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).
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).
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).
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).
In metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmental timetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct) loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia, indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the looping morphogenesis of male terminalia have been unclear. This study shows the role of apoptosis in the organogenesis of male terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerates as development proceeds, and completes a full 360° rotation. This acceleration is impaired when the activity of caspases or JNK or PVF/PVR signaling is reduced. Acceleration is induced by two distinct subcompartments of the A8 segment that form a ring shape and surrounds the male genitalia: the inner ring rotates with the genitalia and the outer ring rotates later, functioning as a 'moving walkway' to accelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation with single-cell tracking shows that the timing and degree of apoptosis correlates with the movement of the outer ring, and upregulation of the apoptotic signal increases the speed of genital rotation. Therefore, apoptosis coordinates the outer ring movement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitalia within a limited developmental time frame (Kuranaga, 2011; full text of article).
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).
During tumorigenesis, pathways that promote the epithelial-to-mesenchymal transition (EMT) can both facilitate metastasis and endow tumor cells with cancer stem cell properties. To gain a greater understanding of how these properties are interlinked in cancers, Drosophila epithelial tumor models were used, that are driven by orthologues of human oncogenes (activated alleles of Ras and Notch) in cooperation with the loss of the cell polarity regulator, scribbled (scrib). Within these tumors, both invasive, mesenchymal-like cell morphology and continual tumor overgrowth, are dependent upon Jun N-terminal kinase (JNK) activity. To identify JNK-dependent changes within the tumors a comparative microarray analysis was used to define a JNK gene signature common to both Ras and Notch-driven tumors. Amongst the JNK-dependent changes was a significant enrichment for BTB-Zinc Finger (ZF) domain genes, including chronologically inappropriate morphogenesis (chinmo). chinmo was upregulated by JNK within the tumors, and overexpression of chinmo with either RasV12 or Nintra was sufficient to promote JNK-independent epithelial tumor formation in the eye/antennal disc, and, in cooperation with RasV12, promote tumor formation in the adult midgut epithelium. Chinmo primes cells for oncogene-mediated transformation through blocking differentiation in the eye disc, and promoting an escargot-expressing stem or enteroblast cell state in the adult midgut. BTB-ZF genes are also required for Ras and Notch-driven overgrowth of scrib mutant tissue, since, although loss of chinmo alone did not significantly impede tumor development, when loss of chinmo was combined with loss of a functionally related BTB-ZF gene, abrupt, tumor overgrowth was significantly reduced. abrupt is not a JNK-induced gene, however, Abrupt is present in JNK-positive tumor cells, consistent with a JNK-associated oncogenic role. As some mammalian BTB-ZF proteins are also highly oncogenic, this work suggests that EMT-promoting signals in human cancers could similarly utilize networks of these proteins to promote cancer stem cell states (Doggett, 2015).
This report has defined the transcriptional changes induced by JNK signaling within both scrib>RasACT and scrib>NACT tumors by carrying out comparative microarray expression arrays. This analysis that JNK exerts a profound effect upon the transcriptional profile of both Ras and Notch-driven tumor types. The expression of nearly 1000 genes was altered by the expression of bskDN in either Ras or Notch-driven tumors, and less than half of these changes were shared between the two tumor types, indicating that JNK signaling elicits unique tumorigenic expression profiles depending upon the cooperating oncogenic signal. Nevertheless, of the 399 JNK-regulated probe sets shared between Ras and Notch-driven tumors, it is hypothesized that these had the potential to provide key insights into JNK's oncogenic activity, and to prioritize these targets, it was considered that the expression of the critical oncogenic regulators would not just be altered by bskDN, but would be normalized to close to wild type levels. This subset of the 399 probe set was identified by comparing the expression profile of each genotype back to control tissue, thereby producing a more focussed JNK signature of 103 genes. Notably, this included previously characterized targets of JNK in the tumors, such as Mmp1,cherand Pax, thereby providing validation of the approach. Also amongst these candidates were 4 BTB-ZF genes; two of which were upregulated by JNK in the tumors (chinmo and fru), and two downregulated (br and ttk) (Doggett, 2015).
Focussing upon chinmo, chinmo overexpression was shown to be sufficient to prime epithelial cells for cooperation with RasACT in both the eye antennal disc and in the adult midgut epithelium, and that chinmo is required for cooperative RasACTor NACT-driven tumor overgrowth, although its function was only exposed when its knockdown was combined with knockdown of a functionally similar BTB-ZF transcription factor, abrupt. This family of proteins is highly oncogenic in Drosophila, since previous work has shown that ab overexpression can cooperate with loss of scrib to promote neoplastic overgrowth, and in these studies, it was also shown that overexpression of a fru isoform normally expressed in the eye disc is capable of promoting cooperation with RasACT and NACT in the eye-antennal disc, in a similar manner to chinmo overexpression. Thus, whether fru also plays a role in driving Ras or Notch-driven tumorigenesis warrants further investigation. Indeed, a deeper understanding of the oncogenic activity of these genes is likely to be highly relevant to human tumors, since of the over 40 human BTB-ZF family members, many are implicated in both haematopoietic and epithelial cancers, functioning as either oncogenes (eg., Bcl6, BTB7) or tumor suppressors (eg., PLZF, HIC1). Furthermore, over-expression of BTB7, can also cooperate with activated Ras in transforming primary cells, and its loss makes MEFs refractory to transformation by various key oncogenes such as Myc, H-rasV12 and T-Ag, suggesting that cooperating mechanisms between BTB-ZF proteins and additional oncogenic stimuli might be conserved (Doggett, 2015).
JNK signaling in Drosophila tumors is known to promote tumor overgrowth through both the STAT and Hippo pathways. Deregulation of the STAT pathway was evident in the arrays through the upregulation of Upd ligands by JNK in both Ras and Notch-driven tumors. In contrast, although cher was identified in the arrays as being upregulated in both tumor types and previous studies have shown that cher is partly required for the deregulation of the Hippo pathway in scrib>RasACT tumors, more direct evidence for Hippo pathway deregulation amongst the JNK signature genes was lacking. In part, this could be due to JNK regulating the pathway through post-transcriptional mechanisms involving direct phosphorylation of pathway components. However, the failure to identify known Hippo pathway target genes, and proliferation response genes in general, may simply highlight limitations in the sensitivity of the array assay and the cut-offs used for determining significance, despite its obvious success in correctly identifying many known JNK targets (Doggett, 2015).
Whether tumor overgrowth through STAT and Yki activity is somehow associated with a stem cell or progenitor-like state remains uncertain. Although imaginal discs exhibit developmental plasticity and regeneration potential, and JNK signaling is required for both of these stem-like properties, there is no positive evidence for the existence of a population of asymmetrically dividing stem cells within imaginal discs. Instead, symmetrical divisions of progenitor cells may be the means by which imaginal discs can rapidly generate enough cells to form the differentiated structures of the adult fly. To date, progenitor cells have only been characterized in the eye disc neuroepithelium. These cells have a pseudostratified columnar epithelial morphology and express the MEIS family transcription factor, Hth, which is downregulated as cells initiate differentiation and begin expressing Dac and Eya. Interestingly, they also require Yki for their proliferation, and can be induced to overproliferate in response to increased STAT activity. However, analysis of cell fate markers indicated that tumor overgrowth was not likley to be solely due to the overproliferation of these undifferentiated progenitor cells. Although scrib>RasACT/NACT tumors, were characterized by the failure to transition to Dac/Eya expression in the eye disc, blocking JNK in scrib > RasACT/NACT tumors did not restore tumor cell differentiation, despite overgrowth being curtailed, and Hth expression was not maintained in the tumors in a JNK-dependent manner. Nevertheless, a JNK-induced gene such as chinmo is likely to be associated with promoting a progenitor-like state, since it is a potential STAT target gene required for adult eye development that is expressed in eye disc progenitor cells in response to increased Upd activity and its overexpression alone is sufficient to block Dac/Eya expression. Furthermore, chinmo is also required for cyst stem cell maintenance in the Drosophila testis, and the current work has shown that chinmo overexpression promotes increased numbers of esgGFP expressing stem cells or enteroblasts in the adult midgut. As the BTB-ZF protein Ab is also highly oncogenic and expressed in the eye disc progenitor cells, it is hypothesize that the JNK-induced expression ofchinmo in scrib>RasACT/NACT tumors could cooperate with Ab to maintain a progenitor-like cell state in the eye disc, and that this is required for scrib->RasACT/NACT tumor overgrowth. However, although Ab was expressed in chinmo-expressing, JNK positive tumor cells, Ab does not appear to be a JNK-induced gene. What JNK-independent mechanisms control ab expression will therefore require further analysis (Doggett, 2015).
Interestingly, previous studies have observed that ab overexpression in eye disc clones upregulates chinmo expression and although the effect of chinmo expression upon ab is yet to be described, the data at least suggest that the control of their expression is interlinked in a yet to be defined manner (Doggett, 2015).
Consistent with Chinmo being important for scrib->RasACT/NACTv tumor overgrowth, chinmo overexpression itself is also highly oncogenic. Over-expression of chinmo with RasACT or NACT drives tumorigenesis in the eye-antennal disc, and also resulted in enlarged brain lobes, presumably due to the generation of overexpressing clones within the neuroepithelium of the optic lobes. In the adult midgut, the overexpression of chinmo with RasACT in the stem cell and its immediate progeny, the enteroblast, promoted massive tumor overgrowth, resulting in esgGFP expressing cells completely filling the lumen of the gut, and eventual host lethality. The luminal filling of esgGFP cells is reminiscent of the effects of RasACT expression in larval adult midgut progenitor cells. Together with the data linking Chinmo function to stem or progenitor cells, these data reinforce the idea that epithelial tumorigenesis can be primed by signals, such as chinmo over-expression, that promote a stem or progenitor cell state (Doggett, 2015).
The function of some Drosophila BTB-ZF proteins including Chinmo and Ab, has also been linked to heterochronic roles involving the conserved let-7 miRNA pathway and hormone signals, to regulate the timing of differentiation. Indeed, Ab can directly bind to the steroid hormone receptor co-activator Taiman (Tai or AIB1/SRC3 in humans), to represses the transcriptional response to ecdysone signaling. Thus, the capacity of BTB-ZF proteins to influence the timing of developmental transitions, particularly if they impede developmental transitions within stem or progenitor cells, could help account for their potent oncogenic activity. Indeed, ecdysone-response genes were repressed by JNK in the tumorigenic state, consistent with the failure of the larvae to pupate and a delay in developmental timing. Whether repressing the ecdysone response cell autonomously might contribute to tumor overgrowth and/or invasion will be an interesting area of future investigation, given the complex role of hormone signaling in mammalian stem cell biology and cancers (Doggett, 2015).
Previous studies have suggested that JNK-dependent tumor cell invasion is developmentally similar to the JNK-induced EMT-like events occurring during imaginal disc eversion. Thus the capacity of JNK to also promote tumor overgrowth is reminiscent of how EMT inducers such as Twist (Twi) and Snail (Sna) are associated with the acquisition of cancer stem cell properties. In Drosophila, however, twi and snawere not induced by JNK in the tumors, although transcription factors involved in mesoderm specification, including the NF-kappaB homologue, dl (a member of the 103 JNK signature), and Mef2 (a member of the 399 JNK signature), were amongst the up-regulated JNK targets. Mesoderm specification is not necessarily associated with a mesenchymal-like cell morphology, however,
dl is involved in the induction of EMT during embryonic development, and both dl and Mef2 act with Twi and Sna to coordinate mesoderm formation. Interestingly, recent studies have identified dl in an overexpression screen for genes capable of cooperating with scrib > in Drosophila tumorigenesis, and Mef2 has been identified as a cooperating oncogene in Drosophila, and possibly also in humans, where a correlation exists between the expression of Notch and Mef2 paralogues in human breast tumor samples. It is therefore possible that dl and Mef2 either act in combination with Twi or Sna, or independently of them but in a similar oncogenic capacity, to promote a mesodermal cell fate in scrib > RasACT/NACT tumors. The potential relevance of this to the mesenchymal cell morphology associated with tumor cell invasion, as well as the acquisition of progenitor states is worthy of further investigation (Doggett, 2015).
In mef2-driven tumors both overgrowth and invasion depend upon activation of JNK signaling, suggesting that Mef2 is not capable of promoting invasive capabilities independent of JNK. In contrast, chinmo+RasACT/NACT tumors appeared non-invasive and retained epithelial morphology despite the massive overgrowth, although closer examination of cell polarity markers will be required to confirm this. Furthermore, the overgrowth of chinmo+RasACT/NACT tumors was not dependent upon JNK signaling, suggesting that the maintenance of a progenitor-like state could be uncoupled from JNK-induced EMT-effectors associated with invasion. Whether clear divisions between mesenchymal behaviour and progenitor states in tumors can be clearly separated in this manner is not yet clear, however, overall, it is likely that multiple JNK-regulated genes will participate in both promoting tumor overgrowth as well as migration/invasion. Although this study used the 103 JNK signature as a means to focus upon potential key candidates, an analysis of the 399 JNK-regulated probe sets common to both Ras and Notch-driven tumours has the potential to provide deeper insights into the multiple effectors of JNK signaling during tumorigenesis. Whilst the individual role of these genes can be probed with knockdowns, the complexity of the response, potentially with multiple redundancies and cross-talk, will ultimately need a network level of understanding to more fully expose key nodes participating in overgrowth and invasion. This approach has considerable potential to further expose core principles and mechanisms that drive human tumorigenesis, since it is clear that many fundamental commonalities underlie the development of tumors in Drosophila and mammals (Doggett, 2015).
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, Parvin, 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).
Aging of a eukaryotic organism is affected by its nutrition state and by its ability to prevent or repair oxidative damage. Consequently, signal transduction systems that control metabolism and oxidative stress responses influence life span. When nutrients are abundant, the insulin/IGF signaling (IIS) pathway promotes growth and energy storage but shortens life span. The transcription factor Foxo, which is inhibited by IIS, extends life span in conditions of low IIS activity. Life span can also be increased by activating the stress-responsive Jun-N-terminal kinase (JNK) pathway. This study shows that JNK requires Foxo to extend life span in Drosophila. JNK antagonizes IIS, causing nuclear localization of Foxo and inducing its targets, including growth control and stress defense genes. JNK and Foxo also restrict IIS activity systemically by repressing IIS ligand expression in neuroendocrine cells. The convergence of JNK signaling and IIS on Foxo provides a model to explain the effects of stress and nutrition on longevity (Wang, 2005).
The data suggest Foxo as a convergence point for IIS and JNK signaling. Through its responsiveness to these two pathways, Foxo is well positioned to integrate information about environmental stress and nutrient availability and to elicit appropriate biological responses. Such a system would ensure that growth could proceed in an unrestrained manner when energy resources are available and the cell is not exposed to external insults (IIS is active, JNK is off, and Foxo is repressed). However, in situations of low food availability or an adverse environment, IIS would cease to signal, or JNK would be activated, resulting in translocation of Foxo to the nucleus. The ensuing Foxo-induced gene expression has several effects at the cell as well as the organism level and is likely to counteract premature senescence. The induction of genes such as thor can reduce cell growth, presumably to limit the cell's anabolic expenses in adverse situations. Other target genes, such as the small heat shock protein l(2)efl, are expected to have a direct role in allaying damage inflicted by environmental insults and may prevent the accumulation of toxic protein aggregates. The suppression of dilp2 expression by JNK and Foxo in insulin-producing cells (IPCs), in contrast, is likely to control growth, metabolism, and stress responses systemically by downregulating IIS in all responsive tissues in a coordinated fashion (Wang, 2005).
The interaction between JNK and Foxo is thus expected to influence stress tolerance and life span at two levels. In peripheral tissues, JNK activates Foxo and prevents senescence cell-autonomously. Such a mechanism is exemplified by the recent finding that Foxo overexpression prevents age-dependent decline of cardiac performance (Wessells, 2004). Systemic control of IIS by JNK-mediated activation of Foxo in IPCs, in contrast, would serve to coordinate cellular responses to changes in the environment throughout the organism. The data indicate that this latter mechanism plays a significant role in the regulation of life span by JNK and Foxo. The identification of this endocrine function of JNK/Foxo signaling supports and extends the proposed role of JNK signaling on longevity and demonstrates a role for IPCs in life span regulation. In addition to controlling growth and metabolism, IPCs may thus act as a coordination point for the organism's stress response by downregulating Dilp production in response to oxidative stress and JNK activation. In target tissues, such a mechanism would induce protective gene expression by the second, cell-autonomous tier of Foxo signaling. Interestingly, the effects of IPC-specific JNK activation on longevity and growth are separable. Life span can be extended by moderately increased JNK activity in IPCs when growth effects are yet not evident. This finding is consistent with observations that the extension of life span in IIS loss-of-function situations is not a mere consequence of small body size (Wang, 2005).
How did such a multilayered regulation of IIS activity by JNK evolve? It is tempting to speculate that localized activation of Foxo is required to prevent cellular damage and ultimately senescence in conditions in which stressful insults are confined to specific tissues. Such localized insults could, for example, be inflicted by reactive oxygen species that are produced in the environment of amyloid deposits in Alzheimer's disease as well as by mechanical and oxidative stress experienced by particularly active tissues such as the heart. Systemic regulation of Foxo activity, in contrast, is expected to be an important response mechanism to coordinate metabolism and stress defenses throughout the organism upon changes in the environment. A good example for such a mechanism is the induction of diapause in invertebrates in response to environmental stress or food deprivation. Accordingly, sensory neurons expressing the insulin-like peptide DAF-28 are required for the induction of the dauer larval stage in response to environmental cues in C. elegans (Wang, 2005).
Systemic and tissue-autonomous effects of JNK/Foxo signaling may be connected in multiple ways. The data indicate that JNK and Foxo interact in IPCs to repress dilp2 expression, ultimately activating Foxo in Dilp2 target tissues in a coordinated fashion. Since JNK was found to be activated in IPCs even under normal culture conditions, it is likely that this systemic control of IIS activity by JNK and Foxo plays a critical role in life span regulation. It is, however, also possible that the cell-autonomous protective function of JNK/Foxo signaling is most critical for the survival of specific tissues as the organism ages, thus extending life of the organism by preventing the loss of indispensable cells or tissues. In addition, stress and the JNK-mediated activation of Foxo in peripheral tissues may signal back to IPCs to initiate a systemic response. In Drosophila, such a mechanism has been documented in the case of the fatbody. Activation of Foxo in this tissue relays a signal to the IPCs, causing them to curb Dilp2 production, a process that has been proposed to require Foxo activity (Hwangbo, 2004). The exact nature of this feedback signaling mechanism in flies is unclear, but it is reminiscent of the complex signaling interactions between β cells and insulin target tissues in mammals. Further studies are required to shed light on the relative contributions of JNK/Foxo signaling in IPCs or Dilp target tissues to life span regulation (Wang, 2005).
JNK-mediated modulation of IIS activity is likely to be evolutionarily conserved. Inhibitory crosstalk from JNK to IIS in mammalian cells has been found to occur by JNK-mediated phosphorylation and inhibition of IRS-1. This interaction is responsible for obesity-induced insulin resistance in mice. Whether mammalian homologs of Foxo take part in this pathology remains to be determined. A second possible mechanism for JNK/IIS pathway interaction is the direct phosphorylation and activation of Foxo by JNK. A recent study supports such a mechanism, showing that in mouse cells JNK can phosphorylate the DFoxo homolog Foxo4 in response to oxidative stress. The physiological relevance of this phosphorylation event has not yet been addressed. The JNK target residues on IRS-1 and Foxo4 are not conserved in the Drosophila homologs Chico and DFoxo, and further studies are thus required to determine whether JNK-Foxo crosstalk in Drosophila is mediated via homologous mechanisms (Wang, 2005).
The systemic regulation of IIS activity by JNK and Foxo appears to be conserved as well. It has been suggested that C. elegans Daf16/Foxo regulates life span (at least in part) by reducing the expression of insulin-like peptides. In mammals, pancreatic β cells (the counterparts of IPCs) reduce their production of insulin in response to oxidative stress-mediated JNK activation. Conversely, dephosphorylation of JNK by MAPK phosphatase 1 can induce insulin expression in these cells. Reducing circulating insulin levels by JNK-mediated Foxo activation may thus be a general mechanism that balances growth and metabolism with stress defense and damage repair (Wang, 2005).
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).
Cell movements represent a major driving force in embryonic development, tissue repair, and tumor metastasis. The migration of single cells has been well studied, predominantly in cell culture; however, in vivo, a greater variety of modes of cell movement occur, including the movements of cells in clusters, strands, sheets, and tubes, also known as collective cell migrations. In spite of the relevance of these types of movements in both normal and pathological conditions, the molecular mechanisms that control them remain predominantly unknown. Epithelial follicle cells of the Drosophila ovary undergo several dynamic morphological changes, providing a genetically tractable model. This study found that anterior follicle cells, including border cells, mutant for the gene hindsight (hnt) accumulated excess cell-cell adhesion molecules and failed to undergo their normal collective movements. In addition, HNT affected border cell cluster cohesion and motility via effects on the JNK and STAT pathways, respectively. Interestingly, reduction of expression of the mammalian homolog of HNT, RREB1, by siRNA inhibited collective cell migration in a scratch-wound healing assay of MCF10A mammary epithelial cells, suppressed surface activity, retarded cell spreading after plating, and led to the formation of immobile, tightly adherent cell colonies. It is proposed that HNT and RREB1 are essential to reduce cell-cell adhesion when epithelial cells within an interconnected group undergo dynamic changes in cell shape (Melani, 2008).
To explore the mechanisms by which HNT affects cluster cohesion and motility, its effects on known signaling pathways were investigated. In the extraembryonic tissue known as the amnioserosa, hnt is a negative regulator of the JNK signaling cascade. Recently, the JNK pathway was shown to be active in the border cells and to affect border cell migration in clusters with reduced PVR activity. In addition, inhibition of the JNK cascade causes a phenotype that strikingly resembles the cluster dissociation phenotype caused by HNT overexpression, suggesting that HNT could be a negative regulator of the JNK pathway or vice versa. By using phospho-Jun antibody staining as a readout of the JNK signaling cascade, the activity of this pathway was seen to be reduced in border cells overexpressing hnt. In clusters in which JNK was reduced by overexpression of either Puckered (the JNK phosphatase) or a dominant-negative form of Basket (Drosophila JNK), cluster disassembly reminiscent of the hnt gain-of-function phenotype was observed. In addition, HNT was upregulated 1.7- and 1.4-fold, respectively. Together, these results indicate that hnt and JNK repress each other. In the embryo, in which HNT also antagonizes JNK, this pathway is required for the turnover of focal complexes and proper dorsal closure. Therefore, HNT appears to play a general role in remodeling of adhesion complexes to facilitate morphogenesis (Melani, 2008).
Although the cluster-disassembly phenotype of HNT could be attributed to effects on JNK signaling, JNK pathway mutations caused milder border cell motility defects than hnt. To determine whether HNT affected, in addition, one of the known border-cell-motility pathways, the effect of hnt on the activity of STAT and its key target SLBO was examined. STAT activation and nuclear translocation is the most upstream event in the differentiation of the border cells and is also required throughout border cell migration. It was found that, in border cells overexpressing HNT, nuclear accumulation of STAT was reduced though not eliminated. In addition, the levels of slbo were dramatically reduced in border cells overexpressing HNT. Because loss of function of either STAT or SLBO causes a dramatic migration defect, the effects of HNT overexpression on STAT and SLBO can account for the severe effect on motility. However, neither stat nor slbo mutant border cells exhibit a cluster-disassembly phenotype. Therefore, it is concluded that HNT mediates its effect on cluster cohesion via JNK and its effect on border cell motility primarily through STAT and SLBO (Melani, 2008).
Although HNT overexpression affects border cell motility via effects on STAT and SLBO, HNT has general effects on cell adhesion and morphogenesis, whereas SLBO appears to be more specific. For example, the effects of hnt on stretched follicle cells and in embryonic tissues are independent of SLBO because this protein is neither expressed nor required in these other cell types. Therefore, it is proposed that HNT plays a general role in regulating cell adhesion and morphogenesis via JNK signaling and a tissue-specific role in motility through STAT and SLBO. In this way, HNT can cooperate with tissue-specific factors to orchestrate a diverse array of collective cell movements (Melani, 2008).
High baselines of transcription factor activities represent fundamental obstacles to regulated signaling. This study shows that in Drosophila, quenching of basal activator protein 1 (AP-1) transcription factor activity serves as a prerequisite to its tight spatial and temporal control by the JNK (Jun N-terminal kinase) signaling cascade. These studies indicate that the novel raw gene product is required to limit AP-1 activity to leading edge epidermal cells during embryonic dorsal closure. In addition, evidence is provided that the epidermis has a Basket JNK-independent capacity to activate AP-1 targets and that raw function is required broadly throughout the epidermis to antagonize this activity. Finally, mechanistic studies of the three dorsal-open group genes [raw, ribbon (rib), and puckered (puc)] indicate that these gene products provide at least two tiers of JNK/AP-1 regulation. In addition to Puckered phosphatase function in leading edge epidermal cells as a negative-feedback regulator of JNK signaling, the three dorsal-open group gene products (Raw, Ribbon, and Puckered) are required more broadly in the dorsolateral epidermis to quench a basal, signaling-independent activity of the AP-1 transcription factor (Bates, 2008).
The initial molecular and genetic studies of the dorsal-open mutant raw revealed it to encode a widely expressed and novel gene product, required for the restriction of JNK/AP-1 activity to LE epidermal cells (Byars, 1999). The Raw protein sequence yielded no insights into its mechanism of function as the Raw sequence harbors none of the canonical motifs that are associated with nuclear localization, phosphorylation, membrane insertion, or protein secretion. Mechanistic studies of a novel protein can be challenging, but this study reports use of a variety of genetic strategies to probe Raw function and test models of AP-1 silencing. In particular (1) the epistatic relationship of raw to genes encoding well-characterized JNK-signaling components was assessed, (2) genes, which have designated the raw group, have been assessed that share an array of loss-of-function phenotypes, (3) the interaction phenotypes among the raw-group loci were determined, and (4) raw transgenics were generated, that were utilized to probe sites of Raw function. These analyses reveal that raw belongs to a small set of dorsal-open group genes that encode JNK/AP-1 pathway antagonists. The characterization of raw, and the raw group more generally, has led to a new appreciation of wide-ranging competence for AP-1 activity in early Drosophila embryos. As signal activation is critical for proper development, so also is its silencing (Bates, 2008).
The current study shows that although raw functions upstream of Jra as an AP-1 antagonist, its action is independent of the bsk-encoded kinase that is required to activate AP-1 activity in LE cells during closure. In addition, raw is required broadly in the epidermis to effect normal dorsal closure. Overall, these studies expose the importance of epidermal AP-1 silencing during embryogenesis and lead to an extension of existing models for dorsal closure, which have largely confined their focus to mechanisms of JNK/AP-1 activation in LE cells. In particular, the data indicate that Raw and the other raw-group gene products (Puckered and Ribbon) function to silence Basket JNK-independent AP-1 activity in the embryonic dorsolateral epidermis. AP-1 silencing, via the combined actions of the raw-group gene products, essentially wipes the epidermal slate clean and primes the system for activation via a still unidentified deterministic signal that acts only in LE cells (Bates, 2008).
The AP-1 abnormality in raw-group mutant embryos has not yet been molecularly defined. Previous studies provide compelling evidence that AP-1 overexpression in Drosophila embryos is not sufficient to disrupt either dorsal closure or development more generally. It seems unlikely, therefore, that elevated levels of the AP-1 transcription factor in raw-group mutants simply override a requirement for kinase activation in initiating an AP-1-dependent program of gene expression. Instead, it is speculated that AP-1 is aberrantly modified in raw-group mutant embryos. It might be that AP-1 escapes inactivation in mutants; either alternatively or additionally, AP-1 in mutants may be inappropriately activated via phosphorylation. In addition to Basket JNK, there are four other Drosophila MAP Kinases (p38a, p38b, Mpk2, and Rolled) that might provide dysregulated kinase activity in mutants. Consistent with this idea is the observation that the oogenesis phenotypes associated with raw (and puc) ectopic expression and mutation have considerable similarity with gain- and loss-of-function phenotypes associated with mutations in the p38 pathway that is required in the germ line for proper oogenesis. Finally, a kinase-dependent activation model for epidermal Jun provides the most parsimonious explanation for ectopic epidermal signaling observed in puc MPK-deficient embryos. From the perspective of regulated signaling more generally, however, lowering an AP-1 activity baseline in wild-type embryos will (1) provide a means for the clean on/off regulation of JNK/AP-1 that has been predicted in computer simulations and (2) make a less strenuous demand on the input activating signal (Bates, 2008).
The discovery that null alleles of raw and puc interact, with double mutants exhibiting an embryonic lethal phenotype distinct from their shared loss-of-function null phenotypes, revealed the independent contributions of raw and puc to embryogenesis, presumably through their effects on AP-1 antagonism. Drosophila overexpression studies have previously implicated several pathways in the parallel control of AP-1 activity, but this analysis represents the first direct demonstration of physiologically relevant, parallel regulatory pathways (Bates, 2008).
The genetic interaction that was documented between null alleles of raw and puc contrasts with the lack of a detectable interaction between null alleles of raw and rib. Moreover, the observation that raw and rib hypomorphs interact genetically during dorsal closure is consistent with previously published data, as well as with findings documenting (1) raw/rib interactions in several other epithelial tissues, including the nervous system, salivary gland, trachea, and gut (Blake, 1998; Blake, 1999) and (2) overlapping raw and rib expression patterns in Drosophila embryos (Byars, 1999). Together, results from these genetic and molecular studies point to roles for raw and rib in a single, previously unrecognized puc-independent AP-1 inactivation system (Bates, 2008).
In addition to providing evidence for raw-mediated global silencing of AP-1, this study underscores a simultaneous requirement for a biologically appropriate activator of JNK/AP-1 signaling. In this regard, expression of raw in LE cells failed to rescue raw-dependent defects in dorsal closure. Even more notable, however, was the observation that overexpression of raw+ in wild-type embryos, and in wild-type LE cells in particular, had no detrimental effects on embryonic development and dorsal closure. From a signaling perspective this result indicates that JNK-dependent AP-1 can be activated despite expression of the wild-type raw gene product, and thus Raw does not function as a binary switch for signaling. Although it is formally possible that LE expression of raw was initiated too late to disrupt JNK/AP-1 signaling and dorsal closure in the LE-gal4/UAS-raw+ transgenics, this interpretation is not favored since the LE-GAL4 driver used in this study has been shown previously to (1) be an effective driver of at least one gene that is required in LE epidermal cells for closure and (2) drive expression of a lacZ reporter in LE cells during dorsal closure (Bates, 2008).
The finding that raw expression in LE cells is not sufficient to inactivate AP-1 activity in a cell-autonomous fashion is consistent with models for independent, developmentally regulated triggers of JNK signaling. Indeed, there is abundant experimental support for developmentally regulated activation of JNK signaling in LE cells. JNK/AP-1 activation likely follows an amalgamation of signals, both from the amnioserosa and the epidermis, both in the form of cytoskeletal components and signaling molecules. Among the best candidates with postulated roles in JNK/AP-1 activation are small GTPases, nonreceptor tyrosine kinases, and integrins. Thus, despite the broad epidermal competence for AP-1 signaling that has been shown in this work, the activation signal is itself limited to only LE cells and functions via an unknown mechanism. Importantly, AP-1 antagonism by raw cannot override its signal-dependent activation in the LE (Bates, 2008).
dpp, when expressed pan-epidermally, leads to a raw-like phenotype: embryonic lethality associated with ventral cuticular defects. In a direct assessment of equivalence of raw loss-of-function and dpp gain-of-function ventral cuticular phenotypes, whether pan-epidermal expression of brinker (brk) can rescue raw-dependent defects in the ventral cuticle was tested. The Dpp signaling modifier Brinker functions by negatively regulating dpp target genes (Bates, 2008).
This study found that although brk is normally expressed in nonoverlapping lateral and ventral domains of the embryonic epidermis, it is undetectable in the epidermis of embryos homozygous for a null allele of raw. It was also found that although brk+ fails to rescue raw-dependent defects in dorsal closure, it does rescue raw-dependent defects in the ventral cuticle. Together, these data point to an important role for dpp, brk, and/or their target genes in development of the ventral epidermis (Bates, 2008).
What cannot be discerned from these studies is (1) how the nonoverlapping epidermal domains of dpp and brk are established and maintained and (2) if and how epidermal dpp and brk interact during normal embryonic development. In this regard, a previous finding that LE dpp is not autoregulatory makes it unlikely that brk functions in direct fashion to set the LE dpp expression boundary. Even more significant is the finding that cuticles derived from Jra raw double mutants exhibit defects in dorsal closure, but not ventral cuticular patterning (BYARS, 1999). Indeed, these data highlight the requirement for functional Jun in generating ventral cuticular defects in raw mutant embryos. Taken together then, these data suggest that the effects of JNK/AP-1-activated dpp in the dorsal epidermis of raw mutant embryos are far reaching, extending even to the most ventral regions of the embryo (Bates, 2008).
Having established a dependence upon Jun for raw-dependent ventral cuticular defects, it is postulated that the absence of brk in raw mutant embryos is a direct consequence of ectopic JNK/AP-1 activity in the dorsal epidermis of these mutants. It is suspected that ectopic JNK/AP-1 activity leads secondarily to ectopic dpp activity, and that in its turn ectopic dpp activity leads finally to brk repression. An alternative view, that raw might have dual regulatory roles in the epidermis, seems less likely although it is not absolutely excluded by this strictly genetic analysis. In this regard, in addition to its function as a JNK/AP-1 antagonist in the embryonic dorsal epidermis, raw might function independently as a trigger of brk expression in the ventral epidermis. Clearly, the mechanism of raw function and the relationship of dpp to brk in eliciting properly formed ventral cuticle warrant further investigation (Bates, 2008).
In Drosophila, as in all animals, signaling pathways are finely regulated at several levels. Although there are multiple tiers of regulation operating on the JNK/AP-1 signaling cascade, surprisingly little of the regulation of this pathway is known. This study of the functions and interactions of a subset of dorsal-open group genes (raw, rib, and puc) has shed some additional light on both old (puc-mediated) and new (raw/rib-mediated) mechanisms of JNK/AP-1 antagonism. These data indicate that Raw functions to silence Basket JNK-independent AP-1-mediated transcription and to set the stage for JNK-dependent regulation of transcription. The suggestion that spatial restriction of the JNK/AP-1 signal requires antagonists, as well as activators, is not without precedent in other signaling systems. Many signaling pathways have already been shown to be multilayered and to depend heavily on negative regulation to terminate developmental events, and/or control both the distance and speed that a signal can move (e.g., Nodal). In addition, and as was suggested is the case for the Drosophila JNK/AP-1 pathway, reducing basal levels of a signaling pathway can augment the effects of its signaling responses (e.g., Hedgehog and Lef1) (Bates, 2008).
Finally, given the numerous associations of improper JNK/AP-1 activity with human disease, it seems apparent that many cell types have the capacity to signal via the JNK/AP-1 pathway. Presumably, this capacity is diminished (and then tightly regulated) during normal vertebrate development and aging. Viewed from this perspective, characterization of Raw as an essential AP-1 antagonist establishes a clear basis for future studies of AP-1 regulation (Bates, 2008).
AP-1, an immediate-early transcription factor comprising heterodimers of the Fos and Jun proteins, has been shown in several animal models, including Drosophila, to control neuronal development and plasticity. In spite of this important role, very little is known about additional proteins that regulate, cooperate with, or are downstream targets of AP-1 in neurons. This paper outlines results from an overexpression/misexpression screen in Drosophila to identify potential regulators of AP-1 function at third instar larval neuromuscular junction (NMJ) synapses. First, >4000 enhancer and promoter (EP) and EPgy2 lines were used to screen a large subset of Drosophila genes for their ability to modify an AP-1-dependent eye-growth phenotype. Of 303 initially identified genes, a set of selection criteria were used to arrive at 25 prioritized genes from the resulting collection of putative interactors. Of these, perturbations in 13 genes result in synaptic phenotypes. Finally, one candidate, the GSK-3α-kinase homolog, shaggy, negatively influences AP-1-dependent synaptic growth, by modulating the Jun-N-terminal kinase pathway, and also regulates presynaptic neurotransmitter release at the larval neuromuscular junction. Other candidates identified in this screen provide a useful starting point to investigate genes that interact with AP-1 in vivo to regulate neuronal development and plasticity (Franciscovich, 2008).
The transcription factor AP-1 is a key regulator of neuronal growth, development, and plasticity, and in addition to cAMP response element binding (CREB) protein, it controls transcriptional responses in neurons during plasticity. Acute inhibition of Fos attenuates learning in mice and in invertebrate models such as Drosophila; AP-1 positively regulates developmental plasticity of motor neurons. Essential to the understanding of AP-1 activity in neurons is the knowledge of other proteins that influence AP-1 function or are downstream transcriptional targets. This study describes a forward genetic screen for modifiers of AP-1 in Drosophila (Franciscovich, 2008).
Using a conveniently scored AP-1-dependent adult-eye phenotype, 4307 EP and EPgy2 lines were screened for genes that modified this phenotype. Several advantages of this screen include: (1) the ease and rapidity of screening as compared to the neuromuscular junction, (2) immediate gene identification, (3) the potential to analyze in vivo phenotypes that arise from overexpression/misexpression, and finally (4) the scope for rapidly generating loss-of-function mutations through imprecise excision of the same P-element. A total of 249 known genes were isolated of which 73 can be directly implicated in eye development. The selection was prioritized using several criteria, to derive a short list of 13 final candidates that were then tested at the NMJ. Future work will focus on other predicted but as yet unstudied genes that are likely to have important functions at the NMJ (Franciscovich, 2008).
The prescreening strategy using the adult eye was successful because (1) almost all the genes selected did not result in eye phenotypes when expressed on their own, but selectively modified a Fbz dependent phenotype (Fbz is a dominant-negative transgenic construct that expresses the Bzip domain of Drosophila Fos); (2) several genes were identified that are known to interact with AP-1 in regulating synaptic phenotypes (these include ras and bsk); (3) multiple alleles of some genes were recovered confirming the sensitivity of the screening technique; (4) several genes involved in eye development were isolated (including cyclinB, which has been shown to be a downstream target of Fos in the regulation of G2/M transition in the developing eye); (5) a large number of putative interactors have connections with neural physiology and/or AP-1 function in other cell types; (6) some candidates with strong phenotypes have previously been shown to play important roles in motor neurons; and finally (7) the majority of candidates (but not all) isolated as enhancers or suppressors of Fbz in the eye exerted a similar effect on AP-1 at the synapse (Franciscovich, 2008).
Although the relative success and merits of a functional screen are considerable, there are a few disadvantages. First, the use of P-element transposons naturally excludes a large fraction of genes that are refractory to P-element transposition events. Second, insertions of EP elements within or in inverse orientation to the gene make it difficult to assign phenotypes to specific genes. Even in instances where overexpression was predicted, it has to be verified that this is indeed the case and also the phenotypes derive from hypomorphic mutations that result from the insertion of the P-element close to the target gene have to be tested. Third, although recover genes that play conserved roles in AP-1 biology is to be expected, those genes that specifically affect synaptic physiology and play no role in the eye will be excluded by this scheme. Finally, this screen will not discriminate between genes that function upstream or downstream of AP-1 in neurons. In spite of these deficiencies, it is believed that candidates identified in this screen provide strong impetus for the investigation of additional factors that are involved in the regulation of synaptic plasticity and development by AP-1 (Franciscovich, 2008).
Following their identification, it was found that several candidates had synaptic functions since several of these genes resulted in significant differences in synaptic size when compared to appropriate controls. This provided the first confirmation of the screening strategy. Next, experiments to determine genetic interaction with AP-1 showed that expression of four genes (pigeon, lbm, Cnx99A, and sty) suppressed the Fbz-dependent small synapse phenotype. Of these, sty had been isolated as an enhancer while the other three similarly suppressed the Fbz-derived eye phenotype, suggesting potentially conserved functions of these genes in the two tissues (Franciscovich, 2008).
Four genes isolated as enhancers, similarly enhanced an Fbz-mediated small synapse (cnk, pde8, fkbp13, and sgg). Notably, expression of these genes also suppressed an AP-1-dependent synapse expansion at the NMJ. These two lines of evidence indicate that these genes are negative regulators of AP-1 function in these neurons. Together with the fact that all four have previously described functions in the nervous system, these observations confirm the validity of the screen and highlight the utility of genetic screens to uncover novel molecular interactions. Further studies will provide a more comprehensive understanding of the interplay between these genes and AP-1 in the regulation of neuronal development and plasticity. For instance, more careful analysis needs to be carried out to discern whether synaptic phenotypes in each of these cases are due to overexpression or potential insertional mutagenesis of specific genes (Franciscovich, 2008).
Although GSK-3β-signaling has been implicated in several neurological disorders such as Alzheimer's disease, it is only recently that neuronal roles for this important kinase have come to light. For instance, several studies have demonstrated the role of GSK-3β in the regulation of long-term potentiation (LTP) in vertebrate hippocampal synapses (Hooper, 2007; Peineau, 2007; Zhu, 2007). In particular, these reports highlight the negative regulatory role of GSK-3β in the induction of LTP or in one case, the switching of long-term depression (LTD) into LTP. Interestingly, LTP induction leads to GSK-3β-inhibition thus precluding LTD induction in the same neurons. In flies, sgg mutations have defects in olfactory habituation, circadian rhythms and synaptic growth. These observations point to a conserved and central role for GSK-3β in neuronal physiology (Franciscovich, 2008).
GSK-3β-dependent modulation of transcriptional responses is widely acknowledged. Among several transcription factors that are known to be regulated by this kinase, are AP-1, CREB, NFAT, c/EBP, and NF-kappaB. In the context of neuronal function, for instance, RNA interference-based experiments in cultured rat cortical neurons have shown that GSK-3β-activity influences CREB and NF-kappaB-dependent transcription. Additionally, two other transcription factors, early growth response 1 and Smad3/4 have been identified in DNA profiling experiments in the same study. Significantly, GSK-3β is also a primary target of lithium, a drug used extensively to treat mood disorders. Lithium treatment has been reported to result in an upregulation of AP-1-dependent transcription, though a role for GSK-3β in this phenomenon has not been tested directly (Franciscovich, 2008).
In Drosophila, recent experiments have described the negative regulation of synaptic growth by the GSK3β-homolog shaggy (Franco, 2004). These studies demonstrate that sgg controls synaptic growth through the phosphorylation of the Drosophila MAP1B homolog futsch. The current studies suggest that Sgg-dependent regulation of synapse size occurs through the immediate-early transcription factor AP-1. GSK-3β is believed to inhibit transcriptional activity of AP-1 in cultured cells by direct inhibitory phosphorylation of c-Jun. Circumstantial evidence also suggests that GSK-3β provides an inhibitory input into AP-1 function in neurons (Franciscovich, 2008).
It was intriguing to find that Sgg inhibition leads to an expanded synapse with reduced presynaptic transmitter release, similar to highwire mutants. Given that in several instances, Sgg-dependent phosphorylation targets a protein for ubiquitination, and that Highwire encodes an E3 ubiquitin ligase, it is conceivable that sgg and hiw function in the same signaling pathway. Consistent with this hypothesis, both hiw and sgg function at the synapse seem to impinge on AP-1-dependent transcription through modulation of the JNK signaling pathway. Considering previous reports of GSK-3β-involvement in multiple signaling cascades, it will be interesting to study how sgg controls multiple aspects of cellular physiology to regulate neural development and plasticity, particularly in the context of brain function and action of widely used drugs such as lithium (Franciscovich, 2008).
Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. This study addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. The increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, evidence is provided that the JAK-STAT and JNK pathways are both required for bacteria-induced stem cell proliferation. Similarly, it was demonstrated that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, it was shown that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity (Buchon, 2009b).
The JAK-STAT and JNK signaling pathways are required to maintain gut homeostasis upon exposure to a broad range of bacteria. In normal conditions, low levels of the indigenous gut microbiota and transient environmental microbes maintain a basal level of epithelium renewal. The increase in gut microbes in old or Imd-deficient flies is associated with a chronic activation of the JNK and JAK-STAT pathways, leading to an increase in intestinal stem cells (ISC) proliferation and gut disorganization. The impact of pathogenic bacteria can have different outcomes on gut homeostasis, depending on the degree of damage they inflict on the host. Damage to the gut caused by infection with E. carotovora is compensated for by an increase in epithelium renewal. Infection with a high dose of P. entomophila disrupts the homeostasis normally maintained by epithelium renewal and damage is not repaired, contributing to the death of the fly (Buchon, 2009b).
Previous studies have shown that the NADPH oxidase Duox plays an essential role in Drosophila gut immunity by generating microbicidal effectors such as ROS to eliminate both invasive and dietary microbes. Ecc15 is a potent activator of Duox, which in turn is important in the clearance of this bacterium. This oxidative burst is coordinated with the induction of many genes involved in ROS detoxification upon Ecc15 ingestion. This study provides evidence that the observed increase in epithelium renewal upon Ecc15 infection is a compensatory mechanism that repairs the damage inflicted to the gut by this oxidative burst. This is supported by the observation that reducing ROS levels by either the ingestion of antioxidants or silencing the Duox gene reduces epithelium renewal. Although ISC proliferation could be directly triggered by ROS, it is more likely a consequence of signals produced by stressed enterocytes. A number of data support this hypothesis: (1) Ingestion of corrosive agents can also induce ISC proliferation, and (2) physical injury is sufficient to induce local activation of the cytokine Upd3, which promotes epithelium renewal. Interestingly, a significant increase in epithelium renewal was observed in Duox RNAi flies at late time points following infection, correlating with damage attributed to the proliferation of Ecc15 in the guts of Duox-deficient flies. While the increase in epithelium renewal observed with Ecc15 is clearly linked to the damage induced by the host immune response, it is likely that effects on epithelium renewal by other pathogens could be more direct and mediated by virulence factors, such as the production of cytolytic toxins (Buchon, 2009b).
The data indicate that the JAK-STAT and JNK pathways synergize to promote ISC proliferation and epithelium renewal in response to the damage induced by infection. The JAK-STAT pathway is implicated in the regulation of stem cells in multiple tissues and is proposed to be a common regulator of stem cell proliferation. The data extend this observation by showing that the JAK-STAT pathway is also involved in ISC activation upon bacterial infection. The cytokine Upd3 is produced locally by damaged enterocytes and subsequently stimulates the JAK-STAT pathway in ISCs to promote their proliferation. The results globally agree with a recent study showing that the JAK-STAT pathway is involved in ISC proliferation upon infection with a low dose of P. entomophila (Jiang, 2009). This work and the current study clearly demonstrate that the JAK-STAT pathway adjusts the level of epithelium renewal to ensure proper tissue homeostasis by linking enterocyte damage to ISC proliferation. The study by Jiang also uncovered an additional role of this pathway in the differentiation of enteroblasts during basal gut epithelium turnover. The implication of the JAK-STAT pathway in differentiation could explain the accumulation of the small-nucleated escargot-positive cells observed in the gut of flies with reduced JAK-STAT signaling in ISCs. The JAK-STAT pathway was also shown previously to control the expression of some antimicrobial peptides such as Drosomycin 3 (Dro3). Therefore, the JAK-STAT pathway has a dual role in the gut upon infection, controlling both the immune response and epithelium renewal (Buchon, 2009b).
The data show that the lack of JNK pathway activity in ISCs results in the loss of ISCs in guts infected with Ecc15, thus preventing epithelium renewal. The findings are consistent with the attributed function of JNK at the center of a signal transduction network that coordinates the induction of protective genes in response to oxidative challenge. This cytoprotective role against ROS would protect ISCs from the oxidative burst induced upon Ecc15 infection, explaining why ISCs die by apoptosis when JNK activity is reduced. It is likely that JNK signaling is required not only to protect ISCs from oxidative stress, but also to induce stem cell proliferation to replace damaged differentiated cells. This is supported by the observation that overexpression of the JNKK Hep in ISCs is sufficient to trigger an epithelium renewal in the absence of infection. In addition, increased JNK activity in ISCs of old flies has been linked to hyperproliferative states and age-related deterioration of the intestinal epithelium. This study shows that JNK signaling is also required for epithelium renewal upon Ecc15 infection. Thus, infection with Ecc15 recapitulates in an accelerated time frame the impacts of increased stress observed in guts of aging flies (Buchon, 2009b).
The inhibition of the dJun transcription factor in ISCs leads to a loss of stem cells in the absence of infection, suggesting that this transcription factor plays a critical role in ISC maintenance in the gut. There is no definitive explanation for why the dJun-IR construct behaves differently than the basket and hep-IR constructs. It is speculated that this could be due to (1) differences in the basal activity of the JNK pathway, which would be blocked only with the dJun-IR that targets a terminal component of the pathway; (2) effects of Jun in ISCs independent of the JNK pathway; or (3) side effects of the dJun-IR construct (Buchon, 2009b).
In contrast to the requirement of the JNK pathway upon Ecc15 infection, it has been reported that oral ingestion with a low dose of P. entomophila still induced mitosis in the JNK-defective mutant hep1. In agreement, this study found that inhibiting the JNK pathway in ISCs did not block the induction of epithelium renewal by a low dose of P. entomophila. This difference in the requirement of the JNK pathway may be explained by the nature of these two pathogens. Whereas Ecc15 damages the gut through an oxidative burst that activates the JNK pathway, the stimulation of epithelium renewal by P. entomophila could be due to a more direct effect of this bacterium on the gut. Altogether, this work points to an essential role of the JAK-STAT pathway in modulation of epithelium renewal activity, while the role of JNK may be dependent on the infectious agent and any associated oxidative stress. While it is known that the JNK pathway is activated by a variety of environmental challenges including ROS, the precise mechanism of activation of this pathway has not been elucidated. Similarly, the molecular basis of upd3 induction in damaged enterocytes is not known. Future work should decipher the nature of the signals that activate these pathways in both ISCs and enterocytes, as well as the possible cross-talk between the JNK and JAK-STAT pathways in ISC control (Buchon, 2009b).
The observation that flies unable to renew their gut epithelium eventually succumb to Ecc15 infection highlights the importance of this process in the gut immune response. It is striking that defects in epithelium renewal are more detrimental to host survival than deficiency in the Imd pathway, even though this pathway controls most of the intestinal immune-regulated genes induced by Ecc15. The results are in agreement with a previous study indicating that, in the Drosophila gastrointestinal tract, the Imd-dependent immune response is normally dispensable to most transient bacteria, but is provisionally crucial in the event that the host encounters ROS-resistant microbes. However, this study demonstrates that efficient and rapid clearance of bacteria in the gut by Duox is possible only when coordinated with epithelium renewal to repair damage caused by ROS. This finely tuned balance between bacterial elimination by Duox activity and gut resistance to collateral damage induced by ROS is likely the reason why flies normally survive infection by Ecc15. Yet, this calibration also exposes a vulnerability that could easily be manipulated or subverted by other pathogens. Along this line, this work also exposes the range of impact different bacteria can have on stem cell activation. It was observed that infection with high doses of P. entomophila led to a loss of gut integrity, including the loss of stem cells. Moreover, the ability of P. entomophila to disrupt epithelium renewal correlates with damage to the gut and the death of the host. Since both JNK and JAK-STAT pathways are activated upon infection with P. entomophila, this suggests that this bacterium activates the appropriate pathways necessary to repair the gut, but ISCs are unable to respond accordingly. Interestingly, a completely avirulent P. entomophila mutant (gacA) does not persist in the gut and does not induce epithelium renewal. In contrast, an attenuated mutant (aprA) somewhat restores epithelium renewal. These observations, along with the dose response analysis using P. entomophila and corrosive agents, suggest that the virulence factors of this entomopathogen disrupt epithelium renewal through excessive damage to the gut. Of note, recent studies suggest that both Helicobacter pylori and Shigella flexneri, two bacterial pathogens of the human digestive tract, interfere with epithelium renewal to exert their pathological effects. This suggests that epithelium renewal could be a common target for bacteria that infect through the gut. In this respect, the host defense to oral bacterial infection could be considered as a bimodular response, composed of both immune and homeostatic processes that require strict coordination. Disruption of either process results in the failure to resolve the infection and impedes the return to homeostasis (Buchon, 2009b).
In contrast to the acute invasion by pathogenic bacteria, indigenous gut microbiota are in constant association with the gut epithelium, and thus may impact gut homeostasis. Using axenically raised flies, it was established that indigenous microbiota stimulate a basal level of epithelium renewal that correlates with the level of activation of the JAK-STAT and JNK pathways. This raises the possibility that both indigenous and invasive bacteria, such as Ecc15, are capable of triggering epithelium renewal by the same process. Additionally, the data support a novel homeostatic mechanism in which the density of indigenous bacteria is coupled to the level of epithelium renewal. This is the first report that gut microbiota affect stem cell activation and epithelium renewal, concepts proposed previously in mammalian systems but never fully demonstrated. This also implies that variations in the level of epithelium renewal observed in different laboratory contexts could actually be due to impacts from gut microbes (Buchon, 2009b).
Importantly, in this context, it was shown that lack of indigenous microbiota reverts most age-related deterioration of the gut. Aging of the gut is usually marked by both hyperproliferation of ISCs and differentiation defaults that lead to disorganization of the gut epithelium. These alterations have been shown to be associated with activation of the PDGF- and VEGF-related factor 2 (Pvf2)/Pvr and JNK signaling pathways directly in ISCs. Accordingly, inhibition of the JNK pathway in ISCs fully reverts the epithelium alterations that occur with aging. This raises the possibility that gut microbiota could exert their effect through prolonged activation of the JNK pathway. Interestingly, immune-deficient flies, lacking the Imd pathway, also display hyperproliferative guts and have higher basal levels of activation of the JNK and JAK-STAT pathways. The observation that these flies also harbor higher numbers of indigenous bacteria further supports a model in which failure to control gut microbiota leads to an imbalance in gut epithelium turnover. Future work should analyze the mechanisms by which gut microbiota affect epithelium renewal and whether this is due to a direct impact of bacteria on the gut or is mediated indirectly through changes in fly physiology. Moreover, the correlation between higher numbers of indigenous bacteria and increased disorganization of the gut upon aging in flies lacking the Imd pathway raises the possibility that a main function of this pathway is to control gut microbiota. This is in agreement with concepts emerging in mammals that support an essential role of the gut immune response in maintaining the beneficial nature of the host-microbiota association. This function also parallels the theory of 'controlled inflammation' described in mammals, where a low level of immune activation is proposed to maintain gut barrier integrity (Buchon, 2009b).
In conclusion, this study unravels some of the complex interconnections between the immune response, invasive and indigenous microbiota, and stem cell homeostasis in the gut of Drosophila. Based on the evolutionary conservation of transduction pathways such as JNK and JAK-STAT between Drosophila and mammals, it is likely that similar processes occur in the gut of mammals during infection. Interestingly, stimulation of stem cell activity by invasive bacteria is proposed to favor the development of hyperproliferative states found in precancerous lesions. Thus, Drosophila may provide a more accessible model to elucidate host mechanisms to maintain homeostasis and the impact of bacteria on this process (Buchon, 2009b).
Holometabolous insects undergo complete metamorphosis to become sexually mature adults. Metamorphosis is initiated by brain-derived prothoracicotropic hormone (PTTH), which stimulates the production of the molting hormone ecdysone via an incompletely defined signaling pathway. This study demonstrates that Torso, a receptor tyrosine kinase that regulates embryonic terminal cell fate in Drosophila, is the PTTH receptor. Trunk, the embryonic Torso ligand, is related to PTTH, and ectopic expression of PTTH in the embryo partially rescues trunk mutants. In larvae, torso is expressed specifically in the prothoracic gland (PG), and its loss phenocopies the removal of PTTH. The activation of Torso by PTTH stimulates ERK phosphorylation, and the loss of ERK in the PG phenocopies the loss of PTTH and Torso. It is concluded that PTTH initiates metamorphosis by activation of the Torso/ERK pathway (Rewitz, 2009).
Many organisms undergo distinct temporal transitions in morphology as a part of their normal life process. In humans, for example, passage through puberty is accompanied by changes in body mass and the acquisition of sexual maturity. Likewise, in all holometabolous insects, metamorphosis transforms the immature larva into a completely new body form that is capable of reproductive activity. In both cases, neuropeptide signaling in response to environmental and nutritional cues triggers the transition process. In insects, the process is initiated by the neuropeptide known as prothoracicotropic hormone (PTTH). PTTH signals to the prothoracic gland (PG), the primary insect endocrine organ, which triggers the production and release of ecdysone, the precursor of the active steroid molting hormone 20-hydroxyecdysone (20E). The increased level of 20E provides a systemic signal that ends the larval growth period and initiates metamorphosis (Rewitz, 2009).
PTTH has been proposed to be structurally similar to certain mammalian growth factors that are ligands for receptor tyrosine kinases (RTKs). Previous studies have also indicated that PTTH signaling results in the phosphorylation of cellular signaling molecules that are linked to the mitogen-activated protein kinase (MAPK) pathway in the PG. In light of the potential involvement of MAPK pathway components in PTTH signaling, the expression of all Drosophila RTKs was examined in the PG to determine whether any showed a tissue-specific expression profile that was consistent with a possible role as a PTTH receptor. It was found that after early embryogenesis, the RTK encoded by torso is expressed specifically in the PG (Rewitz, 2009).
The gene torso belongs to the so-called terminal group of genes that are required for the correct patterning of anterior and posterior structures during early embryogenesis. The presumed ligand for Torso during terminal patterning is Trunk (Trk), which contains a cysteine knot-type motif in the C-terminal region similar to the motif in PTTH. Also like PTTH, Trk is thought to be proteolytically processed from a precursor molecule to generate an active C-terminal fragment that is comparable in length to that of PTTH. Alignment of the protein sequences of Trk and PTTH reveals that they share some conserved structures in the C-terminal region that compose the mature peptide, including all of the six cysteines that are important for intramonomeric bonds of the PTTH homodimeric molecule. Previously, it was noted that Trk is related to Spatzle, but a phylogenetic analysis of different insect cysteine knot-type proteins shows that Trk and PTTH form a separate cluster and that PTTH is the closest paralog of Trk. These results raise the possibility that Trk and PTTH share a conserved three-dimensional structure enabling both to activate Torso despite the modest conservation of primary sequence. Expression of trk is not detected in the wandering third-instar larval (L3) stage using real-time polymerase chain reaction (PCR) (no product after 30 PCR cycles) or by in situ hybridization to the brain-PG complex, supporting the idea that PTTH, and not Trk, is a ligand for Torso in post-blastoderm stages (Rewitz, 2009).
To investigate possible post-embryonic roles of Torso in Drosophila development, RNA interference (RNAi) was used to knock down torso specifically in the PG. The PG-specific phantom (phm)-Gal4 line (phm>) was used to drive expression of RNAi constructs under control of upstream activator sequences (UASs) in the PG. This expression of a torso RNAi construct produced a phenotype that was almost identical to the one created by the loss of PTTH-expressing neurons. Reduction of torso expression in the PG of phm>torso-RNAi larvae delays the onset of pupariation by 5.8 days as compared with the phm> + control animals, similar to the 5.4-day delay of pupariation in animals lacking PTTH. As with the loss of the PTTH-producing neurons, torso silencing in the PG also leads to excessive growth during the prolonged L3 stage, resulting in increased pupal size. To test the specificity of the RNAi, it was confirmed that torso mRNA levels are reduced in phm>torso-RNAi larvae and that the PG cells are morphologically normal, although slightly smaller (Rewitz, 2009).
Because torso is a maternal-effect gene, homozygous mutants derived from heterozygous parents are viable. Therefore, the developmental profile and adult size were examined of animals homozygous and transheterozygous for three different torso mutations. Larvae with mutations in torso exhibited substantial developmental delays, although not as long as those seen by RNAi knockdown, in the time to pupariation as compared with heterozygous controls, and the mutants produced larger adults. The difference in time delay may result from residual maternally loaded torso mRNA. In contrast, trk mutants developed on a normal time scale, and adults were similar in size to heterozygous control adults, demonstrating that the phenotype of torso mutants is independent of early embryonic signaling (Rewitz, 2009).
In animals lacking PTTH-producing neurons, it is the low level of the active molting hormone 20E that causes the developmental delay and tissue overgrowth. To investigate whether the torso loss-of-function phenotype is also caused by low 20E levels, 20E was fed to phm>torso-RNAi larvae. Similar to what was found when the PTTH-producing neurons were removed, feeding these larvae with 20E completely rescued the developmental delay and overgrowth. Taken together, these results demonstrate that reducing Torso signaling in the PG alone phenocopies the loss of PTTH, which is consistent with the notion that Torso mediates PTTH signaling in the PG. If this is the case, it would be expected that the constitutively active torsoRL3 allele might produce precocious pupation, as would overexpression of PTTH. Consistent with this conjecture, it was found, using the daughterless (da)-Gal4 driver (da>), that ubiquitous overexpression of PTTH advances the onset of pupariation by 11.5 hours as compared with (da> +) balancer controls and produces smaller adults. At 25°C, torsoRL3 is activated, and heterozygous torsoRL3/+ animals pupariate 9.2 hours before controls and form smaller adult males (Rewitz, 2009).
To establish whether PTTH can activate Torso in vivo, it was reasoned that if PTTH is a ligand for Torso, then ectopic expression of PTTH in the embryo might elicit partial rescue of trk mutants. To examine this, the maternal nanos (nos)-Gal4 line (nos>) was used to drive ubiquitous early embryonic expression of a UAS-PTTH-hemagglutinin (HA)-tagged transgene in trk mutant embryos. In the blastoderm-stage embryo, activation of Torso by Trk induces expression of the downstream target gene tailless (tll) in the anterior and posterior regions. The inability to activate this target gene in trk or torso mutants leads to the loss of structures posterior to the seventh abdominal segment. Early embryonic expression of PTTH was observed in 13% of blastoderm-stage embryos derived from trk1/trk1; nos>PTTH females. Ectopic expression of PTTH in these embryos was sufficient to activate tll in the posterior part of the embryos. Although PTTH expression did not fully restore wild-type tll expression, the partial rescue elicited by PTTH was sufficient to restore posterior structures, such as the Filzkörper, in several trk mutant embryos. These results provide genetic evidence that PTTH functions as a ligand for Torso in vivo (Rewitz, 2009).
In the embryo, Torso signaling is transduced through the canonical MAPK pathway that includes the Drosophila homologs of Ras (Ras85D), Raf (Draf), MAPK kinase (MEK), and extracellular signal-regulated kinase (ERK). If Torso is indeed the PTTH receptor, it would be expected that disrupting MAPK signaling in the PG would result in a phenotype similar to that resulting from loss of the PTTH-producing neurons and Torso signaling. So far, the role of the MAPK pathway in transduction of the PTTH signal has been determined only by in vitro studies of lepidopteran PG. In Drosophila, the expression of dominant negative forms of Ras and Raf is known to delay development. To further examine the importance of the MAPK pathway in mediating PTTH/Torso signaling, RNAi was used to reduce the expression of several core components of this pathway, including Ras, Raf, and ERK, in the PG. Loss of either Ras, Raf, or ERK delayed pupariation by 4.3, 2.7, and 6.1 days, respectively. ERK silencing in the PG delays pupariation as severely as the reduction of Torso signaling or the complete loss of the PTTH-producing neurons does. The increase in size of phm>ERK-RNAi pupae and adults was also similar to the increase caused by the loss of PTTH or loss of Torso. The developmental delay, as well as the size increase caused by ERK silencing, were negated by 20E feeding. The less-severe phenotypes produced by the loss of Raf and Ras may result from less-efficient knockdown or, in the case of Ras, may reflect partial redundancy with Rap1. Consistent with Ras being downstream of torso, it was also found that expression of constitutively active Ras in the PG completely rescued the torso-RNAi-induced delay and overgrowth phenotype. Taken together, these results indicate that, as during embryonic terminal patterning, Torso regulation of ecdysone production in the PG is primarily mediated by the MAPK pathway, resulting in the activation of ERK (Rewitz, 2009).
To test directly whether stimulation of Torso by PTTH could lead to ERK phosphorylation, a cell culture-based signaling assay was developed. Because active Drosophila PTTH has not been produced in tissue culture, the silkworm Bombyx mori full-length Bombyx torso cDNA was cloned. As in Drosophila, the Bombyx torso ortholog is expressed predominantly in the PG of the final (fifth)-instar larvae. Stimulation of Drosophila S2 cells transfected with Bombyx torso and Drosophila ERK with 10-9 M PTTH led to robust phosphorylation of ERK. PTTH stimulation of ERK phosphorylation was not detected in control S2 cells, either incubated in the absence of PTTH or those stimulated with PTTH but not expressing Bombyx torso. Bombyx PTTH did not stimulate activation of ERK through Drosophila Torso or through the insulin receptor, demonstrating that ERK stimulation by Bombyx PTTH is specific to Bombyx Torso. These results demonstrate that Torso is a functional PTTH receptor that is able to mediate PTTH signaling through the activation of the ERK pathway (Rewitz, 2009).
These observations define another role for the terminal system, which is the initiation of metamorphosis at the end of larval growth. Therefore, insects apparently use the same core system for two developmentally distinct processes: the establishment of terminal cell fate in the embryo and the termination of larval growth at the correct time to ensure an appropriate final adult body size. This identification of the PTTH receptor will facilitate further characterization of the system that determines body size in insects. It will be of interest to ascertain just how similar this system is in overall design to the hypothalamus-pituitary-gonadal axis, which controls the timing of puberty in mammals (Rewitz, 2009).
Axon degeneration is observed at the early stages of many neurodegenerative conditions and this often leads to subsequent neuronal loss. Previous studies have shown that inactivating the c-Jun N-terminal kinase (JNK) pathway leads to axon degeneration in Drosophila mushroom body (MB) neurons. To understand this process, candidate suppressor genes were screened and Wallerian degeneration slow (WldS) fusion protein was found to block JNK axonal degeneration. Although the nicotinamide mononucleotide adenylyltransferase (Nmnat1; see Drosophila Nmnat) portion of WldS is required, its nicotinamide adenine dinucleotide (NAD+) enzyme activity and the WldS/ N-terminus (N70) are dispensable, unlike axotomy models of neurodegeneration. It is suggested that WldS-Nmnat protects against axonal degeneration through chaperone activity. Furthermore, ectopically expressed heat shock proteins (Hsp26 and Hsp70) also protected against JNK and Nmnat degeneration phenotypes. These results suggest that molecular chaperones are key in JNK- and Nmnat-regulated axonal protective functions (Rallis, 2013).
WldS was discovered from the molecular cloning of spontaneously generated slow Wallerian degeneration (WldS) mutant mice that showed a strong capacity to promote axonal survival following acute physical lesion. The WldS protein has neuroprotective effects across different species and in different neurodegeneration models. The WldS gene product results from the fusion of first 70 residues of the UBE4B gene (N70), that is involved in polyubiquitination, with the entire nicotinamide mononucleotide adenylyltransferase protein sequence (Nmnat1) that is involved in nicotinamide adenine dinucleotide (NAD+) biosynthesis. Different portions of WldS can confer neuroprotective function (Coleman, 2010). However, WldS function remains unclear. For example, despite its predominant nuclear localisation, it is axonal localisation that appears to be key to neuroprotection, even though WldS and different Nmnat isoforms have subtle and distinct subcellular locations. Also, while in many neurodegenerative paradigms the Nmnat enzyme activity is essential, it is unclear how the NAD+ pathway contributes to axonal protection. Furthermore, some studies suggest Nmnat neuroprotective functions are enzyme-independent. To date, the relationship between WldS function(s) and axon-neuronal damage and repair also remains unclear, although recent data suggest WldS-Nmnat regulation of mitochondrial motility and calcium buffering functions may underlie key neuroprotective responses to physical injury in Drosophila and mouse axons (Avery, 2012). A further report suggests Drosophila Nmnat (dNmnat or nmnat) also controls axonal mitochondria levels and their availability is key to neuroprotection following acute injury (Fang, 2012). Previous data suggest WldS-Nmnat localisation within mitochondria may also be the underlying basis of axonal neuroprotection (Rallis, 2013).
When tested ectopically, many Nmnat isoforms and homologs show axonal-protective effects even though some appear to be weaker, possibly due to labile effects. However, apart from Drosophila Nmnat, currently only mouse Nmnat2 has an endogenous role in promoting axonal stability. It is important to note, beyond their neuronal roles, Nmnats also have obligate roles in NAD+ metabolism and multiple cellular processes across species. Very recent reports show Nmnat1 mutations cause Leber congenital amaurosis (LCA), highlighting its importance in retinal degenerative diseases in humans (Rallis, 2013).
This study shows that the WldS protein protects against axon degeneration triggered by JNK inactivation. Contrary to previous models, while the Nmnat1 region
is sufficient, this study found that its enzyme activity is dispensable for WldS neuroprotection. The results suggest that Nmnat and JNK axonal-protective functions occur through molecular chaperones (Rallis, 2013).
One previous report showed that Drosophila Nmnat has a non-enzyme function that involves molecular chaperone activity (Zhai, 2008). Drosophila Nmnat was recruited together with the molecular chaperone, Heat shock protein (Hsp) Hsp70 to polyglutamine expanded spinocerebellar ataxin-1 (SCA-1) containing aggregates. Non-enzyme Nmnat functions were involved in regulating protein folding and blocking SCA-1 neurotoxicity. Very recent results show non-enzyme Nmnat also functions to clear tau oligomers in vivo (Ali, 2012). This study tested the effect of Heat shock proteins (Hsps) on the bsk phenotypes in two ways. In bsk-null neuroblast clones, it was found that, like WldS and Nmnats1 and 3, ectopic Hsp70 or Hsp26 also blocked the bsk axon degeneration (Rallis, 2013).
Compared to wild-type axons, bsk axons showed more abnormal protrusions and swellings along the axons and terminals. When Hsp70, WldS, Nmnat and Nmnat enzyme-inactive forms were expressed in these clones, these were reduced suggesting that this phenotype is also linked to Hsps and non-enzyme Nmnat activities (Rallis, 2013).
To further test the neuroprotective activity of Hsps, Nmnat RNAi assays was used. When Nmnat RNAi was expressed in MB neurons, this resulted in a β-axon loss phenotype similar to nmnat1 loss-of-function clones above. Some neuronal loss was visible in newly eclosed adults (1-day-old adults). However, almost all neurons were lost in 7-day-old adults, suggesting that Nmnat is an obligate maintenance factor, consistent with previous reports. The Nmnat RNAi axon and neuronal cell loss was rescued by enzyme-inactive forms of mNmnat1 (H24A) and WldS-dead. Furthermore, Hsp26 and Hsp70 expression also partially suppressed the Nmnat RNAi phenotype. Together, these results suggest non-enzyme Nmnat and chaperone activities are linked to JNK axonal functions (Rallis, 2013).
Using the GAL80ts system to control JNK temporal expression, it has been shown that JNK activity is required throughout development, even though the axon degeneration phenotype occurs mainly at adult stages. To determine Nmnat's temporal requirements, Nmnat RNAi was coupled to GAL80ts control, and the loss-of-function phenotype was induced at various stages of development. It was found that RNAi throughout the development and adult phase caused the strongest neuronal loss phenotype. RNAi induction at pupal or adult stages also caused neuronal loss, albeit at a weaker levels. These results suggest Nmnat is required throughout development as well as adult stages. Even though the Nmnat RNAi phenotype is more severe in adults, as in bsk mutants, unlike bsk, Nmnat's genetic requirements extend beyond the developmental stages and are essential at adult stages. This suggests Drosophila Nmnat may have additional roles at adult stages that may be independent of JNK activity (Rallis, 2013).
Elevated CO(2) levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. This study examined the role of c-Jun N-terminal kinase (JNK) in CO(2) signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO(2) levels rapidly induces activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK requires AMP-activated protein kinase (AMPK) and protein kinase C-zeta leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO(2) levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO(2)-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO(2) signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO(2) signaling in mammals, diptera and nematodes (Vadasz, 2012).
Understanding the mechanism(s) by which dopaminergic (DAergic) neurons are eroded in Parkinson's disease (PD) is critical for effective therapeutic strategies. By using the binary tyrosine hydroxylase (TH)-Gal4/UAS-X RNAi Drosophila melanogaster system, it is reported that p53, basket and ICE gene knockdown in dopaminergic neurons prolong life span and locomotor activity in D. melanogaster lines chronically exposed to (1 microM) paraquat [PQ, oxidative stress (OS) generator] compared to untreated transgenic fly lines. Likewise, knockdown flies displayed higher climbing performance than control flies. Amazingly, gallic acid (GA) significantly protected DAergic neurons, ameliorated life span, and climbing abilities in knockdown fly lines treated with PQ compared to flies treated with PQ only. Therefore, silencing specific gene(s) involved in neuronal death might constitute an excellent tool to study the response of DAergic neurons to OS stimuli. It is proposed that a therapy with antioxidants and selectively 'switching off' death genes in DAergic neurons could provide a means for pre-clinical PD individuals to significantly ameliorate their disease condition (Ortega-Arellano, 2013).
The Drosophila melanogaster homolog of the ced-1 gene from Caenorhabditis elegans is draper, which encodes a cell surface receptor required for the recognition and engulfment of apoptotic cells, glial clearance of axon fragments and dendritic pruning, and salivary gland autophagy. To further elucidate mechanisms of Draper signaling, a genetic screen of chromosomal deficiencies was performed to identify loci that dominantly modify the phenotype of over-expression of Draper isoform II, which suppresses differentiation of the posterior crossvein in the wing. The existence of 43 genetic modifiers of Draper II was deduced. 24 of the 37 suppressor loci and 3 of the 6 enhancer loci have been identified. A further 5 suppressors and 2 enhancers were identified from mutations in functionally related genes. These studies indicated positive contributions to Drpr signaling for the Jun N-terminal Kinase pathway, supported by genetic interactions with hemipterous, basket, jun, and puckered, and for cytoskeleton regulation as indicated by genetic interactions with rac1, rac2, RhoA, myoblast city, Wiskcott-Aldrich syndrome protein, and the formin CG32138, and for yorkie and expanded. These findings indicate that Jun N-terminal Kinase activation and cytoskeletal remodeling collaborate in the engulfment process downstream of Draper activation. The relationships between Draper signaling and Decapentaplegic signaling, insulin signaling, Salvador-Warts-Hippo signaling, apical-basal cell polarity, and cellular responses to mechanical forces are further investigated and discussed (Fullard, 2014).
This study defines TF network that triggers an abnormal gene expression program promoting malignancy of clonal tumors, generated in Drosophila imaginal disc epithelium by gain of oncogenic Ras (RasV12) and loss of the tumor suppressor Scribble (scrib1). Malignant transformation of the rasV12scrib1 tumors requires TFs of distinct families, namely the bZIP protein Fos, the ETS-domain factor Ets21c and the nuclear receptor Ftz-F1, all acting downstream of Jun-N-terminal kinase (JNK). Depleting any of the three TFs improves viability of tumor-bearing larvae, and this positive effect can be enhanced further by their combined removal. Although both Fos and Ftz-F1 synergistically contribute to rasV12scrib1 tumor invasiveness, only Fos is required for JNK-induced differentiation defects and Matrix metalloprotease (MMP1) upregulation. In contrast, the Fos-dimerizing partner Jun is dispensable for JNK to exert its effects in rasV12scrib1 tumors. Interestingly, Ets21c and Ftz-F1 are transcriptionally induced in these tumors in a JNK- and Fos-dependent manner, thereby demonstrating a hierarchy within the tripartite TF network, with Fos acting as the most upstream JNK effector. Of the three TFs, only Ets21c can efficiently substitute for loss of polarity and cooperate with Ras(V12) in inducing malignant clones that, like rasV12scrib1 tumors, invade other tissues and overexpress MMP1 and the Drosophila insulin-like peptide 8 (Dilp8). While rasV12ets21c tumors require JNK for invasiveness, the JNK activity is dispensable for their growth. In conclusion, this study delineates both unique and overlapping functions of distinct TFs that cooperatively promote aberrant expression of target genes, leading to malignant tumor phenotypes.
(Kulshammer, 2015).
Genome-wide transcriptome profiling in the Drosophila epithelial tumor model has generated a comprehensive view of gene expression changes induced by defined oncogenic lesions that cause tumors of an increasing degree of malignancy. These data allowed discovery of how a network of collaborating transcription factors confers malignancy to RasV12scrib1 tumors (Kulshammer, 2015).
This study revealed that the response of transformed RasV12scrib1 epithelial cells is more complex in comparison to those with activated RasV12 alone with respect to both the scope and the magnitude of expression of deregulated genes.
Aberrant expression of more than half of the genes in RasV12scrib1 tumors requires JNK activity, highlighting the significance of JNK signaling in malignancy. Importantly, the tumor-associated, JNK-dependent transcripts cluster with biological functions and processes that tightly match the phenotypes of previously described tumor stages. Furthermore, the RasV12scrib1 transcriptome showed significant overlap (27% upregulated and 15% downregulated genes) with microarray data derived from mosaic EAD in which tumors were induced by overexpressing the BTB-zinc finger TF Abrupt (Ab) in scrib1 mutant clones as well as with a transcriptome of scrib1 mutant wing discs. It is proposed that 429 misregulated transcripts (e.g. cher, dilp8, ets21c, ftz-f1, mmp1, upd), shared among all the three data sets irrespective of epithelial type (EAD versus wing disc) or cooperating lesion (RasV12 or Ab), represent a 'polarity response transcriptional signature' that characterizes the response of epithelia to tumorigenic polarity loss. Genome-wide profiling and comparative transcriptome analyses thus provide a foundation to identify novel candidates that drive and/or contribute to tumor development and malignancy while unraveling their connection to loss of polarity and JNK signaling (Kulshammer, 2015).
In agreement with a notion of combinatorial control of gene expression by an interplay among multiple TFs, this study identified overrepresentation of cis-acting DNA elements for STAT, GATA, bHLH, ETS, BTB, bZIP factors and NRs in genes deregulated in RasV12scrib1 mosaic EAD, implying that transcriptome anomalies result from a cross-talk among TFs of different families. Many of the aberrantly expressed genes contained binding motifs for AP-1, Ets21c and Ftz-F1, indicating that these three TFs may regulate a common set of targets and thus cooperatively promote tumorigenesis. This is consistent with the occurrence of composite AP-1-NRRE (nuclear receptor response elements), ETS-NRRE and ETS-AP-1 DNA elements in the regulatory regions of numerous human cancer-related genes, such as genes for cytokines, MMPs (e.g., stromelysin, collagenase) and MMP inhibitors (e.g., TIMP) (Kulshammer, 2015).
Interestingly, Drosophila ets21c and ftz-f1 gene loci themselves contain AP-1 motifs and qualify as polarity response transcriptional signature transcripts. Indeed, this study has detected JNK- and Fos-dependent upregulation of ets21c and ftz-f1 mRNAs in RasV12scrib1 tumors. While JNK-mediated control of ftz-f1 transcription has not been reported previously, upregulation of ets21c in the current tumor model is consistent with JNK requirement for infection-induced expression of ets21c mRNA in Drosophila S2 cells and in vivo. Based on these data, it is proposed that Ftz-F1 and Ets21c are JNK-Fos-inducible TFs that together with AP-1 underlie combinatorial transcriptional regulation and orchestrate responses to cooperating oncogenes. Such an interplay between AP-1 and Ets21c is further supported by a recent discovery of physical interactions between Drosophila Ets21c and the AP-1 components Jun and Fos (Rhee, 2014). Whether regulatory interactions among AP-1, Ets21c and Ftz-F1 require their direct physical contact and/or the presence of composite DNA binding motifs of a particular arrangement to control the tumor-specific transcriptional program remains to be determined (Kulshammer, 2015).
Importantly, some of the corresponding DNA elements, namely AP-1 and STAT binding sites, have recently been found to be enriched in regions of chromatin that become increasingly accessible in RasV12scrib1 mosaic EAD relative to control. This demonstrates that comparative transcriptomics and open chromatin profiling using ATAC-seq and FAIRE-seq are suitable complementary approaches for mining the key regulatory TFs responsible for controlling complex in vivo processes, such as tumorigenesis (Kulshammer, 2015).
The prototypical form of AP-1 is a dimer comprising Jun and Fos proteins. In mammals, the Jun proteins occur as homo- or heterodimers, whereas the Fos proteins must interact with Jun in order to bind the AP-1 sites. In contrast to its mammalian orthologs, the Drosophila Fos protein has been shown to form a homodimer capable of binding to and activating transcription from an AP-1 element, at least in vitro (Kulshammer, 2015).
The role of individual AP-1 proteins in neoplastic transformation and their involvement in pathogenesis of human tumors remain somewhat elusive. While c-Jun, c-Fos and FosB efficiently transform mammalian cells in vitro, only c-Fos overexpression causes osteosarcoma formation, whereas c-Jun is required for development of chemically induced skin and liver tumors in mice. In contrast, JunB acts as a context-dependent tumor suppressor. Thus, cellular and genetic context as well as AP-1 dimer composition play essential roles in dictating the final outcome of AP-1 activity in tumors (Kulshammer, 2015).
This study shows that, similar to blocking JNK with its dominant-negative form, Bsk, removal of Fos inhibits ets21c and ftz-f1 upregulation, suppresses invasiveness, improves epithelial organization and differentiation within RasV12scrib1 tumors and allows larvae to pupate. Strikingly, depletion of Jun had no such tumor-suppressing effects. It is therefore concluded that in the malignant RasV12scrib1 tumors, Fos acts independently of Jun, either as a homodimer or in complex with another, yet unknown partner. A Jun-independent role for Fos is further supported by additional genetic evidence. Fos, but not Jun, is involved in patterning of the Drosophila endoderm and is required for expression of specific targets, e.g., misshapen (msn) and dopa decarboxylase (ddc), during wound healing. Future studies should establish whether the JNK-responsive genes containing AP-1 motifs, identified in this study, are indeed regulated by Fos without its 'canonical' partner (Kulshammer, 2015).
The current data identify Fos as a key mediator of JNK-induced MMP1 expression and differentiation defects in RasV12scrib1 tumors. Only Fos inhibition caused clear suppression of MMP1 levels and restoration of neurogenesis within clonal EAD tissue, thus mimicking effects of JNK inhibition. Improved differentiation and reduced invasiveness are, however, not sufficient for survival of animals to adulthood, because interfering with Fos function in RasV12scrib1 clones always resulted in pupal lethality (Kulshammer, 2015).
The systems approach of this paper, followed by genetic experiments, identified Ets21c and Ftz-F1 as being essential for RasV12scrib1-driven tumorigenesis. It was further shown that mutual cooperation of both of these TFs with Fos is required to unleash the full malignancy of RasV12scrib1 tumors (Kulshammer, 2015).
TFs of the ETS-domain family are key regulators of development and homeostasis in all metazoans, whereas their aberrant activity has been linked with cancer. ets21c encodes the single ortholog of human Friend leukemia insertion1 (FLI1) and ETS-related gene (ERG) that are commonly overexpressed or translocated in various tumor types. While FLI1 is considered pivotal to development of Ewing's sarcoma, ERG has been linked to leukemia and prostate cancer. As for Ftz-F1 orthologs, the human liver receptor homolog-1 (LRH-1) has been associated with colonic, gastric, breast and pancreatic cancer, whereas steroidogenic factor 1 (SF-1) has been implicated in prostate and testicular cancers and in adrenocortical carcinoma. However, the molecular mechanisms underlying oncogenic activities of either the ERG/FLI1 or the SF-1/LRH-1 proteins are not well understood (Kulshammer, 2015).
This study shows that removal of Ftz-F1 markedly suppressed invasiveness of RasV12scrib1 tumors, restoring the ability of tumor-bearing larvae to pupate. Additionally, and in contrast to Fos, Ftz-F1 inhibition also partly reduced tumor growth in the third-instar EAD and allowed emergence of adults with enlarged, rough eyes composed predominantly of non-clonal tissue. The reduced clonal growth coincided with downregulation of the well-established Yki target, expanded, implicating Ftz-F1 as a potential novel growth regulator acting on the Hpo/Yki pathway. It is further speculated that reduced viability of RasV12scrib1ftz-f1RNAi clones and induction of non-autonomous compensatory proliferation by apoptotic cells during the pupal stage could explain the enlargement of the adult eyes. The precise mechanism underlying compromised growth and invasiveness of RasV12scrib1ftz-f1RNAi tumors and improved survival of the host remains to be identified (Kulshammer, 2015).
In contrast, effects of Ets21cLONG knockdown in RasV12scrib1 tumors appeared moderate relative to the clear improvement conferred by either Fos or Ftz-F1 elimination. ets21cLONG RNAi neither reduced tumor mass nor suppressed invasiveness, and pupation was rescued only partly. However, unlike ftz-f1RNAi, ets21cLONG RNAi significantly reduced expression of dilp8 mRNA. Based on abundance of Ets21c binding motifs in the regulatory regions of tumor-associated genes and the normalized expression of >20% of those genes upon removal of Ets21c, it is further suggested that Ets21c acts in RasV12scrib1 tumors to fine-tune the tumor gene-expression signature (Kulshammer, 2015).
Dilp8 is known to be secreted by damaged, wounded or tumor-like tissues to delay the larval-to-pupal transition. This study has corroborated the role of JNK in stimulating dilp8 expression in RasV12scrib1 tumor tissue, and has further implicated Ets21c and Fos as novel regulators of dilp8 downstream of JNK. However, the data also show that elevated dilp8 transcription per se is not sufficient to delay metamorphosis. Unlike the permanent larvae bearing RasV12scrib1 tumors, those with RasV12scrib1ftz-f1RNAi tumors pupated despite the excessive dilp8 mRNA. Likewise, pupation was not blocked by high dilp8 levels in larvae bearing EAD clones overexpressing Abrupt. As Dilp8 secretion appears critical for its function, it is proposed that loss of Ftz-F1 might interfere with Dilp8 translation, post-translational processing or secretion (Kulshammer, 2015).
Consistent with the individual TFs having unique as well as overlapping functions in specifying properties of RasV12scrib1 tumors, knocking down pairwise combinations of the TFs had synergistic effects on tumor suppression compared with removal of single TF. This evidence supports the view that malignancy is driven by a network of cooperating TFs, and elimination of several tumor hallmarks dictated by this network is key to animal survival. An interplay between AP-1, ETS-domain TFs and NRs is vital for development. For example, the ETS-factor Pointed has been shown to cooperate with Jun to promote R7 photoreceptor formation in the Drosophila adult eye. In mosquitoes, synergistic activity of another ETS-factor, E74B, with the ecdysone receptor (EcR/USP) promotes vitellogenesis. It is thus proposed that tumors become malignant by hijacking the developmental mechanism of combinatorial control of gene activity by distinct TFs (Kulshammer, 2015).
Despite the minor impact of ets21cLONG knockdown on suppressing RasV12scrib1 tumors, Ets21cLONG is the only one of the tested TFs that was capable of substituting for loss of scrib in inducing malignant clonal overgrowth when overexpressed with oncogenic RasV12 in EAD. While invasiveness of such RasV12ets21cLONG tumors required JNK activity, JNK signaling appeared dispensable for tumor growth. Importantly, the overgrowth of RasV12ets21cLONG tumors was primarily independent of a prolonged larval stage, because dramatic tumor mass expansion was detected already on day 6 AEL. How cooperativity between Ets21cLONG and RasV12 ensures sufficient JNK activity and the nature of the downstream effectors driving tumor overgrowth remain to be determined. In contrast, co-expression of either Ftz-F1 or Fos with RasV12 resulted in a non-invasive, RasV12-like hyperplastic phenotype (Kulshammer, 2015).
Why does Ets21cLONG exert its oncogenic potential while Fos and Ftz-F1 do not? Simple overexpression of a TF may not be sufficient, because many TFs require activation by a post-translational modification (e.g., phosphorylation), interaction with a partner protein and/or binding of a specific ligand. Full activation of Fos in response to a range of stimuli is achieved through hyperphosphorylation by mitogen-activated protein kinases (MAPKs), including ERK and JNK. Indeed, overexpression of a FosN-Ala mutated form that cannot be phosphorylated by JNK was sufficient to phenocopy fos deficiency, indicating that Fos must be phosphorylated by JNK in order to exert its oncogenic function. Consistent with the current data, overexpression of FosN-Ala partly restored polarity of lgl mutant EAD cells. It is therefore conclude that the tumorigenic effect of Fos requires a certain level of JNK activation, which is lacking in EAD co-expressing Fos with RasV12. Nevertheless, the absence of an unknown Fos-interacting partner cannot be excluded (Kulshammer, 2015).
Interestingly, MAPK-mediated phosphorylation also greatly enhances the ability of SF-1 and ETS proteins to activate transcription. Two potential MAPK sites can be identified in the hinge region of Ftz-F1, although their functional significance is unknown. Whether Ets21c or Ftz-F1 requires phosphorylation and how this would impact their activity in the tumor context remains to be determined. Genetic experiments demonstrate that at least the overgrowth of RasV12ets21cLONG tumors does not require Ets21c phosphorylation by JNK (Kulshammer, 2015).
In addition, previous crystallography studies revealed the presence of phosphoinositides in the ligand binding pocket of LHR-1 and SF-1 and showed their requirement for the NR transcriptional activity. Although developmental functions of Drosophila Ftz-F1 seem to be ligand independent, it is still possible that Ftz-F1 activity in the tumor context is regulated by a specific ligand. An effect of Ftz-F1 SUMOylation cannot be ruled out (Kulshammer, 2015).
In summary, this work demonstrates that malignant transformation mediated by RasV12 and scrib loss depends on MAPK signaling and at least three TFs of different families, Fos, Ftz-F1 and Ets21c. While their coordinated action ensures precise transcriptional control during development, their aberrant transcriptional (Ets21c, Ftz-F1) and/or post-translational (Fos, Ftz-F1, Ets21c) regulation downstream of the cooperating oncogenes contributes to a full transformation state. The data implicate Fos as a primary nuclear effector of ectopic JNK activity downstream of disturbed polarity that controls ets21c and ftz-f1 expression. Through combinatorial interactions on overlapping sets of target genes and acting on unique promoters, Fos, Ftz-F1 and Ets21c dictate aberrant behavior of RasV12scrib1 tumors. Although originally described in Drosophila, detrimental effects of cooperation between loss of Scrib and oncogenic Ras has recently been demonstrated in mammalian tumor models of prostate and lung cancer. This study and further functional characterization of complex TF interactions in the accessible Drosophila model are therefore apt to provide important insight into processes that govern cancer development and progression in mammals (Kulshammer, 2015).
A fundamental question of biology is what determines organ size. Despite demonstrations that factors within organs determine their sizes, intrinsic size control mechanisms remain elusive. This study shows that Drosophila wing size is regulated by JNK signaling during development. JNK is active in a stripe along the center of developing wings, and modulating JNK signaling within this stripe changes organ size. This JNK stripe influences proliferation in a non-canonical, Jun-independent manner by inhibiting the Hippo pathway. Localized JNK activity is established by Hedgehog signaling, where Ci elevates dTRAF1 expression. As the dTRAF1 homolog, TRAF4, is amplified in numerous cancers, these findings provide a new mechanism for how the Hedgehog pathway could contribute to tumorigenesis, and, more importantly, provides a new strategy for cancer therapies. Finally, modulation of JNK signaling centers in developing antennae and legs changes their sizes, suggesting a more generalizable role for JNK signaling in developmental organ size control (Willsey, 2016).
Two independently generated antibodies that recognize the phosphorylated, active form of JNK (pJNK) specifically label a stripe in the pouch of developing wildtype third instar wing discs. Importantly, localized pJNK staining is not detected in hemizygous JNKK mutant discs, in clones of JNKK mutant cells within the stripe, following over-expression of the JNK phosphatase puckered (puc), or following RNAi-mediated knockdown of bsk using two independent, functionally validated RNAi lines (Willsey, 2016).
The stripe of localized pJNK staining appeared to be adjacent to the anterior-posterior (A/P) compartment boundary, a location known to play a key role in organizing wing growth, and a site of active Hedgehog (Hh) signaling. Indeed, pJNK co-localizes with the Hh target gene patched (ptc). Expression of the JNK phosphatase puc in these cells specifically abrogated pJNK staining, as did RNAi-mediated knockdown of bsk. Together, these data indicate that the detected pJNK signal reflects endogenous JNK signaling activity in the ptc domain, a region of great importance to growth control. Indeed, while at earlier developmental stages pJNK staining is detected in all wing pouch cells, the presence of a localized stripe of pJNK correlates with the time when the majority of wing disc growth occurs (1000 cells/disc at mid-L3 stage to 50,000 cells/disc at 20 hr after pupation, so it is hypothesized that localized pJNK plays a role in regulating growth (Willsey, 2016).
Inhibition of JNK signaling in the posterior compartment previously led to the conclusion that JNK does not play a role in wing development. The discovery of an anterior stripe of JNK activity spurred a reexamination of the issue. Since bsk null mutant animals are embryonic lethal, JNK signaling was conditionally inhibited in three independent ways in the developing wing disc. JNK inhibition was achieved by RNAi-mediated knockdown of bsk (bskRNAi#1or2), by expression of JNK phosphatase (puc), or by expression of a dominant negative bsk (bskDN). These lines have been independently validated as JNK inhibitors. Inhibition of JNK in all wing blade cells (rotund-Gal4, rn-Gal4) or specifically in ptc-expressing cells (ptc-Gal4) resulted in smaller adult wings in all cases, up to 40% reduced in the strongest cases. Importantly, expression of a control transgene (UAS-GFP) did not affect wing size. This contribution of JNK signaling to size control is likely an underestimate, as the embryonic lethality of bsk mutations necessitates conditional, hypomorphic analysis. Nevertheless, hypomorphic hepr75/Y animals, while pupal lethal, also have smaller wing discs, as do animals with reduced JNK signaling due to bskDN expression. Importantly, total body size is not affected by inhibiting JNK in the wing. Even for the smallest wings generated (rn-Gal4, UAS-bskDN), total animal body size is not altered (Willsey, 2016).
To test whether elevation of this signal can increase organ size, eiger (egr), a potent JNK activator, was expressed within the ptc domain (ptc-Gal4, UAS-egr). Despite induction of cell death as previously reporte and late larval lethality, a dramatic increase was observed in wing disc size without apparent duplications or changes in the shape of the disc. While changes in organ size could be due to changing developmental time, wing discs with elevated JNK signaling were already larger than controls assayed at the same time point. Similarly, inhibition of JNK did not shorten developmental time. Thus, changes in organ size by modulating JNK activity do not directly result from altering developmental time. Finally, the observed increase in organ size is not due to induction of apoptosis, as expression of the pro-apoptotic gene hid does not increase organ size. In contrast, it causes a decrease in wing size. Furthermore, co-expression of diap1 or p35 did not significantly affect the growth effect of egr expression, while the effect was dependent on Bsk activity (Willsey, 2016).
In stark contrast to known developmental morphogens, no obvious defects were observed in wing venation pattern following JNK inhibition, suggesting that localized pJNK may control growth in a pattern formation-independent manner. Indeed, even a slight reduction in Dpp signaling results in dramatic wing vein patterning defects. Second, inhibiting Dpp signaling causes a reduction in wing size along the A-P axis, while JNK inhibition causes a global reduction. Furthermore, ectopic Dpp expression increases growth in the form of duplicated structures, while increased JNK signaling results in a global increase in size. Molecularly, it was confirmed that reducing Dpp signaling abolishes pSMAD staining, while quantitative data shows that inhibiting JNK signaling does not. Furthermore, it was also found that Dpp is not upstream of pJNK, as reduction in Dpp signaling does not affect pJNK. Together, the molecular data are consistent with the phenotypic results indicating that pJNK and Dpp are separate programs in regulating growth. Consistent with these findings it has been suggested that Dpp does not play a primary role in later larval wing growth control (Akiyama, 2015). Finally, it was found that inhibition of JNK does not affect EGFR signaling (pERK) and that inhibition of EGFR does not affect the establishment of pJNK (Willsey, 2016).
A difference in size could be due to changes in cell size and/or number. Wings with reduced size due to JNK inhibition were examined and no changes in cell size or apoptosis were found, suggesting that pJNK controls organ size by regulating cell number. Consistently, the cell death inhibitor p35 was unable to rescue the decreased size following JNK inhibition. Indeed, inhibition of JNK signaling resulted in a decrease in proliferation, while elevation of JNK signaling in the ptc domain resulted in an increase in cell proliferation in the enlarged wing disc. Importantly, this increased proliferation is not restricted to the ptc domain, consistent with previous reports that JNK can promote proliferation non-autonomously (Willsey, 2016).
To determine the mechanism by which pJNK controls organ size, canonical JNK signaling through its target Jun was considered. Interestingly, RNAi-mediated knockdown of jun in ptc cells does not change wing size, consistent with previous analysis of jun mutant clones in the wing disc. Furthermore, in agreement with this, a reporter of canonical JNK signaling downstream of jun (puc-lacZ) is not expressed in the pJNK stripe. Finally, knockdown of fos (kayak, kay) alone or with junRNAi did not affect wing size. Together, these data indicate that canonical JNK signaling through jun does not function in the pJNK stripe to regulate wing size (Willsey, 2016).
In search of such a non-canonical mechanism of JNK-mediated size control, the Hippo pathway was considered. JNK signaling regulates the Hippo pathway to induce autonomous and non-autonomous proliferation during tumorigenesis and regeneration via activation of the transcriptional regulator Yorkie (Yki). Recently it has been shown that JNK activates Yki via direct phosphorylation of Jub. To test whether this link between JNK and Jub could account for the role of localized pJNK in organ size control during development, RNAi-mediated knockdown of jub was performed in the ptc stripe, and adults with smaller wings were observed. Indeed, the effect of JNK loss on wing size can be partially suppressed in a heterozygous lats mutant background and increasing downstream yki expression in all wing cells or just within the ptc domain can rescue wing size following JNK inhibition. These results suggest that pJNK controls Yki activity autonomously within the ptc stripe, leading to a global change in cell proliferation. This hypothesis predicts that the Yki activity level within the ptc stripe influences overall wing size. Consistently, inhibition of JNK in the ptc stripe translates to homogeneous changes in anterior and posterior wing growth. Similarly, overexpression or inhibition of Yki signaling in the ptc stripe also results in a global change in wing size (Willsey, 2016).
It is important to note that the yki expression line used is wild-type Yki, which is still affected by JNK signaling. For this reason, the epistasis experiment was also performed with activated Yki, which is independent of JNK signaling. Expression of this activated Yki in the ptc stripe caused very large tumors and lethality. Importantly, inhibiting JNK in this context did not affect the formation of these tumors or the lethality. Furthermore, inhibiting both JNK and Yki together does not enhance the phenotype of Yki inhibition alone, further supporting the idea that Yki is epistatic to JNK, instead of acting in parallel processes (Willsey, 2016).
Mutants of the Yki downstream target four-jointed (fj) have small wings with normal patterning, and fj is known to propagate Hippo signaling and affect proliferation non-autonomously. Although RNAi-mediated knockdown of fj in ptc cells does not cause an obvious change in wing size, it is sufficient to block the Yki-induced effect on increasing wing size . However, overexpression of fj also reduces wing size, which makes it not possible to test for a simple epistatic relationship. Overall, these data are consistent with the notion that localized pJNK regulates wing size not by Jun-dependent canonical JNK signaling, but rather by Jun-independent non-canonical JNK signaling involving the Hippo pathway (Willsey, 2016).
While morphogens direct both patterning and growth of developing organs, a link between patterning molecules and growth control pathways has not been established. pJNK staining is coincident with ptc expression, suggesting it could be established by Hh signaling. During development, posterior Hh protein travels across the A/P boundary, leading to activation of the transcription factor Cubitus interruptus (Ci) in the stripe of anterior cells. To test whether localized activation of JNK is a consequence of Hh signaling through Ci, RNAi-mediated knockdown of ci was performed, and it was found that the pJNK stripe is eliminated. Consistently, adult wing size is globally reduced. In contrast, no change was observed in pJNK stripe staining following RNAi-mediated knockdown of dpp or EGFR. Expression of non-processable Ci leads to increased Hh signaling. Expression of this active Ci in ptc cells leads to an increase in pJNK signal and larger, well-patterned adult wings. The modest size increase shown for ptc>CiACT is likely due to the fact that higher expression of this transgene (at 25 ° C) leads to such large wings that pupae cannot emerge from their cases. For measuring wing size, this experiment was performed at a lower temperature so that the animals were still viable. Furthermore, inhibition of JNK in wings expressing active Ci blocks Ci's effects, and resulting wings are similar in size to JNK inhibition alone . Together, these data indicate that Hh signaling through Ci is responsible for establishing the pJNK stripe (Willsey, 2016).
To determine the mechanism by which Ci activates the JNK pathway, transcriptional profiles of posterior and ptc domain cells isolated by FACS from third instar wing discs were compared. Of the total 12,676 unique genes represented on the microarray, 50.4% (6,397) are expressed in ptc domain cells, posterior cells, or both. Hh pathway genes known to be differentially expressed were identified. It was next asked whether any JNK pathway genes are differentially expressed, and and it was found that dTRAF1 expression is more than five-fold increased in ptc cells, while other JNK pathway members are not differentially expressed (Willsey, 2016).
dTRAF1 is expressed along the A/P boundary and ectopic expression of dTRAF1 activates JNK signaling. Thus, positive regulation of dTRAF1 expression by Ci could establish a stripe of pJNK that regulates wing size. Indeed, Ci binding motifs were identified in the dTRAF1 gene, and a previous large-scale ChIP study confirms a Ci binding site within the dTRAF1 gene. Consistently, a reduction in Ci led to a 29% reduction in dTRAF1 expression in wing discs. Given that the reduction of dTRAF1 expression in the ptc stripe is buffered by Hh-independent dTRAF1 expression elsewhere in the disc, this 29% reduction is significant. Furthermore, inhibition of dTRAF1 by RNAi knockdown abolished pJNK staining. Finally, these animals have smaller wings without obvious pattern defects. Conversely, overexpression of dTRAF1 causes embryonic lethality, making it not possible to attempt to rescue a dTRAF1 overexpression wing phenotype by knockdown of bsk. Nevertheless, it has been shown that dTRAF1 function in the eye is Bsk-dependent. Finally, inhibition of dTRAF1 modulates the phenotype of activated Ci signaling. Together, these data reveal that the pJNK stripe in the developing wing is established by Hh signaling through Ci-mediated induction of dTRAF1 expression (Willsey, 2016).
Finally, localized centers of pJNK activity were detected during the development of other imaginal discs including the eye/antenna and leg. Inhibition of localized JNK signaling during development caused a decrease in adult antenna size and leg size. Conversely, increasing JNK signaling during development resulted in pupal lethality; nevertheless, overall sizes of antenna and leg discs were increased. Together, these data indicate that localized JNK signaling regulates size in other organs in addition to the wing, suggesting a more universal effect of JNK on size control (Willsey, 2016).
Intrinsic mechanisms of organ size control have long been proposed and sought after. This study reveals that in developing Drosophila tissues, localized, organ-specific centers of JNK signaling contribute to organ size in an activity level-dependent manner. Such a size control mechanism is qualitatively distinct from developmental morphogen mechanisms, which affect both patterning and growth. Aptly, this mechanism is still integrated in the overall framework of developmental regulation, as it is established in the wing by the Hh pathway. These data indicate that localized JNK signaling is activated by Ci-mediated induction of dTRAF1 expression. Furthermore,it is not canonical Jun-dependent JNK signaling, but rather non-canonical JNK signaling that regulates size, possibly through Jub-dependent regulation of Yki signaling, as described for regeneration. As the human dTRAF1 homolog, TRAF4, and Hippo components are amplified in numerous cancers, these findings provide a new mechanism for how the Hh pathway could contribute to tumorigenesis. More importantly, these findings offer a new strategy for potential cancer therapies, as reactivating Jun in Hh-driven tumors could lead tumor cells towards an apoptotic fate (Willsey, 2016).
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