Phosphotidylinositol 3 kinase 92E
The human tumor suppressor gene PTEN gets its name from its biochemical function, its domain structure and its chromosomal location: PTEN stands for the combination of phosphatase and tensin homolog on chromosome 10. The lipid phosphatase function of PTEN places it in the middle of the insulin pathway, known to involve lipid signaling (Goberdhan, 1999; Huang, 1999). Drosophila Pten modulates cell size and consequently tissue mass by acting antagonistically to the lipid modifiying enzyme Phosphotidylinositol 3 kinase 92E, also known as Dp110, and its upstream activator Chico, an insulin receptor binding and signal transduction protein. All signals from the insulin receptor can be antagonized by Pten. In terms of its protein phosphatase function, mammalian PTEN targets focal adhesion kinase, a major effector of cytoskeletal function. Overexpression of wild-type mammalian PTEN and mutant PTEN that lacks lipid phosphatase activity can reduce levels of focal adhesion kinase (FAK: see Drosophila Focal adhesion kinase-like)
phosphorylation and the formation of focal adhesions, thereby inhibiting cell migration and invasiveness.
Overexpression of Pten also produces enlargement of wing cells. Wild-type Pten cDNA is overexpressed in particular areas
of the wing using the GAL4-UAS system. Initially flies carrying a dpp-GAL4 construct were used. This drives gene expression in
cells that will normally populate the region between the third and
fourth longitudinal wing veins (LIII and LIV). Overexpression of
Pten reduces the size of these regions by nearly 25% compared
with wild type. This is not a consequence
of a general reduction in wing size in overexpressing flies, since an
adjacent area of the wing between LIV and LV is essentially
unaffected. The effect on wing area is similar to that produced by
overexpression of Dp110D954A, a dominant-negative,
kinase-dead version of Pi3K92E. The
reduction is caused by both a decrease in cell size and cell number
and is opposite of the effect of overexpressing an activated,
membrane-associated form of Pi3K92E, Dp110-CAAX, in the same region.
To test whether Pten's growth regulatory functions are primarily
mediated by its effects on the insulin receptor-Pi3K92E signaling pathway and not by an independent signaling cascade, the
genetic effects of Pten alleles were sought using mutant phenotypes
associated with chico and Pi3K92E (Goberdhan, 1999).
Interestingly, both overexpression of a dominant negative form of
Phosphotidylinositol 3 kinase 92E (also known as Dp110 or Pi3K92E) and mutations
either in the Drosophila insulin receptor
or in chico/IRS1-4 also reduce cell size as well as proliferation. Furthermore, overexpression
of wild-type and activated forms of Pi3K92E produces similar size and
proliferation defects as those seen in Pten mutant cells. These observations are consistent with a model
in which growth is regulated in Drosophila by specific
phosphoinositides whose levels are controlled by the balance of Pten
and Pi3K92E activities. Pten and
Pi3K92E. At 25°C, flies do not survive to adult when
wild-type Pi3K92E is ectopically expressed by means eyeless-GAL4. Interestingly,
this lethality can be rescued by coexpression of Pten.
Furthermore, the small eye phenotype of Pten overexpression
is suppressed by overexpression of wild-type Pi3K92E and
enhanced by overexpression of dominant negative Pi3K92E. These results clearly indicate that Pten and Pi3K92E
function antagonistically in Drosophila.
The recent characterization of chico, a Drosophila IRS1-4
homolog, has shown that chico, Pi3K92E and Insulin receptor(Inr)
act as positive elements in a Drosophila insulin signaling
pathway to regulate cell proliferation and cell size (Huang, 1999).
Class IA phosphoinositide 3-kinases (PI 3-kinases) have been implicated in the regulation of several cellular
processes including cell division, cell survival and protein synthesis. The size of Drosophila imaginal discs (epithelial
structures that give rise to adult organs) is maintained by factors that can compensate for experimentally induced
changes in these PI 3-kinase-regulated processes. However, overexpression of the gene encoding the Drosophila class IA PI
3-kinase, Dp110, in imaginal discs, results in enlarged adult organs. These observations have led to an
investigation of the role of Dp100 and its adaptor, p60, in the control of imaginal disc cell size, cell number and organ size.
Null mutations in Dp110 and p60 were generated and used to demonstrate that these genes are essential genes that are
autonomously required for imaginal disc cells to achieve their normal adult size. In addition, modulating Dp110 activity
increases or reduces cell size in the developing imaginal disc, and does so throughout the cell cycle. The inhibition of
Dp110 activity reduces the rate of increase in cell number in the imaginal discs, suggesting that Dp110 normally
promotes cell division and/or cell survival. Unlike direct manipulation of cell-cycle progression, manipulation of Dp110
activity in one compartment of the disc influences the size of that compartment and the size of the disc as a whole.
It is concluded that during imaginal disc development, Dp110 and p60 regulate cell size, cell number and organ size. These
results indicate that Dp110 and p60 signaling can affect growth in multiple ways: these observations have important implications for
the function of signaling through class IA PI 3-kinases (Weinkove, 1999).
To investigate whether Dp110 and p60 are required within the imaginal discs themselves, mitotic clones of mutant cells were generated in heterozygous Dp110 or p60 flies by using the Flipase (Flp) and Flp recognition target (FRT) site-specific recombination system. Mutant phenotypes were examined in the adult eye, a highly ordered structure made up of repeated units or ommatidia, each of which contains the same pattern of differentiated photoreceptors and accessory cells. Both Dp110- and p60- eye clones form indented patches containing ommatidia that are reduced in size. The internal eye structure was examined using tangential sections in which Dp110- clones (-/-) were distinguished from their corresponding sister clones or twin-spots (+/+) and the surrounding heterozygous cells (+/-) by an absence of red pigment granules (Weinkove, 1999).
Dp110- cells, and to a lesser extent p60- cells, are significantly smaller in both cross-section and longitudinal-section than cells in the surrounding heterozygous and wild-type tissue. Thus, these experiments show that both Dp110 and p60 are autonomously required for eye cells to achieve their normal adult size. In addition, the mutant clones are consistently smaller than their twin-spots, suggesting that cell number as well as cell size might be reduced. Although reduced in size, the mutant photoreceptors can still differentiate and form rhabdomeres (which stain dark blue) by expanding and re-organizing their internal membranes. Furthermore, almost every ommatidium contains the wild-type number of photoreceptors, suggesting that Dp110 and p60 are not essential for photoreceptor differentiation. The characteristic trapezoid arrangement of photoreceptor rhabdomeres within each ommatidium and the orientation of the ommatidia relative to one another are generally wild type in p60- clones, but are sometimes disrupted in Dp110- clones. Together, these data clearly demonstrate that in Drosophila the class IA PI 3-kinase, Dp110, and its adaptor, p60, are required in a cell-autonomous manner for the same process: the attainment of normal cell size. The similarity of the mutant phenotypes for the two genes is consistent with the established role of the adaptor in the activation of class IA PI 3-kinases and represents the first direct comparison of the loss of function phenotypes of a class IA PI 3-kinase and its adaptor (Weinkove, 1999).
Thus Dp110 and p60 are necessary for adult eye cells to achieve their normal final size. A possible explanation for this requirement is that Dp110 drives growth solely in the final stages of eye development when the retinal cells increase in size after proliferation has ceased. Alternatively, Dp110 activity might be required for both proliferating and post-mitotic imaginal disc cells to achieve their normal size. To distinguish between these two possibilities, an investigation was carried out of the effect of modulating Dp110 activity on the size of the proliferating cells of third instar wing imaginal discs. Thus, Dp110 and p60 transgenes under the control of GAL4 upstream activating sequences (UASs) were expressed in clones of cells induced at random locations in wing imaginal discs. signaling through Dp110 was increased by overexpression of wild-type Dp110 and reduced by overexpression of a catalytically inactive and hence dominant-negative version of Dp110 (Dp110D954A). Greater inhibition of Dp110 signaling was achieved by overexpression of wild-type p60 or of p60 with part of the Dp110-binding site deleted. Like the mammalian class IA PI 3-kinase adaptors, p60 and deltap60 inhibit class IA PI 3-kinase signaling when overexpressed, presumably by competing with endogenous Dp110 and p60 complexes for binding sites on upstream activators. Dp110 expression dramatically increases imaginal disc cell size. In contrast, the expression of Dp110D954A slightly reduces cell size whereas the expression of p60 or deltap60 results in a more dramatic reduction in cell size. Thus, modulating Dp110 activity alters the size of proliferating cells in the third instar disc epithelium. To investigate this effect more closely, the sizes of Dp110-expressing and deltap60-expressing cells were examined at different stages of the cell cycle. This analysis demonstrated that Dp110-expressing cells are larger than control cells during both the G1 phase of the cell cycle and the S + G2. Conversely, deltap60-expressing cells are reduced in size both in G1 and S + G2. Thus, Dp110 regulates cell size throughout the imaginal disc cell cycle (Weinkove, 1999).
The increased size of Dp110-expressing cells might result either from an inhibition of cell division, or from accelerated biosynthesis. To distinguish between these two possibilities, the effect of Dp110 expression on cell number was examined. This analysis reveals that Dp110 expression does not alter the rate of increase in cell number. Thus, Dp110-expressing cells are larger because of increased growth and not because of the inhibition of cell division. Furthermore, this result demonstrates that, as far as can be detected with this technique, overexpression of Dp110 is not sufficient to increase cell number. In contrast, deltap60, p60, and to a lesser extent Dp110D954A expression, each reduce the rate of increase in cell number. Thus, attenuating Dp110 activity reduces cell size by reducing growth as opposed to increasing cell division. Taken together, these data demonstrate that the activity of Dp110 is necessary for cells to achieve their normal size and number (Weinkove, 1999).
During flow cytometry analysis, it was noted that, compared with the control cell populations, a greater proportion of the Dp110-expressing cells are in the G2 phase of the cell cycle whereas a greater proportion of the deltap60-expressing cells are in the G1 phase. These observations are consistent with an increase in Dp110 activity increasing the rate of progression of cells through the G1/S transition but not through the G2/M transition of the cell cycle (Weinkove, 1999).
Previous experiments, which utilized GAL4 under the control of the engrailed promotor (En-GAL4) to drive the expression of cell-cycle regulators in the posterior compartment of the wing imaginal disc, have shown that activation or inhibition of cell division in one compartment increases or reduces cell number without affecting the size of that compartment. Rather, that compartment contains more smaller cells or fewer bigger cells and growth is unaffected. These results suggest that imaginal disc growth is not regulated through the cell cycle but by an additional mechanism that is dominant over the cell cycle. Over-expression of Dp110 increases adult wing and eye size, whereas overexpression of Dp110D954A reduces adult wing and eye size. Thus, imaginal disc growth might be controlled by a mechanism that involves Dp110 and p60 (Weinkove, 1999).
To test this hypothesis using experiments directly comparable to the cell-cycle experiments described above, the effect of En- GAL4-driven Dp110 or Dp110D954A expression on the growth of the posterior compartment of the wing imaginal disc was examined. The size of the posterior compartment is significantly increased by Dp110 expression and reduced by Dp110D954A expression. In contrast, the size of the anterior compartment for each genotype is not altered significantly. In addition, when the ratio of anterior to posterior compartment size was calculated for each disc, it was found that Dp110 expression consistently reduces the ratio, whereas Dp110D954A expression consistently increases the ratio. Thus, unlike direct manipulation of the cell cycle, manipulation of activity of Dp110 is sufficient to alter relative compartment size. Together, the experiments described demonstrate that Dp110 modulates both cell size and cell number, and that manipulation of Dp110 activity can override the intrinsic control mechanisms that normally regulate imaginal disc growth and maintain consistent compartment and organ size (Weinkove, 1999).
An important question arising from this work is how does signaling through Dp110 elicit changes at the cellular level that promote growth? Insulin-stimulated mammalian class IA PI 3-kinase activation regulates various processes that influence growth, including glycogen synthesis, glucose uptake and the translation of mRNAs with 5' oligopyrimidine tracts. Furthermore, numerous reports have shown that class IA PI 3-kinases can regulate cell number through effects on cell proliferation and cell survival. Various experiments suggest that manipulating cell division, cell survival or protein synthesis in isolation does not modulate disc size. In contrast, when cell division is induced (by coexpression of cell cycle regulators, and apoptosis is simultaneously inhibited (by co-expression of p35), imaginal disc compartment size is increased. This is accompanied by an extended larval period, which would allow increased biosynthesis, including protein synthesis, to occur. Thus, it is possible that class IA PI 3-kinase activity can modulate organ size because it simultaneously influences cell division, cell survival and biosynthesis (Weinkove, 1999).
Pten, a Drosophila homolog of the mammalian PTEN tumor suppressor gene, plays an essential role in the control of cell
size, cell number, and organ size. In mosaic animals, Pten minus
cells proliferate faster than their heterozygous siblings, show
an autonomous increase in cell size, and form organs of increased size, whereas overexpression of Pten results in opposite
phenotypes. The loss-of-function phenotypes of Pten are suppressed by mutations in the PI3K target Dakt1 and the
translational initiation factor eif4A, suggesting that Pten acts through the PI3K signaling pathway to regulate translation.
Although activation of PI3K and Akt has been reported to increase rates of cellular growth but not proliferation, loss of
Pten stimulates both of these processes, suggesting that PTEN regulates overall growth through PI3K/Akt-dependent and
-independent pathways. Furthermore, Pten does not play a major role in cell survival during Drosophila
development. These results provide a potential explanation for the high frequency of PTEN mutation in human cancer (Gao, 2000).
Genetic interactions between Pten and
PI3K, a component of the insulin signaling pathway in
Drosophila, were examined. Overexpression of the PI3K catalytic subunit,
Dp110, results in increased wing size, while overexpression of a dominant negative
Dp110 construct (PI3KDN) results in the opposite phenotype. These phenotypes are
due to changes in cell size. Coexpression of Pten with
PI3KDN further reduces wing size. In addition,
overexpression of Pten suppresses the increased wing
size resulting from PI3K overexpression. To further examine the genetic interaction between
Pten and PI3K, Pten mutant cells
were examined in a genetic background of overexpressing PI3K or PI3KDN.
When PI3K is overexpressed, Pten
mutant cells are indistinguishable in cell size from their
nonmutant siblings, suggesting that overexpression
of PI3K results in increased PIP3 levels and increased
signaling that cannot be further activated by removing
Pten. Conversely, overexpression of PI3KDN partially
suppresses the increased cell size of Pten mutant cells. Thus, PTEN and PI3K antagonize each other in
regulating cell size (Gao, 2000).
Certain combinations of loss-of-function alleles of the
Drosophila insulin receptor (inr) result in flies with a decreased cell size. This provides an opportunity
to examine the genetic epistasis between Pten and
inr. The increased cell size of Pten mutant
cells can not be reversed in inr mutant animals, and thus loss of Pten function is epistatic to (acts downstream from) mutations
in inr (Gao, 2000).
Previous studies have shown that loss of PTEN function
promotes cell survival in mammals through activation of Akt. In addition, PTEN acts through Akt in metabolic and
longevity control in C. elegans.
Dakt1, a Drosophila homolog of Akt, has been suggested to
play a role in cell survival in embryogenesis (Staveley,
1998) and cell size control (Verdu,
1999). A hypomorphic allele of Dakt1 has been identified
in the large-scale gene disruption project carried out by
the Berkeley Drosophila Genome Project. This allele is semilethal, and homozygous
survivors show reduced body size and cell size, consistent
with a role of Dakt1 in growth control. To examine whether Pten controls cell size through regulating Akt activity, Pten mutant
clones were generated in Dakt1 mutant animals.
Dakt1 mutation completely suppressed the increase of cell size
associated with the Pten mutation. This result
provides strong in vivo evidence that Dakt1 functions
downstream of (or in parallel to) Pten in the control of
cell size. Taken together, these genetic interactions suggest
that the role of Pten in opposing signaling through the
PI3K/Akt pathway is conserved between flies and vertebrates (Gao, 2000).
It is proposed that Pten regulates cell proliferation
by multiple mechanisms, both PI3K-dependent and -independent.
One potential PI3K-independent mechanism is suggested
by the domain in Pten related to tensin, an actin filament
capping protein that localizes to focal adhesions. Overexpression of tensin can suppress
anchorage-independent proliferation of Ras-transformed
3T3 cells, and therefore this domain
may provide a growth-regulatory function in mammalian PTEN as well.
Moreover, in addition to its role as a lipid phosphatase,
PTEN also possesses a dual-specificity protein phosphatase
activity. PTEN has been shown to bind and dephosphorylate
the focal adhesion kinase FAK and to down-regulate the
formation of focal adhesions. Such cell
contacts play a critical role in regulating proliferation in
Drosophila, and the gene products of several Drosophila
tumor suppressors such as expanded, fat, and l(2) discs large all localize to adherens or septate junctions. The results for Pten are thus consistent with a model in which PTEN suppresses cell growth and G1/S progression
by down-regulating the PI3K/Akt pathway and inhibiting the
G2/M transition through an alternative mechanism, perhaps
involving regulation of the cytoarchitecture. The ability
to regulate both growth and cell division may explain
why PTEN is such a common target in advanced tumors.
This model is also consistent with the different mutant
phenotypes between a null Pten allele and an allele that carries a point mutation (Huang, 1999). While the point mutation changes an invariant
amino acid within the phosphatase active site and is likely
to inactivate the lipid phosphatase activity, the other domains
of Pten are still intact. Characterization of Pten
mutants that are specifically defective in cell growth or
proliferation may shed further light on its role in the
control of overall growth (Gao, 2000).
The role of genetic variants that affect cell size and proliferation in the determination of organ size has been investigated. Genetic mosaics of loss or gain of function were used in six different loci, which promoted smaller or larger than normal cells, associated with either smaller or larger than normal territories. These variants have autonomous effects on patterning and growth in mutant territories. However, there is no correlation between cell size or rate of proliferation on the size of the mutant territory. In addition, these mosaics show non-autonomous effects on surrounding wild-type cells, consisting always in a reduction in the number of non-mutant cells. In all mutant conditions the final size (and shape) of the wing is different from normal. The phenotypes of the same variants include higher density of chaetae in the notum. These autonomous and non-autonomous effects suggest that the control of size in the wing is the result of local cell communication defining canonic distances between cells in a positional-values landscape (Resino, 2004).
Size of insect organs is sex- and species-specific. In the
Drosophila wing, where most of the studies on size control
have been carried out, the determination of the size of
imaginal disc is disc-autonomous. Young imaginal discs
transplanted to the abdomens of adult flies grow after
several days of culture, irrespective of hormonal and
nutritional conditions, to a maximal size that corresponds
to that of mature imaginal discs. Minute mosaics and regeneration experiments reveal that
a final normal size is attained irrespective of the rate of cell proliferation. Clonal analysis of cell proliferation in wild-type wings show regional differences related to specification or differentiation, indicative of local as opposed to global control of organ size. Size
of the growing imaginal disc depends on the allocation of
postmitotic cells along the main axes of the wing in regimes
that change with developmental time. There is no indication that cell proliferation or cell allocation relates to the position of cells with respect to distances to compartments boundaries, where postulated diffusible morphogens are at maximal concentration (Resino, 2004).
If control of cell proliferation is local, the question arises as to how this is achieved. Can variations in cell size affect
the final size of the organ or its proliferation parameters?
These variations can be produced using mutations, usually
lethal in organisms, and have to be studied in genetic
mosaics. Mosaics of haploid territories (with half the cell
size of diploid cells) led to bigger territories with more
cells than diploid territories. Male wings have less and smaller cells than females, characteristics that are locally autonomous in gynandromorphs. For
mutations that affect cell size, it has to be considered that they cause different perturbations that may affect other cellular parameters in addition, such as cell viability, proliferation
rate or cell adhesion, which make difficult the interpretation
of the phenotype. Thus, the insufficient function of genes
involved in cell cycle progression, such as string (stg), cdc2 and cyclins or E2F (cycE positive regulator),
may retard the cell cycle and cause cell mortality, an
increase in cell size and smaller mosaic territories in
otherwise apparently normal sized discs. Mutant
cells in these mosaics do not differentiate properly. On the
contrary, over-expression of the same cell cycle genes (i.e.
stg, cycE, cycD-cdk4) or of their activators (i.e., E2F) in imaginal disc clones cause acceleration of their characteristic phases of the cell cycle, as well as a reduction of cell
size (except cycD-cdk4 combination) and an increase in
number of cells of the mutant territory compared with
control cells in apparently normal sized mosaic wing discs. These effects are more extreme in some genetic combinations (e.g., cycE-stg) because they
cause an acceleration of the whole cell cycle. These studies conclude that cell size
reduction/increase is 'compensated' by increment/decrement
in cell number in the mutant territory, as if the organ
would compute a global normal size, because the mutant
wing disc territories have an apparent wild-tupe size. This interpretation is biased by the
fact that those mosaics show high cell mortality. When this
is prevented with the coexpression of P35, the extra growth
of the mutant territories in discs and clones is even higher,
leading to abnormally shaped mutant territories. The over-expression of the cycD-cdk4 combination in the eye reaches the adult stage and causes larger and abnormally shaped mutant territories. These studies have not analyzed non-autonomous effects in non-mutant territories of the same discs (Resino, 2004 and references therein).
Less drastic mutant effects associated with cell viability are obtained with mutant perturbations in the signal transduction and reception of the insulin pathway. As a rule, loss of function of Drosophila Insulin Receptor (Inr), chico or Dp110 causes
reduction in both cell size and cell number of mutant
territories. This is similar to what happens in wild-tupe flies exposed to malnutrition or premature metamorphosis. This
holds for each member of the insulin receptor pathway
except for Drosophila S6 kinase (S6K), because S6K loss
of function only reduces cell size but not cell number. On the contrary, the gain of
function of genes of this pathway causes larger cells and an
increase in the number of cells of the mutant territory in
mosaics. The
loss of function of myc in diminutive mutants leads to
smaller flies, with smaller cells, in addition to poor cell
viability. Its overexpression causes
larger cells but not larger territories, suggesting that in this latter condition (but not the former)
the wing size in globally controlled by a normalizing
compensating mechanism (Resino, 2004 and references therein).
The results show a great heterogeneity
in the response of regional size to genetic perturbations that cause variations in cell size during cell proliferation. In fact, both smaller or larger than normal cell size may accompany normal, larger or smaller mutant territories. In the present paper, the effects on cell proliferation of
mutant conditions in six loci that cause smaller and larger
cell sizes have been studied. Of these, one corresponds to a new gene and five
to previously studied genes that affect cell size. They were
chosen as examples of the cell behavior variants, as
representatives of mutant effects on cell size (larger and
smaller than normal) and rate of proliferation (slower and
faster than normal). The choice was made without
considering the genetic/molecular bases of the corresponding wild-tupe alleles, in any case mechanistically far separated from the analyzed phenotype. Their
autonomous effects in mutant territories and in the mosaic
wing as a whole were studied: nonautonomous
effects were documented as well (Resino, 2004).
Adult cell size is measured by the exposed planar surface
of the cuticle cells. In principle, this may not reflect the size of the proliferating cells, when organ size is determined. However, in some of the cases examined in this study, cell dissociation
has revealed by direct estimation the larger or reduced cell
size in the proliferating wing disc cells. In others, cell size during growth is inferred by the
mutant effects on pattern formation, a process that precedes
final cell differentiation, as in the notum pattern of
microchaetae. This pattern results from the singularization
of sensory organ mother cells (SOMC) in a field of
epidermal cells through a process of lateral inhibition in
a field of proneural clusters. Thus, the final pattern reveals cell-cell interactions or communication, as observed in the form of cell projections emanating from epidermal cells. It holds for all mutant and genetic combinations examined in this study that the pattern, number
and density of chaetae are all altered in the notum (in the mutant
Dmcdc2E1-24 cells fail to differentiate chaetae). In all cases,
chaetae appear more densely spaced (separated by less
epidermal cells) associated with an increase in the total
number of chaetae. These variations to the
wild-tupe condition suggest that mutant cells have impaired
the capacity to signal among themselves to define spaced
SOMC singularization. Whether this is or is not associated
with cell size in individual cases is not known. These pattern
effects reveal abnormal cell communication between cells
during cell proliferation (Resino, 2004).
Although less easy to measure in mosaic nota, there
is a phenotypic association of variable cell size with a
reduction (in l(3)Me10, gigMe109, Dp110D945A) or an
increase (EP(3)3622, fta13, Dp110-CAAX) in notum sizes.
But there is no apparent causal relation between both
parameters of cell size and number of cells making the adult
notum. Perhaps cell viability associated with the mutation, as
in l(3)Me10 and gigMe109, may account for the observed lack
of correlation between both parameters. However, these
effects on notum size in other cases may also reflect failures
in cell-cell communication leading to more or less cell
proliferation (Resino, 2004).
The relationship between cell size and growth can be
more readily measured in the wing. The studied genetic
variants can be grouped, based on variations in these
parameters, as follows:
The autonomous effects on reduced clone size can result
from the poor viability of mutant cells (l(3)Me10 or
Dmcdc2E1-24), as shown in twin clonal analysis and cell
death monitoring. The increased clone size
of EP(3)3622, fta13 or Dp110-CAAX reflects higher than
normal cell proliferation, however there are no correlations
between cell size and clone size. Despite this lack of
correlation it holds for all mutants examined in this study that,
concerning the non-autonomous effects on growth in the
mosaic wing sector: the non-mutant cells of the sector are
always reduced in number. No cases were found in
which the reduction or increase in sector size by the
presence of mutant territories is compensated by wild-tupe
cells to obtain a normal sized sector (Resino, 2004).
The mosaic wings show, in addition to autonomous
effects within mutant sectors, non-autonomous effects in the
rest of the wing. It holds for all cases studied that wings with
entire or mosaic wing sectors show a reduction in the total
area of the wing or more in particular in non-mosaic areas
(sectors or compartments) of the wing. This
phenomenon is designated as 'positive' or 'negative' accommodation,
depending on its correlation with the size of the mutant
region. This phenomenon could be easily trivialized for
mutations that cause size reduction and 'positive accommodation'. It is
arguable that there are not enough cells in the
mutant territories to confront with normal growing cells
abutting the clone, the sector or the mutant compartment.
'Positive accommodation' could result from adjustment
between poorly growing cells and normal ones. However
this large effect hardly explain 'negative accommodation'
for the whole wing. 'Negative accommodation' occurs in
mosaic wings with mutant territories with more cells than
normal, such as EP(3)3622, fta13 or Dp110-CAAX (Resino, 2004).
Reduction in the size of non-mutant territories in mosaic
wings cannot be explained either by delay in development
(mosaic flies hatch at the same time as sib controls) or age of
clone initiation. It cannot be explained either by cell death,
because there is enough time for extraproliferation to reach
normal sized wings, since it occurs in mosaics where cell death
has been massively induced in Gal4 territories. 'Negative accommodation' is surprising because one
would expect that larger than normal mutant territories
should provide adjacent wild-tupe cells with more growth
signals (Resino, 2004).
To account for this 'negative accommodation' it is
postulated that mutant cells do not convey among themselves
and to wild-tupe cells sufficient signals necessary for them to
proliferate. These signals may depend on cell-cell communication. In the notum it has been seen that failures in cell-cell communication may account for abnormal chaetae
patterning and notum size. The same may apply to the wing
blade, although there are not enough pattern elements to
support this inference (Resino, 2004).
A model has been proposed to explain controlled cell
proliferation, based on local cell-cell signalling, as opposite
to reception of graded amounts of morphogens emanating
from compartment boundaries, such as Dpp and Hedgehog or
Wingless.
The Entelechia model (Interactive Fly editor's note: 'Entelechia' is a Greek term coined by Aristotle for the complete reality or perfection of a thing, and refers to the process of coming into being) states that cell proliferation results
from local interactions between neighboring cells. In these
interactions, cells compute positional values, presumably
expressed in the cell membrane. Positional value
discrepancies elicit cell division and readjustment of
positional values of daughter cells to those of neighboring
cells. These values differ along the two main axes of the
wing, A/P and Pr/Ds. Cell proliferation occurs within clonal
boundaries; those of compartments in the early disc and
other boundaries, such as veins, later. In these boundaries
the interchange of some type of signals help to increase
positional values at the border, eliciting cell division,
cascading down to intermediate regions with minimal
values. Cell proliferation is intercalar and driven by
differences in positional values between cells with lower
and higher values. These minimal differences may reflect
canonic efficiencies ('increments') in transduction of signals
(ligands/receptors) between neighboring cells. Cell division
ceases in the anlage when cells in the boundaries reach
maximal values and their increments, between all the cells
of a region become minimal. The anlage has then
reached the Entelechia condition of growth, characteristic of
the organ, the sex and species (Resino, 2004).
An organ such as the wing, grows co-ordinately through
compartments and clonal boundaries because maximal
positional values result from cell interactions at both sides
of the boundaries. In this respect compartments or wing
sectors are not independent units of cell proliferation. This
was first seen in bithorax-Complex (bx-C) mutants, where
either the A or P compartments of the haltere were transformed
to A or P compartments of the wing. The untransformed A or P haltere compartments contain now more cells, and the transformed ones less than a
wild-tupe A or P wing compartment. This
accommodation is explained as due to the reduced extent of the
compartment boundary between apposed mutant and nonmutant
compartments. Similar accommodation effects have
been already reported in other mutant conditions, such as mutants of the EGFR pathway in
extramacrochaetae (emc) and in
nubbin (nub). In the
latter case, the presence of proximal wing mutant territories
causes a distal reduction in growth in all the wing
compartments (Resino, 2004).
The Entelechia model helps to understand the behavior
of mosaic wings for the mutants examined in this study. In all cases,
clones or regions with smaller or larger cells and with less or
more cells than normal, cause autonomous effects on growth
in mutant territories but also a non-autonomous 'accommodation'
in the rest of the wing formed by wild-tupe cells. It should be emphasized that the effects on
proliferation between mutant and non-mutant territories are
reciprocal; the non-mutant territories rescuing proliferation
in the mutant territories and vice versa. It is hypothesized that failures in cell communication of positional values to/from
neighboring mutant or non-mutant cells affect the 'increment'
values of the model. This leads to reduced
proliferation in both genetic territories between cells
because cells cannot generate higher positional values and
thus promote intercalar proliferation. This
finding indicates that the size of territories does not depend
on distances from diffusible morphogen sources, measured
either in physical terms or in number of cells, or on other
postulated parameters such as measuring global cell mass
or wing length. How would
these global dimensions be defined, and how would they be
computed by individual cells? How would one explain that
mosaic territories separated from compartment boundaries
(or morphogen sources) can affect the growth of wild-tupe
territories far away all over the wing? It seems rather that
cell proliferation control depends on local cell interactions
(cell-cell communication) that define positional values
throughout the whole growing organ (Resino, 2004).
All animals coordinate growth and maturation to reach their final size and shape.
In insects, insulin family molecules control growth and metabolism, whereas pulses of the steroid 20-hydroxyecdysone (20E) initiate major developmental transitions. 20E signaling also negatively controls animal growth rates by impeding general insulin signaling involving localization of the transcription factor dFOXO and transcription of the translation inhibitor 4E-BP. The larval fat body, equivalent to the vertebrate liver, is a key relay element for ecdysone-dependent growth inhibition. Hence, ecdysone counteracts the growth-promoting action of insulins, thus forming a humoral regulatory loop that determines organismal size (Colombani, 2005).
In metazoans, the insulin/IGF signaling pathway (IIS) plays a key role in regulating growth and metabolism. In Drosophila, a family of insulin-like molecules called Dilps activates a unique insulin receptor (InR) and a conserved downstream kinase cascade that includes PI3-kinase (PI3K) and Akt/PKB. Recent genetic experiments have established that this pathway integrates extrinsic signals such as nutrition with the control of tissue growth during larval stages. The larval period is critical for the control of animal growth, since it establishes the size at which maturation occurs and, consequently, the final adult size. Maturation is itself a complex process that is controlled by the steroid 20-hydroxyecdysone (20E). Peaks of 20E determine the timing of all developmental transitions, from embryo to larva, larva to pupa, and pupa to adult. Ecdysteroids are mainly produced by the prothoracic gland (PG), a part of a composite endocrine tissue called the ring gland. Final adult size thus mainly depends on two parameters: the speed of growth (or growth rate), which is primarily controlled by IIS, and the overall duration of the growth period, which is limited by the onset of the larval-pupal transition and timed by peaks of ecdysone secretion. Very little is known concerning the mechanisms that coordinate these two parameters during larval development (Colombani, 2005).
To investigate the function of ecdysone in controlling organismal growth, a genetic approach was developed that allowed modulation basal levels of ecdysteroids in Drosophila. The initial rationale was to modify the mass of the ring gland in order to change the level of ecdysteroid production. For this goal, the levels of PI3- kinase activity were manipulated in the PG by crossing P0206-Gal4 (P0206>), a line with specific Gal4 expression in the PG and corpora allata (CA), with flies carrying UAS constructs allowing expression of either wild-type (PI3K) or dominant-negative (PI3KDN) PI3-kinase. As expected, these crosses produced dramatic autonomous growth effects in the ring gland, and particularly in the PG: tissue size was increased upon PI3K activation and decreased upon inhibition. Surprisingly, the changes in ring gland growth were accompanied by opposite effects at the organismal level. P0206>PI3K animals (with large ring glands) showed reduced growth at all stages of development and produced emerging adults with reduced size and body weight (78% of wt). Conversely, reducing PI3K activity in the ring gland of P0206>PI3KDN animals led to increased growth and produced adults with 17% greater weight on average. Adult size increase was attributable to an increase in cell number in the wing and the eye. Adult size reduction was accompanied by a decrease in cell number in the wing and in cell size in the eye (Colombani, 2005).
Importantly, the timing of embryonic and larval development of these animals was comparable to control. Both the L2 to L3 transition as well as the cessation of feeding (wandering) occurred at identical times. Further, animals entered pupal development at the same time, except for P0206>PI3KDN animals, which showed a 1-2 hrs delay intrinsic to the UAS-PI3KDN line itself. The duration of pupal development was slightly modified, however, as adult emergence was delayed in P0206>PI3K animals and advanced in P0206>PI3KDN animals, albeit by less than 4 hours following 10 days of development. In contrast, the speed of larval growth was found to be increased in P0206>PI3KDN animals and decreased in the P0206>PI3K animals background at the earliest stage that could be measured (early L2 instar). Because none of these effects were observed when PI3K levels were modified specifically in the CA using the Aug21- Gal4 driver, it was concluded that the observed phenotypes are solely due to PI3K modulation in the PG. Together, these results demonstrate that manipulating PI3K levels in the PG induces non-autonomous changes in the speed of larval growth (growth rate effects), without changing the timing of larval development (Colombani, 2005).
To investigate whether these effects could be attributed to changes in 20E levels, ecdysteroid titers were measured in third instar larvae of the different genotypes. Early after ecdysis into third instar (74hrs AED) ecdysteroids are present at basal level. They accumulate to an intermediate plateau around 90hrs AED and reach peak levels before pupariation (120hrs AED). Because early L3 levels are below the detection limit of the EIA assay, ecdysteroid titers were measured at the intermediate plateau (90hrs AED). In these conditions, a very modest increase of ecdysteroids was observed in P0206>PI3K animals larvae and a small but significant decrease in P0206>PI3KDN animals animals. This was further confirmed by measuring the transcript levels of a direct target of 20E, E74B, which responds to low/moderate levels of 20E. However, in early L3 larvae with basal ecdysteroid levels (74hrs AED), differences in E74B transcripts were clearly visible, with a 1.9-fold increase seen for P0206>PI3K animals and a 1.7-fold decrease for P0206>PI3KDN animals. This establishes that basal circulating levels of 20E are modified in response to manipulation of PI3K levels in the PG. It also suggests that the differences observed on basal 20E level off with the strong global increase of ecdysteroids in mid/late L3 (Colombani, 2005).
Several related lines of evidence strengthen these results: (1) the increase in growth rate and size observed in P0206>PI3KDN animals can be efficiently reverted by adding 20E to their food; (2) feeding wild-type larvae 20E recapitulates the effects observed in P0206>PI3K animals animals; (3) ubiquitous silencing of EcR using an inducible EcR RNAi construct results in a growth increase similar to that observed in P0206>PI3KDN larvae. Finally, the phantom (phm) and disembodied (dib) genes, which are specifically expressed in the PG and encode hydroxylases required for ecdysteroid biosynthesis, showed 1.65- and 2.2- fold increased expression, respectively, upon PI3K activation in the ring gland. This supports the notion that 20E biosynthesis is mildly induced in these experimental conditions. In line with previous results, neither 20E treatment nor EcR silencing has any effect on developmental timing. Overall, the results indicate that manipulating basal levels of 20E signaling in various ways modifies the larval growth rate without affecting the timing of the larval transitions (Colombani, 2005).
Variations in ecdysone levels in animals with different sized ring glands could be due to changes in the PG tissue mass or, alternatively, to a specific effect of PI3K signaling in the secreting tissue. To distinguish between these two possibilities, extra growth was induced in the PG using either dMyc or CyclinD/Cdk4, two potent growth inducers in Drosophila. Although the size of the larval ring gland was markedly increased under these conditions, no effect on pupal or adult size was observed, implying that the size of the ring gland is not the critical factor in the control of body size. Instead, it is likely that the InR/PI3K signaling pathway can specifically activate ecdysone production from the PG (Colombani, 2005).
The possibility was tested that ecdysone signaling opposes the growth-promoting effects of IIS in the larva. To test this, larvae were fed 20E and xPI3K activity was assessed in vivo using a GFP-PH fusion (tGPH) as a marker. Membrane tGPH localization shows a marked decrease in the fat body of 20E-fed animals, and this effect can be reverted by specifically silencing EcR in the fat body. This indicates that ecdysone-induced growth inhibition correlates with decreased IIS, and is mediated through the nuclear receptor EcR. Conversely, larvae with PI3KDN expression in the PG show a 4.2-fold increase in the global levels of dPKB/Akt activity, as measured by the phosphorylation levels of serine 505. In Drosophila cells (as in other metazoan cells) high levels of PI3K/AKT activity cause the transcription factor dFOXO to be retained in the cytoplasm, while low PI3K/AKT activity allows dFOXO to enter the nucleus where it promotes 4E-BP transcription. In larvae with ectopic PI3K expression in the PG, a strong increase is observed in nuclear dFOXO in fat body cells. Similar results were obtained by feeding larvae with 20E. Conversely, inactivation of EcR signaling in fat body cells was carried out using the clonal over-expression of a dominant-negative form of EcR (EcRF645A). In these conditions, a reduction was observed in the accumulation of dFOXO in the nuclei of EcRF645A-expressing cells. As an expected consequence of the increased nuclear dFOXO, global accumulation of 4E-BP transcripts was consistently higher in P0206>PI3K animals as well as in 20E-fed early L3 larvae as compared to control animals, and reduced in arm>EcR-RNAi animals. Together, these results indicate that ecdysone-dependent inhibition of larval growth correlates with a general alteration of insulin/IGF signaling, and a relocalization of dFOXO into the cell nuclei. To more directly test the role of dFOXO in the growth-inhibitory function of ecdysone signaling, the effects of increasing ecdysone levels were examined in a dFOXOmutant genetic background. Although homozygous dFOXO21 animals do not display a detectable growth phenotype, introducing the dFOXO21 mutation was sufficient to totally revert the growth defects of P0206>PI3K animals animals, either when homozygous or heterozygous. This data establishes that dFOXO is required for 20E-mediated growth inhibition (Colombani, 2005).
The endocrine activities of the brain and the fat body have previously been implicated in the humoral control of larval growth. In order to test for possible roles of these two organs in mediating the systemic growth effects of ecdysone, EcR expression was silenced specifically in the brain cells that produce insulins (IPCs) or in the fat body. While specific expression of EcR RNAi in the IPCs fails to reproduce the overgrowth observed in armGal4>EcR-RNAi animals, EcR silencing in the fat body elicits an acceleration of larval growth and a remarkable increase in pupal size. Moreover, no detectable delay in the larval timing was observed in pplGal4>EcR-RNAi animals. Thus, specifically reducing 20E signaling in the fat body is sufficient to recapitulate the systemic effects of global EcR silencing. This demonstrates that the fat body is a major target for ecdysone, and that this tissue can act to relay the 20E growth-inhibitory signal to all larval tissues (Colombani, 2005).
In summary, these results establish an additional role for 20E in modulating animal growth rates. This function is mediated by an antagonistic interaction with IIS that ultimately targets dFOXO function. A similar antagonistic interaction between 20E and insulin signaling controls developmentally-regulated autophagy in Drosophila larva (Colombani, 2005).
Although a direct effect of ecdysone on the cellular growth rate of all larval tissues cannot be ruled out, the experiments reveal a key role for the fat body in relaying ecdysone-dependent growth control signals. Together with previous work, these data suggest that various inputs such as nutrition and ecdysone converge on this important regulatory organ, which then controls the general IIS to modulate organismal growth (Colombani, 2005).
How then is growth connected to developmental timing? The finding that 20E can modulate growth rates in addition to developmental transitions places this hormone in a central position for coordinating these two key processes and controlling organismal size (Colombani, 2005).
A link exists between natural variation in a Drosophila melanogaster overwintering strategy, diapause, to the insulin-regulated phosphatidylinositol 3-kinase (PI3-kinase) gene, Dp110. Variation in diapause, a reproductive arrest, was associated with Dp110 by using Dp110 deletions and genomic rescue fragments in transgenic flies. Deletions of Dp110 increased the proportion of individuals in diapause, whereas expression of Dp110 in the nervous system, but not including the visual system, decreased it. The roles of phosphatidylinositol 3-kinase for both diapause in D. melanogaster and dauer formation in Caenorhabditis elegans suggest a conserved role for this kinase in both reproductive and developmental arrests in response to environmental stresses (Williams, 2006).
Little is known about genes that harbor ecologically relevant allelic variation in natural populations or the degree of conservation of such variation across species. Arrests in development are widespread. For example, mammals hibernate, insects enter diapause, and nematodes form dauer larvae all in response to adverse conditions. Caenorhabditis elegans arrests development to form dauer larvae in response to harsh environmental conditions such as food depletion and overcrowding; dauer larvae have decreased metabolism and increased fat storage. When confronted with low temperature and short days (SDs), Drosophila melanogaster enters an ovarian reproductive diapause where adult females have immature ovaries and exclusively previtellogenic oocytes. It is not known whether common mechanisms underlie these arrests because, although much is known about the genetic underpinnings of dauer formation in C. elegans, little is known about genes involved in reproductive diapause (Williams, 2006).
Ecological studies show that adult reproductive diapause is a powerful overwintering strategy for many insects including Drosophila. Diapause is advantageous in northern climates because it enables females to survive for several months through harsh winter conditions and then emerge and lay eggs when temperatures increase and days get longer. Arrests in response to adverse conditions are of interest because they can provide us with an evolutionary framework in which to interpret existing genetic variation both within and between species. Diapause is associated with resource allocation trade-offs involved in life history strategies, for example, whether to allocate resources to growth and survivorship or reproduction. D. melanogaster lines that vary in latitudinal origin differ in the proportion of individuals in diapause as well as in a suite of fitness-related traits that are genetically correlated to diapause. These traits include life span, age-specific mortality, fecundity, resistance to cold and starvation stress, lipid content, development time, and egg-to-adult viability. This study asked what gene(s) or gene pathways play a role in natural variation in diapause and potentially underlie this pleiotropy (Williams, 2006).
Two natural diapause variants of D. melanogaster were used as a basis to identify genetic regulators of diapause. The Windsor (W) natural variant from Canada exhibits an autosomal-recessive high-diapause phenotype compared with the Cartersville (C) natural variant from the Southern U.S., which confers a fully dominant low-diapause phenotype. Crosses between the variants suggested that diapause may be influenced by relatively few genes (Williams, 2006).
This study is consistent with a model in which reducing signaling via the insulin/PI3-kinase pathway increases D. melanogaster diapause. The finding that altered PI3-kinase expression in neurons alone can affect fly diapause is reminiscent of the observation that expression of PI3-kinase in C. elegans neurons can restore dauer formation to wild-type levels in age-1 (PI3-kinase) null mutant worms. Furthermore, mutations that lower nematode insulin signaling promote dauer larval formation suggesting the exciting possibility of a shared mechanism for developmental and reproductive arrests in D. melanogaster ovarian diapause and nematode dauer formation between these species' distant relatives. The results suggest that insulin-signaling genes known to play important roles in dauer formation in C. elegans are excellent candidate genes for further investigation into the mechanisms involved in diapause in D. melangaster and the many other species that undergo developmental and reproductive arrests (Williams, 2006).
Various fitness-related phenotypes are genetically correlated to D. melanogaster diapause in nature. This genetic correlation could be explained by pleiotropic effects of the Dp110 gene. Notably, mutational analyses of the insulin-regulated PI3-kinase and other genes in the insulin-signaling pathway in D. melanogaster have revealed affects on lifespan, development, body size and growth, nutrient stress, lipid content, locomotor activity, and egg chamber development. The findings suggest that Dp110 is not only an important regulator of diapause in D. melanogaster but also, through its pleiotropic effects, may influence the suite of life-history trade-offs associated with diapause in natural populations (Williams, 2006).
Few genes are known to affect reproductive diapause. The discovery of a role for Dp110 in reproductive diapause in D. melanogaster is significant because this species is amenable to mutational analyses, transgenic manipulations, and genomic investigations of the various phases of diapause including its photoperiodic induction, maintenance, and termination. Additionally, once genes that affect diapause have been identified, they can be used as candidate genes for the investigation of diapause in other species. From an evolutionary perspective, diapause is an important adaptive trait linked to many life-history parameters important for survivorship; the identification of genes involved in diapause is an entry point into studies of the molecular evolution of these genes. Finally, from an applied perspective, genetic manipulations of diapause can be engineered to control pest species and maximize the efficiency of their biological control agents (Williams, 2006).
Diapause is an adaptive trait critical for survival in temperate climates. The identification of a single major gene regulator of diapause in nature provides a tantalizing example of the small but growing body of literature that reveals that major adaptive phenotypes are affected by variation in genes with large effects (single genes). This finding is in conflict with the historically prominent notion of Fisher, Haldane, and Wright who envisioned hundreds of interactive genes, each having tiny additive effects on a trait. These genes by definition could not be localized or identified because their effect sizes were so small. However, contrary to this model, recent data suggest that genes can have large effects on adaptive phenotypes and that they exist along with genes with small effects. Challenges for the future are to identify both large and smaller effect genes and understand how they interact with the environment to generate variation in traits of adaptive significance (Williams, 2006).
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