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First published online 14 June 2006
doi: 10.1242/dev.02436
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1 Icrea and Institut de Recerca Biomedica, Parc Cientific de Barcelona, Josep
Samitier, 1-5, 08028 Barcelona, Spain.
2 Centro de Biología Molecular `Severo Ochoa'-CSIC, Campus de
Cantoblanco, 28049 Madrid, Spain.
* Author for correspondence (e-mail: mmilan{at}pcb.ub.es)
Accepted 9 May 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key words: Insulin pathway, Growth, Wing, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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;
Stern and Emlen, 1999).
Nutrient availability is one of the major environmental signals that affects
growth and, as such, complex humoral responses ensure that growth and
development are properly coordinated with nutritional conditions.
Multicellular organisms respond to nutrient availability through
cell-autonomous and -non-autonomous mechanisms. Growth regulation through the
latter occurs via the release of insulin-related growth factors from
peripheral tissues. In Drosophila, ablation of several neurosecretory
cells expressing insulin-like peptides causes a growth defect
(Rulifson et al., 2002), and
mice lacking an anterior pituitary expressing growth hormone are dwarves
(Butler and Le Roith, 2001). Studies in Drosophila have highlighted a
crucial role for the insulin receptor (InR) pathway in regulating the
cell-autonomous response to nutrient availability (reviewed by
Goberdhan and Wilson, 2003;
Stocker and Hafen, 2000). The
InR, a tyrosine kinase transmembrane receptor, induces phosphorylation of
insulin receptor substrates (IRS), which activate a cascade of downstream
effectors. In vertebrates, genetic manipulation of several elements of this
pathway modulates tissue growth in vivo thus demonstrating that the
insulin-like growth factor (IGF) pathway is required for growth
(Efstratiadis, 1998;
Shioi et al., 2000;
Shioi et al., 2002).
The InR downstream effectors PI3 kinase/Dp110 and target of rapamycin (TOR)
exert some of their growth effects at the transcriptional level (reviewed by
Neufeld, 2003). In yeast, TOR
controls the expression of a broad group of genes that are involved in
protein, lipid and nucleic acid metabolism
(Beck and Hall, 1999;
Cardenas et al., 1999).
However, in multicellular organisms little is known about effectors of the InR
pathway that are regulated transcriptionally. Here we identify
calderón (cald; orct2-FlyBase), which encodes an
organic cation transporter of the major facilitator superfamily, as a
downstream effector of the InR pathway in Drosophila. Loss of
cald activity mimics the phenotype of mutations in the InR pathway
during embryonic and adult development. Expression of calderón
is positively regulated by the InR downstream effectors PI3 kinase/Dp110 and
TOR, and its activity is required for TOR-mediated growth induction. Thus,
calderón is a target of the PI3 kinase/TOR branch of the InR
pathway required cell autonomously for insulin-mediated cell growth.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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),
patched-Gal4 (ptc-Gal4 in the text)
(Wilder and Perrimon, 1995),
MS-1096-Gal4 (Milan et al.,
1998), Kruppel-Gal4 (Kr-Gal4 in the text)
(Castelli-Gair et al., 1994),
EP1072 (Bloomington Stock Centre), UAS-p35
(Hay et al., 1995),
UAS-Rheb (a gift from Bruce Edgar)
(Saucedo et al., 2003),
UAS-dS6K (a gift from T. P. Neufeld),
UAS-PI3K92E-Dp110D954A (a gift from P. Léopold)
(Leevers et al., 1996), and
brkM12-lacZ (Jazwinska
et al., 1999) were used. H(PDelta2-3)HoP2.1 transposase
and UAS-GFP are described in Flybase. The UAS-cald
transgenic lines were generated by cloning the whole open reading frame [ORF
(isolated by PCR of genomic fly DNA)] into the pUAST vector, using the
BglII/XhoI cloning sites. The constructs were injected into
y w1118 embryos, and stable lines were selected by rescue
of the white phenotype. To distinguish hemizygous or homozygous
mutant embryos from their heterozygous siblings, we used the balancer
TM6b, AbdA-lacZ.
Molecular localization of calderón-Gal4 and P-element mutagenesis
Using plasmid rescue, we cloned and sequenced the flanking genomic sequence
3' of the PGawB element (Brand and
Perrimon, 1993). The insert is located 422 bp upstream of the
CG13610 transcription start. For the generation of P-element excisions, males
homozygous for the pGal4 insertion (Cald-Gal4) were crossed with
females carrying the H(PDelta2-3)HoP2.1 transposase on the CyO
balancer chromosome. Excisions of the PGal4 transposon were selected by the
loss of the w+ eye in the F1 progeny. Individual
revertants were crossed with TM3/TM6B flies and balanced. PCR analysis was
performed with individual stocks corresponding to a new complementation group.
We used one primer located 480 bp downstream of the P-element insertion site
and another primer located 330 bp upstream of this site: downstream primer:
5'-GTCTGTCTGTCGC-AGCGCAGC-3'; upstream primer:
5'-GCAACTGACTTCGTCGA-GTGGCGCCGG-3'.
The mutants recovered corresponded to new insertions of 2.7 Kb (R106), 9 Kb (R107) and 50 bp (R161) in the same place, 11 bp downstream of the original LP1 insertion site.
Analysis of the calderónR161 developmental delay
Embryos that were 0-24 h old were collected, and the development of
heterozygous caldR161/+ and homozygous
caldR161 animals was analyzed. 77 heterozygous
caldR161/+ larvae required 5-6 days to reach the pupal
stage and 5 days to eclose as adults. Ten homozygous
caldR161 larvae required 8-9 days to reach the pupal stage
and 6 days to eclose as adults. Homozygous caldR161
individuals were identified by the absence of the Humeral dominant marker of
the TM6b balancer chromosome.
Genetic mosaics
The following Drosophila strains were used to generate
loss-of-function clones: FRT82B caldR107/TM6B; FRT82B
caldR106/TM6B; FRT82B caldR161/TM6B; FRT82B
caldEP1072/TM6B; hs-FLP122; FRT82B arm-lacZ; hs-FLP122; FRT82B
ubiGFP; ywhs-FLP122; FRT82B arm-lacZ y+ M(3R); yhs-FLP122
tub-Gal4:UAS-GFP; FRTR82B tub-Gal80.
The FLP/FRT technique (Xu and Rubin,
1993) was used to generate loss-of-function clones. Larvae of the
appropriate genotypes were heat shocked for 1 h at 37°C, at different
larval stages. The clones were visualized in discs by either loss of GFP or
ß-Gal expression.
Immunostaining of embryos and discs
Discs were dissected in PBS and fixed in 4% paraformaldehyde for 20 min at
room temperature. They were subsequently washed in PBS, blocked in blocking
buffer (PBS, 0.3% Triton, 1% BSA), and incubated overnight with the primary
antibody diluted in blocking buffer at 4°C. Washes were performed in
blocking buffer, and the appropriate fluorescent secondary antibody was added
for 1 h at room temperature. Following further washes in blocking buffer, the
discs were mounted in Vectashield. Anti-FOXO antibody was kindly provided by
Oscar Puig, anti-ß-Gal (rabbit) and anti-caspase 3 were purchased from
Cappel and from Cell Signalling, respectively. Images were taken in a laser
MicroRadiance microscope (Bio-Rad) and subsequently processed using Adobe
Photoshop. In situ hybridization was performed as described in
(Azpiazu and Frasch, 1993), and
embryos were mounted in Permount (Fisher Scientific). cald antisense
Digoxigenin-labelled RNA probes were generated as described in
(Tautz and Pfeifle, 1989)
using the EST SD08136 (Berkeley Drosophila Genome Project).
Preparation of larval and adult cuticles
Adult flies were prepared by the standard methods for microscopic
inspection. Soft parts were digested with 10% KOH, washed with alcohol and
mounted in Euparal. Embryos were collected overnight and aged an additional 12
h. First instar larvae were dechorionated in commercial bleach for 3 min and
the vitelline membrane was removed using heptano-methanol 1:1. After washes
with 100% methanol and 0.1% Triton X-100, larvae were mounted in Hoyer's
lactic acid (1:1) and allowed to clear at 65°C for at least 24 h.
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RESULTS AND DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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; Herranz and Morata,
2001). We selected this line for further analysis on the basis of
its expression pattern in the developing embryo (see below). The insertion is
located on the right arm of chromosome 3 polytene section 95F8, 422 bp
upstream of the transcription start site of CG13610
(Fig. 1A). We analyzed
CG13610 expression during embryonic and imaginal disc development.
The expression pattern found in LP1-Gal4; UAS-lacZ embryos and
imaginal discs was similar to that of CG13610, as visualized by in
situ hybridization (Fig. 1).
From now onwards we refer to CG13610 as calderón
(cald) and LP1-Gal4 as cald-Gal4. cald started to be expressed ubiquitously at the cellular blastoderm stage (Fig. 1C). At germ band extended stage, cald transcripts were found in a broad region of the dorsal side (Fig. 1F). As development proceeded, cald expression was restricted to amnioserosa cells and the central nervous system (Fig. 1B,G,H; a transverse section of the embryo at this stage showed an expression pattern that resembled the shape of a `calderón', the Spanish name for the music symbol that increases the length of a music note).
cald was ubiquitously expressed in the eye, leg, wing and haltere discs, although with regional modulation (Fig. 1I-N); its expression was stronger in the pouch region of the wing (Fig. 1I,J) and in the region posterior to the morphogenetic furrow in the eye (Fig. 1K,L). The antennal disc showed two rings of higher cald expression (Fig. 1K,L), while expression in the leg disc was greater in the distal domain (Fig. 1M,N).
CG13610 has been mapped cytologically to 95F8, encodes a
567-amino-acid-protein with 11 transmembrane domains, with highest homology
with organic cation transporters of the major facilitator superfamily. Genes
identified as putative homologs have been identified in Homo sapiens, M.
musculus and G. Gallus, among others
(Fig. 1O and data not shown).
Mutations in the human homolog slc22a4 cause rheumatoid arthritis
(Tokuhiro et al., 2003) and
Crohn's disease (Peltekova et al.,
2004), probably because of defects in the transport of organic
metabolites.
Embryonic and imaginal requirement of calderón
The original cald-Gal4 insertion is viable without any overt
phenotype. To perform a functional analysis of cald, we induced
mutations by mobilization of the P-element. We isolated three embryonic lethal
alleles (caldR106, caldR107,
caldR165) and one homozygous viable allele
(caldR161). Sequence analysis revealed that
caldR106 and caldR107 bear truncated
P-elements of 2.7 and 9 Kb, respectively, but 11 bp further downstream from
the cald-Gal4 original insertion site. caldR161
has a P-element fragment of only 50 bp located at the same insertion point as
the two previous lethal alleles. We were unable to characterize the
caldR165 allele. The lethal alleles
caldR165, caldR106 and
caldR107 greatly reduce CG13610 mRNA expression
(compare Fig. 2E with F, and
data not shown). Another independent P-element insertion, EP1072,
located further upstream of the original cald-Gal4 insertion, is
embryonic lethal, strongly reduces the levels of CG13610 mRNA
expression and drives the expression of calderón in a
GAL4-dependent way (Fig. 2G,H
and data not shown). Embryos homozygous for the caldEP1072,
caldR165, caldR106 and caldR107
mutations showed the characteristic U-shape phenotype of embryos unable to
retract the germ band, probably because of abnormal development of the
amnioserosa [compare Fig. 2B,C
with 2A
(Frank and Rushlow, 1996)].
Embryo-mutants for InR present the same phenotype
(Fig. 7F)
(Fernandez et al., 1995).
Development of flies homozygous for the viable allele caldR161 was markedly delayed during larval and pupal development (Fig. 3A). Adult flies were smaller than wild-type animals (fly length ratio of calderón/wild-type animals was 0.9±0.04; Fig. 3B,E). Mutant wings and eyes were also smaller (eye size ratio of calderón/wild-type animals was 0.8±0.05; wing size ratio of calderón/wild-type animals was 0.85±0.04; Fig. 3C-E). Cell density was increased in the absence of cald activity, indicating that cell size but not cell number was affected (wing cell density ratio of calderón/wild-type animals was 1.3±0.16; Fig. 3E).
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)] were
recovered 72 h after induction, demonstrating that these defects are caused by
loss of cald activity.
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;
Moreno et al., 2002). Indeed,
caldR107 clones show increased activity of the effector
caspase drice (Fraser et al.,
1997), a cysteine protease that degrades the cellular substrates
and causes cell death (Fig.
4E,E'). This apoptosis is associated with the upregulation
of brinker (Moreno et al.,
2002). caldR107 clones also exhibit
upregulation of the expression of this gene
(Fig. 4F,F').
To circumvent the problem caused by slow proliferation and apoptosis of
cald mutant cells, and to verify that these cells are lost by cell
competition, we used the Minute technique
(Fig. 5A)
(Morata and Ripoll, 1975) to
increase their proliferation rate. The Minute mutations are defective
in ribosomal proteins, and in heterozygous conditions produce a developmental
delay caused by their low division rate. cald Minute+
clones growing in a Minute/+ background lost the retarding
Minute condition and compensated for the slowdown of proliferation.
Under these conditions, cald Minute+ clones were recovered
(Fig. 5C,E,G). However, they
were smaller than wild-type control clones (compare
Fig. 5F with G). The size ratio
of calderón/wild-type clones was 0.13±0.1, and the
density ratio was 1.24±0.29. Thus, the average number of cells per
clone and the cell size were reduced, indicating that the loss of
cald activity makes cells grow and proliferate at slower rates.
The Minute homozygous condition is cell lethal. Under normal circumstances, Minute homozygous cells resulting from mitotic recombination (Fig. 5A) were not recovered 48 h after induction (Fig. 5D). When cald Minute+ mutant clones were analyzed, the frequency of recovered Minute- clones was much higher than expected (Fig. 5C,E,G-I). These results indicate that Minute- homozygous clones are eliminated from the disc epithelium as a result of cell competition and either the loss of cald activity in neighbouring cells (i.e. the mutant clones), or the cell-autonomous increase in cald activity in the Minute- homozygous cells reduces cell competition and allows Minute- cells to survive longer.
The examination of cald mutant clones in the wing epithelium and adult cuticle revealed that mutant cells were smaller (Fig. 6). caldR107 Minute+ cells were smaller than wild-type cells in the wing epithelium (Fig. 6A,B), and differentiated thinner and shorter bristles at the adult wing margin (Fig. 6E,F) and in the notum (Fig. 6C,D). These results indicate that cald has a cell-autonomous effect on cell size.
Growth stimulation by the InR pathway requires calderón
The cald embryonic phenotype and the cell-autonomous effect on the
rate of cell division are reminiscent of mutations in components of the InR
signalling pathway (Coelho and Leevers,
2000). It is conceivable that cald is a new component or
target of the InR pathway. In response to ligand binding, the InR
phosphorylates the Insulin Receptor Substrate (IRS) proteins (encoded by
chico in Drosophila)
(Böhni et al., 1999),
thereby activating the class I PI 3-kinase (PI3K), which in turn increases the
levels of the second messenger phosphatidylinositol 3,4,5-tryphosphate
(PIP3) at the cell membrane
(Fig. 8). The serine threonine
protein kinase Akt appears to be the major critical target of PIP3
signalling in Drosophila. Two signalling branches downstream of Akt
have been identified. One leads to activation of TOR and S6K through Rheb
kinase activity. The Drosophila Rheb functions in the InR pathway
downstream of Tsc1-Tsc2, and its overexpression causes activation of this
pathway, leading to increased cell proliferation
(Garami et al., 2003;
Zhang et al., 2003). To
determine whether cald is required for Rheb signalling, we used the
MARCM technique to examine the growth properties of clones of cells that lack
cald and overexpress Rheb
(Lee and Luo, 1999).
Rheb overexpression did not induce growth in cald mutant
cells and these clones were lost 72 h after induction (not shown). We
therefore conclude that cald functions downstream of or in parallel
to Rheb signalling.
A major effector of Rheb function is S6K
(Stocker et al., 2003).
Genetic evidence has established that insulin exerts many of its cellular
effects by triggering the activation of S6K (p70 ribosomal S6 kinase). Full
activation of dS6K requires two distinct signals, one in response to growth
factors and another from a nutrient sensing pathway, and thus provides a
mechanism whereby individual cells can coordinate their response to growth
factors with nutrient availability (Zhang
et al., 2003). To determine whether cald is involved in
nutrient sensing, we examined the growth properties of clones of cells that
lack cald and overexpress dS6K. As with Rheb, cells overexpressing
dS6K but lacking cald activity were lost by 72 h after clone
induction (not shown). We conclude that cald is not involved in
nutrient sensing and is epistatic to S6K. Therefore, cald
functions downstream of or in parallel to S6K signalling.
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; Kramer et al.,
2003; Puig et al.,
2003). To determine whether cald is required for dFOXO
inhibition, we examined the subcellular location of dFOXO in clones of
cald mutant cells, and found that dFOXO remains cytoplasmic in these
clones (Fig. 6B). We conclude
that Akt inhibits dFOXO in the absence of cald function. These
results indicate that cald is not required for dFOXO inhibition.
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). TOR controls
expression of a broad group of genes with roles in protein, lipid and nucleic
acid metabolism (Beck and Hall,
1999; Cardenas et al.,
1999). In addition, both TOR and PI3K affect the expression and
activity of the C/EBP family of transcriptional regulators, which regulates
the expression of a number of genes in response to nutrients
(Entingh et al., 2001;
Roesler, 2001). Thus, given
that the InR signalling pathway controls the expression of a diverse spectrum
of metabolism-related genes, we examined whether this pathway activates
cald transcription. To address this issue, cald expression
was first analyzed in the embryo following expression of either S6K
or a dominant-negative form of the Drosophila PI3K
(PI3K92E-Dp110D954A). Expression of S6K in the
patched or Ubx domains induced ectopic cald
expression (Fig. 7C,E, compare
with 7A). When a dominant-negative version of PI3K92E-Dp110
(PI3K92E-Dp110D954A) was expressed in amnioserosa cells under
the control of the Kr-GAL4 driver, cald expression was
reduced in these cells (compare Fig. 7B
with D, red arrows). Furthermore, these embryos did not retract
the germ band (Fig. 7F), as
expected for an embryo that lacks cald expression in the amnioserosa
(Fig. 2B,C). When
PI3K92E-Dp110D954A was expressed in the dorsal side of the
wing imaginal disc under the control of the MS1096-GAL4 driver,
cald expression was reduced (compare
Fig. 7G with H). Expression of
S6K in the patched domain did not increase cald
expression in the wing disc, suggesting that the expression levels of this
gene are saturated in imaginal cells (data not shown). Consistent with this,
over-expression of cald in clones of cells did not cause any overt
phenotype in terms of growth, cell size or cell competition (data not shown).
Taken together, the data indicate that cald expression is regulated
by the InR pathway in the embryo and in imaginal cells.
Concluding remarks
Genetic studies have revealed a key role for InR signalling in coordinating
growth and other nutrition-regulated functions in flies. Although the distinct
elements of this pathway have been well described, the downstream elements
responsible for this task remain to be fully elucitated.
calderón, a gene encoding for an Organic Cation Transporter,
is regulated at a transcriptional level by the PI3-kinase/TOR pathway
(Fig. 8). cald mutant
flies are smaller than wild-type ones and show a developmental delay. Clones
of cells mutant for cald divide more slowly, are smaller than
wild-type cells and are eliminated by cell competition. However, when
cald clones have a proliferative advantage in a Minute
heterozygous background, they are not eliminated and are able to differentiate
normal adult structures, although they are smaller than wild-type ones. Since
cald vertebrate orthologs are involved in carrying organic substrates
across the plasma membrane (Grundemann et
al., 2005), we propose that reduced cald activity in
proliferating cells impairs competition for organic substrates available in
the extracellular media. This impairment may again represent
(Moreno et al., 2002) another
mechanism by which weaker cells are removed from a growing population, and
might serve to regulate cell number and optimise tissue fitness and hence
organ function.
cald activity is required for PI3-kinase/TOR function in inducing growth: up-regulation of this pathway in a cald mutant background does not affect growth. Furthermore, cald expression is dependent on PI3-kinase activity, as embryos overexpressing S6K show increased cald levels, whereas expression of a dominant negative form of PI3K (PI3K92E-Dp110D954A) reduces them. Thus, we propose that cald responds to InR activity levels and that it is required, in a cell-autonomous way, for cell growth and proliferation. We hypothesize that the growth of imaginal cells is then controlled by two distinct, but coordinated, nutrient-sensing mechanisms. A cell autonomous mechanism (e.g. cald) may directly detect nutrient levels in the haemolympha, thus changing the response of cells to insulin. The fat body, functioning as a nutrient sensor of the media, may then regulate the growth of the whole organism, perhaps by modulating the levels of membrane transporters, like Cald, that carry nutrients across the membrane.
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ACKNOWLEDGMENTS |
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