collected in filtration baskets assembled from nylon mesh and modified 50 ml screw-cap Falcon tubes. Embryos were rinsed briefly in room temperature deionized water, and the nylon mesh with embryos was removed and blotted dry by placement on absorbent paper. For embryonic lethality assays, embryos were removed from the mesh and placed in the center of fresh grape juice-agar plates using a fine-tipped artist’s brush. Embryos were placed on the perimeter of the plate in grids of 100 eggs, up to 800 eggs per plate. Any unused eggs were removed from the center of the plate that was then loaded with fresh live yeast paste to attract larvae. Plates prepared in this way were aged for at least 30 hr and were scored before larvae disturbed the unhatched eggs. Unhatched eggs were scored as lethal (these were generally brown and had conspicuous cuticle deposition) or unfertilized (generally white and undeveloped). Embryonic lethality was then calculated as [number of lethal embryos]/([total eggs on plates] – [unfertilized eggs]). Since our hnt stocks are kept using viable X balancer chromosomes, we assume 25% embryonic lethality to be equivalent to 100% lethality of the mutant class. The percent embryonic lethality of the mutant class is thus calculated as the ([embryonic lethality]/ 0.25) ⫻ 100. To test for genetic interactions between hnt and mutations in members of the JNK signaling/ DPP pathway, percent embryonic lethality was measured in crosses of hnt308/FM7 (Table 2). If the test stock is a balanced autosomal lethal, then the measured percent embryonic lethality is a combination of hnt308 embryonic lethality in lethal/⫹ and Balancer/⫹ backgrounds. Assuming that the observed embryonic lethality represents an average of these two classes, then the embryonic lethality of hnt308 in the lethal/⫹ background is ([2 ⫻ observed % embryonic lethality] – [% control value]), where percent control value is equivalent to the percent embryonic lethality of the hnt308/Y; Balancer/⫹ class of progeny. The control value used to calculate the corrected values presented in Table 2 was the measured percent embryonic lethality of hnt308 crossed to Oregon R (40.8%). With respect to genetic suppression of embryonic lethality, this method gives a conservative estimate, since Balancer/⫹ genetic backgrounds tend to give higher hnt308 lethality than ⫹/⫹ backgrounds. For cuticle preparations, lethal embryos were collected from plates similar to those described above and dechorionated using standard bleach treatment. After blotting dry as described above, brown eggs were placed into a drop of (1:1) Hoyer’s mounting medium:lactic acid on a coverslip, mounted on a microscope slide, and heated overnight at 65⬚C.

Immunohistochemistry and microscopy Antibody staining of embryos was performed according to established procedures [44]. Primary antibodies that were used were mouse antiHNT monoclonal antibody 27B8 1G9 (1:20 dilution; [19]), rabbit anti␤-galactosidase (1:200–1:500; Cappel), mouse anti-phosphotyrosine monoclonal antibody (1:500; Upstate Biotechnology), rabbit anti-DJUN (1:200; [23]), and rabbit anti-DFOS (1:400; [15]). Secondary antibodies (all from Jackson Immunoresearch) were alkaline phosphatase-conjugated goat anti-mouse used at 1:500 dilution, horseradish peroxidase (HRP)-conjugated goat anti-rabbit used at 1:200, TRITC-conjugated donkey anti-rabbit used at 1:300, and FITC-conjugated goat anti-mouse used at 1:300. The visualization of filamentous actin (F-actin) using FITCconjugated phalloidin (Sigma) was performed according to [3]. Light microscopy was carried out on a Zeiss Axioplan microscope, and images were captured with a Spot digital camera (Diagnostic Instruments). Confocal microscopy used a Leica TCS 4D confocal microscope with “scanware” software. All confocal images presented are single sections. Images were processed and displayed using Photoshop and Illustrator software (Adobe).


Molecular mapping and analysis of P element mutants hnt308 and hntEP55

To find the insertion site of P element P[ry⫹ boss]308, PCR was performed on genomic DNA extracted from adult wild-type or P[ry⫹ boss]308 flies, using a combination of P element- and hnt-specific primers. Approximatley 700-bp bands were observed with either 5⬘-end P element-specific primer: P3 (5⬘-TCCAGTCACAGCTTTGCAGC-3⬘) or P2 (5⬘-AAGTGTATACTTCGGTAAGC-3⬘) in combination with the hindsight (hnt)-specific primer hnt-41 (5⬘-TGCGACGGCATTCCAACTCC-3⬘). The band obtained with P2/hnt-41 was sequenced to determine the exact point of insertion of P[ry⫹ boss]308. P[ry⫹ boss]308 is located 509 bp upstream of the 5⬘-end of hnt cDNA NB701 [19], oriented such that the 5⬘-end of the P element is closest to the first exon of hnt. We henceforth refer to P[ry⫹ boss]308 as hnt308.

9. 10.

11. 12.

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Using a similar approach, we mapped the insertion site of the EP element in P{EP}EP55 flies [29]. We found an ⵑ800-bp PCR band with the 3⬘-end P element-specific primers: P1 (5⬘-AGCATACGTTAAGTGG ATGTC-3⬘) and P4 (5⬘-GAGTTAATTCAAACCCCACGGACATGC-3⬘) in combination with hnt-41. The P1/hnt-41 band sequence reveals the EP element to be inserted 585 bp upstream of the 5⬘-end of the NB701 cDNA, with the 3⬘-end of the EP element closest to the first hnt exon. To confirm that P{EP}EP55 could direct ectopic hnt gene expression, we crossed females that contain the GAL4 driver, pnrGAL4 [30] to P{EP}EP55 males. We immunostained embryos obtained from this cross with antiHNT monoclonal antibody 27B8 1G9 (see above) to confirm ectopic expression of HNT in the lateral epidermis (data not shown). We refer to P{EP}EP55 as hntEP55.

Acknowledgements The following kindly provided reagents used in this study: S.L. Zipursky, J. Duffy, W. Gelbart, D. Bohmann, P. Rorth, M. Mlodzik, P. Simpson, L. Dobens, and the Bloomington Drosophila Stock Center. We appreciate critical comments on the manuscript from J. Brill, N. Harden, H. Krause, M. Lamka, and A. Pickup. In particular, we thank N. Harden for numerous useful discussions during the course of this research. R.W. was supported in part by an Ontario Graduate Scholarship and a studentship from the Research Institute of the Hospital for Sick Children. This research was supported by an operating grant to H.D.L. from the National Cancer Institute of Canada, with funds from the Canadian Cancer Society.

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