hemipterous
In embryos mutant for hemipterous, dorsal closure [Image] is defective. Spreading of the dorsal epithelia toward the midline is blocked. Cell sheet movement can be arrested at any time, indicating hep is
required in maintaining epithelial spreading. Cuticular preparations of dead embryos show a large gap in dorsal structures. There are in addition striking deletions of adult structures such as wing, dorsal mesothorax, eye and metathoracic leg (Glise, 1995).
In addition to affecting dorsal closure, hep mutation also disturbs head involution. The anterior-most stripe of engrailed expressing cells marks a gnathal-derived structure, the dorsal ridge; its formation relies on the fusion of its two lateral edges at the onset of dorsal closure. Once formed, the dorsal ridge slides anteriorly and delimits a dorsal fold at a point where head structures normally sink during head involution. The dorsal ridge in hep mutants remains as a cleft as a consequence of the motionless phenotype of hep. The absence of a functional dorsal ridge in mutants may well explain the disorganized head-derived strutures, suggesting that the effect of hep mutations on head involution is indirect (Glise, 1995).
Evidence that misshapen (msn) and basket
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 hemipterous,
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).
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).
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).
TGF-ß activated kinase 1
is required during morphogenetic changes and the fusion of the epithelial wing disc cell layers that takes place in thoracic closure, acting in the context of JNK signaling. JNK signaling is required in thoracic closure. The notum of the adult animal is formed by tissue of the two collateral wing imaginal discs, which undergo extensive morphogenetic rearrangements during metamorphosis. LOF in hep/JNKK and kayak/D-Fos results in aberrant wing disc morphogenesis and failure of wing disc fusion, giving rise to a thoracic cleft along the dorsal midline in the adult. To test whether Tak1 can also act in this context, DN (kinase dead) forms of Tak1
(UAS-Tak1K46R or
UAS-Tak1D159A) were overexpressed with ap-GAL4 and pnr-GAL4 in the thoracic parts of the wing discs. Examination
of such flies shows incomplete closure of the thorax, giving rise to
a mild thoracic cleft at the dorsal midline of the notum. Although this phenotype is relatively weak, it has a very high penetrance of 91% and is highly
reminiscent of that observed in hypomorphic allelic combinations of
either hep/JNKK or kay/d-fos. This is also in agreement with the phenotypic result of expressing Puckered, a negative regulator of JNK signaling at the time when wing disc fusion occurs. Puc overexpression driven by
pnrGAL4 leads to the same phenotypic thorax cleft defects. These observations suggest that Tak1 is required during morphogenetic changes and the fusion of the epithelial wing disc cell layers, acting in the context of JNK
signaling (Mihaly, 2001).
An examination was carried out to see whether directed
overexpression of Tak1 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 Drosophila development, the Jun N-terminal kinase signal transduction pathway regulates morphogenetic tissue closure movements that involve cell shape changes and reorganization of the actin cytoskeleton. The genome-wide transcriptional response to activation of the JNK pathway has been analyzed in the Drosophila embryo by serial analysis of gene expression (SAGE) and loci encoding cell adhesion molecules and cytoskeletal regulators as JNK responsive genes have been identified. The role of one of the upregulated genes, chickadee (chic), encoding a Drosophila profilin, in embryogenesis was analyzed genetically. chic-deficient embryos fail to execute the JNK-mediated cytoskeletal rearrangements during dorsal closure. This study demonstrates a transcriptional mechanism of cytoskeletal regulation and establishes SAGE as an advantageous approach for genomic experiments in the fruitfly (Jasper, 2001).
With SAGE, virtually every transcript in a sample RNA population can be identified and quantitated by generating specific 14 bp sequence tags from a defined position. Concatemers of such tags are then sequenced, and the frequency with which a given tag is detected represents a direct measure of the abundance of the corresponding mRNA (Jasper, 2001).
SAGE in Drosophila has become particularly powerful with the availability of the Drosophila genomic sequence. Due to the comparatively small size of the fly genome (1.2 x 108 bases of euchromatin), the 14 bases of a SAGE tag (2.7 x 108 possibilities) are typically sufficient to locate the respective transcript in the genome without having to rely on further information (Jasper, 2001).
The dorsal closure process takes place between embryonic stages 13 and 16, corresponding to 10-16 hr of development at 25°C. To capture the relevant changes of gene expression involved in setting up and executing dorsal closure, SAGE libraries were generated from 4-16 hr old Drosophila embryos in which the JNK pathway was either repressed by the ubiquitous expression of a dominant-negative form of Basket (BskDN) or ubiquitously activated due to expression of a constitutively active form of Hemipterous (Hepact). A library from wild-type embryos was prepared as a reference. The expression of the transgenes was dependent on Gal4 that was expressed ubiquitously under the control of the armadillo promoter starting at around 4.5 hr after egg laying. The effect of the BskDN and Hepact molecules on the transcription of target genes was therefore limited to the period of embryogenesis relevant for dorsal closure (Jasper, 2001).
Among the upregulated genes identified, several were known from previous studies to interact genetically or biochemically with components of the JNK pathway or to be required for embryonic dorsal closure. However, dpp and puc, until now the only known JNK-responsive genes, were not among them. Despite a significant upregulation of both dpp and puc in Hepact-expressing embryos as detected by RT-PCR, significant numbers of the corresponding tags were not obtained in the SAGE experiment. The absence of dpp and puc in this analysis is due to the low expression levels of these regulatory transcripts. To generate statistically relevant SAGE data for such rare messages, greater numbers of tags will have to be sequenced (Jasper, 2001).
Consistent with the proposed role of JNK signaling in reorganization of the actin cytoskeleton, this analysis identified several cytoskeletal regulators as JNK target genes. One example is the Drosophila homolog of the profilin gene, chickadee, which is strongly upregulated in Hepact-expressing embryos. Although mutants for chic have been described as defective in a number of actin-dependent processes, a role in dorsal closure has not yet been reported. Mutants lacking chic function were examined. In addition to pleiotropic defects observed in cuticle preparations, about 30% of these embryos secreted a very thin cuticle with obvious dorsal closure defects. The lateral epidermis failed to stretch normally during the closure process, confirming that the defects detected were not secondary effects caused by the weak cuticle (Jasper, 2001).
To further investigate the proposed role of chic downstream of JNK signaling in dorsal closure, whether chic and hep mutations interact genetically was examined. When female flies homozygous for the X-linked hypomorphic mutation hep1 are crossed to wild-type males, the male offspring die with mild dorsal closure defects: only 30% of these embryos are completely open, whereas in the remaining 70%, the segments a5-a8 close normally. In contrast, when hep1 homozygous females are crossed to chic221 heterozygous males, the dorsal open phenotype is enhanced and the number of completely open embryos in the offspring is increased to around 65%. Thus, the gene dose of chic becomes critical in embryos with compromised hep function. In an independent experiment, it was found that in chic heterozygous embryos the phenotypic effects of Hepact expression are suppressed. Together with the molecular data, these genetic interactions suggest that chic is required downstream of hep for normal dorsal closure (Jasper, 2001).
To understand the cellular function of Hep-induced chic expression in the embryo in more detail, the actin cytoskeleton in the relevant genotypes was examined. Hepact-expressing embryos display ectopic foci of actin polymerization among the lateral epithelial cells and show increased actin polymerization in leading edge cells, resulting in a stronger actin cable compared to wild-type embryos. In contrast, the leading edge of embryos lacking JNK activity shows a less prominent actin cable overall, which is occasionally disrupted. Significantly, the same phenotype can be observed in chic mutant embryos, consistent with Chic and Hep acting in the same pathway (Jasper, 2001).
Actin-based filopodia that extend dorsally from wild-type leading edge cells have been proposed to mediate the movement and proper alignment of the lateral epidermal sheets during dorsal closure. In hep-deficient embryos, these structures do not form. chic mutants share this phenotype and show an almost complete lack of these filopodia, indicating that this defect is a consequence of insufficient profilin expression in JNK pathway mutants (Jasper, 2001).
Eiger, the first invertebrate tumor necrosis factor (TNF) superfamily ligand that can induce cell death, was identified in a large-scale gain-of-function screen. Eiger is a type II transmembrane protein with a C-terminal TNF homology domain. It is predominantly expressed in the nervous system. Genetic evidence shows that Eiger induces cell death by activating the Drosophila JNK pathway. Although this cell death process is blocked by Drosophila inhibitor-of-apoptosis protein 1 (DIAP1, Thread), it does not require caspase activity. Genetically, Eiger has been shown to be a physiological ligand for the Drosophila JNK pathway. These findings demonstrate that Eiger can initiate cell death through an IAP-sensitive cell death pathway via JNK signaling (Igaki, 2002).
Many mammalian TNF superfamily proteins activate both the NF-kappaB and the JNK pathway, and activation of the latter pathway facilitates cell death (Davis, 2000). To examine whether Eiger activates the JNK pathway, the genetic interactions of Eiger with the components of the Drosophila JNK cascade were examined. The reduced-eye phenotype induced by Eiger is strongly suppressed in basket (bsk), a heterozygous mutant of Drosophila JNK. In addition, overexpression of a dominant-negative form of Bsk almost completely suppresses the eye phenotype. Moreover, heterozygosity at the hemipterous (hep) locus, which encodes Drosophila JNKK, suppresses the reduced-eye phenotype much as does bsk, and its hemizygosity (null background) rescues the phenotype almost completely. Furthermore, the co-expression of a dominant-negative form of dTAK1 TGF-ß activated kinase 1; Drosophila JNKKK) also rescues the Eiger-induced phenotype completely. Misshapen Puckered (Puc) is a dual-specificity phosphatase, the expression of which is induced by the Drosophila JNK pathway to inactivate Bsk, so that puc expression can be used to monitor the extent of activation of the JNK pathway. To confirm that the JNK pathway is actually activated by Eiger, puc expression level was assessed in the eye disc of GMR>regg1GS9830 flies using a puc-LacZ enhancer-trap allele. The strong induction of puc-LacZ was observed in the region posterior to the morphogenetic furrow of the eye disc compared with the control eye disc. Furthermore, Western blot analysis with an anti-phospho-JNK antibody has revealed that Bsk is phosphorylated by Eiger overexpression. These genetic and biochemical data led to a model in which Eiger activates Msn, thereby triggering the JNK signaling pathway, sequentially stimulating dTAK1, Hep and Bsk. Using RT-PCR analysis, whether Eiger could stimulate the NF-kappaB pathway was tested; however, no obvious upregulation of the antimicrobial peptide genes, the target genes of the Drosophila NF-kappaB pathway, was detected (Igaki, 2002).
Two Drosophila tumor necrosis factor receptor-associated factors (TRAFs), Traf1 and Traf2, are proposed to have similar functions with their mammalian counterparts as a signal mediator of cell surface receptors. However, as yet their in vivo functions and related signaling pathways are not fully understood. Traf1 is shown to be an in vivo regulator of c-Jun N-terminal kinase (JNK) pathway in Drosophila. Ectopic expression of Traf1 in the developing eye induces apoptosis, thereby causing a rough-eye phenotype. Further genetic interaction analyses reveal that the apoptosis in the eye imaginal disc and the abnormal eye morphogenesis induced by Traf1 are dependent on JNK and its upstream kinases, Hep and TGF-ß activated kinase 1. In support of these results, the Traf1-null mutant shows a remarkable reduction in JNK activity with an impaired development of imaginal discs< and a defective formation of photosensory neuron arrays. In contrast, Traf2 was demonstrated as an upstream activator of nuclear factor-kappaB (NF-kappaB). Ectopic expression of Traf2 induces nuclear translocation of two Drosophila NF-kappaBs, Dif and Relish, consequently activating the transcription of the antimicrobial peptide genes diptericin, diptericin-like protein, and Drosomycin. Consistently, the null mutant of Traf2 shows immune deficiencies in which NF-kappaB nuclear translocation and antimicrobial gene transcription against microbial infection were severely impaired. Collectively, these findings demonstrate that Traf1 and Traf2 play pivotal roles in Drosophila development and innate immunity by differentially regulating the JNK- and the NF-kappaB-dependent signaling pathway, respectively (Cha, 2003).
To investigate the consequences of ectopic expression of Traf1 in the developing Drosophila eye, Traf1 was overexpressed by using an eye-specific gmr-GAL4 driver. The eyes of adults carrying one copy each of both gmr-GAL4 and Traf1 show a rough-eye surface with disorganized arrays of ommatidia, whereas the eyes of flies carrying either one copy of gmr-GAL4 or one copy of Traf1 alone appear normal. Examination of the retinal sections of adults carrying both gmr-GAL4 and Traf1 reveals the number of ommatidia to be reduced and the number and shape of the photoreceptor cells in each ommatidium also to be abnormal, compared to the controls, which carry only the gmr-GAL4 driver (Cha, 2003).
When two copies of Traf1 were overexpressed in the eye, it displayed a more severe phenotype and a reduced number of ommatidia, resulting in a size reduction of the compound eye, and some ommatidia were fused with each other. In contrast, ectopically expressed Traf2 had no effect on the eye development; ommatidial array, bristles, and compound eye size were all found to be normal (Cha, 2003).
To determine which signaling pathway is activated and induces malformation of the optic system by ectopic expression of Traf1 in vivo, the genetic interactions were tested between mutants of various signaling pathways and a Traf1-overexpressing line (gmr>Traf1/+). Included in this screen were UAS lines that activate extracellular signal-regulated kinase (ERK), p38 MAP kinase, and JNK signaling pathway, respectively. Among the various overexpression lines tested, only Hemipterous (Hep; Drosophila homolog of MKK7 encoded by hemipterous [hep]) and Basket were found to interact genetically with Traf1. The UAS-hep or UAS-bsk itself under the control of gmr-GAL4 driver had no effect on eye morphogenesis. Coexpression of Bsk with Traf1 increased the disturbance of the ommatidial array in the compound eye in comparison to the eye phenotype resulting from one copy overexpression of Traf1. In addition, when Hep was coexpressed with Traf1, the number of ommatidia and the size of the compound eye were reduced more dramatically, which is very similar to the Traf1 two-copy expression phenotype. To further examine whether Traf1 signaling is mediated by Hep, Traf1 was expressed under a hemizygous hep mutant background. As a result, the abnormal ommatidial array of the compound eye was recovered to the level of the wild-type eye (Cha, 2003).
Interestingly however, Traf2, did not display any interaction with the ERK or the p38 MAP kinase pathway components, nor even with JNK pathway components. Tests were performed to see whether Traf2 exerts its effect on the eye development by interacting with Traf1. Coexpression of Traf2 with Traf1 in the Drosophila compound eye did not alter the rough-eye phenotype caused by a sole overexpression of Traf1. These results strongly support the view that Traf1 can activate the JNK signaling cascade in vivo and that Traf2 is not correlated with Traf1 signaling at all, at least during eye development (Cha, 2003).
Based upon the result that Traf1 is involved in JNK signaling, attempts were made to find out the signaling components between Traf1 and Hep by genetic interaction studies. Various kinases, such as Misshapen (msn), Slipper (slpr), and Drosophila Transforming growth factor ß-activated kinase 1 (Tak1), are known to be the upstream kinases for Hep in the eye development. Among these kinases, Tak1 synergistically increased the roughness of the compound eye surface and also reduced the eye size when coexpressed with Traf1. Moreover, Tak1-null mutation (Tak11), which has no effect on the eye morphology, is able to block the rough-eye phenotype caused by Traf1 overexpression. These data suggest that Traf1 activates the JNK signaling pathway via Tak1 and Hep (Cha, 2003).
In order to further confirm that Traf1 activates the JNK kinase signaling pathway at a molecular level, JNK activity in the eye discs was examined by two different experimental approaches -- an immunohistochemical assay with anti-phospho-specific JNK antibody and a puckered-LacZ reporter assay. JNK phosphorylation is highly induced by overexpression of Traf1 compared to the control. In addition, expression of puckered (puc), a well-known downstream target of JNK, is also highly induced by Traf1 compared to the control. Collectively, the results clearly demonstrated that Traf1 activates the JNK signaling pathway in vivo (Cha, 2003).
Apoptosis can be induced by the activation of the JNK pathway. Because ectopic coexpression of Traf1 with Hep has a synergistic effect on the reduction of ommatidia number and eye size, the rough-eye phenotype induced by Traf1 overexpression seemed to be a result of the Hep/JNK signaling-dependent apoptotic cell death. Therefore, whether overexpression of Traf1 can induce apoptosis in the eye disc cells was investigated by using TUNEL assay. In the discs of wild-type third-instar larvae, there are few apoptotic cells. In contrast, the eye imaginal discs from transgenic flies overexpressing Traf1 reveal a highly increased number of apoptotic cells in the region posterior to the morphogenetic furrow in a gene dosage-dependent manner. However, ectopic expression of Traf2 failed to induce apoptotic cell death, which is consistent with its inability to activate the JNK pathway (Cha, 2003).
Since hemizygous hep mutation (hep1/Y) is sufficient to suppress the rough-eye phenotype caused by Traf1 overexpression, whether hep mutation can inhibit the Traf1-induced apoptotic cell death in the eye imaginal discs was examined. When TUNEL assay was performed against the eye discs of hep1/Y; gmr>Traf1/± larvae, the number of apoptotic cells was dramatically decreased to almost wild-type levels. These results prove that Traf1 overexpression can activate the Hep/JNK signaling pathway and consequently induce apoptosis (Cha, 2003).
Traf1ex1, a loss-of-function allele for Traf1 gene, was generated through imprecise excision of the P-element in EP(2)0578 fly. RT-PCR analysis clearly demonstrate that the homozygous Traf1ex1 mutant fails to produce Traf1 mRNA, indicating that Traf1ex1 is a null allele for Traf1. The endogenous puckered transcription level was examined in the mutant larvae by RT-PCR analysis; the amount of puckered gene transcript was severely decreased in Traf1ex1 mutant in comparison to wild-type larvae. This strongly implies the reduced JNK activity in Traf1ex1 mutant (Cha, 2003).
In order to confirm this, the genetic interaction between the Traf1-null fly and a transgenic fly for JNK (ap>JNKDN) was examined by observing the thorax closure phenotype. Thorax closure, the joining of the parts of the two wing imaginal discs during metamorphosis, is tightly controlled by the Drosophila JNK signaling pathway. When the activity of JNK pathway is downregulated by expressing a dominant-negative form of JNK on the thorax in ap>JNKDN flies, the joining process is impaired and a cleft is formed at the dorsal midline in a gene dosage-dependent manner. Strikingly, a reduction of Traf1 gene dosage in heterozygous Traf1ex1 dramatically enhances the thorax closure defect in ap>JNKDN flies by expanding the cleft and also disrupting its notum structure, suggesting that Traf1 mutation leads to a greater reduction of the endogenous JNK activity in ap>JNKDN flies. These data strongly support the critical roles of Traf1 in Drosophila development by positively modulating JNK signaling activities (Cha, 2003).
It is concluded that Traf1 can specifically activate the JNK signaling cascade. This conclusion is based on four lines of evidence: (1) ectopic expression of Bsk, Hep, or Tak1 with Traf1 exerts a highly synergistic effect on the rough-eye phenotype of Traf1 overexpressing flies; (2) disruption of hep or Tak1 function sufficiently suppresses the Traf1-induced eye defects; (3) histochemical analysis with either an anti-phospho-specific JNK antibody or the puckered-LacZ reporter system provides direct molecular evidence that Traf1 can induce phosphorylation and consequent activation of JNK; (4) Traf1 deficiencies in Traf1ex1 mutants generate the same phenotypes detected in the loss-of-function mutants of the JNK signaling pathway, such as increased thorax closure defects and reduced puckered transcription (Cha, 2003).
Innate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. These transcriptional patterns can be dissected into distinct groups. In addition to signaling through the Toll/NFkappaB or Imd/Relish pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, the results may imply a close link between the control of tissue repair and antimicrobial processes (Boutros, 2002).
Lipopolysaccharides (LPS) are the principal cell wall components of gram-negative bacteria. In mammals, exposure to LPS causes septic shock through a Toll-like receptor TLR4-dependent signaling pathway. LPS treatment of Drosophila SL2 cells leads to rapid expression of antimicrobial peptides, such as Cecropins (Cec). SL2 cells resemble embryonic hemocytes and have also been used as a model system to study JNK and other signaling pathways. LPS-responsive induction of the antimicrobial peptides AttacinA (AttA), Diptericin (Dipt), and Cec relies on IKK and Relish. In order to obtain a broad overview on the transcriptional response to LPS in Drosophila, genome-wide expression profiles of SL2 cells were generated at different time points following LPS treatment. Altered expression of 238 genes was detected (Boutros, 2002).
In time-course experiments, a complex pattern of gene expression was observed that can be separated into different temporal clusters. A first group, with peak expression at 60 min after LPS, primarily consists of cytoskeletal regulators, signaling, and proapoptotic factors. This group includes cytoskeletal and cell adhesion modulators such as Matrix metalloprotease-1, WASp, Myosin, and Ninjurin, proapoptotic factors such as Reaper, and signaling proteins such as Puckered and VEGF-2. A second group, with peak expression at 120 min, includes many known defense and immunity genes, such as Cec, Mtk, and AttA, but not the gram-positive-induced peptide Drs. Interestingly, this cluster also includes PGRP-SA, which is a gram-positive pattern recognition receptor in vivo, suggesting possible crossregulation between gram-positive- and gram-negative-induced factors. A third group is transiently downregulated upon LPS stimulation. This cluster includes genes that play a role in cell cycle control, such as String and Rca1. Altogether, these results show that, in response to LPS, a defined gram-negative stimulus, cells elicit a complex transcriptional response (Boutros, 2002).
In the Rel-independent group, several transcripts were identified that are indicative of other signaling events. For example, puc is transcriptionally regulated by JNK signaling during embryonic development. Therefore, the effect of removing SAPK/JNK activity was tested on LPS-induced transcripts. mkk4/hep dsRNA-treated cells lose the ability to induce the Rel-independent cluster, indicating that LPS signaling branches downstream of Tak1 into separate Rel- and JNK-dependent branches. To validate the results obtained from the microarray experiments, quantitative PCR (qPCR) was performed using puc and cec mRNA levels as indicators for Imd/Rel- or Mkk4/Hep-dependent pathways. Additionally, the effect of removing imd, which, in vivo, acts upstream of Tak1, was tested to clarify whether, in addition to Tak1, other known upstream components of a gram-negative signaling pathway are required for both Rel- and Mkk4/Hep-dependent pathways. These qPCR experiments confirm that cec is dependent for its expression on Imd, Tak1, Rel, and Key, whereas LPS-induced puc expression is dependent on Imd, Tak1, and Mkk4/Hep. Hence, the immunity signaling pathway in response to LPS bifurcates downstream of Imd and Tak1 into Rel- and SAPK/JNK-dependent branches. Both the Rel and SAPK/JNK pathways regulate different functional groups of downstream target genes (Boutros, 2002).
While both Rel and Mkk4/Hep pathways are downstream of Imd and Tak1 in response to LPS, the two downstream branches elicit different temporal expression patterns. It was then asked whether the first transcriptional response is controlled by downstream targets that might negatively feed back into the signaling circuit. puc was a candidate for such a transcriptionally induced negative regulator. Expression profiles of cells depleted for puc were tested before and after a 60 min LPS treatment. These experiments showed that transcripts dependent on the Mkk4/Hep branch of LPS signaling are upregulated, even without further LPS stimulus. In contrast, Rel branch targets are not influenced. puc dsRNA-treated cells show loss of the typical round cell shape. These cells appear flat and have a delocalized Actin staining, consistent with a deregulation of cytoskeletal modulators in puc-deficient cells (Boutros, 2002).
The analysis of expression profiles shows that, while SAPK/JNK and Rel signaling are controlled by the same Imd/Tak1 cascade, they appear to have different feedback loops. Whereas Rel signaling induces Rel expression and thereby generates a self-sustaining loop, possibly leading to the maintenance of target gene expression, the SAPK/JNK branch induces an inhibitor and thereby establishes a self-correcting feedback loop. These results may explain how a single upstream cascade can lead to different dynamic patterns (Boutros, 2002).
Since the expression of cytoskeletal genes after LPS stimulation is dependent on a JNK cascade, whether removing JNK activity in vivo affects the induction of fln was examined. In Drosophila, JNK signaling pathways have been previously implicated in epithelial sheet movements during embryonic and pupal development, a process that has been likened to wound-healing responses. hep1 (JNKK) mutants, which are impaired in JNK signaling, the induction of fln is diminished, whereas the expression of the antimicrobial peptide dipt is not affected. A test was performed to see whether fln induction in Tl loss-of-function alleles is affected. These experiments show that fln expression is lost in Tl mutants, suggesting that Toll acts upstream of a JNK pathway to induce septic injury-induced target genes (Boutros, 2002).
NFkappaB pathways play a central role for innate and adaptive immune response in mammals. In innate immune responses, TLRs on dendritic cells recognize microbial agents and activate NFkappaB, leading to the expression of proinflammatory cytokines and other costimulatory factors required to initiate an adaptive immune response. Additionally, other signaling pathways have been implicated at later stages during immune responses in mammals, but their physiological role in innate immunity remains rather poorly understood. For example, several cytokines, such as IL-6 and IL-11, signal through a JAK/STAT pathway to induce the expression of acute phase proteins. Similarly, JNK pathways are activated in response to TNF and IL-1, may lead to the expression of immune modulators, and are required for T cell differentiation. In Drosophila, studies have investigated two distinct NFkappaB-pathways --Toll and Imd/Rel -- that have been shown to mediate gram-positive/fungal and gram-negative responses. Both pathways induce specific antimicrobial peptides and thereby focus the response on the invading microbial agent. Genetic analysis has shown that functions of the NFkappaB-pathways are separable; flies that are mutant for only one of these pathways are susceptible to subgroups of pathogens. Could the contribution of NFkappaB-dependent and, possibly, other signaling pathways be identified by examining global expression profiles? The obtained data set demonstrates that NFkappaB-independent signaling pathways contribute to the transcriptional patterns observed after microbial infection. Both in cells and in vivo, JNK-dependent targets precede the peak expression of antimicrobial peptides that require NFkappaB. JAK/STAT targets are induced with a distinct temporal pattern that shows late, but only transient, expression characteristics. The stereotyped pathway patterns after microbial challenge suggest that the correct temporal execution of signaling events, similar to signaling during development, may play an important role in the regulation of homeostasis (Boutros, 2002).
Strikingly, cytoskeletal gene expression during innate immune responses is controlled by JNK through the same upstream signaling cascade that activates NFkappaB pathways. JNK pathways act downstream of microbial stimuli, both in vivo and in cells, to induce cytoskeletal regulators. In SL2 cells, JNK signaling is required for the induction of a cluster of cytoskeletal, cell adhesion regulators and proapoptotic factors. Interestingly, both NFkappaB and JNK branches share the same upstream components, Tak1 and Imd, indicating that the activation of both processes are tightly linked. MMP-1, a matrix metalloproteinase that is one of the most markedly upregulated genes after LPS stimulation, has been implicated in wound-healing responses in mammals. Compared with experiments in cells, the situation in vivo after septic injury is likely more complex. Gene expression profiling in whole organisms likely has a lower sensitivity for transcriptional changes that occur in rather small numbers of cells. Also, tissue-specific differences in signaling pathway activity may not reflect the transcriptional changes observed in the cell culture model. Muscle-specific cytoskeletal factors, possibly because they were injected into the thoracic muscle, are not inducible in a JNK-deficient genetic background. However, since it was necessary to remove both Mkk4 and Hep (Mkk7) in cells to deplete JNK pathway activity, an experiment that cannot be performed in vivo because of the lack of an Mkk4 mutant, these experiments might not have uncovered all JNK-dependent transcripts. SAPK/JNK modules can also be linked to different upstream activating cascades. For example, a recent study reported the activation of p38a through a cascade involving Toll, TRAF6, and TAB. Similarly, during innate immune responses JNK pathways can be activated by both Toll and Imd pathways in vivo (Boutros, 2002).
The activation of JNK signaling is reminiscent of signaling during dorsal and thorax closure. In dorsal closure, SAPK/JNK signaling controls cytoskeletal rearrangements that lead to the epithelial sheet movements of the embryonic epidermis. SAGE analysis of embryos with activated SAPK/JNK signaling has shown an induction of cytoskeletal factors. Also, dorsal closure movements are proposed to be similar to the reepithelization that occurs during wound healing. In other developmental contexts, SAPK/JNK signaling has been implicated in cytoskeletal rearrangements and cell motility, such as the generation of planar polarity in Drosophila and convergent-extension movements in vertebrates. A common theme of SAPK/JNK pathways might be their control of cytoskeletal regulators for diverse biological processes. The finding that, in response to LPS, SAPK/JNK and NFkappaB targets are coregulated through the same intracellular pathway suggests a close linkage of directed antimicrobial activities and tissue repair processes (Boutros, 2002).
In conclusion, genome-wide expression profiling was employed to examine the contribution of different signaling pathways in complex tissues and to assign targets to candidate pathways. Both a cell culture model system and an in vivo analysis were used to show the temporal order of NFkappaB-dependent and -independent pathways after septic injury. An interesting question that remains is, how do the extracellular events leading to pathway activation reflect the nature of the pathogen? Clean injury experiments induce a largely overlapping set of induced genes, but to a lower extent than septic injury. This is consistent with experiments showing that septic injury with only gram-negative E. coli induces both anti-gram-negative and anti-gram-positive responses. These results can be interpreted to suggest that wounding, in itself, might be sufficient to induce a transient (and unspecific) innate immune response. However, further studies are needed to understand the nature of the inducing agent (Boutros, 2002).
Tensin is an actin-binding protein that is localized in focal adhesions. At
focal adhesion sites, tensin participates in the protein complex that
establishes transmembrane linkage between the extracellular matrix and
cytoskeletal actin filaments. Even though there have been many studies on
tensin as an adaptor protein, the role of tensin during development has not
yet been clearly elucidated. Thus, this study was designed to dissect the
developmental role of tensin by isolating Drosophila tensin mutants
and characterizing its role in wing development. The Drosophila
tensin loss-of-function mutations results in the formation of blisters in the
wings, that are due to a defective wing unfolding process. Interestingly,
by1 -- the mutant allele of the gene blistery
(by) -- also shows a blistered wing phenotype, but fails to complement
the wing blister phenotype of the Drosophila tensin mutants, and
contains Y62N/T163R point mutations in Drosophila tensin coding
sequences. These results demonstrate that by encodes
Drosophila Tensin protein and that the Drosophila tensin
mutants are alleles of by. Using a genetic approach, it has been demonstrated that Tensin interacts with integrin and also with the components
of the JNK signaling pathway during wing development; overexpression of
by in wing imaginal discs significantly increases JNK activity and
induces apoptotic cell death. Besides the defects in wing cell adhesion process, another distinct mutant phenotype was observed in the by mutants; they lay rounded eggs due to defective oocyte elongation during oogenesis. Collectively, these data suggest that Tensin
relays signals from the extracellular matrix to the cytoskeleton through
interaction with integrin, and through the modulation of the JNK signal
transduction pathway during Drosophila wing development (Lee, 2003).
In mammalian cells, tensin has been implicated in signal transduction
related to cell adhesion such as Src, JNK and PI3K. To examine
the role of tensin in the signaling processes related to wing development, the in vivo interaction between tensin and signaling molecules
including rl/Erk, Src, JNK and PI3K was investigated. Interestingly, it was found that the JNK signaling pathway is tightly correlated with tensin in the wing development, while other signaling molecules including rl/Erk do not show any interactions with tensin. Homozygous by2 mutants with heterozygotic mutations of the JNK signaling components bsk1 or hep1 (the
loss-of-function mutants for Drosophila JNK and MKK7,
respectively) display a highly severe blistered wing phenotype, compared with
either homozygous by2, heterozygous
bsk1 or heterozygous hep1 mutants. Notably, the rate of flies, which show Class II blistered wings,
increases from 46.5% to 70% for these double mutants compared with homozygous
by2 mutants, and about 15% of these flies had multiple
blisters in their wings. Furthermore, the double homozygotic mutants for
by2 and hep1 die at pharate adult
stage. The lethality of these double mutants may be due to an impairment of essential in vivo interactions between tensin and the JNK signaling pathway in Drosophila (Lee, 2003).
Next, whether overexpressed by also interacts with the components of the JNK signaling pathway was tested. Overexpression of by using MS1096-GAL4 driver turns the adult wings into a convex shape with a smaller overall size, and this phenotype becomes more severe when two copies of the by gene are overexpressed. Simultaneous overexpression of bsk or hep with by results in a severely curled wing phenotype, which is fully penetrant, whereas overexpression of bsk or hep alone by MS1096-GAL4 driver did not induce any detectable phenotypes in the wing. Collectively, these data suggest that tensin activity is highly related to the JNK signaling pathway during wing development in Drosophila (Lee, 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).
Drosophila imaginal discs are monolayered epithelial invaginations that grow during larval stages and evert at metamorphosis to assemble the adult exoskeleton. They consist of columnar cells, forming the imaginal epithelium, as well as squamous cells, which constitute the peripodial epithelium and stalk (PS). A new morphogenetic/cellular mechanism for disc eversion has been uncovered. Imaginal discs evert by apposing their peripodial side to the larval epidermis and through the invasion of the larval epidermis by PS cells, which undergo a pseudo-epithelial-mesenchymal transition (PEMT). As a consequence, the PS/larval bilayer is perforated and the imaginal epithelia protrude, a process reminiscent of other developmental events, such as epithelial perforation in chordates. When eversion is completed, PS cells localize to the leading front, heading disc expansion. The JNK pathway is necessary for PS/larval cells apposition, the PEMT, and the motile activity of leading front cells (Pastor-Pareja, 2004).
One of the processes that best exemplify the dramatic changes that shape organisms is insect metamorphosis. In Drosophila and other holometabolous insects, most of the larval structures are replaced with new tissues that will give rise to the adult or imago. In particular, the adult epidermis with the exception of the abdominal structures develops from imaginal epithelial discs. During larval stages, the primordia of imaginal discs, set during embryogenesis, invaginate and grow to become flattened sacs arranged in a monolayer epithelium connected to the larval epidermis by a stalk. The mature discs contain two populations of cells, a columnar epithelium that will give rise to most of the adult structures and a thinner and more squamous peripodial epithelium (PE) with a reduced contribution to adult tissues. Upon metamorphosis, the imaginal discs undergo striking morphological changes, everting, expanding, and fusing to ipsilateral and contralateral adjacent discs generating the adult exoskeleton (Pastor-Pareja, 2004).
The process of movement and sealing of imaginal discs and, in general, epithelial sheets can be subdivided into three sequential steps: (1) leading cells are specified and brought into position; (2) cells execute coordinated forward movements by changing shape and/or migrating over a substratum, and (3) sheets merge and fuse. Most recent work on disc morphogenesis has focused on the cellular and molecular events underlying their late expansion and fusion, while the mechanisms involved in disc eversion have been poorly explored in vivo (Pastor-Pareja, 2004).
In late third instar larvae, the steroid molting hormone 20-hydroxyecdysone is believed to coordinate the almost simultaneous eversion of all discs by inducing a contraction of the PE. This is thought to drive movement of the appendages to the outside of the larval epidermis through relaxed and widened disc stalks. This classical view is supported by in vitro studies showing that treatment of cultured discs with ecdysone is sufficient to induce eversion and that contraction of an intact PE is necessary to achieve this goal. These descriptive reports have led to the proposition that cell shape changes (longitudinal contraction in the PE and circumferential elongation at the disc stalks) are sufficient for imaginal disc eversion. However, there are as yet no data to confirm that this mechanism exists in vivo and no convincing explanation on how a stalk of no more than ten cells in diameter could achieve the width required to allow the entire disc (more than 60,000 cells) to pass through. Further, this accepted view neglects earlier proposals suggesting a different eversion mechanism mediated by the rupture of the PE. A model supported by fate maps has been developed for the PE of Calliphora, a related dipteran, imaginal wing discs (Pastor-Pareja, 2004).
Several studies have revealed a requirement for cytoskeletal components and a number of signal transduction molecules for imaginal disc morphogenesis during the first hours of metamorphosis. The latter include the Drosophila AP-1 transcription factors, D-Jun and D-Fos (Kayak [Kay]), and an upstream kinase cascade homologous to the Jun-NH2-terminal kinase (JNK) pathway in mammals. The core of this cascade is formed by the stress-activated kinases JNKK and JNK. In Drosophila, JNKK and JNK homologs are encoded by the genes hemipterous (hep) and basket (bsk). JNK signaling mutant larvae do not spread their discs in the process of thorax closure. This phenotype is accompanied by a loss of puckered (puc) expression in the disc stalk and the PE. Puc is a dual-specificity phosphatase that selectively inactivates Bsk and, thus, is thought to act in a negative feedback loop. JNK activity is necessary to maintain the adhesion of the imaginal leading edge cells to their larval substrate and to promote actin dynamics (lamellipodia and filopodia formation). It has been shown that this signaling cascade also regulates the process of embryonic dorsal closure, where the embryonic epidermis fuses along the dorsal midline. Based on these similarities, it has been suggested that a conserved mechanism regulates the spreading and fusion of epithelial sheets (Pastor-Pareja, 2004).
A new morphogenetic/cellular mechanism has been uncovered for disc eversion based on histological sections and direct observation of imaginal morphogenesis in vivo. At the onset of metamorphosis, imaginal discs coordinately appose their peripodial sides and stalks (PS cells) to the larval epidermis. Then, eversion proceeds through the progressive invasion of the larval epidermis by PS cells undergoing a pseudo-epithelial-mesenchymal transition (PEMT). Multiple perforations in the peripodial/larval bilayer are thus generated: these coalesce with the disc stalk into a single hole, widening the gap and allowing disc evagination. When eversion is complete, the PS cells localize to the leading front of the discs, spearheading their expansion over larval cells. The roles of the JNK pathway at discrete steps of disc morphogenesis progression have been analyzed. The JNK cascade functions to promote the apposition of PS and larval cells, to determine the degree of PEMT and the motility of leading edge/PS cells, and to maintain the adhesion between the larval and imaginal tissue. It is proposed that this molecular mechanism can be relevant to morphogenetic processes of perforation of transient epithelia in different phyla (Pastor-Pareja, 2004).
The current view of imaginal disc eversion asserts that the externalization of appendage primordia proceed through widened discs' stalks during early pupal development. However, a detailed analysis of PS cell markers appears to challenge this simple inversion mechanism (Pastor-Pareja, 2004).
In early third instar imaginal wing discs, the gene puc is expressed at high levels in stalk cells and some PE cells. This expression evolves through the third instar until all PS cells (about 700 in the mature wing disc) express puc at the white prepupa stage (0-1 hr hours after puparium formation [APF]). These dynamic changes of puc expression are also observed in leg, haltere, and eye discs. The PS expression of puc strictly depends on JNK activity, and it is abolished from mutant hep (JNKK) larvae or after Puc overexpression. Thus, a JNK signaling feedback loop, first described during embryonic dorsal closure, is shared by PS cells at the onset of the eversion and closure of the discs. During wing disc eversion, only cells found at the edge of the hole through which the disc everts and at the leading front mediating fusion to adjacent prothoracic, mesothoracic, and metathoracic discs show puc expression, and hence JNK activity. Importantly, marking all cells that have expressed puc as well as their descendants shows that puc-expressing cells do not change their identity, nor do they die or get excluded from the epithelium until the end of the disc fusion process, when most of these cells are lost. Hence, all PS cells are recruited to the front edge during disc eversion (Pastor-Pareja, 2004).
These findings lead to a topological dilemma. In order to reach their final position at the leading front, the PS cells would need to reposition themselves within the epithelia. Although this rearrangement just could be achieved through a massive constriction of the PE, a complementary mechanism has been uncovered, which involves larval epidermis perforation and PE cells intercalation (Pastor-Pareja, 2004).
At third instar larval stages, the wing disc obliquely hangs from the larval epidermis, which is separated from the peripodial surface of the disc at the notum level by several larval muscles and tracheal tubules. The disc and the larval epidermis are isolated by their corresponding extracellular basal lamina. During late third instar stages and the first hours APF, the notum-wing side of the disc folds progressively to acquire the adult organ shape. At the initiation of pupariation, the disc affixes to the larval epidermis through its peripodial side. At 3 hr APF, the PS cells lose their squamous shape to adopt a more rounded one and are found in close contact with the larval epidermal cells via their basal surfaces. Multiple actin-rich protrusions lead this apposition. At this step, the basal lamina in between both layers degrades, leading to an intimate adhesion (Pastor-Pareja, 2004).
Once imaginal discs appose the larval epidermis, PS cells, mostly around the disc stalk, invade the larval epithelium, gradually replacing the larval cells at the pupal surface without compromising the integrity of the peripodial sheet. Several holes are opened in the peripodial/larval bilayer, which within a few minutes converge with the original stalk into a single aperture. Interfering with apoptosis by overexpressing the P35 cell death inhibitor in imaginal and larval tissues does not affect epithelial perforation and disc eversion (Pastor-Pareja, 2004).
Following coalescence, the progressive widening of the hole by intercalation of PS cells at the leading front was observed. A cell lineage analysis was performed and multiple clones of PS cells were found, that remain compact up to the third instar larval stage in the PE and lose cohesion during eversion. Thus, PS cells appear to change neighbors, become extremely active, and emit and retract filopodia and lamellipodia at their front and rear ends. They squeeze in between themselves and the rest of the epithelium (planar intercalation), migrating to and expanding the front of the disc, and leading the migration over the larval tissue (Pastor-Pareja, 2004).
Simultaneous to wing disc eversion, all legs and haltere discs evert using the same mechanism (Pastor-Pareja, 2004).
One hallmark of epithelial cells is their distinct apico-basal cell polarity. This polarity depends on a set of intercellular connections, which encircle epithelial cells at the border of the apical and basal-lateral membrane domains. The cells in insect epithelial tissues are interconnected by zonula adherens (ZAs), which function in both cellular adhesion and signaling. DE-cadherin is the major constituent of the ZAs in a complex with Armadillo (Arm, ß-catenin) and Dalpha-catenin. In addition, epithelia of flies and other invertebrates exhibit septate junctions, which are located basally to the ZAs. Septate junctions prevent diffusion through the pericellular space and are functionally equivalent to vertebrate tight junctions (Pastor-Pareja, 2004).
All imaginal disc cells at the third instar larval stage presented ZAs in an apical belt. During disc eversion, however, it was found that ZAs components delocalize from the free edges of the PS cells, remaining cytoplasmic at the edges of the perforations arising through the PS/larval bilayer and in those PS cells leading the spreading of the discs over the larval tissues. As a consequence, ZAs are lost in these cells . Moreover, septate junction components, such as Coracle and Disc Large are also found to be missing from the membranes of leading front cells (Pastor-Pareja, 2004).
The loss of apico/basal polarity and adhesion of the PS cells during disc eversion is reminiscent of an epithelial-mesenchymal transition (EMT), as described for mesoderm and neural crest cells in vertebrates, and for the acquisition of the invasive phenotype in carcinomas (Pastor-Pareja, 2004).
The JNK signaling cascade dictates the expression of puc in all PS cells but their early specification appears not to be affected by lowering the level of JNK activity, since the complete absence of Hep function did not alter either their number or morphology in third instar larval discs. However, several mutant phenotypes have provided strong evidence for a leading role of the JNK pathway in imaginal disc fusion and disc eversion; e.g., hep mutants occasionally show uneverted wing discs lying inside the body of the pupa. When and how is JNK signaling needed? Transversal semi-thin sections, at 6 ± 1 hr APF, long after closure is completed in wild-type, of hepr75 (a strong hypomorphic mutation) pupae show a range of phenotypic defects (classes I to III). Class I corresponds to a complete failure of PS/larval apposition (40% of individuals); in class II, discs apposed to the larval tissue but did not complete their eversion (50%); the mildest condition, class III, refers to discs that everted completely and advanced to some extent but were unable to fuse (10%). By the complete inactivation of the JNK signaling cascade through the ubiquitous overexpression of puc (from 48-60 hr before puparium formation onward), a fully penetrant failure was found of disc apposition to the larval epidermis (class I phenotype). A delayed or reduced (in a puc heterozygous background) overexpression of puc produced less severe class II and III phenotypes. Thus, the JNK cascade appears to be essential for PS and larval cell apposition and, as suggested by the observed phenotypic progression, may also be involved in later steps during eversion (Pastor-Pareja, 2004).
JNK activity levels also affect the degree of PEMT in PS cells. ZAs are absent from leading front cells and the membrane localization of DE-cadherin and Arm is progressively lost, as PS cells moved closer to the free edge. However, in hepr75 mutant pupae (class III), the cells at the leading front of the disc do not delocalize either Arm or DE-cadherin in the free edge, suggesting that partial loss of JNK signaling blocks the correct transition of these cells from immotile epithelial to migrating and invading leading front cells. Further, a surplus of JNK activity in PS cells in pucE69F-GAL4 mutants conveyed the transition of an excess of PS cells to a mesenchymatic phenotype. Hence, an adequate balance of JNK activity is key to control the level of PEMT. Too few mesenchymal-like PS cells restrain the ability of discs to evert and spread, while too many transformed cells affect the ability of discs to appropriately fuse. Further, pucE69F-GAL4 mutants also showed enhanced cell motility and massive cell detachment from the free edges of the epithelium. These cells adopted a rounded shape but remained in close proximity, establishing transient contacts. Conversely, leading cells in hepr75 mutants do not show any migratory activity. Hence, the JNK pathway regulates not only the adhesive properties of PS cells, but also their motility (Pastor-Pareja, 2004).
In summary, the JNK signaling cascade participates in four key steps in the process of disc eversion: (1) the expression of puc in PS cells; (2) the apposition of PS and larval cells; (3) the regulation of the adhesive and motile properties of PS cells as they undergo PEMT, and (4) the maintenance of the adhesion between the larval and imaginal tissue (Pastor-Pareja, 2004).
Thus, within the first 5 hr after puparium formation, the precursors of the adult structures evert. Multiple evidences show that eversion is mediated by actin microfilaments contraction, which modulate a general change of morphology of PS and larval cells driving the inside-out eversion of the disc. Several observations, however, suggest that other morphogenetic mechanisms are also involved (Pastor-Pareja, 2004).
(1) An imaginal disc is a rigidly determined primordium, which allows the construction of fate maps. Surprisingly, peripodial fate maps of Calliphora, a related diptera, show that adjacent territories develop into nonadjacent adult pleural structures, suggesting that the peripodial layer splits during metamorphosis (Pastor-Pareja, 2004).
(2) PS cells expressing puc relocate during eversion to the leading front. Thus, intercalation of PS cells appears to be concurrent to eversion (Pastor-Pareja, 2004).
(3) Pupal serial sectioning shows that, at eversion, imaginal discs appose to the larval epidermis through their peripodial side. Just before eversion, PS cells lose their basal lamina and detach from the extracellular matrix (Pastor-Pareja, 2004).
(4) Preceding disc eversion, in vivo time-lapse reveals the opening of larval/peripodial gaps, which are the outcome of the invasive behavior and planar intercalation (PEMT) of PS cells (Pastor-Pareja, 2004).
In summary, the evagination of imaginal disc can be divided into the following sequential steps: (1) an overall positional change of the imaginal discs leading to the confrontation and apposition of the PS and the larval epidermis; (2) a regulated modulation (PEMT) of PS cells, which involves the downregulation of their cell-cell adhesion systems and allows them to move into their local neighborhood and invade the larval epithelium; (3) the fenestration of the peripodial/larval bilayer and the formation of an unbound peripodial leading front, which will direct imaginal spreading by planar cell intercalation, and (4) a bulging of the imaginal tissue (Pastor-Pareja, 2004).
Once the hole is opened, the planar intercalation of PS cells ensures that, first in the hole and later in the leading front, all four dorsal, ventral, anterior, and posterior compartments of the wing disc are represented. This mechanism also guarantees the maintenance of a continuous epithelial barrier (Pastor-Pareja, 2004).
In Drosophila, the axons of retinal photoreceptor cells extend to the first optic ganglion, the lamina, forming a topographic representation. DWnt4, a secreted protein of the Wnt family, is the ventral cue for the lamina. In DWnt4 mutants, ventral retinal axons misproject to the dorsal lamina. DWnt4 is normally expressed in the ventral half of the developing lamina and DWnt4 protein is detected along ventral retinal axons. Dfrizzled2 and dishevelled, respectively, encode a receptor and a signaling molecule required for Wnt signaling. Mutations in both genes caused DWnt4-like defects, and both genes are autonomously required in the retina, suggesting a direct role of DWnt4 in retinal axon guidance. In contrast, iroquois homeobox genes are the dorsal cues for the retina. Dorsal axons accumulate DWnt4 and misproject to the ventral lamina in iroquois mutants; the phenotype is suppressed in iroquois:Dfrizzled2 double mutants, suggesting that iroquois may attenuate the competence of Dfrizzled2 to respond to DWnt4 (Sato, 2005).
JNK signaling is known to act downstream of the noncanonical Wnt pathway in many developmental contexts. The involvement of JNK signaling was examined by expressing puckered (puc), which encodes a JNK phosphatase, and a dominant-negative form of JNK encoded by basket (bsk) to block JNK signaling in the retina. Defects were observed only rarely, and it was next asked whether genetic interactions exist between hemipterous (hep) encoding a JNK kinase and DWnt4 or Dfz2. In a strong hep mutant background, or in DWnt4, DWnt4 or Dfz2 heterozygous backgrounds, little or no ventral-to-dorsal misrouting was observed. However, a reduction in the dosage of DWnt4 or Dfz2 in the hep background resulted in a marked increase in the ventral-to-dorsal phenotype. These findings provide some support for the idea that JNK signaling is involved in the DWnt4/Dfz2 pathway in retinal axon guidance. Since iro expression and ommatidial chirality were normal in retinae expressing the dominant-negative form of bsk and in hep hemizygotes in combination with DWnt4/+ and Dfz2/+, the misrouting of ventral axons observed in the brain mutant for JNK signaling appears to be caused by a failure in axon guidance and independent of the dorsoventral cell specification or PCP signaling in the retina. Note that mutations in JNK pathway components alone have no PCP phenotype (Sato, 2005).
The PcG proteins function through cis-regulatory elements called PcG response elements (PREs), which enable them to bind and to maintain the state of transcriptional silencing over many cell divisions. PcG proteins operate in two key evolutionarily conserved chromatin complexes, and reduced expression of these complexes, as found in PcG mutants, results in the derepression of PRE-controlled genes. To determine whether PcG silencing is modulated in regenerating tissue, the FLW-1 line, which contains a lacZ reporter gene under the control of the Fab7 PRE, was used. Prothoracic leg discs silent for lacZ expression were fragmented and transplanted into the abdomen of host flies. Flies were fed with 5-bromodeoxyuridine (BrdU) to mark the regenerated tissue (the blastema). In uncut discs, there was little proliferation and expression of lacZ was undetectable. On fragmentation, however, lacZ was expressed in the blastema. To confirm that this derepression was due to a reduction in PcG silencing and not simply to massive proliferation at the wound site, the line LW-1 was used; this line lacks the Fab7 PRE and is normally silent, but it can be activated by induction of GAL4. Neither uncut nor cut leg discs of the LW-1 line showed expression of lacZ after transplantation (Lee, 2005).
To show that transdetermination takes place only in cells with downregulated PcG function, fragmented leg discs of the FLW-1 line were stained for lacZ expression and for Vg in order to visualize the transdetermination to wing fate. It was consistently observed that the Vg staining lay within the lacZ expression domain, suggesting that PcG genes are downregulated in the blastema, enabling PRE-silenced genes to be reactivated according to new morphogenetic cues (Lee, 2005).
To investigate direct targets of PcG regulation that, when reactivated, might contribute to transdetermination, the PREs predicted at the wg and vg genes were tested and both were found to be controlled by PcG proteins. The fact that both the transgenic vg-lacZ reporter construct (which lacks the PRE) and the endogenous vg gene were upregulated in the blastema suggests that PcG proteins may affect vg expression both indirectly (for example, through wg) and directly by means of the vg PRE (Lee, 2005).
JNK signalling in Drosophila is crucial for wound healing and is implicated in many different developmental processes, such as dorsal and thorax closure. hemipterous encodes the JNK kinase (JNKK) that activates the Drosophila JNK Basket. Products of DJun and kayak (the Drosophila homologue of Fos) form the AP-1 transcription factor. A downstream target of JNK signalling is puckered (puc), which encodes a phosphatase that selectively inactivates Basket and thus functions in a negative feedback loop. The expression of puc thus mirrors JNK activity. Because wound healing takes place after fragmentation, it was reasoned that activation of the JNK pathway might be causing the downregulation of PcG proteins in the blastema. The pucE69 line, which carries a P(lacZ) insertion at the puc locus, was used to monitor JNK activity. During the third-instar larval stage puc is not expressed and thus JNK signalling was not activated in leg discs. As expected, however, puc was expressed on fragmentation in all cells at the annealing cut edge (Lee, 2005).
To check whether cells that have activated the JNK pathway also show transdetermination, fragmented leg discs of flies carrying the puc-lacZ reporter and vgBE-Gal4; UAS-GFP constructs were transplanted. In these flies, cells that adopted a wing fate were identified by their expression of green fluorescent protein (GFP). Two days after fragmentation, weak residual puc-lacZ staining was still visible in the central region of the disc. puc-lacZ staining is known to decline rapidly after wound healing is completed. It was found that stronger staining was visible along the cut site, probably owing to ongoing wound healing. On comparison of puc-lacZ staining and GFP fluorescence, JNK-active cells showed a substantial overlap with transdetermined cells; thus, it is concluded that JNK signalling is activated in cells that undergo transdetermination (Lee, 2005).
JNK signalling affects the transcription of numerous genes, including those encoding chromatin regulating factors. Therefore whether JNK signalling can downregulate the PcG proteins required for transdetermination was examined. A constitutively active form of hep was overexpressed in UAS-hepact; hsGal4 flies by a heat-shock pulse. Activating the JNK pathway caused a downregulation of some PcG genes, such as Pc, ph-p and E(Pc). No downregulation of these genes was observed in wild-type larvae before and after heat shock, indicating that this was not an unspecific heat-shock response. Expression was examined of two genes of the Trithorax group (ash1 and brm) that function antagonistically to PcG proteins, but found no upregulation on JNK induction (Lee, 2005).
To show further that JNK has a specific effect on PcG proteins, the analogous experiment was carried out in mammalian cells. The JNK pathway can be activated in mouse embryonic fibroblasts by exposing the cells to ultraviolet light. The expression of MPh2 (mouse polyhomeotic2) was examined because this mammalian PcG gene is expressed in these cells. The expression of MPh2 was decreased on JNK induction, but after treatment with a specific JNK inhibitor it was partially restored. In addition, to show that the downregulation of PcG genes is directly controlled by AP-1, chromatin immunoprecipitation was carried out using antibodies against Fos on chromatin from UAS-hepact; hsG4 and kay1 mutant flies. Enrichment of Fos on the promoter region of ph-p was observed, but no enrichment in chromatin from flies lacking Fos. This finding suggests that AP-1 binds directly to this region to regulate negatively the transcription of ph-p (Lee, 2005).
If activation of JNK signalling in the blastema indeed leads to a downregulation of PcG genes, then impairment of the JNK pathway should result in reduced efficiency of transdetermination. The transdetermination behaviour of wild-type discs was compared with that of discs bearing mutations in the JNKK hep. The transdetermination events were classified into three categories: large regions, small regions, and no regions of transdetermination. In wild-type discs only large regions were detected. In males hemizygous for hep1 (a weak hypomorphic allele), most transplanted leg discs had large transdetermined regions; however, a substantial proportion showed only small regions of transdetermination and a few showed no transdetermination event. In flies heterozygous for hepr75 (a null allele which is hemizygous lethal), most discs showed no or only small regions of transdetermination, and large regions were rarely seen. The morphology of the regenerated discs seemed unaffected in these mutants, indicating that the decline of transdetermination efficiency was not due to inefficient wound healing (Lee, 2005).
This study has shown that PcG genes are downregulated by JNK signalling. Because many developmental regulators need to be switched, the role of PcG downregulation may be to render the cells susceptible to a change in cell identity by shifting the chromatin to a reprogrammable state. Transdetermination has been ascribed to the action of ectopic morphogens, which induce cells to activate incorrect gene cascades. Without doubt, wg and decapentaplegic signalling must be crucially involved in this process, because transdetermination does not result from any random cut but occurs preferentially when cuts are made through particular regions of the disc called 'weak points', which are regions of high morphogen. Inappropriate or overextreme downregulation of the PcG system by JNK in sensitive cells of the weak points thus may create such aberrant local patterns. Indeed, the data indicate that at least the two patterning genes, wg and vg, may be direct targets of the PcG. Notably, hyperactive Wnt signalling can also induce a switch in lineage commitment in mammals, implying that signalling pathways are a potent inducer of cell fate changes in many organisms (Lee, 2005).
Another study has shown that regenerating and transdetermining cells in the blastema have a distinct cell-cycle profile in contrast to the surrounding normal disc cells. It has been proposed that this change in cell-cycle regulation is a prerequisite for the change in cell fate. Indeed, PcG targets include genes involved in cell-cycle regulation, suggesting that this initial step is part of the complete reprogramming cascade required for the regenerating cells to achieve multipotency. Downregulation of PcG silencing by JNK seems to be a fundamental, evolutionarily conserved mechanism of cell fate change and thus may also have implications for studies of stem cell plasticity and tissue remodelling (Lee, 2005).
The follicle cells of the Drosophila egg chamber provide an excellent model in which to study modulation of the cell cycle. During mid-oogenesis, the follicle cells undergo a variation of the cell cycle, endocycle, in which the cells replicate their DNA, but do not go through mitosis. Previously, it was shown that Notch signaling is required for the mitotic-to-endocycle transition, through downregulating String/Cdc25, and Dacapo/p21 and upregulating Fizzy-related/Cdh1.
In this paper, it is shown that Notch signaling is modulated by Shaggy and temporally induced by the ligand Delta, at the mitotic-to-endocycle transition. In addition, a downstream target of Notch, tramtrack, acts at the mitotic-to-endocycle transition. It is also demonstrated that the JNK pathway is required to promote mitosis prior to the transition, independent of the cell cycle components acted on by the Notch pathway. This work reveals new insights into the regulation of Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).
Notch controls the mitotic-to-endocycle transition in follicle epithelial cells; Notch pathway activity arrests mitotic cell cycle and promotes endocycles by downregulating string/cdc25 and dacapo/p21, and upregulating fzr/Cdh1. This study identified components regulating this transition, Delta, Shaggy, and Tramtrack. Shaggy and Delta are required for the activation of Notch protein. However, Delta is sufficient to activate Notch in this process, since premature expression of Delta in the germline stops mitotic division of the follicle cells. This study identified Tramtrack as a connection between Notch and the cell cycle regulators stg, fzr, and dap. Loss of Tramtrack function phenocopies the Notch and Su(H) phenotypes; overproliferation and misregulation of cell cycle components. However, high FAS3 expression, indicative of differentiation defects in Notch clones, is not observed in ttk clones, suggesting that Tramtrack might regulate a branch of the Notch pathway specific for cell cycle control. It was also shown that the JNK-pathway is a critical mitosis promoting pathway in follicle cells. Loss of JNK(bsk) or JNKK(hep) activities stop follicle cell mitotic cycles, while loss of JNK promotes premature endocycles. In addition, loss of the negative regulator of the pathway, the phosphatase Puckered, results in a lack of endocycles. However, the Notch-responsive cell cycle targets that, in combination, can induce the mitotic-to-endocycle transition, stg, fzr, and dap, are not regulated by the JNK-pathway (Jordan, 2006).
Notch signaling is highly regulated throughout development. The Notch receptor can be regulated by glycosylation of the extracellular domain, as well as by endocytosis and degradation of the intracellular domain, thus affecting the activity of the pathway. Shaggy has been shown to phosphorylate and thus affect the stability of Notch protein. Normal processing and clearing of Notch protein from the apical surface of follicle cells upon Notch activation does not occur in shaggy clones, indicating that Notch is not normally activated and therefore regulation of the downstream targets does not take place (Jordan, 2006).
In many organisms and tissues the Notch ligands are ubiquitously expressed and thus not likely to regulate Notch pathway activation. However, at the mitotic to endocycle transition, Delta is upregulated in the germline, making ligand expression a likely candidate for regulation of Notch activity. Premature expression of Delta in the germline can cause mitotic division to stop at least one stage earlier than in control ovarioles. Nonetheless, this effect is seen in only half of the ovarioles. Therefore, it is possible that yet another process is regulating Notch activity at the transition in addition to Delta expression. Further testing will determine if endocytosis of Notch might also regulate Notch activity at the mitotic-to-endocycle transition. One possible protein is Numb, which regulates Notch in human mammary carcinomas, indicating that Numb may have a more general role in cell cycle control than just the division of the sensory organ precursors (Jordan, 2006).
The fact that Notch overrides the mitotic activity of the JNK pathway by acting on cell cycle regulators that can induce the mitotic-to-endocycle transition puts further demand on understanding the connection between Su(H) and cell cycle regulators. One such component, the transcription factor Tramtrack, has been identified. Two Tramtrack proteins exist, Ttk69 and Ttk88, both of which are affected by the allele used in these studies. However, staining with antibodies specific to the two forms reveals that only Ttk69 is detectable in the follicle cells and downregulated in Notch clones (Jordan, 2006).
Ttk69 can control proliferation in glial cells, strengthening its candidacy for a critical component between Notch and cell cycle controllers in follicle epithelial cells. In addition, the Ttk-like BTB/POZ-domain zinc-finger transcription repressor in humans is Bcl-6, a protein associated with B-cell lymphomas (Jordan, 2006).
Ttk function in the follicle cell mitotic-to-endocycle transition was analyzed and it has been shown that the Notch-responsive cell cycle components stg, dap, and fzr are responsive to Ttk function. Interestingly, Ttk69 controls the string promoter in the Drosophila eye discs. In the future, it will be important to determine whether Ttk DNA binding sites are found in the Notch-responsive stg promoter as well. In addition, the binding sites of transcription factors that can interact with Ttk will be of interest, since Ttk can act as a DNA binding or non-binding repressor (Jordan, 2006).
Previous work revealed that the JNK pathway is closely connected to cell cycle control. For example, in fibroblasts the JNK pathway is critical for cdc2 expression and G2/M cell cycle progression. In the case of the follicle cell mitotic-to-endocycle transition, it was shown that the JNK pathway is a critical positive controller of the mitotic cycles. Lack of JNK activity leads to a block in mitosis and initiation of premature endocycles. Conversely, lack of the negative regulator of the JNK-pathway, the phosphatase Puckered, results in a loss of endocycles. However, puc mutant clones do not consistently support extra divisions but might induce apoptosis as shown recently in disc clones (Jordan, 2006).
These data are interesting in light of the results showing that the JNK pathway does not control the same cell cycle targets as the Notch pathway, and could be explained by the following hypothesis: the JNK-pathway positively regulates the mitotic cycles prior to stage 6 in follicle epithelial cells. This positive action on mitotic cycles is negatively short-circuited by the direct control of cell cycle regulators by the Notch pathway at stage 6 in oogenesis, resulting in the mitotic-to-endocycle transition. Premature termination of the JNK pathway is sufficient to induce mitotic-to-endocycle transition. However, prolonged JNK activity, while disrupting endocycles, cannot maintain mitotic cycling efficiently, due to Notch action on string, dacapo, and fzr (Jordan, 2006).
What then terminates JNK-pathway activity at stage 6 in oogenesis? Prolonged JNK activity (puc mutant clones) affects endocycles and the expression of pJNK and Puc subsides at stages 6-7; results that both suggest the downregulation of JNK activity at the mitotic-to-endocycle transition. One possibility is that Notch activity downregulates the JNK pathway. However, at least Su(H)-dependent Notch activity does not regulate the JNK pathway, since no effect on puckered expression was observed in Su(H) mutant clones. It is plausible that Su(H)-independent Notch activity regulates the JNK pathway in this context, as has been shown to be the case in dorsal closure. Interestingly, Deltex might play a role in this Su(H)-independent Notch activity (Jordan, 2006).
An important question in analyzing the developmental control of cell cycle is whether the same signaling pathways control both differentiation and cell cycle, and if so, how the labor is divided. The Notch-dependent mitotic-to-endocycle transition is an example of such a question; Notch action in stage 6 follicle cells is critical for the cell cycle switch and for at least some aspects of differentiation. This work reports the first component that separates Notch dependent cell cycle regulation from Fas3 marked differentiation; Ttk. In the ttk mutant clones, upregulation of FAS3, characteristic for Notch clones, is not observed. Therefore, Ttk constitutes a branch of Notch activity that might be solely required for cell cycle control in this context. However, Ttk's independent function cannot yet be rule out. In the future, it will be important to understand whether signaling pathways in general show a clear separation of differentiation and cell cycle control on the level of downstream transcription factors. Importantly, these and previous results have revealed the essential cell cycle regulators and their roles in controlling the Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).
Mixed Lineage Kinases (MLKs) function as Jun-N-terminal kinase (JNK) kinase kinases to transduce extracellular signals during development and homeostasis in adults. slipper (slpr),
which encodes the Drosophila homolog of mammalian MLKs is implicated in
activation of the JNK pathway during embryonic dorsal epidermal closure. To further define the specific functions of Slpr, the phenotypic consequences of slpr loss- and gain-of function was studied throughout development, using a semi-viable maternal effect allele and wildtype or dominant negative transgenes. From these analyses it was confirmed that failure of dorsal closure is
the null phenotype in slpr germline clones. In addition, there is a functional maternal contribution, which can suffice for embryogenesis in the zygotic null mutant, but rarely suffices
for pupal metamorphosis, revealing later functions for slpr as the maternal contribution is depleted. Zygotic null mutants that eclose as adults display an array of morphological defects, many of which are shared by hep mutant animals, deficient in the JNK kinase (JNKK/MKK7) substrate for Slpr, suggesting that the defects observed in slpr mutants primarily reflect loss of hep-dependent JNK activation. Consistent with this, the maternal slpr contribution is sensitive to the dosage of positive and negative JNK pathway regulators, which attenuate or potentiate Slpr-dependent signaling in development. Though Slpr and TAK1, another JNKKK family member, are differentially used in dorsal closure and TNF/Eiger-stimulated apoptosis, respectively, a Tak1 mutant shows dominant genetic interactions with slpr suggesting potential
redundant or combinatorial functions. Finally, it was demonstrated that SLPR overexpression can induce ectopic JNK signaling and that the Slpr protein is enriched at the epithelial cell cortex (Polaski, 2006).
Previous genetic studies have established a role for SLPR/MLK in JNK pathway activation during embryonic tissue closure. This study characterizes the phenotype of an allele affecting postembryonic development as well as protein products encoded by wildtype and mutant alleles. slprBS06 is a newly isolated null allele that encodes an early nonsense mutation
and consequently, no protein product is detected in mutant tissue clones or by Western immunoblot. Phenotypic comparison between the null allele and existing alleles confirms the
role for SLPR in dorsal closure, clarifies that slpr has a maternal contribution and that the prior
two alleles encode dominant negative proteins, and uncovers additional roles for Slpr in metamorphosis of the adult (Polaski, 2006).
The severe dorsal open phenotype of slprBS06 germline clones, maternally and zygotically mutant, indicates that dorsal closure is the earliest requirement for Slpr in embryogenesis and
that a failure of dorsal closure is the null phenotype, consistent with the phenotype of the previously characterized 921 and 3P5 slpr alleles. In contrast though, most slprBS06 zygotic
mutants survive embryogenesis and adult mutant males are recovered at a low frequency. These males display several visible morphological phenotypes of variable penetrance, presumably as a
consequence of the eventual depletion of functional maternal product. These observations indicate that the maternal slpr gene product is nearly sufficient for embryogenesis in the absence
of zygotic product, but rarely provides enough function for metamorphosis, revealing additional roles for Slpr in postembryonic processes (Polaski, 2006).
Defects observed in mutant adults implicate Slpr function for proper metamorphosis of the genital discs, dorsal abdomen and notum, maxillary palps, and wing. Females show somatic defects during oogenesis demonstrating that Slpr is required for proper morphology of the
chorionic dorsal appendages. Many of the defects, including those affecting the thorax, genitals and dorsal appendages, have been documented previously to result from loss of JNK signaling. Thus, the data reported here implicate Slpr as the upstream JNKKK family member required for JNK activation in these processes. The current study also
suggests that slpr function is mediated primarily, if not entirely, via HEP/MKK7 and the JNK pathway, as evidenced by the fact that hep mutants share in common all of the defects observed in slpr mutants and that reducing the dosage of two known negative regulators of JNK
signaling, puc and raw, suppresses the slpr phenotypes. In light of these results, it will be informative to systematically test whether, in vivo, the mammalian MLK proteins activate alternative substrates and pathways as has been suggested from tissue culture studies (Polaski, 2006).
Given that the slpr921 and slpr3P5 alleles are phenotypically more severe in zygotic mutants than the null allele indicates that the encoded products have dominant negative activity, which interferes with the functional pool of maternal slpr gene product. This is consistent with
the molecular nature of the alleles, which predict that full length (921) or partial (3P5) protein product would be expressed in the mutants. Indeed, clonal analysis and immunofluoresence
staining confirm the expression of mutant protein in slpr921 animals. Protein levels in slpr3P5 mutant tissue appear reduced relative to wildtype, but the encoded fragment retains the SH3 domain and most of the kinase domain, each of which, if folded properly, could engage in protein-protein interactions. Similarly, the catalytically inactive, full length protein encoded by slpr921 retains several functional protein interaction motifs, which could account for the antimorphic properties of the protein (Polaski, 2006).
What candidates are known to interact with the various regions of the Slpr protein to account for dominant negative activity? By analogy with the mammalian MLK proteins, at least three recognized domains have potential protein binding activity. The leucine zipper mediates
homodimerization, which is requisite for autophosphorylation and substrate activation. Mutant Slpr protein in slpr921 cells might
trap wildtype protein in unproductive dimers. The CRIB domain binds to the activated form of the small GTPases, Cdc42 and Rac1, both implicated in
dorsal closure. Titration of these GTPases by non-catalytic Slpr921 protein could also contribute to dominant interference of the wildtype Slpr protein. Finally, the N-terminal SH3 domain, retained in the proteins encoded by both slpr3P5 and slpr921, has the potential to engage
in both intra- and intermolecular interactions. The SH3 domain of mammalian MLK3 can bind to a region between the LZ and CRIB domains through a critical proline residue that is conserved in Drosophila Slpr. The postulated intramolecular binding is thought to negatively regulate MLK activation by locking the protein in a closed conformation (Polaski, 2006).
This type of autoinhibition has been demonstrated for other modular kinases, such as Src tyrosine kinase. Also, the SH3 domain may serve as a docking site for upstream activating kinases of the Ste20 family, for which titration by an SH3-containing protein fragment could impair signal relay to the JNK pathway. Therefore, the modular domain organization of the Slpr protein with the potential for multiple regulatory protein interactions is likely to explain why residual mutant protein is more detrimental than complete loss of protein in the null mutant (Polaski, 2006).
Why then does overexpression of an engineered kinase dead Slpr transgene that is
functionally equivalent to the protein encoded by slpr921 have such mild consequences in the embryo? Evidence suggests that it is due to a substantial functional maternal component, in addition to the zygotic contribution, because reducing the maternal pool in embryos derived from slpr-/+ heterozygous mothers exacerbates the effect of dominant negative Slpr transgene
expression. Further support for the function of the maternal gene product is demonstrated by the sensitivity of the maternal contribution to the dosage of additional positive and negative JNK pathway regulators, which has been monitored as the extent of recovery of slprBS06 mutant adult males (Polaski, 2006).
Given that the maternal product is nearly sufficient for mutant males to survive to adulthood, it is curious that immunodetection of endogenous Slpr protein in the embryo has been difficult relative to the ease with which transgenic protein can be detected. This may suggest that the maternal pool of slpr gene product is largely mRNA rather than protein, that an active
mechanism exists to maintain low levels of embryonic protein, or that protein complexes mask the ability to detect the epitope on the Slpr protein, either of which could be overcome by the abundant expression of exogenous transgenic protein. Though the mechanism is not clear, the genetic loss-of-function and overexpression data together indicate that certain cell types or developmental contexts are sensitive to the levels of Slpr protein in modulating JNK signaling.
For example, while exogenous Slpr can induce JNK signaling in embryonic dorsal ectoderm cells, normally limited for JNK activity, not all cells are equally inducible, suggesting there may be other limiting components or brakes that modulate the precise levels of JNK activity in cells.
Inferring function from overexpression experiments in the absence of loss-of-function data can
be misleading however, because wildtype transgene expression can stimulate JNK signaling promiscuously, or at least where the endogenous protein appears not to be required. For
example, transgenic expression of either Slpr or TAK1 can induce JNK signaling ectopically in the embryonic dorsal ectoderm under the control of pnr-GAL4,
even though endogenous levels of TAK1 cannot provide enough JNK signaling activity in slpr null embryos to rescue dorsal closure. Moreover, Tak1 mutants are viable providing corroborating evidence that Tak1 is not required for dorsal closure. In sum, JNK signaling activity may be at a threshold level in most cells, easily overactivated by expression of many different upstream regulators, but whose selective use in physiological circumstances is only revealed through analysis of loss-of-function (Polaski, 2006).
The combined gain- and loss- of function analysis for Slpr described here supports two proposed mechanisms of signaling specificity among JNKKK proteins; first, that individual family members are used selectively in particular contexts and second, that potential combinatorial or redundant functions may exist among members with common substrates. With respect to TNF/Eiger signaling, both loss-of-function analysis and dominant
negative constructs consistently implicate TAK1 rather than Slpr in this JNK-dependent response. In addition, JNK-dependent
developmental morphogenetic events, in particular dorsal closure in the embryo, selectively require Slpr. It was consistently observed that the slprBS06, Tak1 double mutant is more severe than either single mutant alone, suggesting that there are likely to be additional, perhaps redundant, functions of Slpr and TAK1 that are only revealed in the double mutant. These functions have yet to be investigated in detail (Polaski, 2006).
At face value, genetic analysis has allowed the assignment of Slpr to mediate many JNK-dependent morphogenetic events and TAK1 to mediate JNK-dependent homeostatic responses including apoptosis and immunity. However, it is still unclear whether the selective functions reflect differential transcriptional responses or whether cell and developmental context shapes what appear to be quite different cellular behaviors. In other words, though the
developmental defects that arise as a consequence of loss of Slpr function may suggest a common failure in cell shape change or cytoskeletal functions, similar to the defects that underlie the failure of embryonic dorsal closure in the mutants, that assumption may be too simplistic. It
is a formal possibility that Slpr could regulate additional or alternative JNK-dependent cell responses in distinct contexts. For example, though Slpr appears not to mediate TNF-induced apoptosis in the Drosophila eye under conditions where Eiger is overexpressed, the male genital misrotation phenotype observed in slpr mutants may be linked to JNK-dependent developmental
programmed cell death. Defective genital rotation is observed in certain viable alleles of hid, encoding a protein with proapoptotic function. The basis of the rotation defect may be due to an excess of genital disc cells, similar to
the embryonic defects in head involution, the namesake phenotype of hid (Polaski, 2006).
Thus, JNK signaling and HID function are both required for proper genital rotation and interestingly, there is precedent for hid being a transcriptional target of the JNK pathway downstream of Eiger. Thus, it will be important to determine specifically whether Slpr mediates JNK-dependent HID expression or even apoptosis in imaginal discs, or whether the requirement for Slpr in male genital rotation is unrelated to apoptosis. More generally, a full understanding of Slpr function will require systematic definition of the molecular and cellular mechanisms that underlie the morphological defects. If Slpr functions to regulate different outputs in different contexts, determining what regulates a selective response will be of considerable interest for future studies (Polaski, 2006).
The mammalian GADD45 (growth arrest and DNA-damage inducible) gene family is composed of three highly homologous small, acidic, nuclear proteins: GADD45α, GADD45β, and GADD45γ. GADD45 proteins are involved in important processes such as regulation of DNA repair, cell cycle control, and apoptosis. Annotation of the Drosophila genome revealed that it contains a single GADD45-like protein (CG11086; D-GADD45). As its mammalian homologs, D-GADD45 is a nuclear protein; however, D-GADD45 expression is not elevated following exposure to genotoxic and nongenotoxic agents in Schneider cells and in adult flies. The D-GADD45 transcript increased following immune response activation, consistent with previous microarray findings. Since upregulation of GADD45 proteins has been characterized as an important cellular response to genotoxic and nongenotoxic agents, the effect of D-GADD45 overexpression on Drosophila development was characterized. Overexpression of D-GADD45 in various tissues led to different phenotypic responses. Specifically, in the somatic follicle cells overexpression caused apoptosis, while overexpression in the germline affected the dorsal-ventral polarity of the eggshell and disrupted the localization of anterior-posterior polarity determinants. This article focused on the role of D-GADD45 overexpression in the germline and it was found that D-GADD45 caused dorsalization of the eggshell. Since mammalian GADD45 proteins are activators of the c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) signaling pathways, a genetic interaction was tested in Drosophila. It was found that eggshell polarity defects caused by D-GADD45 overexpression are dominantly suppressed by mutations in the JNK pathway, suggesting that the JNK pathway has a novel, D-GADD45-mediated, function in the Drosophila germline (Peretz, 2007).
The GADD45 gene family is composed of three highly homologous (55-58% overall identity at the amino acid level), small, acidic, nuclear proteins: GADD45α, GADD45β (MyD118), and GADD45γ (CR6, cytokine response gene 6). In recent years, evidence has emerged that the proteins encoded by these genes play similar but not identical roles in terminal differentiation and negative growth control, including growth suppression and apoptotic cell death (Peretz, 2007 and references therein).
One of the well-described responses to genotoxic and nongenotoxic stresses is the rapid upregulation of different GADD45 proteins, which in turn affect cell-cycle regulation, cell survival, and cell death. It has been shown that all the GADD45 proteins mediate cell-cycle regulation through interactions with PCNA, the cyclin-dependent kinase inhibitor p21, and the Cdk/cyclin B complex. The potential role of GADD45 proteins in apoptosis emanates from the observation that GADD45 expression is enhanced during apoptosis following induction by a variety of genotoxic agents. Several studies have shown that GADD45 proteins may play a role in apoptosis via activation of the c-Jun N-terminal kinase (JNK) and/or p38 mitogen-activated protein kinase (MAPK) signaling pathways. GADD45 proteins physically interact with the MAPKKK, MTK1 (synonym MEKK4), and the ensuing interactions result in the activation of MTK1. Activated MTK1 is thought to further activate its downstream targets JNK and p38. It was shown that the N-terminal of MTK1 auto-inactivates its kinase activity and binding of GADD45 proteins to MTK1 relieves this inhibition. It has been proposed that in response to genotoxic stress, p53 is activated, which causes transcriptional upregulation of GADD45α, and GADD45α interacts with MTK1 to initiate the JNK/p38-mediated apoptotic pathway (Peretz, 2007 and references therein).
Several model systems have been used to analyze the role of GADD45 proteins during development. GADD45α-null mice exhibit several phenotypes including genomic instability, increased radiation carcinogenesis, and a low frequency of exencephaly. GADD45γ-deficient mice develop normally and are indistinguishable from their littermates, possibly due to functional redundancy among the GADD45 family members. In the fish, Oryzias latipes, ectopic expression of GADD45γ leads to cell cycle arrest without inducing apoptosis. Loss of function of GADD45γ causes a significant increase in apoptosis, suggesting that GADD45γ is an important component of the molecular pathway that coordinates cell cycle vs. apoptosis decisions during vertebrate development. The zebrafish GADD45β genes were found to be periodically expressed as paired stripes in the anterior presomitic mesoderm. Both knockdown and overexpression of GADD45β genes caused somite defects with different consequences for marker gene expression, indicating that the regulated expression of GADD45β genes is required for somite segmentation. The possible functional redundancy among the GADD45 proteins in these model systems makes the analysis of the molecular function of GADD45 difficult. Annotation of the Drosophila genome revealed that it contains only one GADD45-like protein (Peretz, 2007).
Since upregulation of GADD45 proteins may affect cell cycle regulation, cell survival, and cell death, the effect was studied of D-GADD45 overexpression on D. melanogaster oogenesis. Overexpression of D-GADD45 in the somatic follicle cells led to apoptosis of the entire egg chamber. In contrast, overexpression of D-GADD45 in the germline did not cause apoptosis but affected the dorsal-ventral polarity of the eggshell. Moreover, D-GADD45 also affected anterior-posterior polarity determinants. However, anterior oocyte nuclear migration and bcd localization were unaffected. Finally, it was found that mutations in the MAPK-JNK pathway dominantly suppressed the egg asymmetric defects in D-GADD45 overexpression ovaries, suggesting a novel, D-GADD45-mediated function for the JNK pathway in the germline (Peretz, 2007).
In Drosophila D-GADD45 preserves the nuclear localization property, but unlike its mammalian homologs its expression is not elevated following exposure to different stress stimuli. This result is supported by the Drosophila whole genome microarray analysis which did not identify D-GADD45 as a gene whose expression is increased following various genotoxic and nongenotoxic treatments. Although a number of stress treatments were tried, it is possible that D-GADD45 expression would rise only following exposure to as yet untested stressful conditions (Peretz, 2007).
D-GADD45 was identified as a gene whose expression is induced following microbial infection. It was also shown that D-GADD45 expression may be regulated by the NF-BkappaB-like transcription factor, Dorsal, which has an optimal binding site 3 kb upstream to D-GADD45 transcription start site. These results are consistent with published results further strengthen a possible function for D-GADD45 in the immune response. Given that Drosophila is devoid of an adaptive immune system and relies only on innate immune reactions for its defense, D-GADD45 may play an important role during infection (Peretz, 2007).
Ubiquitous overexpression of D-GADD45 was lethal, most likely due to apoptosis, as was directly demonstrated in the follicle cells. However, the results suggest that apoptosis induced by overexpression of D-GADD45 is tissue specific since overexpression of D-GADD45 in other somatic tissues, such as the eye and wing, did not lead to apoptosis. Also, overexpression in the germline did not cause cell death; rather, it affected egg chamber asymmetric development. The apparent phenotypic differences in overexpression of D-GADD45 in the germline as opposed to somatic derived tissues probably reflect the complexity of the biological functions of GADD45, which may be subject to tissue- and/or signal-specific regulation that ultimately dictate their output. Similarly, it has been shown that individual members of the GADD45 family play critical roles in negative growth control in some tissues while in others they are associated with uncontrolled cell growth and tumor development. GADD45α was identified as an important mediator of tumor suppression in human ovarian cancer cells. While in pancreatic ductal adenocarcinoma GADD45α was found to be overexpressed at the mRNA and protein level. Downregulation of GADD45α by means of RNAi reduced proliferation and induced apoptosis in pancreatic cancer cells implying that GADD45α contributes to pancreatic cancer cell proliferation and viability (Peretz, 2007).
Overexpression of D-GADD45 in the germline results in dorsalization of the chorion due to defects in grk localization and translation. The posterior markers osk and Kin:β-gal were mislocalized during mid-oogenesis. In contrast, D-GADD45 overexpression does not affect the localization of anterior end markers such as bcd and Nod:β-gal and also the anterior oocyte nuclear migration is unaffected. Similar results have reported in mutants of squid (sqd) which encodes a heterogeneous nuclear ribonucleoprotein (hnRNP). In these mutants grk mRNA is mislocalized along the anterior ring, leading to dorsalization of the eggshell. Furthermore, loss of sqd function causes an aberrant localization of osk and Kin:β-gal, but does not affect bcd localization and oocyte nucleus migration. It was shown that in sqd mutant oocytes short microtubules (MTs) around the entire oocyte cortex are retained, including at the posterior pole, unlike wild-type MTs which emanate mostly from the anterior. It has been suggested that the primary MT defect in sqd mutants is the failure to eliminate cortical sites of MT nucleation beyond stage 7. It is possible that D-GADD45 overexpression also affects MT organization in the oocyte. This possibility is further supported by the finding that GADD45α interacts with elongation factor 1α (EF-1α), a microtubule-severing protein that plays an important role in maintaining microtubule cytoskeletal stability. To test whether D-GADD45 affects MT organization, the ovaries were stained with anti-tubulin. Using this tool, no gross morphological changes were detected in the MT network. Given that the patterning defect seen in D-GADD45 overexpression is weaker than that in sqd mutants, it could be that this kind of staining is not sensitive enough to identify the MT network alterations in D-GADD45 overexpression flies (Peretz, 2007).
A genetic interaction was found between D-GADD45 and proteins of the MAPK-JNK pathway. Mutations in the JNKK, hemipterous, dominantly suppres the dorsalized eggshell phenotype. This genetic interaction is supported by the finding that in human cells GADD45 proteins act as initiators of JNK/p38 signaling via their interaction with the MAPKKK, MTK1 (Takekawa, 1998). It was shown that the N-terminal of MTK1 inhibits its C-terminal kinase domain by preventing the kinase domain from interacting with its substrate, MKK6, and binding of GADD45 proteins relieves this auto-inhibition (Peretz, 2007).
Up until now the only roles attributed to the JNK pathway during oogenesis were in the follicle cells and included morphogenesis of the dorsal appendages and the micropyle. It was also reported that the JNK pathway is involved in the morphogenetic process of dorsal closure during embryogenesis. Surprisingly, it was found that eggshell patterning defects caused by D-GADD45 overexpression are dominantly suppressed in a hep deficient background suggesting an additional role for the JNK pathway in the germline. This novel function may have gone unnoticed in the past while studying JNK loss-of-function alleles due to redundancy with some other pathway. In this study, overexpression of the JNK activator, D-GADD45, may have unmasked this new role during oogenesis (Peretz, 2007).
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, 2009).
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, 2009).
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, 2009).
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, 2009).
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, 2009).
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, 2009).
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, 2009).
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, 2009).
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, 2009).
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, 2009).
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, 2009).
Regeneration and tissue repair allow damaged or lost body parts to be replaced. After injury or fragmentation of Drosophila imaginal discs, regeneration leads to the development of normal adult structures. This process is likely to involve a combination of cell rearrangement and compensatory proliferation. However, the detailed mechanisms underlying these processes are poorly understood. A system was established to allow temporally restricted induction of cell death in situ. Using Gal4/Gal80 and UAS-rpr constructs, targeted ablation of a region of the disc could be performed and regeneration monitored without the requirement for microsurgical manipulation. Using a ptc-Gal4 construct to drive rpr expression in the wing disc resulted in a stripe of dead cells in the anterior compartment flanking the anteroposterior boundary, whereas a sal-Gal4 driver generated a dead domain that includes both anterior and posterior cells. Under these conditions, regenerated tissues were derived from the damaged compartment, suggesting that compartment restrictions are preserved during regeneration. These studies reveal that during regeneration the live cells bordering the domain in which cell death was induced first display cytoskeletal reorganisation and apical-to-basal closure of the epithelium. Then, proliferation begins locally in the vicinity of the wound and later more extensively in the affected compartment. Finally, regeneration of genetically ablated tissue was shown to require JNK activity. During cell death-induced regeneration, the JNK pathway is activated at the leading edges of healing tissue and not in the apoptotic cells, and is required for the regulation of healing and regenerative growth (Bergantiños, 2010).
Two main conclusions can be drawn from this work: (1) that genetically induced regeneration entails compartment-specific proliferation; and (2), that this type of regeneration requires JNK signalling for early regeneration events (Bergantiños, 2010).
This study established that the proliferation response to ptc>rpr induction is concentrated in the A compartment and consists of two activities: a local and a compartment-associated response. The local proliferation response resembles the activity of blastemas, a feature found in discs after fragmentation and implantation. The late compartment-restricted proliferation could be indicative of a reutilisation of developmental programs. The entire A compartment responds to the lack of the original ptc region by reactivating proliferation in order to achieve the final organ size. Thus, it is concluded that genetically induced regenerating discs restore the overall organ size by activation of proliferation, not only near the wound, as in fragmented and implanted discs, but also in the whole affected compartment. Thus, it is believed that the local proliferation is a fast and early response to the lost structures and that the later compartment-associated proliferation is a response to adjust the size of the tissue (Bergantiños, 2010).
ptc and sal were selected because of the precise removal of cells and also because they enabled testing whether both A and P compartments are involved in regeneration. The results suggest that when the A compartment is damaged (ptc>rpr), the P compartment only responds to the injury by sealing the gap that separates it from the A compartment through the generation of F-actin-rich cell extensions. These are projected to anchor the extensions from the cells at the edge of the A compartment as they proceed towards recovery of the intact cell sheet. In this situation, the regenerated tissue is derived exclusively from the A compartment. By contrast, when cells from both the A and P compartments are killed (sal>rpr), proliferation increases in both compartments. The boundaries between compartments are rapidly re-established after injury and prevent cells from crossing into adjacent compartments. Thus, boundaries are respected and compartments act as units of growth during regeneration (Bergantiños, 2010).
Following genetic ablation driven by either the ptc or sal drivers, healing starts at the DV boundary and spreads laterally towards the proximal regions, which are the last to close the wound. Cells at the DV boundary are arrested in G1-S, through a mechanism based on Notch and wg signalling. These arrested cells are the first to respond to healing and drive the cytoskeletal machinery for tissue reorganisation. This is consistent with the idea that the requirements for cell proliferation and for cell shape changes that occur during normal fly and vertebrate development and wound repair place incompatible demands on the cytoskeletal machinery of the cell. Another issue to be considered is that the DV boundary is the first zone of closure for F-actin extensions. This is reminiscent of Drosophila embryonic dorsal closure and wound repair, in which matching filopodia on both sides of the opening are recognised by the code of segment polarity genes in each parasegment. In addition, mechanical forces may be involved in tissue reorganisation. Stretching forces could be altered upon the induction of cell death, and they could have an important role in mounting a quick healing response. For example, mechanical forces, which have been proposed to act in the developing wing disc and compress the tissue through the central region, could stretch it towards the DV border after ablation of the ptc domain. Thus, either by matching affinities or by stretching forces, wound repair spreads from the apical DV border to basal and proximal domains (Bergantiños, 2010).
It has been shown in the Drosophila wing disc that massive loss of cells after irradiation gives rise to apparently normal adult wings as a result of compensatory proliferation driven by surviving cells. Experiments involving irradiation or induction of apoptosis in a p35 background have suggested that this compensatory proliferation is controlled by signals, including JNK, emerging from cells that have entered apoptosis, and that cell-death regulators, such as p53 and the caspase Dronc (Nedd2-like caspase), function as regulators of compensatory proliferation and blastema formation in the surviving cells. By contrast, the results show that proliferation is compartment specific and occurs independently of the dead tissue following targeted ablation. Two observations strongly support this interpretation. First, puc expression, as a marker of JNK activity, is concentrated in a narrow strip of apical cells, suggesting that JNK signalling is activated in the leading edges during wound closure. This again resembles other repair mechanisms described not only in imaginal discs, but also in other healing tissues, and reiterates epithelial fusion events observed in embryogenesis. Second, perturbation of the JNK pathway within the dying domain has no effect on either healing or regeneration. Even the early peak of localised mitosis near the wound and the later A-compartment-associated mitoses are present when UAS-bskDN and UAS-puc are driven in the dying domain. Effects on healing and regeneration are found only in hep mutant backgrounds, when JNK is impaired in the whole epithelium and not only in the dead domain. This requirement for the JNK pathway at the edges of the wound has also been found in studies of microsurgically induced regeneration. Cell lineage analysis of puc-expressing cells near the wound has shown that puc sets the limits of a blastema and that puc derivatives are able to reconstitute most of the missing tissue (Bergantiños, 2010).
Finally, whether JNK is required for healing alone or also functions as a signal for proliferation remains an open issue. Rapid local proliferation is affected in unhealed hep heterozygotes. Also, salPE>rpr wing regeneration cannot be achieved after 10 hours induction in a hepr75 background. Reduced proliferation could be due to a lack of healing or to loss of JNK activity. The possibility canot be ruled out that the JNK cascade, through the active AP-1 (Kayak and Jun-related antigen -- FlyBase) transcription factor complex, targets not only genes required for healing and epithelial fusion, but also those required for regenerative growth. In mammals, inhibition of the JNK pathway or lack of c-Jun results in eyelid-closure defects and also impairs proliferation by targeting Egfr transcription. Reconstruction of normal pattern and size might also require multiple signals. It has recently been found that regenerative growth induced by cell death requires Wnt/Wg signalling to increase dMyc stability, suggesting the involvement of other signalling pathways and also cell competition. It is very likely that an integrated network of signals and cell behaviours is necessary to reconstitute the damaged tissue (Bergantiños, 2010).
Taken together, these results suggest a model for cell-induced regeneration that includes two phases. The first, which occurs near the wound edges, involves JNK activity and is important for healing and rapid local proliferation. The second involves proliferation to compensate for the lost tissue and is extended throughout the damaged compartment. As in normal development, the regenerative growth that occurs in this second phase requires the reconstitution of morphogenetic signals that drive proliferation (Bergantiños, 2010).
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hemipterous:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 15 December 2010
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