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First published online 25 July 2007
doi: 10.1242/dev.02876
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Institut de Génétique Humaine, CNRS UPR 1142, 141, rue de la Cardonille, 34396 Montpellier Cedex, France.
Author for correspondence (e-mail:
jmdura{at}igh.cnrs.fr)
Accepted 4 June 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Mushroom body, Developmental genetics, Drosophila, Receptor tyrosine kinase, derailed (linotte), Wnt5 signaling, Brain development, Ryk ortholog
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Connoly et al., 1996; de Belle and
Heisenberg, 1994). It seems clear that the harmonious formation of
a brain structure requires both intrinsic clues, i.e. molecular information
provided by the cells belonging to the structure, and extrinsic clues provided
by cells located outside the structure. The development and anatomy of
Drosophila MBs have been rather well documented compared with other
brain areas (Strausfeld et al.,
2003), and a number of gene products have been described as being
autonomously required to different extents for proper MB development
(Lee et al., 2000;
Reuter et al., 2003;
Nicolai et al., 2003). While
these gene studies included some cases of non-autonomy
(Ng et al., 2002;
Wang et al., 2002), these
cases were interpreted as reflecting a strong community effect in axon
guidance and branching within the MB neurons rather than genuine extrinsic
molecular information.
The derailed (drl) gene, which is also known as
linotte (lio), was first isolated based on its role in
olfactory learning and memory (Dura et al.,
1993). The DRL protein was subsequently shown to be a receptor
tyrosine kinase (RTK) (Dura et al.,
1995; Callahan et al.,
1995) belonging to the RYK subfamily of RTKs
(Halford et al., 1999;
Hovens et al., 1992).
drl mutants present structural brain defects in the MBs
(Moreau-Fauvarque et al.,
1998; Moreau-Fauvarque et al.,
2002; Simon et al.,
1998). However, one major unsolved question about the drl
MB mutant phenotype is whether it is due to intrinsic (i.e. cell-autonomous)
or extrinsic (i.e. non-cell-autonomous) factors. With respect to the former
possibility, an enhancer trap line within the drl gene has been
recovered that shows a weak adult MB expression pattern. This, and the weak
anatomical rescue seen with a GAL4 line that is expressed (although
not exclusively) in the MBs, has led us to favor the cell-autonomous
hypothesis in the past (Moreau-Fauvarque
et al., 1998). Alternatively, the finding that DRL is expressed in
interhemispheric glial cells supports the non-cell-autonomous hypothesis
(Simon et al., 1998).
In a detailed analysis, we show here that DRL is not expressed within
developing MB cells and that this is true as early as the embryonic stage and
remains so throughout development. These results are supported by a clonal
analysis of the drl loss-of-function (LOF) mutation, which shows that
the gene is not required intrinsically within MB cells. Our results strongly
support a neuronal requirement for drl close to, but not within, MB
cells. Further, it has been shown that in the embryonic nervous system, DRL
keeps axons out of the posterior commissure by acting as a receptor for WNT5,
a member of the Wnt family of signaling molecules
(Yoshikawa et al., 2003;
Fradkin et al., 2004).
Interestingly, the function and signaling mechanism of WNT proteins appear to
be highly conserved (Fradkin et al.,
2005). In particular, the mammalian Ryk-Wnt pair plays a role in
neurite outgrowth and axon guidance (Lu et
al., 2004; Keeble et al.,
2006). We show here that Wnt5 mutants have MB defects
that can be rescued by expressing Wnt5 cDNA specifically in MB cells.
Strikingly, LOF Wnt5 MB phenotypes resemble those induced by
drl gain of function (Taillebourg
et al., 2005) (this study), and we show that Wnt5 and
drl interact genetically. We propose that the gene pair drl
and Wnt5 work to build adult MBs, with Wnt5 being required
within MB neurons and drl being required within non-MB neurons.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Moreau-Fauvarque et al.,
2002), drlR343 and
drlRed2
(Callahan et al., 1996;
Bonkowsky and Thomas, 1999).
The Wnt5 null allele used was Wnt5400
(Fradkin et al., 2004). The
same mutant phenotypes were obtained with Wnt5D7
(Yoshikawa et al., 2003).
fzh51, fz2C1 and fz3G10
(Srahna et al., 2006) were
used. Enhancer-trap or regulatory region-GAL4 construct lines used were
lio1 (Dura et al.,
1993; Simon et al.,
1998), GAL4-c739
(Yang et al., 1995),
GAL4-7B (Ferveur et al.,
1995; Moreau-Fauvarque et al.,
1998; Nicolai et al.,
2003), GAL4-OK107
(Connolly et al., 1996;
Adachi et al., 2003),
GAL4-C155 (Lee and Luo,
1999), GAL4-247 (Zars
et al., 2000) and GAL4-442
(Hitier et al., 2000). For the
rescue experiments, UAS-drl
(Callahan et al., 1996) and
UAS-Wnt5 (Fradkin et al.,
2004) were used. UAS-nls-lacZ, UAS-tau, and
UAS-mCD8-GFP were used as reporter genes. Wild-type Canton-S was used
as a control line. All the stocks were raised on standard corn medium at
25°C.
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), rabbit anti-ß-galactosidase (1/5000) (Cappel) and goat
CY3-coupled anti-HRP (1/200) (Jackson ImmunoResearch). Secondary antibodies
were goat anti-mouse and goat anti-rabbit coupled with Alexa 488, Alexa 568,
Alexa 647 (Molecular Probes) or HRP (Jackson ImmunoResearch) used at a 1/1000
dilution. HRP staining in embryos and first instar larval brains was revealed
using the DAB substrate (Sigma). Antibody stainings and dissections at all
stages were performed as previously published
(Patel, 1994;
Nicolai et al., 2003).
Fluorescent samples were mounted in Fluoromount vectashield mounting medium.
DAB-revealed samples were mounted in 90% glycerol.
MARCM mosaic analysis
For the drl clonal analysis (MARCM), the lio2
mutation was recombined with the FRT40A chromosome and the clones
generated by crossing this line with GAL4-C155, hs-FLP, UAS-mCD8-GFP;
tubP-GAL80 FRT40A flies. A tubP-GAL80 drlR343
FRT40A chromosome was also engineered. Wnt5 clones were induced
in w hs-FPL tub-GAL80 FRT19A/w Wnt5400 FRT19A; GAL4-c739
UAS-mCD8-GFP/+ individuals. Clones were induced in late-stage
embryos/first instar larvae by applying a 1 hour heat shock at 37°C, as
previously described (Lee et al.,
1999; Lee and Luo,
1999). fz clones were induced in y w hs-FLP;
GAL4-c739 UAS-mCD8-GFP/+; tub-GAL80 FRT2A/fzh51
fz2C1 FRT2A individuals. In that case the heat shock was
applied for an hour on 24- to 32-hour-old pupae.
Microscopy and image processing
Fluorescent samples were analyzed using a confocal microscope (LSCM 1024
BIORAD and a LEICA SP2). Image reconstruction was performed using NIH ImageJ
and Photoshop software. DAB-revealed samples were observed under a LEICA
microscope. Pictures were acquired on slide film, developed and subsequently
digitized.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lee et al., 1999;
Kurusu et al., 2002), is a
continuous process that starts at the end of embryogenesis and continues until
metamorphosis. Hence, it is possible that the anatomical defects in the MBs
that have been previously described in drl (lio) LOF adult
flies arise during early stages of development. In order to define at which
developmental stage the defects are manifest, we analyzed MB morphology in
drl LOF individuals at three developmental stages: adult, late third
instar larva and newly hatched first instar larva. The drl mutant
flies were obtained by crossing two independent null alleles,
lio2 and drlR343. To label the MB
axons at all stages, we used GAL4-OK107 UAS-mCD8-GFP. We first
confirmed the adult MB defects that had been previously characterized using
other techniques: overextension of the medial lobes and reduction or
disappearance of the vertical lobes (compare A with D in
Fig. 1). Second, we observed
that similar defects are already present both at the end of larval life
(compare B with E in Fig. 1)
and immediately after larval hatching (compare C with F). Interestingly, at
the earliest developmental stage analyzed, the reduction or disappearance of
the vertical larval 
lobes was much less pronounced than at later
stages. In conclusion, our results show that drl function is required
at early stages of MB development, raising the possibility that the defects
observed in adult MBs are a consequence of earlier-arising defects.
Clonal analysis reveals a non-cell-autonomous requirement for drl function during MB development
The MB defects displayed by drl LOF individuals could be due
either to a need for the gene product in the Kenyon cells (KCs) themselves
(cell-autonomous requirement) or to a requirement for drl expression
in surrounding cells (non-cell-autonomous requirement). In order to determine
which of these two hypotheses is correct, we took advantage of the MARCM
technique (Lee et al., 1999;
Lee and Luo, 1999), which
allows the generation of homozygous drl LOF KC clones in an otherwise
heterozygous genetic background. Mitotic recombination was induced in
late-stage embryos/early first instar larvae and the clones analyzed at the
adult stage. We obtained 19 MB lio-/- neuroblast clones
that include all three types of MB neurons. The drl LOF KC clones
displayed an axonal morphology that was identical to that in wild-type clones
(compare A with B in Fig. 2).
These results demonstrate that the MB defects observed in the drl LOF
individuals are not due to a cell-autonomous requirement for the drl
gene product in the KCs.
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DRL expression during embryonic brain development
We first confirmed the specificity of the anti-LIO antibody in the null
combination lio2/drlR343. No crossreaction with
another protein was detected in null individuals (compare
Fig. 3A with 3B). As the
drl LOF MB defects are already present in newly hatched larvae, we
analyzed the expression pattern of the DRL protein in the developing embryonic
brain. The protein was expressed from the onset of brain commissure formation
in a subset of axonal projections linking the two hemispheres (data not
shown). At later stages, two axonal tracts in the brain commissure expressed
DRL (see Fig. 4A-F).
Colocalization with an antibody raised against HRP, which is neuron-specific,
confirmed the neuronal identity of the DRL-expressing tracts (see
Fig. 4B,E).
In order to more clearly define the subsets of axonal tracts expressing the
DRL protein, we performed double-labeling experiments using the anti-FASII
antibody, the staining pattern of which has been previously well characterized
in the embryonic brain (Noveen et al.,
2000; Kurusu et al.,
2002). No apparent colocalization was observed (see
Fig. 4C,F). We next analyzed
the embryonic pattern of DRL expression relative to the MB primordium.
GAL4-OK107 (Connolly et al.,
1996) allows the visualization of the MB neuroblasts and siblings
from early stages of development (Adachi et
al., 2003). At late embryonic stage 16, the KC axons have already
formed the pedunculus, while the dorsal and medial lobes are not yet formed
(Fig. 4H). While the DRL
protein was not present on the KC axons, their distal tips, most probably the
growth cones, were in close contact with DRL-labeled axons
(Fig. 4H). In conclusion, our
overall results showed that the DRL protein is expressed by two commissural
tracts during embryonic brain development, and that the axonal growth cones of
embryonic MBs terminate close to these tracts in the neuropile before
extending the dorsal and medial larval lobes. A schematic
representation of MB primordia in the embryonic brain, such as
Fig. 10A of Kurusu et al.
(Kurusu et al., 2002), may
help the reader understand the relationship between commissural tracts and MB
axons.
|
). We
first confirmed that adult drlRed2/drlR343
flies display an MB phenotype identical to that of null drl flies
(data not shown). Then, we analyzed the expression pattern of the reporter
gene in the embryonic brain. At late embryonic stage 16, the
Tau-ß-galactosidase protein was detectable in neuronal cells that send
their axons along two commissural tracts (see
Fig. 5A). Colocalization
experiments demonstrated that the two commissural tracts were those expressing
the DRL protein (data not shown). Analysis of the morphology of the two
commissural tracts in drlRed2/drlR343 embryos
revealed no differences compared to heterozygous embryos (compare A with B in
Fig. 5). Finally, we showed
that FASII-expressing axonal tracts, which do not express the DRL protein,
were also unaffected by the drl LOF mutation (compare C with D and E
with F in Fig. 5). In
conclusion, our analysis failed to identify any obvious defects in the
embryonic brain of drl LOF individuals.
DRL expression in the third instar larval brain
Previous studies characterized the expression pattern of DRL protein in the
third instar larval brain, revealing the antibody pattern by enzymatic
reaction (Simon et al., 1998).
The obtained results, combined with enhancer trap data and transgenic reporter
gene constructs (Hitier et al.,
2000), showed an expression pattern in four cells located in the
interhemispheric region. The shape of these cells, as well as their lack of
ELAV immunoreactivity, led the authors to assume that they were glial cells.
The authors also described, although in less detail, additional,
uncharacterized cells that send cellular processes across the brain
commissure. In order to gain a better understanding of the drl
expression profile in the third instar larval brain, we performed a confocal
analysis using different markers. We first positively confirmed that the four
interhemispheric cells expressing ß-galactosidase in the original
enhancer-trap line lio1 are indeed glial cells, as they
express the REPO transcriptional factor (see below).
Next, we analyzed the DRL expression profile relative to the MB axonal
tracts, which were labeled by anti-FASII immunoreactivity. Our data were
consistent with previously published results: the DRL protein is expressed in
four cells located in the interhemispheric region (see
Fig. 6F), which, based on their
size and position, are most likely the same glial cells expressing the
ß-galactosidase reporter gene in the lio1
enhancer-trap line. The cellular processes of these cells enwrap the medial
larval KC lobes (see Fig.
6B,C). We confirmed with the GAL4-442 construct line,
which was previously described as being expressed in the interhemispheric
glial cells (Hitier et al.,
2000), that glial cells processes enwrap medial larval MB lobes
(not shown). Interestingly, we were able to detect DRL expression in many
cells found all over the surface of the brain (see
Fig. 6A-E). Most of them
expressed DRL in cellular processes crossing the brain commissure. This
pattern of expression is reminiscent of that described in the embryonic brain
(see above), and we speculate that these cells are neurons. No colocalization
was detectable between the DRL labeling and the FASII-positive MB axons (see
Fig. 6A-E). We also detected
intense DRL expression in the thoracic and abdominal ganglia (see
Fig. 6A-E). This expression
profile, which was stronger in the thoracic segments, is similar to that
observed in the brain, although no glial cells expressed DRL in the midline
region. Finally, strong DRL expression was observed in the optic lobe region.
In conclusion, the overall results show that the DRL protein is expressed in
more cells than previously characterized. The majority of them, very likely
neurons, send their axons across the brain commissure. Interhemispheric glial
cells also express the DRL protein, while KC axons appear to be
DRL-negative.
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) by the pan neural elav driver GAL4-C155
was able to almost completely rescue the adult MB defects in a drl
LOF genetic background (Fig.
7). The rescuing effect was directly due to the
UAS-drl construct, as the GAL4-C155 line alone had
no effect (Fig. 7). These
experiments provide evidence that the drl gene is required in
neuronal cells for correct MB axonal development, in apparent contradiction to
previously published results linking the phenotype to DRL expression in
interhemispheric glial cells (Simon et
al., 1998). In order to verify that GAL4-C155 expression
is indeed exclusively neuronal, as is commonly believed, we performed
double-labeling experiments in the third instar larval brain using a
UAS-nls-lacZ reporter and the anti-REPO antibody. Surprisingly,
although mainly expressed in REPO-negative cells, the ß-galactosidase
expression driven by the GAL4-C155 line was also detected in
REPO-positive cells located close to the brain commissure (see
Fig. 8D-F). The size and
position of these cells were similar to those in cells expressing the
ß-galactosidase reporter gene in lio1 individuals
(compare panels A-C with D-F in Fig.
8).
In order to test whether expressing drl in glial interhemispheric
cells can rescue the drl LOF MB phenotype, we used the
GAL4-442 line. Confocal analysis revealed that GAL4-442
induces expression of the GAL4 protein in more cells than previously published
(data not shown). In addition to the interhemispheric glial cells, expression
was detected in many cortical and perineural glial cells. No rescue of the
drl LOF phenotype was observed when the UAS-drl transgene
was expressed under the control of GAL4-442 (see
Fig. 7). These last results,
combined with those obtained with GAL4-C155, suggested that the
drl transgene product might need to be expressed by both neuronal and
glial cells in order to rescue the drl LOF MB defects. In order to
test this hypothesis, we characterized the expression pattern of the
GAL4-7B line, which was previously shown to be able to rescue very
well the drl LOF MB defects when combined with the UAS-drl
transgene (Moreau-Fauvarque et al.,
1998). Confocal analysis of the GAL4-7B line expression
pattern together with REPO antibody staining revealed no positive REPO cells
that also expressed the reporter gene under GAL4 control (see
Fig. 8G-I). These data suggest
that neuronal drl expression is sufficient to allow appropriate MB
development. Formally we cannot exclude that the interhemispheric glial cells
indeed have a role in the normal situation, but what the GAL4 rescue
experiments tells us is that this glial cell expression seems neither
necessary nor sufficient for appropriate MB development.
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|
). While a
mild rescue effect was, however, induced by GAL4-OK107 in combination
with the UAS-drl transgene, a similar rescue was obtained using
GAL4-OK107 alone. The obtained values were not significantly
different (P>0.5), suggesting that a dominant effect of the
OK107 insertion site might be responsible for the mild rescue (see
Fig. 7). In conclusion, our
functional rescue approach suggests that the drl LOF MB defects are
linked to a lack of expression of the drl RTK in neuronal cells that
are extrinsic to the MBs.
Role of WNT5 and its genetic interaction with the DRL receptor during MB development
As the WNT5 protein has been shown to act as a repulsive ligand for the DRL
receptor in the embryonic central nervous system (CNS)
(Yoshikawa et al., 2003), we
wondered whether WNT5 might have a role in MB development. The Wnt5
null MB phenotype was visualized directly using an anti-FASII antibody or
using the GAL4-c739 line combined with UAS-mCD8-GFP. We
consistently observed a predominant mutant phenotype characterized by an
absence of 
and ß lobes (about 75% of 30 to 50 MBs), indicating a
complete arrest of axonal growth at the level of the peduncle
(Fig. 9A). The other mutant MBs
were variably distributed among three classes: no lobes, no ß
lobes and `wild-type-looking'. Strikingly, these Wnt5 mutant
phenotypes resemble those obtained as a consequence of drl
pan-neuronal overexpression [see Fig.
2B,C and Fig. 3C of
Taillebourg et al. (Taillebourg et al.,
2005)] suggesting an antagonistic interaction between
Wnt5 and drl. To confirm that this drl
gain-of-function (GOF) phenotype is still present when the expression is
mainly restricted to the MB, we overexpressed two doses of UAS-drl
and two doses of UAS-drl
intra with
GAL4-OK107. We looked, in the two different genetic combinations with
the anti-FASII labeling, to the MB phenotype: UAS-drl: 58 MB (all
lobes missing: 0/58; some lobes missing: 2/58; no lobes missing or
wild-type-looking: 56/58), UAS-drlintra: 62 MB (all
lobes missing: 27/62; some lobes missing: 23/62; no lobes missing or
wild-type-looking: 12/62). As was already seen
(Taillebourg et al., 2005),
the GOF effect is much more effective when the RTK is deleted of its
intracellular domain. Therefore, clear MB axon pathfinding defects are
obtained when drlintra is overexpressed mainly in the
MB cells. This result is in good accordance with a titrating role of ectopic
drl receptor domain on the WNT5 ligand and renders unlikely a
negative regulation effect of ectopic drl on Wnt5
transcription, as described in the embryonic CNS
(Fradkin et al., 2004).
|
|
).
However, WNT5 is a secreted protein, and as such, removal of Wnt5
function in MB clones should not affect axonal outgrowth because these neurons
should still be able to receive WNT5 signal from neighboring MB neurons.
Wnt5400 homozygous MB neuroblast clones were obtained with
the MARCM technique. Eighteen clones were produced and all look like wild
type. An attractive hypothesis would be that axons grow under intrinsic
Wnt5 signaling during normal development and stop growing at the
interhemispheric region because WNT5 becomes trapped by the extrinsic DRL
receptor. One can propose that during the growing process, the secreted WNT5
protein is activating an MB intrinsic receptor, which may be of the Frizzled
type (Logan and Nusse, 2004)
in order to activate axonal growth. We were not able to uncover any
involvement of fz, fz2 or fz3. We looked, in different
genetic combinations with the anti-FASII labeling, to the MB phenotype.
Wnt5400/+: 60 MB (all wild type), fzh51
fz2C1/+: 70 MB (69 wild type, 1 without lobe),
Wnt5400/+; fzh51 fz2C1/+:
64 MB (62 wild type, 2 without lobe),
Wnt5400/fz3G10: 66 MB (all wild type).
The UAS-fz2GPI bears a dominant-negative form of FZ2 and was
associated with the GAL4-OK107 line and assessed with anti-FASII: 36
MB (36 wild type). Finally, we induced 13 homozygous fzh51
fz2C1 MB clones (neuroblast or multiple single-cell), which
all looked wild type, with the MARCM technique. These results indicate that
another type of receptor for this unusual Wnt5 signaling pathway
might be at work. This hypothesis also predicts that Wnt5 and
drl should genetically interact during MB development. This is indeed
the case, as drl overexpression mimicked Wnt5 mutant MB
phenotypes (see above), and reduced expression of
lio+/drl+ associated with overexpression of
Wnt5+ within the MBs gradually led to a drl-like
phenotype (Fig. 11). This last
result indicates a strong antagonistic genetic interaction between the two
proteins, where overproduction of WNT5 leads to a diminution of the activity
of DRL, very likely by a titration mechanism. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We
can hypothesize that the MB defects displayed by drl LOF adult flies
are at least partially due to aberrant early MB development. The DRL protein
is not expressed within the MB intrinsic neurons at any developmental stage
that we have analyzed. This result is strengthened by our clonal analysis
experiments, which showed that the early removal of the wild-type drl
gene in a subset of the three classes of MB intrinsic neurons does not alter
their axonal morphology. The clonal analysis results demonstrate sensu stricto
a non-cell-autonomous requirement for the drl gene in the MB
intrinsic neurons; it does not, however, completely exclude the possibility
that drl mutant clones develop properly due to the expression of the
DRL protein in MB intrinsic neurons outside the clones. This would imply that
the DRL expression level in the MBs is below the level required by the
detection method used in our study. However, restoring the expression of the
drl gene solely in a subpopulation of MB intrinsic neurons with the
GAL4-247 line, or even in most if not all MB intrinsic neurons with
GAL4-OK107 (see Fig.
7), was insufficient to rescue the MB defects induced by the
drl LOF mutation. The partial rescue obtained previously with the
GAL4-c739 line (Moreau-Fauvarque
et al., 1998) is likely to be due to some transient expression
outside the MBs during development
(Nicolai et al., 2003). The
fact that the mutant phenotype cannot be rescued by two other GAL4 lines that
are expressed either more specifically (GAL-247) or in more MB
neurons (GAL-OK107) is ruling out a role of the MB expression of
GAL-c739 in the weak rescuing effect. Based on the overall results
obtained, we favor the hypothesis that drl gene function is required
extrinsically by MBs for their proper development. Finally, the MARCM
technique allowed us to visualize the morphology of single-side median MB
axons in drl LOF individuals. This analysis revealed that the mutant
phenotype is not simply a fusion of the median contralateral lobes at the
midline, but rather a real crossing of the axons, which then intermingle with
their contralateral equivalents.
|
This neuronal hypothesis is particularly attractive when we take into
account the Wnt5 results. We tested Wnt5 mutants because
WNT5 was described as being a ligand for the DRL receptor in the ventral CNS
(Yoshikawa et al., 2003;
Fradkin et al., 2004). We
found clear MB phenotypes in Wnt5 mutant brains. The Wnt5 MB
mutant phenotype is most consistent with WNT5 being required for neurite
outgrowth. It is striking that these mutant phenotypes resemble those
described for lio+/drl+ overexpression
(Taillebourg et al., 2005)
(this study). We propose that this GOF phenotype is due to
lio+/drl+ expression within or close
to MB cells, where the ectopic DRL protein can bind to the WNT5 protein and
prevent its function. Therefore, we can propose a general model for the role
of the Wnt5-drl pair in building normal MBs: WNT5 is
expressed and required within MB cells in order to insure proper axonal
growth. One can propose that during this process the secreted WNT5 activates
an MB intrinsic receptor, which seems not to be of the fz type, in
order to activate axonal growth. When WNT5 is absent, e.g. in a Wnt5
mutant MB, then the axons fail to grow properly. In the normal situation,
these MB intrinsic axons will stop growing at the midline when they reach
extrinsic axons expressing DRL, because WNT5 is trapped by the DRL receptor.
In drl mutant individuals, however, the MB axons will continue to
grow, because WNT5 is not trapped by the DRL receptor. Although the
biochemical relationship between the ligand and receptor is conserved from the
embryonic ventral CNS to the adult brain, it should be stressed that MB
development involves neurons that express WNT5 and not DRL, which is exactly
opposite to the case in the embryo, where the mutant phenotype involves
neurons that express the drl gene and not Wnt5. This is why
drl and Wnt5 mutants have the same phenotype in the
embryonic ventral CNS but have opposite phenotypes in adult MBs.
The genetic control of brain development requires both intrinsic and extrinsic clues. The perfect crosstalk between both types of molecular information, coming from neurons of different types of brain substructures, ultimately ensures the development of a harmonious and functional brain. It is central for neurobiology to decipher these interacting and developing neuronal networks at the cellular and molecular levels. Here, we describe a clear case in which drl, a receptor tyrosine kinase, is required within the brain for the normal development of MBs, although it is neither expressed nor required intrinsically within the MB neurons. Further, we propose that the WNT5 signaling molecule is the intrinsic MB axon target that needs to interact with the extrinsic DRL receptor in order to construct proper MBs within the brain.
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ACKNOWLEDGMENTS |
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Footnotes |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 test comparisons. Only
the GAL4-c155 was able to rescue; the other GAL4 lines (247,
OK107 and 442) were unable to rescue the drl MB
phenotypes. These results indicate that the drl MB defects are linked
to the lack of expression of the drl RTK in neuronal cells extrinsic
to the MBs.
