basket/JNK
basket mutants were originally described as generating a defect in dorsal closure (Nüsslein-Volhard, 1994). Mutants reveal an aberrant dorsal cuticle. The severly deformed cuticles show a dramatic reduction in dorsal ridge formation, with head and anterior segments exposed. In other embryos, a hole in the dorsal epidermis is observed. The size of the hole varies, from small to one large enough to cover most of the dorsal region. Both the amnioserosa and epidermis are present, suggesting that dorsoventral patterning is not defective. The hole in the dorsal cuticle is a result of defective dorsal closure during midembryogenesis (Sluss, 1996).
basket mutants were stained with antibodies against Spectrin and Coracle. Coracle is the Drosophila homolog of the vertebrate band 4.1 cytoskeletal protein, while Spectrin is an integral component of the cortex cytoskeleton situated immediately under the cell surface membrane. The marginal cells at the leading edge during dorsal closure change shape and elongate, but the process fails to achieve complete closure, because the first rows underneath the leading edge cells either show only a partial change in shape, or fail to change cell shape completely. It is as if the defect in basket affects the communication between leading edge and following cells. In wild-type embryos, the epidermal cells underneath the leading edge change shape and elongate after the leading edge cells change shape. In mutants, the spreading defect of the cells is more pronounced in anterior cells. In the posterior part of the embryo in weak mutants, some stretching epidermal cells meet at the dorsal midline and effect closure, but not in the anterior part. As a consequence, the anterior portion remains open and the forming head structures and mouthparts are thrust forward, together with parts of the gut and the CNS. The graded series of bsk mutant phenotypes argues for a sustained requirement of bsk throughout the process of dorsal closure: reductions in the amount of protein are reflected in a concomitant reduction in the extent of cell elongation (Riego-Escovar, 1996). See Hemipterous for more discussion of the dorsal closure phenotype.
Dorsal closure requires two signaling pathways: the
Drosophila Jun-amino-terminal kinase (DJNK) pathway and the Decapentaplegic pathway. The changes in cell shape in the lateral epidermis occur in two phases. In the first phase, the cells of the leading edge begin to stretch dorsally. In a second phase, the remaining cells ventral to the first row change shape. DJNK, known as Basket, controls dorsal closure by
activating DJun and inactivating the ETS repressor Aop/Yan by phosphorylation. The role of Aop/Yan is to hold decapentaplegic transcriptionally silent until Aop/Yan is inactivated by phosphorylation. These phosphorylation events regulate dpp expression in the most dorsal row of cells. Interestingly, mutants in components of the DJNK and Dpp pathways affect the two phases of dorsal closure differently. Whereas loss-of-function mutations in either DJNK or DJun block the cell shape changes of all cells, mutations in thick veins and punt block only the second phase. Thus it is concluded that Dpp
functions to instruct more ventrally located cells to stretch. These results provide a causal link
between the DJNK and Dpp pathways during dorsal closure (Riesgo-Escovar, 1997).
The tissue polarity genes of Drosophila are required for correct establishment of planar polarity in
epidermal structures, which in the eye is shown in the mirror-image symmetric arrangement of ommatidia,
relative to the dorsoventral midline. Mutations in the genes frizzled, dishevelled and
prickle-spiny-legs (pk-sple) result in the loss of this mirror-image symmetry. Little
is known of the signaling pathway(s) involved other than that Dsh acts downstream of Fz. Mutations have been identified in Drosophila Rho1; by analysis of their phenotypes it has been shown that Rho1 is required for the generation of tissue polarity. Genetic interactions indicate a role for Rho1 in signaling mediated by Fz and Dsh. Overexpression of Fz in a subset of photoreceptor precursors gives ommatidial polarity defects, characterized by misrotations and incorrect chiralities. Consistent with this phenotype representing overactivation of the Fz signaling pathway, Rho1 phenotypes are dominantly suppressed by fz mutations. The phenotype due to overexpression of Fz is also dominantly suppressed by dsh. Overexpressed Dsh is dominantly suppressed by Rho1 mutations. Given the similarity of the mutant phenotypes of fz, dsh and Rho1, as well as these genetic interactions, it is proposed that Rho1, like Dsh, acts downstream of Fz in the generation of tissue polarity. Mutations in the gene basket, which encodes a JNK/SAPK homolog, suppress the Fz overexpression phenotype and the Dsh overexpression phenotype to a similar extent as does mutation of Rho1. The embryonic phenotypes of Rho1 and bsk mutants are similar, supporting a role for Bsk in Rho signaling. Eye clones have been generated for bsk hypomorphic mutations, and these do not show an ommatidial polarity defect (or any other significant eye phenotype). However, the phenotype of bsk null clones is not known. These data are consistent with a Fz/Rho1 signaling cascade analogous to the yeast pheromone signaling pathway. This pathway has been proposed as an activator of the serum response factor (SRF) in vertebrate cells (Strutt, 1997).
During patterning of the Drosophila eye, the Notch-mediated cell fate decision is a critical step that determines the identities of the R3/R4 photoreceptor pair in each ommatidium. Depending on the decision taken, the ommatidium adopts either the dorsal or ventral chiral form. This decision is directed by the activity of the planar polarity genes, and, in particular, higher activity of the receptor Frizzled confers R3 fate. Evidence is presented that Frizzled does not modulate Notch activity via Rho GTPases and a JNK cascade as previously proposed. The planar polarity proteins Frizzled, Dishevelled, Flamingo, and Strabismus adopt asymmetric protein localizations in the developing photoreceptors. These protein localizations correlate with the bias of Notch activity between R3/R4, suggesting that they are necessary to modulate Notch activity between these cells. Additional data support a mechanism for regulation of Notch activity that could involve direct interactions between Dishevelled and Notch at the cell cortex. In the light of these findings, it is concluded that Rho GTPases/JNK cascades are not major effectors of planar polarity in the Drosophila eye. A new model is proposed for the control of R3/R4 photoreceptor fate by Frizzled, whereby asymmetric protein localization is likely to be a critical step in modulation of Notch activity. This modulation may occur via direct interactions between Notch and Dishevelled (Strutt, 2002).
A number of lines of evidence have previously suggested that Rho/Rac GTPases and the JNK cascade are required for ommatidial polarity decisions and, in particular, the R3/R4 fate decision. These include the following: overexpression of Fz or Dsh in the eye gives a polarity phenotype that is dominantly suppressed by RhoA, bsk, hep, and Djun; RhoA clones or expression of dominant-active/negative RhoA or Rac1 gives ommatidial polarity phenotypes; overexpression of dominant-active/negative JNK pathway components and human Jun elicits ommatidial polarity defects, and expression of a Dl enhancer trap is altered by overexpression of either fz or dsh or by activated human Jun, Hep, RhoA, or Rac1. These observations led to the hypothesis that higher levels of Fz/Dsh signaling in R3 result in higher activation of Dl transcription in R3 via a Rho GTPase/JNK cascade, biasing the N/Dl feedback loop to produce high N in R4 (Strutt, 2002).
However, the data do not support the hypothesis that activation of Dl transcription via Rho GTPases/JNK cascade is the primary mechanism for biasing N activity in R3/R4 during normal eye development. Reexamination of RhoA phenotypes indicates that these rarely affect R3/R4 fate and ommatidial chirality, and RhoA activity is not required for N repression in R3. No role is found for Rac in this process, a finding confirmed by the recent report that deletion of all three Rac homologs in the Drosophila genome has no effect on planar polarity. In addition, notwithstanding the observation that loss of Djun activity can result in ommatidial polarity defects in 2%–3% of ommatidia, mosaic ommatidia where the presumptive R3 lacks Djun activity have wild-type levels and polarity of Notch signaling, so no evidence is found that Djun is directly modulating N activity in R3/R4. It is also interesting to note that although a double mutant combination of the JNK pathway components hemipterous and puckered produces an ommatidial polarity phenotype, this apparently consists entirely of rotation and not chirality defects (Strutt, 2002).
One factor not directly investigated is the STE20 kinase homolog encoded by msn. Loss of function analysis of msn does reveal some ommatidial chirality defects, albeit rarely. More compellingly, mosaic analysis suggests that if one cell of the R3/R4 pair is msn-, this cell will generally (but not exclusively) take the R4 fate. This suggests a role for Msn in repression of N in R3, although the apparent rarity of chirality defects in ommatidia lacking msn function suggests this is a nonessential pathway (Strutt, 2002).
Taken together, the phenotypic evidence from loss-of-function studies does not support a primary role for Rho GTPases/JNK cascades in the R3/R4 fate decision. But the weight of genetic evidence does support a secondary role for some of the proposed pathway components, possibly in the augmentation of polarity decisions driven largely by asymmetric localization of polarity proteins and direct repression of N activity. In addition, the observation that RhoA mutations result largely in defects in ommatidial rotation supports the hypothesis that RhoA acts downstream of the planar polarity genes in regulating this aspect of ommatidial polarity (Strutt, 2002).
For some years, the standard view of planar polarity gene function has been that this is mediated by Rho GTPases and a JNK kinase cascade, most likely leading to a transcriptional response. In particular, it was thought that this pathway controlled R3/R4 photoreceptor fate in the developing eye, via transcriptional activation of Dl. Although Rho GTPases do regulate one aspect of planar polarity in the eye (ommatidial rotation), they do not appear to be the primary determinant of R3/R4 fate. Therefore, it is concluded that Rho GTPases/JNK cascades are not the major effectors of planar polarity activity in this context. Furthermore, it has been demonstrated that several planar polarity proteins adopt asymmetric subcellular localizations in the eye that correlate with N activation and R3/R4 fate. Therefore, an alternative model has been proposed for modulation of N activity via direct interactions with planar polarity proteins (and most probably Dsh) at the cell cortex (Strutt, 2002).
Frizzled family proteins have been described as receptors of Wnt signaling molecules. In Drosophila, the
two known Frizzled proteins are associated with distinct developmental processes. Genesis of epithelial
planar polarity requires Frizzled, whereas Dfz2 affects morphogenesis by wingless-mediated signaling.
Dishevelled is required in both signaling pathways. Genetic and overexpression assays have been used to
show that Dishevelled activates JNK cascades. In contrast to the action of wingless-pathway components, mutations in rhoA, hemipterous, basket, and jun as well as deficiencies removing the Rac1 and Rac2 genes show a strong dominant suppression of a Dishevelled overexpression phenotype in the compound eye. In an in vitro assay, expression of Dsh has been shown to induce phosphorylation of Jun, indicating that Dsh is a potent activator of the JNK pathway. Whereas the PDZ domain of Dsh, known to be required in the transduction of the wingless signal, is dispensable for signal-independent induction of Jun phosphorylation, the C-terminal DEP domain of Dsh is found to be essential. The planar polarity-specific dsh1 allele is found to be mutated
in the DEP domain. These results indicate that different Wnt/Fz signals activate distinct intracellular
pathways, and Dishevelled discriminates among them by distinct domain interactions (Boutros, 1998).
How can Fz/Dsh signaling be linked to small GTPase and JNK/MAPK pathways? Recent studies provided evidence
that links G protein-coupled receptors, which share structural features with Fz proteins, to MAPK signaling through
heterotrimeric G proteins and PI-3 kinases. It is intriguing to speculate that a subset of Fz proteins might signal through a
similar pathway. It was also shown recently that XWnt5A and rFz2, in a heterologous assay, increase intracellular
calcium via G proteins and phosphoinositol signaling. A mutation in the beta-subunit of a heterotrimeric G protein in C. elegans prevents correct spindle orientation, a process that is believed to be dependent on a Wnt and a Fz receptor, but not on Arm. Further studies regarding a possible involvement of PI-3K and G proteins in planar
polarity signaling may provide additional insight to the diversity of Fz-related signaling pathways (Boutros, 1998 and references).
Evidence that msn and bsk
function in the same pathway comes from the observation that some
embryos doubly heterozygous for msn and bsk display
a dorsal open phenotype. About 10% of embryos derived from
a cross between msn/+ and
bsk/+ flies exhibit a dorsal open phenotype. The defect in
dorsal closure in embryos doubly heterozygous for msn and
bsk is not a dominant effect of either gene; a defect in
dorsal closure was very rarely observed when
msn/+ or bsk/+
flies are crossed with wild-type flies. The presence of such
defects in doubly heterozygous flies strongly suggests that the genes function in the same pathway. Moreover, the severity of the phenotype correlates with the strength of the bsk allele with which the
msn/+ flies were crossed. To confirm genetically that msn also functions upstream of hep,
embryos doubly heterozygous for msn and hep were examined. hep is
on the X chromosome, and both maternal and zygotic hep
contribute to dorsal closure. To obtain flies doubly heterozygous for
msn and hep, msn/+ males were
crossed with hepr75/+ females. About 35% of the flies obtained from this cross display a
defect in dorsal closure. This finding is very close to the predicted
frequency of 37% for embryos with a reduction in both the maternal and
zygotic dosage of hep and the zygotic dosage of msn. A defect in dorsal closure is not observed when
hep/+ females are crossed with
+/Y males, indicating that embryos with the dorsal
closure defect carry the msn mutation. Reduced dosage of both
zygotic and maternal hep is required for the zygotic
lethality of heterozygous msn/+ embryos. The viability of msn/+ flies lacking one copy of
zygotic hep is reduced by >80% (Su, 1998).
The Ral GTPase is activated by RalGDS, which is one of the effector proteins for Ras. Previous studies have suggested that Ral might function to regulate the cytoskeleton; however, its in vivo function is unknown. A Drosophila homolog of Ral has been identified that is widely expressed during embryogenesis and imaginal disc development. Two mutant Drosophila Ral (DRal) proteins, DRal(G20V) and DRal(S25N), were generated and analyzed for nucleotide binding and GTPase activity. The biochemical analyses demonstrated that DRal(G20V) and DRal(S25N) act as constitutively active and dominant negative mutants, respectively. Overexpression of the wild-type DRal does not cause any visible phenotype, whereas DRal(G20V) and DRal(S25N) mutants cause defects in the development of various tissues, including the cuticular surface, which is covered by parallel arrays of polarized structures such as hairs and sensory bristles. The dominant negative DRal protein causes defects in the development of hairs and bristles. These phenotypes are genetically suppressed by loss of function mutations of hemipterous and basket, encoding Drosophila Jun NH(2)-terminal kinase kinase (JNKK) and Jun NH(2)-terminal kinase (JNK), respectively. Expression of the constitutively active DRal protein causes defects in the process of dorsal closure during embryogenesis and inhibits the phosphorylation of JNK in cultured S2 cells. These results indicate that DRal regulates developmental cell shape changes through the JNK pathway (Sawamoto, 1999a).
Misshapen
acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings.
Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation.
Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the
planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This
demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase
family in higher eukaryotes. Msn acts in EPP signaling through the JNK
(Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no
apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein
kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, evidence is provided for an involvement of the related MAP kinases of the p38 subfamily in planar polarity
signaling downstream of Msn (Paricio, 1999).
Although there is accumulating evidence that JNK-type MAPK modules are involved in planar polarity signaling, the analysis of mutant clones of either hep or bsk alleles shows no or weak phenotypes in imaginal discs. These observations suggest a high degree of redundancy at this level in the polarity signaling pathway. To address this issue further, a potential involvement of related kinases that could account for the proposed redundancy was examined. The recently described Drosophila kinases, belonging to the JNK/p38 class within the MAPK modules were examined for genetic interactions with the planar polarity phenotypes of sev-Dsh and sev-msn. These are obvious candidates to be cooperating with Hep and Bsk in polarity generation. At the level of Hep/JNKK (an MKK7 homolog), two other MKKs have been reported (DMKK3 and DMKK4). Similarly, at the level of Bsk/JNK, two p38-like kinases were isolated (Dp38a and Dp38b). Since no mutants have yet been isolated for these genes, whether deficiencies removing these kinases would show an interaction with sev-Dsh was examined. DMKK3 maps in the vicinity of hep: deficiencies removing DMKK3, Df(X)G24 and Df(X)H6, also remove hep. These deficiencies show externally a very strong suppression of sev-Dsh with a marked decrease of misrotated ommatidia as observed in tangential sections. Deficiency Df(3R)p13 removes the DMKK4 locus and also dominantly suppresses sev-Dsh. Similarly, deficiencies removing either Dp38a, Df(3L)crb87-4 and Df(3L)crbF89-4, or Dp38b, Df(2L)b80e3 and Df(2L)b87e25, are suppressors of sev-Dsh. Whether the respective deficiencies showed an interaction with sev>msn was also examined, and it was found that all of them act as dominant suppressors of this genotype as well. It is interesting to mention that the Msn-induced defects in rhabdomere morphology are also suppressed by those deficiencies. These interactions suggest that the p38 kinases are redundant with JNK in the context of planar polarity signaling (Paricio, 1999).
Although genetic evidence suggests an involvement of bsk (JNK) and hep (JNKK) in polarity signaling, phenotypic analyses suggest that the JNK module components are highly redundant in this process. It is interesting to note that all phenotypic defects of sev>Msn were dominantly suppressed by mutations in both components of the JNK and the p38 kinase module. In contrast to these interactions, tissue culture experiments in mammalian cells have shown that NIK overexpression leads to JNK phosphorylation, but no detectable p38 activation was observed. This difference can be explained by cell- and tissue-specific requirements, e.g. in Drosophila during dorsal closure, JNK activation downstream of Msn is not redundant, while redundancy and p38 interactions are observed in polarity signaling. Thus, it is tempting to speculate that both JNK and p38 kinases cooperate in polarity generation (Paricio, 1999).
The reported promiscuity of the kinases at both the MKK and the MAPK levels could account for the redundancy. The Drosophila MKKs and JNK/p38 MAPKs also appear to act (at least partially) on overlapping downstream targets. Whereas DMKK3 appears rather specific for p38 activation (although it activates both p38s), DMKK4 and Hep (the MKK7 cognate) both activate Bsk/JNK. Similarly, Bsk/JNK and both Dp38s can phosphorylate the downstream targets dJun and ATF2. Thus, a potential downstream target can still be phosphorylated when one of the upstream kinases is removed, and likewise for their upstream activators. An even more complicated picture may emerge when all relevant kinases are identified. Other examples of redundancy are described in yeast MAP kinases. Although KSS1 and FUS3 normally have specific roles in different pathways, it has been shown that they are redundant in the process of mating and in this case KSS1 replaces Fus3 when the latter is not present. The isolation and analysis of all the respective kinases and their mutants will be necessary to understand fully the contribution of each single kinase in planar polarity signaling (Paricio, 1999).
Jun acts as a signal-regulated transcription factor in many
cellular decisions, ranging from stress response to
proliferation control and cell fate induction. Genetic
interaction studies have suggested that Jun and JNK
signaling are involved in Frizzled (Fz)-mediated planar
polarity generation in the Drosophila eye. However, simple
loss-of-function analysis of JNK signaling components does
not show comparable planar polarity defects. To address
the role of Jun and JNK in Fz signaling, a
combination of loss- and gain-of-function studies has been used. Like Fz,
Jun affects the bias between the R3/R4 photoreceptor pair
that is critical for ommatidial polarity establishment.
Detailed analysis of jun- clones reveals defects in R3
induction and planar polarity determination, whereas gain
of Jun function induces the R3 fate and associated polarity
phenotypes. Affecting the levels of JNK
signaling by either reduction or overexpression leads to
planar polarity defects. Similarly, hypomorphic allelic
combinations and overexpression of the negative JNK
regulator Puckered causes planar polarity eye phenotypes,
establishing that JNK acts in planar polarity signaling. The
observation that Delta transcription in the early R3/R4
precursor cells is deregulated by Jun or Hep/JNKK
activation, reminiscent of the effects seen with Fz
overexpression, suggests that Jun is one of the transcription
factors that mediates the effects of fz in planar polarity
generation (Weber, 2000).
Jun, as a member of the AP-1 family, is activated by many
distinct extracellular stimuli and acts downstream of several
signaling pathways. Besides its
involvement in stress response, Jun has been implicated in the
control of proliferation, apoptosis, morphogenesis and cell fate
induction. In Drosophila, Jun is critical for the
process of dorsal closure in embryogenesis acting downstream
of the JNK module. It has also been implicated
in cell fate induction downstream of Ras/ERK signaling in the
eye. This analysis has shown that Jun also acts downstream of Fz in planar polarity
signaling in the eye. It is the first transcription factor implicated
in Fz/planar polarity signaling. Fz signaling also requires a
JNK (or related kinase) module, and thus in the
eye imaginal disc Jun acts downstream of both ERK and JNK.
How does Jun achieve a specific response in this context?
The S/T residues that are phosphorylated in Jun are the same
for both ERK and JNK. Thus, although differences in phosphorylation level
and/or preference for any of the serine/threonine target residues
cannot be excluded in vivo, differential phosphorylation is
unlikely to create specificity. A potential mechanism for
specificity might be provided by other transcription factors that
cooperate with Jun in the different processes. This is supported
by the observation that the sev-JunAsp (expression of a constitutively active Jun) phenotype is a composite
of two events, photoreceptor recruitment and ommatidial
polarity generation. These two effects can, however, be
separated by the reduction of specific interacting partners. In
the process of Ras/ERK signaling in photoreceptor induction
Jun interacts and synergizes with the ETS domain transcription
factor Pointed (Pnt). Pnt has been
characterized as a target of the ERK/Rl kinase in Drosophila
in all ERK-dependent processes analyzed. However, it has not been linked to any JNK-mediated process.
Removing one dose of pnt strongly suppresses the Ras/ERK-related
extra photoreceptor phenotype of sev-JunAsp, whereas
the polarity defects persist and thus are more prominent. This observation indicates that,
in the absence of normal Pnt levels, sev-JunAsp specifically
affects polarity, suggesting that the interaction with Pnt is
important for its role in the ERK-mediated induction. It is
likely that for its planar polarity function other specific
transcription factors provide the specificity cues (Weber, 2000).
Although all components of the JNK module tested genetically
interact with sev-Fz and sev-Dsh, analysis of existing loss-of-function
mutants did not show defects in planar polarity
establishment, suggesting a redundant role. Even null alleles of
the Drosophila homolog of JNKK hep have no effects on
planar polarity (Weber, 2000).
However, expression of a dominant negative (kinase dead) isoform of
Bsk interferes with planar polarity, giving rise to
typical polarity phenotypes, implying that Bsk and
JNK signaling are important in this process. Consistently,
homozygous mutant clones of the deficiency Df(2R)flp170B
that removes bsk and other neighboring loci (a deficiency considered to be a true null for bsk), show a mild
polarity phenotype in the eye, including the presence of
symmetrical ommatidia (Weber, 2000).
What are the redundant kinases in this process? Genetic
interaction analysis with sev-Msn (Misshapen expressed in a Sevenless pattern) has shown that, besides hep
and bsk, deficiencies affecting other MKKs and the Drosophila
p38a and p38b loci suppress the sev-Msn phenotype. This suggested that the p38 kinase module [related to JNK and has been shown to have (at least partially)
overlapping phosphorylation targets] might be responsible for the redundancy in this process. The
analysis with the dominant negative (DN) Bsk isoform and the
respective deficiencies suggests that the p38 kinase(s) are
contributing to this redundancy, because they enhance the DN-Bsk
phenotype in a manner very similar to that of the bsk deficiency. The
identification of specific mutant alleles of p38a/b and double
mutant analysis with bsk will be necessary to further clarify
this issue (Weber, 2000).
The available results indicate that the level of JNK/p38
signaling in planar polarity establishment is important, but that
the removal of a single kinase does not significantly affect this
level. In support, the observation that an allelic combination of
hep and puc hypomorphic alleles can give rise to planar
polarity eye phenotypes suggests that the balance
between negative and positive regulators of JNK and related
kinases is critical. Similarly, overexpression of the negative
JNK regulator Puc, a dual specificity phosphatase, causes typical polarity defects similar to
those of fz or dsh mutants. It is likely that this phosphatase
negatively regulates all JNK-related kinases and thus reduces
the overall signaling more than the lack of a single kinase (Weber, 2000).
In summary, these data indicate that the transcriptional
events downstream of Fz in R3 specification and chirality
establishment (e.g. regulation of Dl) are mediated by Jun. The
factors with which Jun is redundant in the imaginal discs are
not yet identified. It is possible that other members of the AP-1
family are also involved in planar polarity signaling, since they
are related to Jun and could dimerize with it via the leucine-zipper
motif. A potential candidate is Fos, because like Jun, Fos is
required downstream of JNK in the process of dorsal closure
in the embryo. Similarly, the ETS domain protein Yan acts as a
negative regulator in dorsal closure and is inactivated by JNK
in the process. However, these factors do not show informative
planar polarity phenotypes in clones and thus their
involvement in this process remains unclear. Although AP-1
and ETS family members are attractive candidates,
transcription factors belonging to other families cannot be
excluded in this context (Weber, 2000).
An examination was carried out to see whether directed
overexpression of TGF-ß activated kinase 1 in the eye imaginal disc of third instar
larvae (at the time of planar polarity Fz/JNK signaling) can
interfere with the correct establishment of planar polarity. To this
end UAS-Tak1 was expressed in photoreceptor precursors R3/R4
in the eye imaginal disc (under the sev-enhancer GAL4 driver: sev>Tak1).
This type of overexpression creates specific eye planar polarity
phenotypes with Fz, Dsh and other components of planar polarity signaling. Weak Tak1 expression (by rearing the flies at 18°C) causes a specific phenotype reminiscent of that caused by the components of planar polarity signaling, with polarity defects affecting both rotation and chirality, and also some loss of photoreceptors. This phenotype is already evident with the appropriate markers (e.g. svp-lacZ) at the time of planar polarity establishment in the third instar eye disc, indicating that it is a primary defect in polarity establishment, and not due to late differentiation defects (Mihaly, 2001).
The GOF sev>Tak1 phenotype provides a tool to test for genetic interactions with mutations in components of the Fz/planar polarity pathway and other signaling cascades. In such genetic interaction assays, it was found
that reducing the dosage of the JNK signaling components (hep,
bsk and D-jun) causes a strong suppression of the
sev>Tak1 phenotype. These
results are consistent with Tak1 acting upstream of the JNK module in
polarity signaling, and support the notion that Tak1 can act
generally upstream of JNK signaling (Mihaly, 2001).
During Drosophila oogenesis, the formation of the egg respiratory appendages and the micropyle require the shaping of anterior and dorsal follicle cells. Prior to their morphogenesis, cells of the presumptive appendages are determined by integrating dorsal-ventral and anterior-posterior positional information provided by the epidermal growth factor receptor (EGFR) and Decapentaplegic (Dpp) pathways, respectively. Another signaling pathway, the Drosophila Jun-N-terminal kinase (JNK) cascade, is essential for the correct morphogenesis of the dorsal appendages and the micropyle during oogenesis. Mutant follicle cell clones of members of the JNK pathway, including DJNKK/hemipterous (hep), DJNK/basket (bsk), and Djun, block dorsal appendage (DA) formation and affect the micropyle shape and size, suggesting a late requirement for the JNK pathway in anterior chorion morphogenesis. In support of this view, hep does not affect early follicle cell patterning as indicated by the normal expression of kekkon (kek) and Broad-Complex (BR-C), two of the targets of the EGFR pathway in dorsal follicle cells. Furthermore, the expression of the TGF-ß homolog dpp, which is under the control of hep in embryos, is not coupled to JNK activity during oogenesis. hep controls the expression of puckered (puc) in the follicular epithelium in a cell-autonomous manner. Since puc overexpression in the egg follicular epithelium mimics JNK appendages and micropyle phenotypes, it indicates a negative role of puc in their morphogenesis (Suzanne, 2001).
The making of a mature egg is a multistep process during
which the oocyte differentiates, grows, acquires polarity,
and is finally embedded into a shell secreted by the overlaying
follicle cells. During this maturation process, the activities of several signaling cascades are required and coordinated, with some of them, like the
Egfr pathway, being used reiteratively. The JNK pathway is
required during late oogenesis for the morphogenesis of the
DA and micropyle, thus adding a new player in the signaling
machinery underlying egg formation. Since the other
two MAPK pathways (ERK and p38) have also been shown
to be involved in oogenesis, the Drosophila egg chamber represents a paradigm for the study of multiple MAPK signaling pathways during development (Suzanne, 2001).
The outer follicular epithelium surrounding each oocyte
secretes the chorionic envelope to protect the mature egg
from external aggressions. During the late stages of its development, the follicular epithelium undergoes extensive morphogenesis in its anterior
region, resulting in the decoration of the egg with few
stereotyped structures. These include the DA, the operculum,
and the micropyle, which are all essential for the egg.
The micropyle allows sperm entry and fertilization, the
operculum provides an exit for the hatching larvae, and the
two DA serve as floating and breathing devices when the
egg is covered by liquid. Interestingly, the DA show an
extreme variation in their shape and number in different
Drosophila species and the Egfr pathway may provide
the molecular basis for this variability (Suzanne, 2001).
The analysis of hep, bsk, and Djun mutant clones indicates
that JNK pathway activity is crucial in the follicular
epithelium for DA morphogenesis. The observation of a
complete series of phenotypes, ranging from reduced,
'paddle-less' to completely nonelongated appendages, suggests
that the JNK pathway plays a role in the elongation
and shaping of these structures. As shown by the normal
expression of two targets of the ERK pathway, kek and BR-C, hep does not affect the patterning or development of the appendages during early and midoogenesis. It is proposed that the JNK pathway plays a previously unknown role in late oogenesis for appropriate morphogenesis of the DA and micropyle. The unique phenotype of JNK pathway mutants may thus provide a link between pattern formation and morphogenesis in the egg chamber (Suzanne, 2001).
hep and the JNK pathway lie downstream of both the Egfr and Dpp pathways in DA formation during late oogenesis. One interesting question is
whether or not JNK activation is directly mediated by the
ERK and/or Dpp pathways. Since both the ERK and JNK
pathways are required for appendage formation, it is tempting
to speculate that they may converge and their inputs
integrate at the molecular level. One good candidate for
such an integrating element is the AP-1 (activating
protein-1) transcription factor, whose levels of expression
and activity are regulated by both the ERK and JNK pathways in vertebrate cells. As their vertebrate counterparts, the Drosophila Djun and Dfos homologs are also part of the JNK pathway, and these factors may also interact with the ERK cascade in the eye. Although the level of Dfos protein is normal in hep mutant follicle cells, analyzing the pattern of AP-1 activation in the egg chamber will be of particular interest to understand the relative contributions of the two MAPK pathways to appendage morphogenesis (Suzanne, 2001).
It has been observed that ectopic ERK activation in the
posterior region of the egg can induce the formation of
appendage-like material. However, this material does not
fully elongate as normal appendages do, but remains very
rudimentary, as observed in hep, bsk and Djun mutant
clones. A possible explanation for this 'incompetence'
to normally elongate is that the JNK pathway may not be
activated or fully inducible in the posterior part of the egg,
due to the lack of some component(s) of the JNK pathway.
In this respect, it is worth noting that puc expression escapes hep control in the posterior part of the follicular epithelium (Suzanne, 2001).
The hep chorionic phenotype is accompanied by a loss of
puc expression in the anterior stretched and main body
follicle cells, suggesting that these cells are important for
JNK-dependent morphogenesis of the appendages. This is
supported by overexpression of puc in subsets of columnar
follicle cells. The use of a slboGAL4 line allows for the exclusion of
a role for centripetal cells in DA formation. These
observations suggest that anterior main body follicle cells,
including appendage precursor cells, and stretched cells,
require hep function for DA elongation. Morphogenesis of
the DA may thus require JNK activity in the two adjacent
epithelia (stretched and columnar). For micropyle formation,
the use of a slboGAL4 line has identified the centripetal
cells as the ones requiring JNK activity for normal micropyle
development. The absence of any obvious migratory
defect in mutant border and centripetal cells excludes the possibility that
micropyle shape defects are due to an early aberrant behavior
of these two cell types. As for the DA, it is proposed that the micropyle is assembled in two steps: a hep-independent step requiring border and centripetal cells during early migration, and a hep-dependent step that takes place during late stages (stage 11 onward) of oogenesis (Suzanne, 2001).
Epithelia are components of many different tissues,
which they shape and make functional through elaborate
morphogenesis. Different cellular mechanisms underlie the
movement of epithelia, including folding (gastrulation),
branching (tracheal development), or migration of entire
sheets (dorsal closure, imaginal discs morphogenesis,
wound-healing). One important goal is to identify the molecular mechanisms underlying these different behaviors, and understand how these are modulated to
contribute to the diversity encountered in developing animals.
One way to understand the basis of cell movement
diversity is to compare related processes controlled by a
single signaling cascade, like the JNK pathway. The comparison
of dorsal closure and imaginal disc morphogenesis,
which both are controlled by the JNK pathway in flies,
allows the proposal of a model for the morphogenesis of
symmetrical epithelia containing 'free margins'. In this model, the morphogenesis
or movement of bilateral epithelial sheets, like those taking
place during dorsal closure, is driven by the activation of
the JNK pathway in particular sites called margins. Interestingly,
these margins are morphologically distinguishable,
delineating two adjacent populations of cells: a columnar
epithelium and a stretched one. In flies, several tissues
show such an organization, including the lateral ectoderm
in embryos and the imaginal discs. Strikingly, JNK activity
in the egg chamber is essential for structures originating
near such a boundary between a columnar (the main body
or centripetal follicular epithelium) and a squamous epithelium
(stretched cells), and may share several features with
apparently different morphogenetic processes, like dorsal
and thorax closures. Based on the current understanding of
the JNK pathway in Drosophila, it is also tempting to
speculate that every epithelium showing a discontinuity
(i.e., the juxtaposition of a columnar and a stretched epithelium)
may use the JNK pathway for its morphogenesis (Suzanne, 2001).
All the processes involving the JNK pathway also require
a normal dpp activity, suggesting that these two pathways
are intimately linked during epithelial morphogenesis in
flies. During dorsal closure, but not during thorax closure, the JNK pathway controls the expression of dpp in leading edge cells. Interestingly, during oogenesis, dpp expression is not under the control of the JNK pathway, as it is during dorsal closure. Thus, based on the presence or the absence
of a transcriptional coupling between JNK and dpp, it is
possible to define two different types of epithelial morphogenesis.
In this respect, the way the JNK and dpp pathways
are set up in the ovary is more similar to the situation found
in imaginal discs. The study of JNK signaling in these
different but related processes in Drosophila thus represents
a unique system to study the molecular origin of
diversity in epithelial morphogenesis (Suzanne, 2001).
\
Eiger, the first invertebrate tumor necrosis factor (TNF) superfamily ligand that can induce cell death, was identified in a large-scale gain-of-function screen. Eiger is a type II transmembrane protein with a C-terminal TNF homology domain. It is predominantly expressed in the nervous system. Genetic evidence shows that Eiger induces cell death by activating the Drosophila JNK pathway. Although this cell death process is blocked by Drosophila inhibitor-of-apoptosis protein 1 (DIAP1, Thread), it does not require caspase activity. Genetically, Eiger has been shown to be a physiological ligand for the Drosophila JNK pathway. These findings demonstrate that Eiger can initiate cell death through an IAP-sensitive cell death pathway via JNK signaling (Igaki, 2002).
Many mammalian TNF superfamily proteins activate both the NF-kappaB and the JNK pathway, and activation of the latter pathway facilitates cell death (Davis, 2000). To examine whether Eiger activates the JNK pathway, the genetic interactions of Eiger with the components of the Drosophila JNK cascade were examined. The reduced-eye phenotype induced by Eiger is strongly suppressed in basket (bsk), a heterozygous mutant of Drosophila JNK. In addition, overexpression of a dominant-negative form of Bsk almost completely suppresses the eye phenotype. Moreover, heterozygosity at the hemipterous (hep) locus, which encodes Drosophila JNKK, suppresses the reduced-eye phenotype much as does bsk, and its hemizygosity (null background) rescues the phenotype almost completely. Furthermore, the co-expression of a dominant-negative form of dTAK1 TGF-ß activated kinase 1; Drosophila JNKKK) also rescues the Eiger-induced phenotype completely. Misshapen
Puckered (Puc) is a dual-specificity phosphatase, the expression of which is induced by the Drosophila JNK pathway to inactivate Bsk, so that puc expression can be used to monitor the extent of activation of the JNK pathway. To confirm that the JNK pathway is actually activated by Eiger, puc expression level was assessed in the eye disc of GMR>regg1GS9830 flies using a puc-LacZ enhancer-trap allele. The strong induction of puc-LacZ was observed in the region posterior to the morphogenetic furrow of the eye disc compared with the control eye disc. Furthermore, Western blot analysis with an anti-phospho-JNK antibody has revealed that Bsk is phosphorylated by Eiger overexpression. These genetic and biochemical data led to a model in which Eiger activates Msn, thereby triggering the JNK signaling pathway, sequentially stimulating dTAK1, Hep and Bsk. Using RT-PCR analysis, whether Eiger could stimulate the NF-kappaB pathway was tested; however, no obvious upregulation of the antimicrobial peptide genes, the target genes of the Drosophila NF-kappaB pathway, was detected (Igaki, 2002).
Two Drosophila tumor necrosis factor receptor-associated factors (TRAFs), Traf1 and Traf2, are proposed to have similar functions with their mammalian counterparts as a signal mediator of cell surface receptors. However, as yet their in vivo functions and related signaling pathways are not fully understood. Traf1 is shown to be an in vivo regulator of c-Jun N-terminal kinase (JNK) pathway in Drosophila. Ectopic expression of Traf1 in the developing eye induces apoptosis, thereby causing a rough-eye phenotype. Further genetic interaction analyses reveal that the apoptosis in the eye imaginal disc and the abnormal eye morphogenesis induced by Traf1 are dependent on JNK and its upstream kinases, Hep and TGF-ß activated kinase 1. In support of these results, the Traf1-null mutant shows a remarkable reduction in JNK activity with an impaired development of imaginal discs< and a defective formation of photosensory neuron arrays. In contrast, Traf2 was demonstrated as an upstream activator of nuclear factor-kappaB (NF-kappaB). Ectopic expression of Traf2 induces nuclear translocation of two Drosophila NF-kappaBs, Dif and Relish, consequently activating the transcription of the antimicrobial peptide genes diptericin, diptericin-like protein, and Drosomycin. Consistently, the null mutant of Traf2 shows immune deficiencies in which NF-kappaB nuclear translocation and antimicrobial gene transcription against microbial infection were severely impaired. Collectively, these findings demonstrate that Traf1 and Traf2 play pivotal roles in Drosophila development and innate immunity by differentially regulating the JNK- and the NF-kappaB-dependent signaling pathway, respectively (Cha, 2003).
To investigate the consequences of ectopic expression of Traf1 in the developing Drosophila eye, Traf1 was overexpressed by using an eye-specific gmr-GAL4 driver. The eyes of adults carrying one copy each of both gmr-GAL4 and Traf1 show a rough-eye surface with disorganized arrays of ommatidia, whereas the eyes of flies carrying either one copy of gmr-GAL4 or one copy of Traf1 alone appear normal. Examination of the retinal sections of adults carrying both gmr-GAL4 and Traf1 reveals the number of ommatidia to be reduced and the number and shape of the photoreceptor cells in each ommatidium also to be abnormal, compared to the controls, which carry only the gmr-GAL4 driver (Cha, 2003).
When two copies of Traf1 were overexpressed in the eye, it displayed a more severe phenotype and a reduced number of ommatidia, resulting in a size reduction of the compound eye, and some ommatidia were fused with each other. In contrast, ectopically expressed Traf2 had no effect on the eye development; ommatidial array, bristles, and compound eye size were all found to be normal (Cha, 2003).
To determine which signaling pathway is activated and induces malformation of the optic system by ectopic expression of Traf1 in vivo, the genetic interactions were tested between mutants of various signaling pathways and a Traf1-overexpressing line (gmr>Traf1/+). Included in this screen were UAS lines that activate extracellular signal-regulated kinase (ERK), p38 MAP kinase, and JNK signaling pathway, respectively. Among the various overexpression lines tested, only Hemipterous (Hep; Drosophila homolog of MKK7 encoded by hemipterous [hep]) and Basket were found to interact genetically with Traf1. The UAS-hep or UAS-bsk itself under the control of gmr-GAL4 driver had no effect on eye morphogenesis. Coexpression of Bsk with Traf1 increased the disturbance of the ommatidial array in the compound eye in comparison to the eye phenotype resulting from one copy overexpression of Traf1. In addition, when Hep was coexpressed with Traf1, the number of ommatidia and the size of the compound eye were reduced more dramatically, which is very similar to the Traf1 two-copy expression phenotype. To further examine whether Traf1 signaling is mediated by Hep, Traf1 was expressed under a hemizygous hep mutant background. As a result, the abnormal ommatidial array of the compound eye was recovered to the level of the wild-type eye (Cha, 2003).
Interestingly however, Traf2, did not display any interaction with the ERK or the p38 MAP kinase pathway components, nor even with JNK pathway components. Tests were performed to see whether Traf2 exerts its effect on the eye development by interacting with Traf1. Coexpression of Traf2 with Traf1 in the Drosophila compound eye did not alter the rough-eye phenotype caused by a sole overexpression of Traf1. These results strongly support the view that Traf1 can activate the JNK signaling cascade in vivo and that Traf2 is not correlated with Traf1 signaling at all, at least during eye development (Cha, 2003).
Based upon the result that Traf1 is involved in JNK signaling, attempts were made to find out the signaling components between Traf1 and Hep by genetic interaction studies. Various kinases, such as Misshapen (msn), Slipper (slpr), and Drosophila Transforming growth factor ß-activated kinase 1 (Tak1), are known to be the upstream kinases for Hep in the eye development. Among these kinases, Tak1 synergistically increased the roughness of the compound eye surface and also reduced the eye size when coexpressed with Traf1. Moreover, Tak1-null mutation (Tak11), which has no effect on the eye morphology, is able to block the rough-eye phenotype caused by Traf1 overexpression. These data suggest that Traf1 activates the JNK signaling pathway via Tak1 and Hep (Cha, 2003).
In order to further confirm that Traf1 activates the JNK kinase signaling pathway at a molecular level, JNK activity in the eye discs was examined by two different experimental approaches -- an immunohistochemical assay with anti-phospho-specific JNK antibody and a puckered-LacZ reporter assay. JNK phosphorylation is highly induced by overexpression of Traf1 compared to the control. In addition, expression of puckered (puc), a well-known downstream target of JNK, is also highly induced by Traf1 compared to the control. Collectively, the results clearly demonstrated that Traf1 activates the JNK signaling pathway in vivo (Cha, 2003).
Apoptosis can be induced by the activation of the JNK pathway. Because ectopic coexpression of Traf1 with Hep has a synergistic effect on the reduction of ommatidia number and eye size, the rough-eye phenotype induced by Traf1 overexpression seemed to be a result of the Hep/JNK signaling-dependent apoptotic cell death. Therefore, whether overexpression of Traf1 can induce apoptosis in the eye disc cells was investigated by using TUNEL assay. In the discs of wild-type third-instar larvae, there are few apoptotic cells. In contrast, the eye imaginal discs from transgenic flies overexpressing Traf1 reveal a highly increased number of apoptotic cells in the region posterior to the morphogenetic furrow in a gene dosage-dependent manner. However, ectopic expression of Traf2 failed to induce apoptotic cell death, which is consistent with its inability to activate the JNK pathway (Cha, 2003).
Since hemizygous hep mutation (hep1/Y) is sufficient to suppress the rough-eye phenotype caused by Traf1 overexpression, whether hep mutation can inhibit the Traf1-induced apoptotic cell death in the eye imaginal discs was examined. When TUNEL assay was performed against the eye discs of hep1/Y; gmr>Traf1/± larvae, the number of apoptotic cells was dramatically decreased to almost wild-type levels. These results prove that Traf1 overexpression can activate the Hep/JNK signaling pathway and consequently induce apoptosis (Cha, 2003). Traf1ex1, a loss-of-function allele for Traf1 gene, was generated through imprecise excision of the P-element in EP(2)0578 fly. RT-PCR analysis clearly demonstrate that the homozygous Traf1ex1 mutant fails to produce Traf1 mRNA, indicating that Traf1ex1 is a null allele for Traf1. The endogenous puckered transcription level was examined in the mutant larvae by RT-PCR analysis; the amount of puckered gene transcript was severely decreased in Traf1ex1 mutant in comparison to wild-type larvae. This strongly implies the reduced JNK activity in Traf1ex1 mutant (Cha, 2003).
In order to confirm this, the genetic interaction between the Traf1-null fly and a transgenic fly for JNK (ap>JNKDN) was examined by observing the thorax closure phenotype. Thorax closure, the joining of the parts of the two wing imaginal discs during metamorphosis, is tightly controlled by the Drosophila JNK signaling pathway. When the activity of JNK pathway is downregulated by expressing a dominant-negative form of JNK on the thorax in ap>JNKDN flies, the joining process is impaired and a cleft is formed at the dorsal midline in a gene dosage-dependent manner. Strikingly, a reduction of Traf1 gene dosage in heterozygous Traf1ex1 dramatically enhances the thorax closure defect in ap>JNKDN flies by expanding the cleft and also disrupting its notum structure, suggesting that Traf1 mutation leads to a greater reduction of the endogenous JNK activity in ap>JNKDN flies. These data strongly support the critical roles of Traf1 in Drosophila development by positively modulating JNK signaling activities (Cha, 2003).
It is concluded that Traf1 can specifically activate the JNK signaling cascade. This conclusion is based on four lines of evidence: (1) ectopic expression of Bsk, Hep, or Tak1 with Traf1 exerts a highly synergistic effect on the rough-eye phenotype of Traf1 overexpressing flies; (2) disruption of hep or Tak1 function sufficiently suppresses the Traf1-induced eye defects; (3) histochemical analysis with either an anti-phospho-specific JNK antibody or the puckered-LacZ reporter system provides direct molecular evidence that Traf1 can induce phosphorylation and consequent activation of JNK; (4) Traf1 deficiencies in Traf1ex1 mutants generate the same phenotypes detected in the loss-of-function mutants of the JNK signaling pathway, such as increased thorax closure defects and reduced puckered transcription (Cha, 2003).
Changes in the genetic makeup of an organism can extend lifespan significantly if they promote tolerance to environmental insults and thus prevent the general deterioration of cellular function that is associated with aging. This study introduces the Jun N-terminal kinase (JNK) signaling pathway as a genetic determinant of aging in Drosophila. Based on expression profiling experiments, it is demonstrated that JNK functions at the center of a signal transduction network that coordinates the induction of protective genes in response to oxidative challenge. JNK signaling activity thus alleviates the toxic effects of reactive oxygen species (ROS). In addition, flies with mutations that augment JNK signaling accumulate less oxidative damage and live dramatically longer than wild-type flies. This work thus identifies the evolutionarily conserved JNK signaling pathway as a major genetic factor in the control of longevity (Wang, 2003).
JNK phosphorylates a variety of transcription factors and enhances their transcriptional activation potential. Thus, insight into the biological consequences of stress-activated JNK signaling might be gained by analyzing the relevant downstream genetic programs. Drosophila was chosen as a model organism for such studies, since its JNK pathway is genetically very tractable. The multiplicity of homologous mammalian kinases that are functionally, at least partially, redundant (three JNK genes produce at least ten protein isoforms), has impeded similar analyses in mammals. The genomic response to JNK signaling has been mapped in the Drosophila embryo using serial analysis of gene expression. Among the genes induced in embryos with increased JNK signaling, a group was identified with tentative functions in cellular stress responses as well as several genes that are known to be activated in response to oxidative damage. In an independent experiment, similar genes were found to be upregulated in response to JNK signaling in differentiating photoreceptors. These findings suggested that JNK signaling activates a gene expression program that confers tolerance to oxidative stress in a variety of cell types. To test this hypothesis, the expression was monitored, in the adult fly using quantitative real-time RT-PCR, of four representative genes (hsp68, gstD1, fer1HCH, and mtnA), which were identified as JNK dependent in the SAGE experiments. The induction of the respective mRNAs in response to oxidative stress, artificially brought on by treatment with the drug paraquat, was measured in wild-type flies and in hemizygotes for hep1, a hypomorphic allele of the Drosophila JNKK gene, hemipterous (hep). Paraquat, a compound widely used to apply oxidative stress to cells and organisms, leads to continuous intracellular generation of O2.- radicals. It efficiently activates JNK in the fly, as indicated by the transcriptional activation of puckered (puc), one of the prototypical target genes of JNK signaling in Drosophila. puc encodes a JNK-specific phosphatase that downregulates the pathway, thus establishing a negative feedback loop. RT-PCR data show that JNK signaling is required for the induction of the four tested genes in response to oxidative stress, supporting the notion that flies react to oxidative challenge with a protective gene expression program dependent, at least in part, on JNK signal transduction (Wang, 2003).
To examine the relevance of JNK signaling for the sensitivity of the organism to oxidative stress, adult flies were exposed to paraquat for a prolonged period of time and their survival was monitored. Compared to wild-type animals, flies with decreased JNK signaling potential (hemizygotes for hep1, or heterozygotes for a hypomorphic allele of the Drosophila JNK gene basket, bsk2) were more sensitive to moderate doses of paraquat. Conversely, flies gained resistance to paraquat when signal flow through the kinase cascade was promoted by overexpression of Bsk or Hep. Similarly, boosting JNK signal transduction by reducing the gene dose of puc, conferred strong paraquat resistance in a hep- and bsk-dependent fashion. Flies heterozygous for puc exhibit elevated levels of JNK activity, as inferred by the dosage sensitivity of JNK-mediated apoptotic phenotypes in the developing wing, as well as by rescue of developmental defects normally observed in flies carrying hep and kay mutations. Constitutive overexpression of one of the identified JNK-inducible stress response genes, Hsp68, also protects flies against oxidative stress, suggesting that JNK's downstream genetic program mediates the observed protection. The observed differences in sensitivity to paraquat were not due to feeding abnormalities or a general tolerance to toxic compounds of the tested genotypes, since they are similarly sensitive to G418 toxicity (Wang, 2003).
Tissue-specific overexpression of superoxide dismutase (SOD) in motorneurons increases the resistance to oxidative stress and extends the lifespan of Drosophila. This result suggests neurons as the 'weakest link' in the organism's tolerance to oxidative insults and as a cell type in which protective mechanisms would be most critical. To investigate whether JNK signaling in neurons could play a role in such mechanisms, fly strains were examined in which Hep overexpression was directed either to the nervous system or to muscle tissue in an RU486-inducible manner (using the 'gene-switch Gal4' driver). The toxicity of paraquat was reduced significantly when Hep was expressed in the nervous system (ELAV Gal4 drives expression in all cells of the peripheral and central nervous systems but not when it was expressed in the musculature). This result highlights a specific function of JNK signaling in the protection of neurons against oxidative stress. While it cannot be ruled out that JNK may also act protective in nonneuronal cells (for instance in muscle cells), such protection seems not to be sufficient for the organism's survival, indicating that protection of neurons is critical (Wang, 2003).
Importantly, the inducibility of the JNK effect by RU486 in this system rules out variations in the genetic background as an explanation for differences in paraquat sensitivity (Wang, 2003).
According to the free radical theory of aging, one genetic determinant for the lifespan of an organism is its sensitivity to oxidative stress. It was asked whether the protection against oxidative damage that is brought about by an increase in JNK signaling potential might be sufficient to extend Drosophila's life expectancy. Flies heterozygous for puc were examined to test this hypothesis, since the experiments demonstrated that the tolerance of flies to oxidative stress increases with decreasing gene dose of puc. Flies heterozygous for either one of two different loss-of-function alleles of puc (pucA251.1 or pucE69) showed dramatic extensions of median and maximum life expectancy compared to wild-type flies and to flies of an isogenic control strain. The difference in the degree of lifespan extension by the two alleles correlates well with their described allelic strength. The results thus suggest a direct relationship between the decrease of Puc activity in the mutants and the resulting lifespan extension. Since biochemical and genetic data indicate that the activity of Puc is limited to the JNK signaling pathway (as opposed to other MAPK pathways, the lifespan extension in puc mutants is likely to be caused by higher levels of JNK signaling. The requirement for a functional JNK pathway in the longevity of puc mutants was tested directly by comparing the lifespan of pucE69 heterozygous males in a wild-type background to pucE69 heterozygotes in a hep1 hemizygous backgound. Heterozygosity for puc leads to an only modest increase in mean and maximum lifespan of hep1 hemizygous flies, indicating that a functional JNK cascade is required for efficient lifespan extension in puc mutants. These results strongly support the notion that the longevity phenotype observed in puc mutants is due to an increase in JNK signaling activity (Wang, 2003).
The genomic experiments suggested that elevated JNK signaling activity causes higher basal levels of protective genes. Whether constitutive overexpression of one of the identified JNK target genes, hsp68, would be sufficient to extend lifespan of Drosophila was tested. In agreement with the hypothesis, small but significant increases were observed in mean and maximum lifespan in flies that overexpress hsp68 compared to isogenic wild-type controls. This experiment is consistent with observations that increased expression of chaperones extend the lifespan of Drosophila (Wang, 2003).
Providing higher JNK signaling levels in neuronal tissue is sufficient to increase oxidative stress tolerance. To test whether neuronal-specific protection would also be sufficient to extend lifespan of the organism, survival was monitored of flies that overexpress Hep constitutively in neuronal tissue under the control of ELAV Gal4. Neuronal overexpression of Hep extended lifespan significantly, indicating that the level of JNK activity in neuronal tissue determines not only the fly's oxidative stress tolerance, but also its lifespan. Importantly, these results confirm, independently of puc mutations, that JNK signaling promotes longevity (Wang, 2003).
Several genetically determined changes in physiology have been associated with extended lifespan in Drosophila. Such changes include reduced reproductive activity, dwarfism, delays in development, as well as stress tolerance. Whether the JNK pathway might affect parameters indicative of such physiological changes was examined. puc heterozygotes and wild-type controls exhibit roughly equivalent sizes (as determined by body weight), reproductive activities (fecundity), as well as developmental timing. In contrast, oxidative stress tolerance and tolerance to starvation differ markedly between wild-type and puc heterozygous flies. Importantly, 10-day-old puc heterozygotes contain significantly decreased levels of oxidized proteins. The quantity of protein oxidation products, such as polypeptides carrying carbonyl groups, is a measure for the accumulated oxidative damage suffered by an organism. Taken together, these results suggest that increased JNK signaling is sufficient to reduce oxidative damage throughout the lifetime of a fly and that this beneficial effect may be the cause of the longevity phenotype of gain-of-function mutants for this signaling pathway (Wang, 2003).
This work identifies the JNK signaling pathway as a significant genetic determinant of longevity in Drosophila. Activation of JNK in response to oxidative challenge and to other environmental insults has been well described in a number of model systems and was proposed to trigger the expression of genes that could mediate protective functions on the organism at least in certain cell types. Against this backdrop, resulting in the prediction that JNK signaling would protect the organism from oxidative challenge, it may seem surprising that, until now, no evidence has been produced that links JNK signaling to an extended lifespan. Evidently, experimental limitations of mammalian systems, including increased functional complexity and genetic redundancy, have precluded clear-cut experiments to address this question (Wang, 2003).
While unidentified functions of JNK signaling that might be relevant to the aging process cannot be excluded, it seems plausible (and the free radical theory of aging would predict) that the observed protection against oxidative insults decisively delays aging and thus causes the longevity phenotype of puc heterozygotes. Earlier observations, as well as the current experiments, support this notion: Hsp70, and its JNK-inducible relative Hsp68, have been shown to extend lifespan when overexpressed in Drosophila. These chaperones have been implicated in oxidative stress resistance and may have repair functions downstream of JNK signaling. The reduced level of oxidative damage in aging puc heterozygotes further supports this view. JNK signaling thus emerges as an evolutionarily conserved gene-regulatory network that limits oxidative damage in the organism and its impact on aging (Wang, 2003).
Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. C-terminal Src kinase (Csk) encodes a critical negative regulator of Src family kinases. The Drosophila melanogaster Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Genetic analysis indicates that the dCsk–/– overgrowth phenotype results from activation of Src, Jun kinase, and STAT signal transduction pathways. In particular, blockade of STAT function in dCsk mutants severely reduced Src-dependent overgrowth and activated apoptosis of mutant tissue. The data provide in vivo evidence that Src activity requires JNK and STAT function (Read, 2004).
Partial reduction of Src64B, Src42A, or Btk29A activity suppresses the dCsk–/– phenotype, providing functional data to support the view that the imaginal disc overgrowth, defective larval and pupal development, and lethality of dCsk–/– mutants results from inappropriate activation of the Src-Btk signal transduction pathways. Mutations in Btk29A more strongly suppress dCsk phenotypes than either Src42A or Src64B mutations, perhaps reflecting that (1) Src paralogs act redundantly to each other in Drosophila as in mammals and (2) Btk29A has been shown to act downstream of Src family kinases (SFK) in flies and in mammals. In vivo evidence is provided that loss of Csk function hyperactivates Btk to drive cell cycle entry in development, demonstrating that Tec-Btk family kinases are critical to SFK-mediated proliferation. The data raise the possibility that partial reduction of Tec-Btk kinase activity could reduce proliferation in other cellular contexts in which overgrowth is driven by hyperactivated SFKs, such as in colon tumors (Read, 2004).
Tissue culture models show that constitutively activated SFK signal transduction modulates the function of numerous downstream effector molecules and pathways. Using a loss-of-function approach to identify effectors that mediate the dCsk overgrowth phenotypes, some of these pathways were not implicated in dCsk function. For example, SFKs up-regulate the SOS-Ras-ERK pathway in multiple tissue culture studies and Drosophila overexpression models. However, although dRas1 signaling is active throughout retinal development, reduced dEGFR, Sos, and Jra (c-jun) gene dosage failed to affect the dCsk phenotype. dCsk mutations also failed to modify a hypermorphic allele of dEGFR. Levels of doubly phosphorylated and activated ERK appeared unaltered in dCsk–/– tissue. Moreover, the dCsk phenotype failed to phenocopy defects caused by Ras pathway hyperactivation. For example, constitutively active dRas1 causes increased cell size and patterning defects in the developing imaginal discs, defects that were not observed in dCsk mutant eye tissues. These data argue that not every signal transduction pathway implicated in SFK tissue culture models necessarily functions as predicted within a developing epithelial tissue (Read, 2004).
These studies emphasize the importance of two signaling pathways in dCsk and SFK function. Since certain defects in dCsk–/– animals, such as a split notum, resembled those of hep (JNKK) mutants, it is suspected that JNK pathway activity is involved in dCsk function. Phenotypic and FACS analysis established that reduced JNK (bsk) function suppresses the phenotypes and cell cycle defects caused by loss of dCsk. These results confirm studies indicating that JNK functions downstream of the Src-Btk pathway in Drosophila and mammalian tissue culture cells. Components of the JNK pathway are required for Src-dependent cellular transformation, but the exact role of JNK in these cells is unknown. Importantly, the data show that the JNK pathway mediates proliferative responses to Src signaling in vivo. Further work will be needed to precisely understand its role in proliferation (Read, 2004).
Genetic studies also highlight the importance of the Jak/Stat signal transduction pathway. dCsk proves a negative regulator of Jak/Stat signaling; for example, dCsk mutant tissues show up-regulation of Stat92E protein, a hallmark of Jak/Stat activation in Drosophila. Stat92E, the sole Drosophila STAT ortholog, is most similar to mammalian STAT3. In mammalian cells, Src directly phosphorylates and activates STAT3 and STAT3 function and activation are required for Src transforming activity. Conversely, overexpression of Csk blocks STAT3 activation in v-Src transformed fibroblasts. Activating mutations in STAT3 can also promote oncogenesis in mice. However, the physiological significance of these interactions within developing epithelia remains unclear (Read, 2004).
dCsk; Stat92E double mutant clones reveal that blockade of STAT function in dCsk mutants severely reduces Src-dependent overgrowth and promoted apoptosis of mutant tissue. dCsk–/–; Stat92E–/– EGUF adult eyes (the EGUF method produces genetically mosaic flies in which only the eye is exclusively composed of cells homozygous for the mutation) are nearly identical to phenotypes caused by overexpression of Dacapo, the fly ortholog of the cdk inhibitor p21, and PTEN, a negative regulator of cell proliferation and growth. Importantly, removing Stat92E function in dCsk mutant tissue led to a synthetic small eye phenotype and did not simply rescue the dCsk–/– proliferative phenotype. This outcome distinguishes Stat92E from mutations in Src64B, Btk29A, or bsk, which rescue dCsk-mediated defects toward a normal phenotype. The loss of tissue in dCsk–/–; Stat92E–/– clones indicates that Src-Btk signaling provokes apoptosis in the absence of Stat92E function. Consistent with this interpretation, reduced Btk29A function rescued the dCsk–/–; Stat92E–/– EGUF phenotype to a more normal phenotype, demonstrating that the reduced growth and increased apoptosis observed in the dCsk–/–; Stat92E–/– tissues is indeed Src-Btk pathway dependent (Read, 2004).
The data suggest the existence of a Src-dependent proapoptotic pathway that is normally suppressed by STAT. One possible component of this pathway is JNK, given that JNK signaling is an important activator of apoptosis in both flies and mammals. Perhaps Src-dependent hyperactivation of Bsk (JNK) in dCsk–/–; Stat92E–/– tissue contributes to cell death in the absence of proliferative and/or survival signals provided by Stat92E. However, a number of other candidate pathways may also mediate this response. The further characterization and identification of these pathways may have important implications for interceding in Src-mediated oncogenesis (Read, 2004).
Together, these observations indicate that, in tissue that contains hyperactive Src or reduced Csk, blocking STAT function is sufficient to trigger apoptosis and decrease proliferation in the absence of any further mutations or interventions. Reduced STAT3 function can promote apoptosis within breast and prostate cancer cells that show elevated SFK activity, but the molecular pathways driving apoptosis in these cells are unknown. These cells may require survival signals provided by STAT3 to counteract apoptosis due to chromosomal abnormalities or other defects. Alternatively, these cells may die because of proapoptotic signals provided by hyperactive SFKs in the absence of STAT3 function. The data argue that the latter may be true, which suggests the intriguing possibility that therapeutic blockade of STAT function in tumors with activated Src may actively provoke Src-dependent apoptosis and growth arrest in tumor tissues (Read, 2004).
Cell adhesion and migration are dynamic processes requiring the coordinated action of multiple signaling pathways, but the mechanisms underlying signal integration have remained elusive. Drosophila embryonic dorsal closure (DC) requires both integrin function and c-Jun amino-terminal kinase (JNK) signaling for opposed epithelial sheets to migrate, meet, and suture. PINCH (Steamer duck), a protein required for integrin-dependent cell adhesion and actin-membrane anchorage, is present at the leading edge of these migrating epithelia and is required for DC. By analysis of native protein complexes, RSU-1, a regulator of Ras signaling in mammalian cells, has been identified as a novel PINCH binding partner that contributes to PINCH stability. Mutation of the gene encoding Drosophila RSU-1 results in wing blistering in Drosophila, demonstrating its role in integrin-dependent cell adhesion. Genetic interaction analyses reveal that both PINCH and RSU-1 antagonize JNK signaling during DC. These results suggest that PINCH and RSU-1 contribute to the integration of JNK and integrin functions during Drosophila development (Kadrmas, 2004).
To determine if PINCH contributes to DC, its localization was examined in stage 14 embryos. PINCH and ß-PS integrin colocalize in both the LE and the amnioserosa, consistent with PINCH's established role as an integrin effector. The amnioserosa is an extraembryonic tissue present on the dorsal surface of the embryo. Since it has been established that coordinated signaling between the amnioserosa and migrating epithelium is key to formation of LE focal complexes, PINCH could exert an effect in the LE epithelium, the amnioserosa, or both tissues. stck homozygous mutant embryos rescued with a PINCH:GFP transgene under the control of the endogenous PINCH promoter display PINCH-GFP at the LE of the advancing epithelial sheets. Within the LE, PINCH is precisely localized to areas of active phosphotyrosine signaling at triangular nodes corresponding to apical adherens junctions (Kadrmas, 2004).
Zygotic stck mutants proceed normally through DC with complete lethality arising at the embryo-to-larval transition. When maternal PINCH contribution is eliminated, only 12% of cuticles have wild-type morphology. Dorsal puckers and dorsal holes characteristic of aberrant DC are observed at a 36% and 23% frequency, respectively, indicating that maternally inherited PINCH is a key contributor to the process of DC. Moreover, in the absence of maternal PINCH, epithelial defects are observed in ventral patterning and head involution, indicating that PINCH may have additional functions in the developing embryo. Cuticles from embryos lacking both maternal and zygotic PINCH have the same array of phenotypes (Kadrmas, 2004).
PINCH is composed of five LIM domains, each of which could serve as a protein-binding interface. The SH2-SH3 adaptor protein, Nck2, has been reported to interact with mammalian PINCH. This association is intriguing because the Drosophila homologue of Nck2, Dreadlocks, interacts directly with Misshapen (Msn), a MAP4K in the JNK signaling cascade. As with other components of the JNK pathway, null mutations in msn result in embryonic lethality due to failure of DC. Although no direct binding of PINCH to Dreadlocks was observed in Drosophila, this study uncovered a link between PINCH's role in DC and the JNK cascade by testing for genetic interaction between stck and msn. Reduction of PINCH protein levels by introduction of a single copy of the loss-of-function allele, stck18, into the msn102 homozygous null background allows partial restoration of DC, suggesting that PINCH functions as a negative regulator of JNK signaling (Kadrmas, 2004).
Puckered (Puc) is a JNK phosphatase whose expression is up-regulated in response to JNK activation, setting up a negative feedback loop. During DC, JNK-regulated expression of a Puc-LacZ fusion reporter is restricted to the LE cells. In embryos lacking maternal PINCH, expression of the Puc-LacZ fusion protein is disorganized and present in an expanded number of cells, including those beyond the LE border. This phenotype is similar to Puc-LacZ expression observed in puc loss-of-function mutants and further supports a role for PINCH in the negative regulation of the JNK cascade (Kadrmas, 2004).
Thorax closure is a post-embryonic developmental process with features common to DC, including migration of epithelial sheets and a dependence on JNK signaling. Within the wing disc, cells of the stalk region are functionally similar to LE cells during DC. These cells comprise the eventual fusion site for adjacent imaginal discs and are active in JNK signaling. Spatially restricted JNK signaling in the stalk of wing disc can be visualized via a Puc-LacZ reporter, and PINCH expression overlaps with Puc-LacZ in this area of active JNK signaling. Therefore, as in DC, PINCH is properly positioned to act as a regulator of the JNK cascade (Kadrmas, 2004).
Although msn null mutations are embryonic lethal due to DC failure, flies homozygous for the hypomorphic allele msn3349 are semi-viable and a large proportion of the eclosing adults have thorax closure defects. These observations underscore the similarities between thorax closure and DC. In a stck18 heterozygous background, a greater percentage of msn3349 homozygotes are able to eclose, supporting the hypothesis that PINCH is a negative regulator of the JNK pathway in both dorsal and thorax closure (Kadrmas, 2004).
Drosophila PINCH was purified in complex with its binding partners using tandem affinity purification (TAP)–tagged PINCH (TAP-PINCH). stck homozygous mutant embryos rescued with a TAP:PINCH transgene driven by the endogenous stck promoter to wild-type levels afford material for purification of soluble, cytoplasmic TAP-PINCH complexes in the absence of endogenous PINCH protein. Three partners that copurified stoichiometrically with TAP-PINCH from embryos, as well as in complex with TAP-PINCH from cultured Drosophila S2R+ cells, were identified by mass spectrometric analysis. Consistent with what is observed in mammalian cells, ILK copurified with PINCH. The Drosophila homologue of the parvin/actopaxin family of proteins, CG32528, is also present in PINCH protein complexes. Parvin is known to bind ILK and actin in mammalian systems, but the isolated Parvin/ILK/PINCH complexes are the first to be described in Drosophila. Additionally, a novel 31-kD protein was identified as Drosophila CG9031. The CG9031 protein is 55% identical and 74% similar to human RSU-1, a leucine-rich repeat containing protein first identified as a suppressor of cell transformation by v-Ras and subsequently implicated in regulation of MAP kinase signaling, specifically the JNK and ERK cascades, when overexpressed in cultured cells. Despite its potent ability to act as a tumor suppressor, little is known about the mechanism of action of RSU-1. Its partnership with the PINCH protein allows placement of RSU-1 in a molecular pathway that is linked to integrins (Kadrmas, 2004).
To assess the specificity and nature of the interaction between PINCH and RSU-1, domain-mapping studies were performed in cell culture and in yeast two-hybrid assays. Drosophila RSU-1 copurifies with full-length His-tagged PINCH, but not with a truncated His-tagged PINCH containing only LIM1–3, confirming the specificity of the interaction and suggesting LIM4 and/or 5 is the site of binding. ILK, which binds LIM1 of PINCH, copurifies with both full-length and the truncated LIM1–3 version of His-tagged PINCH, serving as a positive control. Both PINCH and ILK are copurified with His-tagged RSU-1. Moreover, endogenous PINCH and RSU-1 can be coimmunoprecipitated. The site of RSU-1 binding to PINCH was further mapped using yeast two-hybrid analysis. Only cells expressing LIM5 bait/RSU-1 prey activated all three reporters, indicating LIM5 is the site of RSU-1 binding. Consistent with the view that they interact in vivo, PINCH:GFP and RSU-1 are prominently colocalized at integrin-rich muscle attachment sites in Drosophila embryos (Kadrmas, 2004).
Drosophila RSU-1, which displays seven leucine-rich repeats with high sequence similarity to small GTPase regulators, is encoded by the CG9031 locus. A P-element insertion allele was characterized that disrupts the RSU-1 coding sequence. Flies homozygous for this mutation within CG9031 are viable and fertile, and lack RSU-1 protein as indicated by Western analysis with multiple anti-RSU-1 antibodies. PINCH and RSU-1 are both expressed in larval wing discs and similar to stck wing clones, the mutation within CG9031 produces flies with wing blisters at 60% penetrance. These data are consistent with PINCH and RSU-1 acting in concert to support integrin-dependent adhesion. The CG9031 gene was named icarus (ics) after the son of Daedalus who had unstable wings (Kadrmas, 2004).
Although elimination of RSU-1 function does not result in dorsal or thorax closure defects, the role of RSU-1 in these processes was evaluated by testing for genetic interactions between ics and msn. Similar to what occurs with reduction of stck dosage, homozygous mutation of ics suppresses DC defects observed in msn102 mutant embryos. Absence of RSU-1 also increases eclosure rates of msn3349 hypomorphs and completely suppresses the thorax defects present in msn3349 animals, suggesting that like PINCH, RSU-1 can function as a negative regulator of JNK signaling. To confirm that the suppression of msn DC defects by ics mutation is mediated by the JNK signaling cascade, RSU-1 was eliminated in basket (bsk) embryos that lack zygotic JNK, the terminal kinase in this cascade. Homozygous ics mutation suppresses the DC defects of bsk1 mutants, confirming that ics loss-of-function mutations affect DC by influencing the JNK cascade. Moreover, wing discs isolated from ics mutants display a 30% increase in active phospho-JNK relative to wild type, providing direct biochemical confirmation that RSU-1 influences JNK activation state in vivo. Although no localized accumulation of RSU-1 during DC was detected, RSU-1 is readily detected by Western analysis in stage 13 embryos that are undergoing DC. Thus, the temporal pattern of RSU-1 expression is consistent with genetic results that highlight its role in regulation of JNK-dependent morphogenesis (Kadrmas, 2004).
Analysis of PINCH and RSU-1 levels in wild-type versus stck or ics mutant embryos provided insight into the physiological significance of their association. In embryos mutant for both maternal and zygotic stck, RSU-1 is dramatically reduced relative to wild-type levels. Likewise, in ics embryos, PINCH levels are also decreased. These observations suggest that PINCH and RSU-1 are reciprocally dependent on each other for maximal expression and/or stability. The mechanism for coordinate regulation of RSU-1 and PINCH remains to be determined. Because the phenotypes associated with loss of RSU-1 represent a subset of stck phenotypes, the processes disturbed in ics mutants may be exquisitely sensitive to PINCH levels. Alternately, RSU-1 may have functions that are independent of its role in PINCH stabilization (Kadrmas, 2004).
The data are consistent with a model in which PINCH could modulate JNK signaling in two distinct ways. (1) PINCH is present at areas where JNK is active and antagonizes JNK signaling. This behavior is reminiscent of Drosophila Puc, a phosphatase regulator of the JNK cascade that establishes a negative feedback loop. PINCH has no intrinsic catalytic activity, but might recruit proteins that could alter the availability or activity of JNK signaling components. Like Puc, PINCH expression is up-regulated in response to constitutive JNK signaling. Availability of RSU-1 at these sites of active JNK signaling could independently regulate JNK signaling or modulate the effects of PINCH on JNK through regulation of PINCH stability. (2) PINCH and RSU-1 are required for integrin-dependent adhesion. PINCH links integrins to the actin cytoskeleton via ILK and Parvin, and these connections could influence both integrin-dependent adhesion and signaling. Integrin signaling, through a variety of tyrosine kinases and Rac, stimulates the JNK cascade; therefore, PINCH may also exert an influence on JNK signaling via integrin. The current findings illustrate that the cellular concentration of PINCH affects the level of RSU-1 and vice versa. Thus, modulation of the ratio of RSU-1 to PINCH could provide a mechanism to regulate JNK signaling during DC and thorax closure in Drosophila. It is hypothesized that PINCH/RSU-1 complexes fine-tune and integrate the JNK and integrin signaling cascades required during morphogenesis, highlighting the potential role of integrin-associated apical junctional complexes as signal coordination points for epithelial morphogenesis (Kadrmas, 2004).
In Drosophila, the axons of retinal photoreceptor cells extend to the first optic ganglion, the lamina, forming a topographic representation. DWnt4, a secreted protein of the Wnt family, is the ventral cue for the lamina. In DWnt4 mutants, ventral retinal axons misproject to the dorsal lamina. DWnt4 is normally expressed in the ventral half of the developing lamina and DWnt4 protein is detected along ventral retinal axons. Dfrizzled2 and dishevelled, respectively, encode a receptor and a signaling molecule required for Wnt signaling. Mutations in both genes caused DWnt4-like defects, and both genes are autonomously required in the retina, suggesting a direct role of DWnt4 in retinal axon guidance. In contrast, iroquois homeobox genes are the dorsal cues for the retina. Dorsal axons accumulate DWnt4 and misproject to the ventral lamina in iroquois mutants; the phenotype is suppressed in iroquois:Dfrizzled2 double mutants, suggesting that iroquois may attenuate the competence of Dfrizzled2 to respond to DWnt4 (Sato, 2005).
JNK signaling is known to act downstream of the noncanonical Wnt pathway in many developmental contexts. The involvement of JNK signaling was examined by expressing puckered (puc), which encodes a JNK phosphatase, and a dominant-negative form of JNK encoded by basket (bsk) to block JNK signaling in the retina. Defects were observed only rarely, and it was next asked whether genetic interactions exist between hemipterous (hep) encoding a JNK kinase and DWnt4 or Dfz2. In a strong hep mutant background, or in DWnt4, DWnt4 or Dfz2 heterozygous backgrounds, little or no ventral-to-dorsal misrouting was observed. However, a reduction in the dosage of DWnt4 or Dfz2 in the hep background resulted in a marked increase in the ventral-to-dorsal phenotype. These findings provide some support for the idea that JNK signaling is involved in the DWnt4/Dfz2 pathway in retinal axon guidance. Since iro expression and ommatidial chirality were normal in retinae expressing the dominant-negative form of bsk and in hep hemizygotes in combination with DWnt4/+ and Dfz2/+, the misrouting of ventral axons observed in the brain mutant for JNK signaling appears to be caused by a failure in axon guidance and independent of the dorsoventral cell specification or PCP signaling in the retina. Note that mutations in JNK pathway components alone have no PCP phenotype (Sato, 2005).
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