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First published online March 2, 2007
Journal of Experimental Biology 210, 956-963 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.001586
The role of larval fat cells in adult Drosophila melanogaster
1 School of Life Sciences, University of Nevada Las Vegas, 4505 Maryland
Parkway, Las Vegas, NV 89154, USA
2 UNLV Institute for Cell and Molecular Medicine, University of Nevada Las
Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154, USA
Author for correspondence (e-mail:
dkhosh{at}clark.nscee.edu)
Accepted 4 January 2007
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Key words: cell death, fat body, starvation resistance, autophagy
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Introduction |
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) and accumulation of nutrient reserves primarily
in the larval fat body, a single-cell thick tissue composed of fat cells.
These larval fat cells serve as an energy reservoir to support the animal
through the subsequent non-feeding period. An important but somewhat
overlooked aspect of Drosophila development is that this non-feeding
period includes a period of time both before and after metamorphosis. Prior to
metamorphosis, the larva ceases feeding and `wanders' for 12–24 h in
search of a pupation site (Riddiford,
1993). After eclosion, the newly emerged adult remains inactive
for approximately 8 h until the wings expand and the cuticle tans
(Chiang, 1963) (J.R.A. and
D.K.H., unpublished data). Larvae must therefore acquire enough nutrients not
only to fuel developmental reorganization but also to survive the late larval
and early adult periods. In nature, the new adult may also need to seek a new
food source, if the fruit or other substrate upon which it developed is no
longer available. Thus, sufficient larval-derived nutrients must be stored and
remain available for use by the adult.
The unusual developmental history of the larval fat body complicates our
understanding of its role as an energy reservoir and its effects on the
overall physiology of the animal. During metamorphosis, most larval tissues
undergo autophagy and cell death, whereas the adult progenitor cells, i.e.
imaginal discs and histoblasts, undergo cell proliferation, differentiation
and organogenesis to give rise to the adult structures
(Bainbridge and Bownes, 1981;
Bodenstein, 1950;
Robertson, 1936). The fat
body, however, is refractive to cell death, but does undergo an unusual
transformation from an organized tissue to a loose association of individual
fat cells (Hoshizaki, 2005;
Nelliot et al., 2006). The
phenomenon of fat-body tissue dissociation has been documented in Diptera
(D. melanogaster and Sarcophaga peregrina) and Lepidoptera
(Calpodes ethlius) and is likely to be a common feature of
holometabolous insects (reviewed by
Hoshizaki, 2005).
In D. melanogaster, the individual cells of the larval fat body
persist throughout metamorphosis as freely floating fat cells dispersed
throughout the body cavity of the pupa
(Butterworth, 1972;
Hoshizaki, 2005;
Nelliot et al., 2006). The
newly eclosed adult contains freely floating fat cells that are likely to be
larval-derived fat cells. These cells later undergo cell death and are
replaced by sheets of fat cells recognized as the adult fat body. The adult
fat cells are most likely derived from cells embedded within the larval body
wall and from adepithelial cells associated with imaginal discs
(Hoshizaki et al., 1995).
Fully differentiated adult fat cells are not easily recognized within the
abdomen of the adult until 3–4 days post-eclosion. Although the adult
fat cells are derived from a distinct and separate cell lineage from the
larval fat body, both tissues share an important energy storage function.
Our focus in this study is the role of larval energy stores in the adult fly. Using cell markers we have identified the free-floating fat cells in the young adult as larval fat cells and experimentally extended their lifespan. We hypothesized that larval fat cells function in the young adult as `meals-ready-to-eat' until the animal is flight-ready and successfully feeds. To test this hypothesis, we compared the ability of adults to resist starvation in the absence or presence of larval fat cells. Young adults harboring larval fat cells are nearly three times as resistant to starvation as older adults. The half-life of the larval fat cells is 9 h, and unfed adults begin to die from starvation once 85% of the larval fat cells have undergone cytolysis. We experimentally manipulated the lifespan of the larval fat cells and found that unfed adults are more starvation resistant when death of these cells is blocked. These data suggest that nutrients acquired by the larva and stored within the larval fat cells can contribute to adult stress resistance. Thus, larval fat cells have a fundamental role in post-metamorphic energy metabolism and provide an effective energy reserve important to the young adult animal.
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The stocks (a) y w; P{w[+mC]=UAS-n-syb.eGFP}3,
(b) y w; P{Lsp2-GAL4.H}, (c) w;
P{w[+mC]=UAS-p35.H}BH2, (d) w;
P{w[+mC]=UAS-diap1.H}3 and (e) w;
P{w[+mC]=UAS-diap1.H}1 were obtained from the
Bloomington Stock Center (Bloomington, IN, USA). The protein trap line G000343
was identified as part of a screen for proteins expressed in the larval fat
body and salivary glands (Andres et al.,
2004; Morin et al.,
2001) and was generously provided by L. Cooley (Yale University,
New Haven, CT, USA). The artificial exon encoding green fluorescent protein
(GFP) in G000343 is inserted in-frame with a gene coding for a larval protein
localized to polytene chromosomes (Andres
et al., 2004) and is within chickadee but on the opposite strand,
i.e. in the opposing reading frame (L. Cooley, unpublished data).
In separate experiments, we used the GAL4/UAS system of Brand and Perrimon
to restrict expression of GFP to larval fat body cells
(Brand and Perrimon, 1993).
Briefly, the GAL4/UAS system is a bipartite system composed of a GAL4
driver (GAL4 transgene) and a UAS responder gene
(UAS transgene). The GAL4 driver in this case is
Lsp2-GAL4 (P{Lsp2-GAL4.H}3), a chimeric transgene composed
of the promoter from the larval serum protein 2 (Lsp2) gene
and the coding sequence of the yeast Saccharomyces cerevisiae GAL4
gene (C. Antoniewski, unpublished data).
Because the Lsp2-GAL4 transgene contains the Lsp2 promoter, it recapitulates the expression pattern of the endogenous Lsp2 gene, which is expressed solely in larval fat body cells beginning early in the third larval instar (B. Hassad, personal communication to FlyBase). Thus, Gal4 protein encoded by Lsp2-GAL4 is produced only in the larval fat body cells in the identical temporal and spatial pattern of the endogenous LSP2 protein. Gal4 is a DNA-binding protein that recognizes a 17-basepair sequence that functions as an upstream activation sequence designated UAS. Binding of Gal4 protein to the UAS sequence is sufficient to activate transcription of a downstream gene. Thus, in animals carrying both Lsp2-GAL4 and a chimeric gene containing a UAS promoter region fused to the coding sequence for GFP, i.e. UAS-GFP, (P{w[+mC]=UAS-n-syb.eGFP}3), the expression of the GFP gene occurs strictly in the larval fat body cells.
Standard genetic crosses were performed to recombine UAS-GFP, which serves as a cell marker, and the larval fat-cell driver transgene Lsp2-GAL4 onto the same chromosome. The final stock is homozygous for the genotype y w; P{Lsp2-GAL4.H}, P{w+mc=UAS-n-syb.eGFP}3 and is abbreviated as Lsp2-GAL4::UAS-GFP. This stock specifically marks the larval fat body cell with GFP and is used in conjunction with other UAS transgenes to target expression to this tissue.
Two different cell death inhibitor genes, p35 and Drosophila
inhibitor of apoptosis 1 (diap1), were employed to block cell
death in the larval fat cells. Ectopic expression of p35 or
diap1 was achieved using the GAL4/UAS system
(Brand and Perrimon, 1993).
Individuals carrying a UAS transgene for either p35 or
diap1, i.e. UAS-p35 (P{w[+mC]=UAS-p35.H}BH2) or
UAS-diap1 (either P{w[+mC]=P{UAS-DIAP1.H}3 or
P{w[+mC]={UAS-DIAP1.H}1), were crossed with
Lsp2-GAL4::UAS-GFP to drive ectopic expression of either p35
or diap1 to the larval fat cells and thus block cell death in these
cells.
Quantitative analysis of larval fat cells
Two methods were used to quantify the number of larval fat cells in the
adult. In the first method the abdomens of Lsp2-GAL4::UAS-GFP females
were gently teased open and the free-floating larval fat cells were released
into 1x Dulbecco's phosphate buffered saline (DPBS) (52 mmol
l–1 NaCl; 40 mmol l–1 KCl; 10 mmol
l–1 Hepes; 1.2 mmol l–1 MgSO4;
1.2 mmol l–1 MgCl2; 2 mmol l–1
Na2HPO4; 0.4 mmol l–1
KH2PO4; 1 mmol l–1 CaCl2; 45
mmol l–1 sucrose; 5 mmol l–1 glucose, pH
7.2) on a 25x75 mm glass slide. Cells were examined by light and
fluorescence microscopy to confirm that all larval fat cells expressed the GFP
cell marker. A micro-grid and a counter were used to physically count the
number of larval fat cells in the abdomen.
In the second method, larval fat cells were quantified by GFP fluorescence. Intact Lsp2-GAL4::UAS-GFP aged females were mounted dorsal-side down onto 25x75 mm glass slides using GelMount (Sigma, St Louis, MO, USA). GFP fluorescence was measured using a Typhoon 8600 Variable Mode Imager and the intensity of the phosphoimage (in pixels) quantified using ImageQuant software.
Starvation resistance
For each genotype, newly eclosed females were collected immediately upon
eclosion (0–10 min) and further identified by their deflated wings that
have the appearance of flattened raisins. These adults were immediately
assayed for starvation resistance or placed on food supplemented with yeast
until tested. For starvation experiments, flies were divided into groups of 10
and starved in 47 mm plastic Petri dishes containing a disc of Whatman #42
ashless filter paper soaked with 650 µl of deionized water. Flies were
maintained at 25°C, and mortality rates were determined by counting the
number of dead flies every three hours. The starvation graphs are the average
percent survival for N groups of 10 animals over time and error bars
represent standard deviations.
Fluorescent and confocal imaging
Fluorescent and confocal microscopy was performed in the Nevada INBRE
Center for Biological Imaging using a Zeiss LSM-510 microscope and LSM-510
Axioplan 2 Imaging software. Freely floating fat-body cells were obtained from
Lsp2-GAL4::UAS-GFP females and mounted in 1x DPBS. Cells were
analyzed within an hour after slide preparation.
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;
Hoshizaki, 2005;
Nelliot et al., 2006). We
re-examined the origin of the freely floating fat cells in the adult because
it is important to our understanding of the energy flow that supports the
young adult and in defining the underlying basis of the higher starvation
resistance of newly eclosed adults.
To experimentally establish the origin of these cells in the young adult,
we took advantage of a GFP protein trap line for a polytene
chromosome-associated protein (Andres et
al., 2004). Polytene chromosomes are a hallmark of larval tissues
including the fat body. We used this cell marker to distinguish between adult
tissues that contain mitotic chromosomes and larval polytenized tissues. As
expected, the free-floating fat cells in the newly eclosed adult were
GFP-labeled, thus confirming their larval origin
(Fig. 2).
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).
Thus, in the adult the only GFP-positive cells are the fat cells from the
dissociated larval fat body. We determined the rate at which these cells were
lost in the adult by following the loss of GFP fluorescence by measuring
phosphoimage intensity (Fig.
3). GFP fluorescence was quantified for individual aged female
adults and compared with the number of larval fat cells obtained by dissection
of individual animals (Fig. 4);
GFP fluorescence was proportional to the number of larval fat cells. Thus, by
measuring GFP fluorescence we can monitor the transient presence of larval fat
cells in the adult. We found that within 
9 h post-eclosion, 50% of the
larval fat cells have undergone cytolysis
(Fig. 4, and see
Fig. 6).
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Larval fat cells increase starvation resistance in the adult
To directly test whether larval fat cells contribute to adult starvation
resistance, we inhibited the normal cell death of the larval fat cells. We
employed both the Drosophila inhibitor of apoptosis 1 (DIAP1) protein
and the baculovirus p35 protein, both of which directly inhibit the caspase
cascade leading to apoptotic cell death
(Wang et al., 1999;
Wilson et al., 2002). Ectopic
expression of either p35 or diap1 in the larval fat cells
was accomplished using the larval fat-cell driver Lsp2-Gal4 (i.e.
Lsp2-GAL4::UAS-GFP) and either the UAS-p35 or
UAS-diap1 transgene. As a control we tested whether the inhibition of
cell death in the fat body affects the total number of fat cells. We compared
the number of larval fat cells present in the newly eclosed control adults
(Lsp2-GAL4::UAS-GFP) with the number of larval fat cells in the
experimental adults (Lsp2-GAL4::UAS-GFP + UAS-diap1)
(Fig. 7); we found that an
equal number of fat cells was present.
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70% of the fat cells have normally undergone
cytolysis, only 38% of fat cells were absent in the adults in which cell death
was blocked. The increased survivorship of fat cells in the experimental
adults was also detected at 48 h, when cytolysis of the larval fat cells is
normally complete. In the cell death-blocked animals, 40% of the fat cells
were still present. Finally, at 72 h experimental adults began to succumb to
starvation while 22% of the larval fat cells remained (compare
Fig. 7A with
Fig. 8).
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). Most
larval tissues undergo autophagy leading to cell death, thereby allowing bulk
recycling of components; however, the fat body undergoes tissue remodeling
leading to the dissociation of the fat body
(Nelliot et al., 2006). In
addition to supporting pupal development, sufficient larval energy stores must
also be in reserve to support the newly eclosed adult until a suitable
foraging site is located. We present here the first experimental evidence that
the energy reservoirs acquired during the larva feeding period are carried
into the adult by free-floating cells derived from the dissociated fat body.
By employing GFP cell markers, we demonstrated that the free-floating fat
cells are larval in origin and have established a profile measuring the loss
of these cells in the young adult. Correlated with the loss of larval fat
cells is an increased sensitivity to starvation. By genetic manipulation, we
have inhibited cell death of the larval fat cells in the adult and have
correspondingly increased starvation resistance. These data demonstrate that
the larval fat cells serve as `meals-ready-to-eat' for young adults and are of
importance for individuals that have developed on ephemeral breeding sites and
which must relocate to new feeding sites.
Larval fat cells in the adult
Through the use of cell markers, we have demonstrated that the
free-floating fat cells in the adult are the dissociated cells from the larval
fat body (Fig. 2). We have
determined the number of free-floating fat cells in the abdomen of the newly
eclosed female adults to be 766 (N=49; s.d.=49), which is in contrast
to the 1052 cells (N=8; s.d.=177) estimated by Butterworth
(Butterworth, 1972). We believe
the discrepancy between our results and those of Butterworth lies in our
improved ability to identify larval fat cells. In our in situ counts,
the fat cells express GFP, thereby allowing easy identification of the cells
from other free-floating cells and debris. By contrast, Butterworth examined
unstained samples and, as noted by Butterworth
(Butterworth, 1972), the in
situ counts are likely to include cells from other tissues.
It has been estimated that the female larval fat body is made up of 2500
fat cells (Rizki, 1969). After
tissue dissociation during metamorphosis, 20% of the fat cells are thought to
reside in the pupal head, with some cells in the thorax
(Rizki, 1969). Based on these
estimations, approximately 2000 fat cells should be present in the abdominal
region of the pupa. In newly eclosed adults, however, far fewer fat cells were
recovered (Butterworth, 1972)
(this study). This discrepancy might reflect partial elimination of larval fat
cells during pupal development
(Butterworth, 1972), or the
estimated distribution of fat cells in the pupa might not be correct. Our
recent descriptive analysis of fat-cell dissociation in the early pupa
indicates that a substantial proportion of the fat cells reside in the thorax
[fig. 1 in Nelliot et al. (Nelliot et al.,
2006)]. We estimate that in the early stage pupa, at least half of
the fat cells reside in the pupal head and thorax. Therefore, the pupal
abdomen should contain approximately 1250 cells. Our mean number of cells
recovered from the adult abdomen was 766, only 60% of the predicted number of
cells.
It is possible that a portion of the fat cells undergo cell death during pupal development, but we believe this to be unlikely for two reasons. First, we have measured the number of fat cells at the beginning of pupal development using the GFP-assay and find that this number remains the same between white prepupae and newly eclosed adults (data not shown). Second, the inhibition of apoptotic cell death by expression of diap or p35 did not change the number of fat cells recovered in the newly eclosed adult. These data indicate that few larval fat cells are eliminated during pupal development. The discrepancy in the predicted cell number in the adult abdomen might be due in part to the incomplete efficiency in recovering the abdominal fat cells for in situ counts and/or distribution of fat cells in the early pupa might be altered during later pupal development.
Mechanism of larval fat cell cytolysis in the adult
During metamorphosis the fat body is refractive to cell death and does not
begin to undergo cytolysis until after eclosion. Based on our measurements,
cytolysis is essentially complete by 48 h of adult development
(Fig. 6). The factors that
control or trigger fat-cell cytolysis and the underlying mechanism by which
cell death is achieved are not known. It has been suggested that juvenile
hormone and the gene apterous might participate in triggering
programmed cell death in the fat cells
(Butterworth, 1972;
Postlethwait and Jones, 1978),
but a reassessment of the apterous mutant
(Richard et al., 1993)
suggests otherwise (reviewed by Hoshizaki,
2005). We suggest that the cytolysis signal is also not likely to
be a nutritional cue because we did not observe an accelerated rate of larval
fat cell loss in starved adults.
We note that in adults in which fat-cell death is blocked, expression of GFP in the fat cells does not correspond to the in situ number of fat cells (Fig. 7). We surmise that the ectopic activity of the Lsp2-GAL4 is compromised in the adult and does not allow for maintenance of GFP beyond 48 h. Under normal conditions, this is not a concern for the GFP-based assay because removal of fat cells is complete by this time. If the activity of Lsp2-GAL4 is compromised, then it follows that the expression of the UAS-diap1 would also be compromised. If induction of cell death occurs immediately after eclosion, then expression of cell death inhibitors, such as diap1 and p35, during this window should be sufficient to prevent loss of fat cells. The nature of subsequent removal of the remaining larval fat cells at 72 to 96 h post-eclosion is not known and is currently under investigation.
The programmed cell death of the larval fat cells is the final and normal
step in the developmental history of this tissue. Two major classes of
programmed cell death, type 1 (apoptotic) and type 2 (autophagic), are
recognized as normal processes for remodeling tissues, controlling cell number
and eliminating abnormally damaged cells. Apoptotic cell death is
characterized by cellular and nuclear shrinking, association of chromatin with
the nuclear periphery, DNA fragmentation, formation of apoptotic bodies,
caspase activation and the engulfment and lysosomal degradation of the dying
cell by a phagocyte (Kerr et al.,
1972). Autophagic cell death, however, is a membrane trafficking
process involving autophagosomes that engulf cytosol and organelles and then
are fused with lysosomes to form autolysosomes in which the cargo undergoes
hydrolysis (Yoshimori,
2004).
The major signal that triggers metamorphosis and larval tissue histolysis
is the high titer pulse of ecdysone that occurs at puparium formation, i.e.
the larval-pupal transition. Most larval tissues undergo histolysis, with the
notable exception of the fat body, which is remodeled from an intact tissue to
detached cells (Nelliot et al.,
2006). Larval histolysis is associated with formation of acidic
autophagic vesicles consistent with an autophagic cell death response.
However, histolysis is also accompanied by hallmarks of apoptosis. The
degenerating prothoracic and labial glands of the tobacco horn worm
Manduca sexta, for example, are accompanied by highly condensed
chromatin indicative of apoptosis (Dai and
Gilbert, 1997; Jochova et al.,
1997), whereas the D. melanogaster salivary glands are
characterized by DNA fragmentation (Jiang
et al., 1997). Furthermore, inhibition of caspase activity by p35
blocks DNA fragmentation and salivary gland cell death
(Jiang et al., 1997;
Lee and Baehrecke, 2001) and
expression of diap1 (a direct inhibitor of caspase activity) in the
salivary glands is required throughout larval development to inhibit
reaper- and head involution defective-triggered apoptotic
cell death (Yin and Thummel,
2004). Based on these observations, we surmise that larval tissue
histolysis might be accompanied by autophagy to allow efficient recycling of
larval cellular components during metamorphosis and in the young adult, and
that the final destruction of the cell in the aged adult is dependent upon
apoptotic cell death.
A developmental conundrum, however, is presented by the larval fat body. Ecdysone signaling that triggers histolysis in most larval tissue triggers fat-cell dissociation but not cell death, which is delayed until adult stage. The final destruction of the fat cells, however, is also inhibited by expression of diap1 and p35, thereby suggesting that fat cell death is through a process similar to that used to remove the other larval tissues. Further studies are needed to understand why the fat body is initially refractive to cell death while other larval tissues are destroyed, and the relationship between apoptotic cell death and recycling of cellular components (macroautophagy) in larval fat cells of the adult.
Importance of larval energy stores for adult performance
The natural feeding and oviposition site of D. melanogaster,
rotting fruit, is an ephemeral resource. Eclosing flies may have no food
available, but their ultimate evolutionary success depends upon finding a
foraging and breeding site that leads to successful reproduction. The larval
fat cells may therefore contribute to the success of the adult by serving as a
reserve energy source in case foraging is delayed (e.g. by the deterioration
of the pupal development site or by inclement weather). It is also important
to note that energy expenditure during pupation and early adulthood will vary
according to temperature. Drosophila habitats can vary widely in
temperature, on timescales of minutes to days
(Feder et al., 1997;
Gibbs et al., 2003), so a
reserve of larval-derived energy may prove essential for adult success.
Although larval-derived energy may be essential for the success of
individual adults, selection experiments indicate there is a trade-off between
energy storage and other life history parameters. Starvation-selected
populations of D. melanogaster store more energy in the larval stage
than unselected control populations, but they develop more slowly and their
egg-to-adult viability is lower
(Chippindale et al., 1996;
Chippindale et al., 1998).
Similar patterns can be found in desiccation-selected lines, in which larval
accumulation of water and glycogen leads to slower development
(Chippindale et al., 1998;
Gefen et al., 2006).
At the organismal level, our most surprising finding is that starvation
resistance decreased during the first 3 days of adult life, despite the fact
that flies were able to feed and presumably store energy. Similar results have
been obtained for several other Drosophila species
(Sevenster and Vanalphen,
1993), although not all (Baldal
et al., 2004). A likely explanation for this phenomenon is
allocation of resources to reproduction during early adulthood. Once these
resources are committed to gonad development they might not be readily
available to the soma to support the animal during starvation. When D.
melanogaster are provided with a high-protein diet, energy storage
declines as fecundity and metabolic rates increase
(Simmons and Bradley, 1997).
Resources acquired during the first few days of adult life may be
preferentially directed to reproduction, rather than stored as an energy
reserve. This is in accordance with D. melanogaster being considered
a `fast' species (Sevenster and Vanalphen,
1993) that develops and breeds rapidly at the expense of adult
survival.
Conclusion
Nutrient stores acquired by the larva are transferred to the adult in the
dissociated cells of the larval fat body. These larval fat cells appear to be
a very efficient source of nutrients compared with the adult fat cells, based
on the observation that newly eclosed adults are nearly three times as
resistant to starvation as older fed flies. The ability of newly eclosed
adults to resist starvation, however, goes beyond their access to fat-cell
energy stores left over from pupal development. By blocking cytolysis of the
larval fat cells, starvation resistance can be further increased by more than
24 h. This increase is not because of an increase in the number of larval fat
cells in the newly eclosed adult. One possible explanation is that energy
stores contained within the fat cells are more easily mobilized to support the
starving animal than energy stores previously released by cell death or
autophagy and distributed in other tissues or hemolymph. Thus, not all energy
stores in the adult fly may be equally accessible.
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Acknowledgments |
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Footnotes |
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References |
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