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First published online 26 November 2008
doi: 10.1242/dev.029975
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Department of Biochemistry and Molecular Biophysics, Integrated Program in Cellular, Molecular, Structural and Genetic Studies, Columbia University, 701 W. 168th Street, HHSC 1104, New York, NY 10032, USA.
Author for correspondence (e-mail:
rsm10{at}columbia.edu)
Accepted 27 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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24 hours earlier than the telopodite progenitors. Together,
these findings provide a complete and high-resolution fate map of the
Drosophila appendage primordia, linking the primary domains to
specific cis-regulatory elements in Dll.
Key words: Distalless, Drosophila melanogaster, Leg, Limb primordium
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) (reviewed by Boxshall,
2004). The coxopodite, which includes the coxa, the most proximal
leg segment, is thought to have been derived as an outgrowth of the body wall
and may have been the ancestral, unsegmented appendage. The telopodite, or leg
proper, includes all of the more distal leg segments, and is thought to have
evolved subsequently, to allow more sophisticated leg movements by bendable
joints that separate each leg segment. However, despite the existence of this
primary subdivision, and its importance in arthropod evolution, a molecular
understanding of this process is lacking.
Most of the molecular dissection of arthropod leg development has come from
studying the leg imaginal discs of the fruit fly, Drosophila
melanogaster. These studies suggest that the formation of the telopodite
is under the control of the Hedgehog (Hh) signaling pathway, whereas the
coxopodite forms independently of this pathway
(Gonzalez-Crespo et al., 1998;
Gonzalez-Crespo and Morata,
1996). For the telopodite to form, Hh induces the expression of
two downstream signals, Wingless (Wg), ventrally, and Decapentaplegic (Dpp),
dorsally (Basler and Struhl,
1994). The combinatorial action of Wg plus Dpp creates the PD axis
of the leg by activating target genes such as Distalless
(Dll) and dachshund (dac)
(Campbell et al., 1993;
Diaz-Benjumea et al., 1994;
Estella and Mann, 2008;
Lecuit and Cohen, 1997). Based
on these studies, the sum of the Dll and dac expression
domains in a mature leg imaginal disc may correspond to the telopodite, a
conclusion that is supported by studies in other arthropods
(Abzhanov and Kaufman, 2000).
By contrast, there is no clear molecular marker for the coxopodite. Initially,
the presence of nuclear Extradenticle (nExd), a homeodomain protein that
requires the co-expression of homothorax (hth) for nuclear
localization, was proposed to be a marker for the coxopodite in the leg
imaginal disc (Gonzalez-Crespo and Morata,
1996; Rieckhof et al.,
1997). However, a true coxopodite gene should not be expressed
distal to the coxa, and Hth-nExd are also expressed in the next-most distal
leg segment, the trochanter (Abu-Shaar and
Mann, 1998). The molecular definition of these two domains is also
complicated by the observation that the relative expression patterns of Dll
and Hth-nExd change over time. When Dll, the earliest marker of the
leg primordium, is first activated in embryogenesis, all
Dll-expressing cells co-express Hth-nExd in circular domains
comprising 20 cells per thoracic hemisegment
(Gonzalez-Crespo et al.,
1998). Slightly later Hth-nExd are no longer expressed in a
central subset of the Dll domain, but the three proteins remain co-expressed
in the remaining cells (Bolinger and
Boekhoff-Falk, 2005; Mann and
Abu-Shaar, 1996). Eventually, in the third instar leg imaginal
disc, the expression domains of Hth-nExd and Dll are mutually exclusive except
for a thin ring of cells that co-express these genes and gives rise to the
trochanter (Abu-Shaar and Mann,
1998; Gonzalez-Crespo and
Morata, 1996).
We reasoned that insights into how the telopodite and coxopodite are
specified might come from characterizing the cis-regulatory elements that
regulate Dll in the embryonic leg primordia. Dll is
initially activated at 6 hours of embryonic development under the control
of an early-acting enhancer called Dll304
(Vachon et al., 1992). Wg
provides the anteroposterior (AP) positional cue that activates
Dll304 (Cohen et al.,
1993). Two other signaling pathways, Dpp and Epidermal growth
factor receptor (Egfr) signaling, limit the leg progenitor domain dorsally and
ventrally, respectively (Goto and Hayashi,
1997; Kubota et al.,
2000). Furthermore, although the Wg, Dpp and EGFR signals are
deployed similarly in all embryonic trunk segments, Dll expression is
limited to the thoracic segments by the abdominal Hox genes that directly
repress Dll304 activity in the abdomen
(Gebelein et al., 2002;
Vachon et al., 1992).
Although Dll304 is activated by Wg and repressed by Dpp,
Dll expression in the imaginal disc is activated by both signals
(Campbell et al., 1993;
Diaz-Benjumea et al., 1994),
implying that additional Dll regulatory elements must exist.
Recently, such a leg disc regulatory element, termed LT for
`leg-trigger', has been described (Estella
et al., 2008). Unlike Dll304, LT continuously requires Wg
and Dpp input for its activity in the leg disc. Although LT (also
called Dll215) has been reported to be active in late stage embryos
(Castelli-Gair and Akam, 1995;
Cohen et al., 1993), its
spatial relationship compared to Dll304 and its regulation by Wg and
Dpp during embryogenesis has not been described. In addition, the lineages
that Dll304- and LT-expressing cells give rise to have not
been examined and may help inform how the coxopodite and telopodite are
specified.
Another important unresolved set of questions concerns the relationship
between the development of the adult and larval legs. As a holometabolous
insect, Drosophila undergoes complete metamorphosis, meaning that the
tissues that give rise to the adult structures, the imaginal discs, grow
within the larva but do not contribute to the larval body plan. Nevertheless,
Drosophila has rudimentary larval appendages called Keilin's organs
(KOs) that serve as thoracic-specific sensory organs. KOs are intimately
associated with the developing leg imaginal disc
(Lakes-Harlan et al., 1991;
Madhavan and Schneiderman,
1977) and, like the adult telopodite, require Dll to form
(Cohen and Jurgens, 1989).
Although a group of cells within the Dll-expressing leg primordia
express neural markers and is therefore thought to give rise to the KOs
(Bolinger and Boekhoff-Falk,
2005; Cohen,
1993), its relationship to other Dll-dependent lineages
has not been clearly defined.
Here we compare the spatial relationships, subsequent lineages and genetic
inputs that regulate three Dll cis-regulatory elements, Dll304,
LT, and a newly defined element, DKO, dedicated to the formation
of the KOs. We show that when the leg primordia are first allocated,
coincident with the activity of Dll304, this domain is multipotent
and has the potential to give rise not only to the entire telopodite,
coxopodite and KO, but also to dorsal (e.g. wing) appendage fates. A few hours
later, Dll304 activity fades, and LT and DKO are
activated in mutually exclusive subsets of the Dll304-expressing
domain. In contrast to the multipotency of the Dll304 expression
domain, LT-expressing cells give rise to the entire telopodite and
only the telopodite, while DKO-expressing cells give rise to the KO.
As in the leg imaginal discs, LT requires both Wg and Dpp to be
activated during embryogenesis. In addition, we show that the telopodite fate
is repressed by the KO fate, suggesting that these two sets of progenitor
cells are mutually antagonistic. Surprisingly, we also find that the onset of
coxopodite growth is advanced relative to the onset of telopodite growth,
which begins only after Hth-nExd are turned off in these cells at 60
hours of development. These experiments thus provide a complete description of
all the cell types within the Drosophila leg primordia, their
temporal development, and the subsequent structures they generate.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
DKO fragment was selected based on sequence conservation to other
Drosophilids (Vista Genome Browser), and cloned by PCR (details are
available upon request) into the hs43-nuc-lacZ vector
(Estella et al., 2008). The
DKO and 304 enhancers were also cloned in pChs-Gal4.
Immunostaining
Discs and embryos were stained using standard procedures. The primary
antibodies used were, rabbit and mouse anti-β-Gal (Cappell and Promega),
mouse anti-Wg, mouse anti-Cut, mouse anti-En, mouse anti-Dac and mouse
anti-Elav (Developmental Studies Hybridoma Bank, University of Iowa). Guinea
pig anti-P-Mad (a gift from E. Laufer and T. Jessell), guinea pig anti-Dll
(generated against the full-length protein), rabbit anti-Hth generated against
the full-length protein, mouse anti-Ubx
(Crickmore and Mann, 2006),
guinea pig anti-Tsh (a gift from G. Struhl).
Drosophila stocks and mutant analysis
lacZ lines, Gal4 lines and lineage analysis
esg-lacZ (Hayashi et
al., 1993), esg-Gal4
(Goto and Hayashi, 1999),
Dll-Gal4 (line MD 23) (Calleja et
al., 1996), Dll-Gal4 (line 212)
(Gorfinkiel et al., 1997),
tsh-Gal4 (Wu and Cohen,
2000), hth-Gal4 (GETDB-Gal4 Enhancer Trap Insertion Data
Base), prd-Gal4 (Gebelein et al.,
2004). Two Dll-Gal4 lines were used: MD 23 recapitulates
all Dll expression whereas em 212 does not capture early
(Dll304) expression.
Lineage analyses and neutral clones used act5C>stop>lacZ;
UAS-flp (Struhl and Basler,
1993). In the tsh-Gal4 and hth-Gal4 lineage
experiments flies of the genotype tub-Gal80ts; tsh-Gal4;
tub-Gal80ts (Zirin and
Mann, 2007) were crossed to act5C>stoP>lacZ;
UAS-flp at 18°C. At various time points, 12-hour collections were
transferred to 29°C. Female larvae were dissected at the third instar.
Developmental stage at time of transfer to the restrictive temperature was
determined based upon the amount of time spent at 29°C before dissection.
tub-Gal80ts was also used to show that limiting
LT-Gal4 activity to the embryo is sufficient to label the entire
telopodite. For the Minute experiment we generated wild-type clones
in a Minute heterozygous background in telopodite precursor cells
using the LT-Gal4; UAS-flp.
Mutant lines
DllSA1 (Vachon et
al., 1992), Df(1)sc-B57
(Dominguez and Campuzano,
1993), btdXG81 and Df(1)C52
(Cohen, 1990) were used. The
wg temperature-sensitive experiments (WgIL114)
were performed as previously described
(Cohen et al., 1993) with
modifications. Embryos were kept at the permissive temperature (18°C)
until Dll expression is initially activated (10-14 hours), then
transferred to the restrictive temperature (29°C) until stage 14.
UAS lines
UAS-hid (Zhou et al.,
1997), UAS-ase (Brand
and Dormand, 1995), UAS-arm (delta N)
(Chan et al., 1997),
UAS-tkvQD (Abu-Shaar
and Mann, 1998), UAS-dad
(Tsuneizumi et al., 1997),
UAS-brk (Jazwinska et al.,
1999), UAS-Dll
(Gorfinkiel et al., 1997),
UAS-btd (Schock et al.,
1999) and UAS-p35 (Bloomington Center) were used.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), we
examined the activity of this enhancer relative to Dll and
Dll304 during embryonic stages. Dll protein in the thorax was first
detected during embryonic stage 11 (Fig.
1B), and continued to be visualized in this region until the end
of embryogenesis (Fig. 1C; data
not shown). Although a Dll304-lacZ transgene recapitulated
the initial pattern of Dll expression
(Fig. 1D), the activity of this
enhancer decayed within a few hours (Cohen
et al., 1993). By contrast, an LT-lacZ reporter gene
became active in Dll-expressing cells of the thorax after germ-band
retraction, with robust expression by stage 14
(Fig. 1E). Importantly, the
LT enhancer was not active in all of the Dll-expressing
cells of the thorax. LT-lacZ was expressed in the outermost ring of
15 cells of the Dll clusters
(Fig. 1G). At this stage,
LT-lacZ-expressing cells also expressed hth, esg and
teashirt (tsh; Fig.
1H,I, and data not shown). Because esg is required for
the maintenance of diploidy, it has been suggested that
esg-expressing cells give rise to the imaginal discs
(Hayashi et al., 1993). By
contrast, the Dll-expressing LT-lacZ-nonexpressing cells
within the LT ring did not express esg. Instead, these cells
expressed cut (ct), which encodes a transcription factor
required for the development of external sensory organs
(Bodmer et al., 1987).
Accordingly, these cells may give rise to the KO
(Bolinger and Boekhoff-Falk,
2005; Cohen et al.,
1993).
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Existence of different cell fates in the ventral limb primordia
To determine whether the two cell types defined above give rise to
different fates, we performed lineage-tracing experiments with a panel of
Gal4 drivers, including drivers made with these enhancer elements. In
general, lineages were analyzed by using these Gal4 drivers to express the
yeast recombinase Flp, which deleted a transcriptional stop cassette from an
actin-lacZ transgene (actin>stop>lacZ; see Materials
and methods for details).
Lineage tracing using Dll304-Gal4 labeled cells in both the dorsal
and ventral appendages (Fig.
2A), indicating that this enhancer was active prior to the
separation of leg and wing fates, consistent with previous cell-lineage
analyses (Wieschaus and Gehring,
1976). Within the leg, cells were labeled in both the coxopodite
and telopodite, unlike the distal-only pattern of Dll immunostaining in mature
third instar leg discs (Fig.
2A). This pattern matches that generated by Dll-Gal4, an
enhancer trap into the Dll locus (line MD 23, see Material and
methods; see Fig. S2A in the supplementary material)
(Campbell and Tomlinson,
1998).
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LT-expressing cells of the embryonic limb primordia give rise to the
entire telopodite. This is surprising given that, in the embryo, these cells
also express hth and tsh
(Fig. 1H), which are genes that
are expressed only in the proximal domain of the third instar leg disc and
have therefore been considered to be coxopodite markers.
To confirm that the telopodite progenitor cells also express hth
and tsh, we performed lineage-tracing experiments using
tsh-Gal4 and hth-Gal4. Because the entire thoracic ectoderm
expresses tsh and hth prior to the initiation of
Dll expression, the tub-Gal80ts transgene was
used to control the activity of these Gal4 drivers. Raising the
animals at the temperature where Gal80ts was active for all of
development (the permissive temperature) resulted in no lacZ
expression (data not shown), confirming the efficacy of the Gal80ts
protein. Switching the animals to the nonpermissive temperature at the
beginning of the second larval instar (48 hours) resulted in
lacZ expression throughout the entire leg disc
(Fig. 2C), indicating that
tsh was active in both coxopodite and telopodite progenitors long
after LT activation in the embryo. By contrast, switching the animals
to the nonpermissive temperature at the beginning of the third larval instar
(72 hours) consistently labeled the coxopodite, but rarely labeled the
telopodite (Fig. 2D). Similar
results were obtained using hth-Gal4 instead of tsh-Gal4
(data not shown).
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Because the Dll- and DKO-lacZ-expressing cells also
express ct and elav, but not esg
(Fig. 1G-J; see Fig. S1A in the
supplementary material), these cells were predicted to be the progenitors of
the larval KO. To test this, we carried out lineage tracing using a
DKO-Gal4 transgene. One-third (n=40) of these third instar
leg discs had no lacZ expression, demonstrating that
DKO-expressing cells did not contribute to imaginal disc fates.
Approximately one third of the discs contained small numbers of
lacZ-positive cells that co-expressed the neural marker Elav
(Fig. 2F, see inset). These
neurons may be the same as previously described, embryonically born neurons
that persist until larval stages (Tix et
al., 1989). The cell bodies of these neurons reside in the leg
imaginal disc and project dendrites to the KO in the larval epidermis
(Tix et al., 1989). Finally,
approximately one-third of the discs had lacZ-expressing clones
present in the disc epithelia. Because the DKO element is expressed
in Dll-negative cells (see above), these clones probably result from
the spurious activity of this enhancer. Altogether, these data are consistent
with an earlier report (Bolinger and
Boekhoff-Falk, 2005) and support the conclusion that the
Dll-positive, Ct-positive cells in the center of the leg primordia,
previously considered to be the progenitors of the telopodite, are the
progenitors of the Keilin's organ and do not contribute to the imaginal disc.
These conclusions were further confirmed by using these Gal4 drivers to
express the proapoptotic gene hid
(Zhou et al., 1997) to induce
cell death (see Fig. S2C in the supplementary material).
In summary, when Dll304 is first activated, Dll-positive cells have the potential to give rise to all regions of the dorsal and ventral appendages. A few hours later three cell types are defined: the KO progenitors [Dll(DKO)-positive, esg-, hth- and tsh-negative], the telopodite progenitors [Dll(LT)-positive, esg-, hth- and tsh-positive], and the coxopodite progenitors (Dll-negative, esg-, hth- and tsh-positive). Together, these cells comprise the entire thoracic ventral limb primordia (Fig. 1F).
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),
LT is activated in the embryo in cells that receive both inputs, as
monitored by anti-Wg and anti-PMad staining
(Fig. 3A). To determine whether
wg is required for LT activity, we used a
temperature-sensitive allele of wg to allow earlier Dll
activation (Cohen et al.,
1993). Switching the embryos to the restrictive temperature at
stage 11 resulted in the absence of LT activity, despite the presence
of Dll protein (probably derived from Dll304 activity;
Fig. 3C). In addition, ectopic
activation of the wg pathway [using an activated form of
armadillo (arm*)] resulted in more
LT-lacZ-expressing cells (Fig.
3D).
Like wg, the dpp pathway is necessary for
LT-lacZ expression in leg discs. Paradoxically, dpp
signaling represses Dll in the embryo because dpp mutants
show an expansion in Dll304-lacZ expression (data not shown)
(Goto and Hayashi, 1997). By
contrast, LT-lacZ is not expressed in dpp null embryos (data
not shown). LT-lacZ, but not Dll protein, was also repressed by two
dpp pathway repressors, Dad and brk
(Campbell and Tomlinson, 1999;
Jazwinska et al., 1999;
Minami et al., 1999;
Tsuneizumi et al., 1997)
(Fig. 3E; and data not shown).
Conversely, stimulation of the dpp pathway [using an activated form
of the Dpp receptor (TkvQD)] resulted in ectopic activation of
LT ventrally (Fig.
3F).
Taken together, these data demonstrate that LT is activated by Wg and Dpp in the embryonic limb primordia, just as it (and Dll) is in the leg disc. Similarly, DKO activity also requires Wg and Dpp input (see Fig. S3D,E in the supplementary material).
Dll and btd confer ventral thoracic-specificity to LT expression
Although LT is activated by wg and dpp in the
leg primordia, these signals are also present in each abdominal segment.
Consequently, there must be additional factors that restrict LT
activity to the thorax. One possibility is that LT is repressed by
the abdominal Hox factors, such as Dll304
(Gebelein et al., 2002;
Vachon et al., 1992).
Alternatively, LT might be regulated by Dll, itself
(Castelli-Gair and Akam,
1995). We found that in Dll null embryos LT-lacZ
was initially expressed in a stripe of cells instead of a ring, but then
expression decayed (Fig. 4C).
Ectopic expression of Dll resulted in weak ectopic expression of
LT-lacZ in the thorax and abdomen (see Fig. S3A in the supplementary
material). These data suggest that LT activity is restricted to the
thorax in part because of the earlier restriction of Dll304 activity
to the thorax.
The related zinc-finger transcription factors encoded by
buttonhead (btd) and Sp1 are also expressed in the
limb primordia and are also required for ventral appendage specification
(Estella et al., 2003). In
strong btd hypomorphs, the activity of LT was still detected
but the number of cells expressing LT-lacZ was decreased and its
pattern was disrupted (see Fig. S3C in the supplementary material).
LT-lacZ expression was completely eliminated in animals bearing a
large deficiency that removes both btd and Sp1
(Fig. 4B). By contrast,
Dll304 was activated normally in these animals (data not shown).
Importantly, LT-lacZ expression was rescued by expressing
btd in these deficiency embryos
(Fig. 4B). By contrast,
expressing Dll, tkvQD, or arm* did not
rescue LT expression in these deficiency embryos (data not shown). Ectopic
expression of btd resulted in weak ectopic activation of
LT-lacZ in cells of the thorax and abdomen (see Fig. S3B in the
supplementary material). Strikingly, the simultaneous expression of
Dll and btd resulted in robust ectopic expression of
LT-lacZ in abdominal segments in the equivalent ventrolateral
position as the thoracic limb primordia
(Fig. 4D). btd and
Dll were not sufficient to activate LT in wg null
embryos (data not shown). These data indicate that the thoracic-specific
expression of the LT enhancer is controlled by the combined
activities of btd and/or Sp1, Dll and the wg and
dpp pathways.
Proneural genes activate DKO and repress LT
Although the above data suggest that LT is activated by a
combination of Wg, Dpp, Btd and Dll, these activators are also present in the
precursors of the KO, which activate DKO instead of LT.
Because the KO is a sensory structure, we tested the role of members of the
achaete-scute complex (ASC) that are expressed in these cells
(Bolinger and Boekhoff-Falk,
2005). In embryos hemizygous for a deficiency that removes the
achaete-scute complex, LT-lacZ expression was expanded at the expense
of the Ct-expressing cells (Fig.
4E). Consistently, ectopic expression of the ASC gene
asense (ase) repressed LT and increased the number
of Ct-expressing cells (Fig.
4F). These data suggest that there is a mutual antagonism between
the progenitors of the telopodite and those of the KO. We also found that
DKO-lacZ expression in the leg primordia was lost in Dll or
btd null embryos, consistent with the loss of KOs in these mutants
(Cohen and Jurgens, 1989;
Estella et al., 2003) (data
not shown). DKO activity was also lost from the limb primordia in
embryos deficient for the ASC (Fig.
4H). These results indicate that DKO is activated by the
same genes that promote LT expression but, in addition, requires
proneural input from the ASC.
Distinct cell proliferation dynamics in the coxopodite and telopodite
To follow the development of the telopodite, we performed a time-course
experiment to visualize LT-expressing cells throughout larval
development. esg-lacZ was used to identify all imaginal disc cells
and LT-Gal4, UAS-GFP was used to mark the progenitors of the
telopodite. We estimate that there are 15 progenitor cells each for the
telopodite and coxopodite in stage 14 embryos
(Fig. 5A). Previous clonal
analyses suggested that the cells of the leg primordia stop dividing during
embryogenesis and resume proliferation at approximately 48 hours
(Bryant and Schneiderman,
1969). Consistent with these studies, the number of Esg-positive
cells began increasing at the beginning of the second larval instar, about 48
hours AEL (Fig. 5A).
Surprisingly, at 60 hours, the coxopodite progenitors (Esg-positive,
LT- and Dll-negative cells) far outnumbered the telopodite
progenitors (LT- and Dll-positive). In addition, the nuclei of the
telopodite progenitor cells were larger than those of the coxopodite
progenitor cells in these early leg discs. The telopodite progenitors also
appear to be tightly associated with the larval epidermis, and they continued
to express hth and tsh, in addition to Dll and
esg (Fig. 5A,B). By
the end of the second larval instar stage, between 60 and 72 hours AEL, the
entire leg disc invaginated from the larval cuticle, and the number of
LT-positive cells was dramatically increased
(Fig. 5B). At this stage, these
cells no longer expressed tsh or hth
(Fig. 5B; and data not
shown).
These data suggest that there is a difference in the time when the progenitor cells of the coxopodite and the telopodite begin to proliferate. By direct observation, we estimate that the coxopodite progenitors begin to divide between 12 and 24 hours earlier than those of the telopodite. To rule out that LT-positive cells start to proliferate at the same time, and LT is rapidly shut off in some progeny, we repeated the LT-Gal4 lineage analysis, comparing at early time points the number of cells in which LT had been active with the number that continued to express LT. Fig. 5C shows that these two cell populations are identical, arguing that the progenitors of the telopodite rarely proliferate prior to this time.
We confirmed the delay in the onset of telopodite proliferation by inducing marked clones in both domains between 12 and 24 hours AEL, and quantifying the location and number of cells 36 and 48 hours later. For these experiments, we defined the coxopodite as being Hth- or Tsh-positive and LT-GFP-negative. Conversely, the telopodite progenitors were defined as being LT-GFP-positive. At the 36-hour time point, the average number of cells in telopodite clones was 1.3 (n=31; Fig. 5D; see Fig. S4A in the supplementary material). By contrast, the average number of cells in coxopodite clones was 3.2 (n=51; Fig. 5D; see Fig. S4A in the supplementary material). All KO clones (LT-GFP-negative and Hth/Tsh-negative) remained as single cells (n=11). When measured 48 hours after clone induction, the average number of cells in telopodite clones was 4.6 (n=9) while the average number of cells located in coxopodite clones was 5.6 (n=20; Fig. 5D; see Fig. S4B in the supplementary material). These data suggest that the progenitors of the coxopodite resume proliferating approximately one to two cell divisions earlier than the progenitors of the telopodite.
Interestingly, we found that telopodite and coxopodite clones stayed within
their respective domains (LT-GFP expressing or nonexpressing,
respectively) (see Fig. S4A,B in the supplementary material). When clones
(n=25) induced between 12 and 24 hours were allowed to grow to the
third instar, their progeny continued to demonstrate a restriction in lineage
(Fig. 5E). However, both sets
of clones could enter the trochanter, which expresses both hth and
Dll (see Fig. S5A,B in the supplementary material). By contrast,
clones induced prior to stage 14 (5.5-7 hours AEL) occasionally spanned both
the coxopodite and telopodite (19%, n=32; see Fig. S4C in the
supplementary material). These data suggest that there is a lineage
restriction along the PD axis of the developing leg that forms at stage 14,
about the same time that LT is activated in the limb primordia. This
lineage restriction does not constitute a compartment boundary, however,
because when cells were given a growth advantage using the Minute
technique (Morata and Ripoll,
1975) we observed clones that did not respect this boundary (data
not shown). Moreover, this restriction is not a discreet border, but is
instead defined by a region (the trochanter), that expresses both telopodite
(Dll) and coxopodite (Hth) markers.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Gonzalez-Crespo and Morata,
1996). As a result, and partly because of the analogy with the
third instar leg disc, the predominant view of this fate map became that the
Dll-positive, Hth-nExd-negative cells of the embryonic primordia gave rise to
the telopodite, while the surrounding Hth-positive cells gave rise to the
coxopodite (reviewed by Morata,
2001). The expression pattern of esg, a gene required for
the maintenance of diploidy, was also misinterpreted as being a marker
exclusively of proximal leg fates (Goto
and Hayashi, 1997; Kubota et
al., 2000; Kubota et al.,
2003). Counter to these earlier studies, our experiments
unambiguously show that the Dll-positive, Hth-nExd-negative cells in the
center of the primordia give rise to the KO, the ring of Dll-positive,
Esg-positive, Hth-nExd-positive cells gives rise to the telopodite, and the
remaining Esg-positive, Dll-negative cells give rise to the coxopodite
(Fig. 6). The spurious expression of DKO-lacZ in Dll-non-expressing cells outside the leg primorida complicates the interpretation of several experiments. Attempts to refine DKO activity by changing the size of the cloned fragment proved unsuccessful. Nevertheless, our evidence supports the idea that DKO-positive, Dll-positive cells of the leg primordia give rise to the Keilin's organ, and not the adult appendage.
Regulation of proliferation along the PD axis of the developing leg
The progenitors of the coxopodite begin to proliferate at approximately 48
hours of development, consistent with previous measurements of leg imaginal
disc growth, whereas the progenitors of the telopodite do not resume
proliferating for an additional 12 to 24 hours. According to estimates of the
cell cycle time in leg discs
(Postlethwait, 1978), this
difference in the onset of proliferation results in one to two additional cell
divisions in the coxopodite, consistent with images of late second instar leg
discs presented here. Why might the telopodite and coxopodite begin
proliferation at different times? One possibility is that the cells of the
coxopodite give rise to the peripodial epithelium that covers the leg imaginal
disc, and therefore require additional cell divisions relative to the
telopodite. It is also possible that the telopodite is delayed because the
neurons of the Keilin's organ serve a pathfinding role for larval-born neurons
that innervate the adult limb (Jan et al.,
1985). Perhaps this pathfinding function requires that the KO and
telopodite remain associated with each other through the second instar.
Consistently, the leg is the only imaginal disc that has not invaginated as a
sac-like structure in newly hatched first instar larvae
(Madhavan and Schneiderman,
1977).
A possible explanation for the delay in the onset of telopodite
proliferation is the persistent co-expression of hth and Dll
in these cells; hth (and tsh) expression is turned off in
these cells at about the same time they begin to proliferate. Consistent with
this idea, maintaining the expression of hth throughout the primordia
blocks the proliferation of the telopodite (see Fig. S5C in the supplementary
material) (Azpiazu and Morata,
2002). Also noteworthy is the finding that the genes no
ocelli and elbow have been shown to mediate the ability of Wg
and Dpp to repress coxopodite fates (Weihe
et al., 2004). Together with our findings, it is possible that the
activation of these two genes in the LT-expressing progenitors is the
trigger that turns off hth and tsh in these cells.
Restriction of cell lineage between coxopodite and telopodite
Our experiments suggest that once LT is activated, and under
normal growth conditions, there is a lineage restriction between the
telopodite and coxopodite. By contrast, previous lineage-tracing experiments
using tsh-Gal4 concluded that the progeny of proximal cells could
adopt more distal leg fates (Weigmann and
Cohen, 1999). However, these authors were unaware that
tsh is still expressed in the telopodite progenitors far into the
second instar, providing an explanation for their results. In contrast to this
early restriction, there is no evidence for a later lineage restriction within
the telopodite. For example, the progeny of a Dll-positive cell can lose
Dll expression and contribute to the dac-only domain
(Gorfinkiel et al., 1997).
Interestingly, the lineage restriction between coxopodite and telopodite is
not defined by the presence or absence of Hth-nExd or Tsh because both
progenitor populations express hth and tsh after their fates
have been specified. By contrast, when these two domains are specified, the
telopodite expresses Dll, while the coxopodite does not, suggesting
that Dll may be important for the lineage restriction. However, later in
development, some cells in the telopodite lose Dll expression and
express dac, but continue to respect the coxopodite-telopodite
boundary. Thus, either Dll expression in the telopodite is somehow
remembered or the telopodite-coxopodite boundary can be maintained by
dac, which is expressed in place of Dll immediately adjacent
to the telopodite-coxopodite boundary. Also noteworthy is our finding that
clones originating in the coxopodite can contribute to the trochanter, the
segment inbetween the proximal and distal components of the adult leg that
expresses both Dll and hth in third instar imaginal discs
(Abu-Shaar and Mann, 1998).
However, the progeny of such clones do not contribute to fates more distal
than the trochanter. Likewise, a clone originating in the telopodite can also
contribute to the trochanter, but will not grow more proximally into the coxa
(see Fig. S5A,B in the supplementary material). Thus, the lineage restriction
uncovered here seems to be determined by distinct combinations of
transcription factors expressed in the coxopodite and telopodite progenitors
at stage 14. The progeny of cells that express Dll, tsh and
hth can populate the telopodite or trochanter, whereas the progeny of
cells that express tsh and hth, but not Dll, can
populate the coxopodite or trochanter. In light of the Minute-positive
results, however, the lineage restriction between coxopodite and telopodite
does not satisfy the classical definition of a compartment boundary. A similar
non-compartment lineage restriction has also been documented along the PD axis
of the developing Drosophila wing
(Zirin and Mann, 2007).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/1/61/DC1
|
Footnotes |
|---|
* These authors contributed equally to this work 
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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