1 Department of Biological Sciences, Stanford University, Stanford, CA 94305,
USA
2 Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto
University, Sakyo-ku, Kyoto 606-8507, Japan
3 Core Research for Evolutional Science and Technology (CREST), Japan Science
and Technology, Institute for Virus Research, Kyoto University, Kyoto
606-8507, Japan
Author for correspondence (e-mail:
lluo{at}stanford.edu)
Accepted 26 November 2002
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SUMMARY |
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Key words: Axon, Dendrite, Polarity, Cytoskeleton, Pleiotropy
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INTRODUCTION |
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In theory, forward genetic screens can be used to identify genes essential
for all aspects of neuronal morphogenesis. However, many genes important for
the morphological development of neurons may also be required for similar
processes in other cells. Thus, in a multicellular organism that is homozygous
mutant for a pleiotropic gene, defects in many cell types and developmental
processes are to be expected. It may be difficult to distinguish whether
defects in a particular cell type are caused by autonomous or non-autonomous
gene disruption. Moreover, development may be arrested in early embryonic
stages before neuronal development occurs. Screening genetic mosaics, in which
only a small fraction of cells are homozygous mutant for a gene of interest
(Xu and Rubin, 1993;
Newsome et al., 2000
), helps
overcome difficulties associated with a traditional screen.
The MARCM (mosaic analysis with a repressible cell marker) system allows
positive labeling of mutant cells in mosaic animals thus facilitating genetic
analysis of neuronal morphogenesis (Lee
and Luo, 1999). We have used the Drosophila mushroom body
(MB), the insect center for olfactory-mediated learning and memory
(Davis and Han, 1996
;
Heisenberg, 1998
), as a model
system for such studies. In Drosophila, there are three major classes
of mushroom body intrinsic neurons called Kenyon cells or MB neurons:
,
'/ß', and
/ß neurons
(Crittenden et al., 1998
). Each
hemisphere contains four MB neuroblasts, with each neuroblast giving rise to
all three major types of neurons (Ito et
al., 1997
) that are distinguished by morphological characteristics
and by birth order during development. MB neurons born in embryos and early
larvae belong to the
class; in late larvae to the
'/ß' class; and after puparium formation to the
/ß class (Lee et al.,
1999
). Each MB neuron sends an initial process that gives rise to
dendritic branches in the calyx. The MB axon then fasciculates tightly with
other MB axons in the peduncle that extends towards the anterior brain, where
each axon then bifurcates, sending one branch towards the midline and another
branch dorsally (Fig. 1A)
(Lee et al., 1999
). During
metamorphosis,
class MB neurons prune their larval-specific axon
branches and re-extend only the medial branches that give rise to the adult
lobe (Fig. 1A). MB
neurons have been used as a model system to analyze candidate genes for their
function in different aspects of neuronal morphogenesis (for reviews, see
Lee and Luo, 2001
;
Jefferis et al., 2002
).
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We describe a genetic screen to isolate genes required for neuronal morphogenesis in larval MB neurons. From examining MARCM clones generated on chromosome arm 2R in 4600 independent lines mutagenized with EMS, we identified 33 mutations that cause defects in neuroblast proliferation, cell size, membrane trafficking, and axonal and dendritic morphogenesis. Our study provides new insights into the functions of three previously identified genes and starting points to study axonal and dendritic morphogenesis further using newly identified genes.
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MATERIALS AND METHODS |
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We then screened another 3600 chromosomes using the GAL4-201Y driver,
which specifically labels MB
neurons in larvae
(Lee et al., 1999
). Males
carrying isogenized second chromosomes homozygous for FRTG13, GAL4-201Y,
UAS-mCD8-GFP were treated with 25-40 mM EMS and mated according to the
genetic scheme illustrated in Fig.
1B. After a pre-laying period of 3 days, flies were transferred to
fresh wet-yeasted vials. Eight hours later, the parents were transferred back
to their original vials and kept at 18°C for eventual recovery of
mutagenized chromosomes exhibiting MB clonal phenotypes. Twenty-four hours
later, newly hatched larvae in the new vials were heat shocked for 60 minutes
at 37°C. Brains were dissected from wandering third instar larvae and
viewed in a Nikon E-600 compound fluorescence microscope.
We used a third pan-MB GAL4 driver, GAL4-OK107
(Lee et al., 1999), to retest
putative mutants. All clones shown in this paper were visualized using the
GAL4-OK107 driver.
Deficiency mapping and complementation testing
Although MBs are not required for viability, it is likely that genes
involved in MB neuronal morphogenesis are required in other neurons that are
essential for viability. We made the assumption that mutations in such genes
would be homozygous lethal and thus amenable to complementation mapping.
Indeed the vast majority of our mutations were homozygous lethal. This allowed
us to map these mutations using the 2R deficiency kit from the Bloomington
Stock Center, followed by mapping with small deficiencies, and eventually with
candidate genes in the region of the lethal mutation. It is important to note
that there could be other background lethal mutations on the mutagenized
chromosome that are unrelated to the mutation that causes the observed
phenotype.
Non-complementation screen for new alleles of heron and
kali
To identify additional alleles of heron and kali, we
screened mutagenized lines for failure to complement the original mutants.
Males homozygous for cn, bw were mutagenized (*) as described above
and mated en masse to y, w; Pin/CyO, P[y+] virgins. F1
males of the genotype cn, bw, */CyO, P[y+] were mated to either
w; FRTG13, UAS-mCD8-GFP, hrn1/CyO or y, w; FRTG13,
GAL4-201Y, UAS-mCD8-GFP, kali1/CyO, P[y+] virgin females.
Four lines (out of 1133 tested) failed to complement hrn1. These fell into three lethal complementation groups. When recombined with FRTG13, only one complementation group containing a single allele recapitulated the phenotype of reduced cell number and overextended dendrites. We named this allele hrn2. Based on its rate of recombination with FRTG13 (12/66=18.2%), we estimate (with 95% confidence) that hrn is located between 48E and 55A. As hrn1 was complemented by all available deficiencies in that region, we presume that it is located in one of the regions not covered by the deficiency kit: 48C-48E, 50C-51A, 52F-54B or 54C-54E.
Five lines (out of 1683 lines screened) failed to complement kali1. None showed the dendritic overextension phenotype after clonal analysis. One possibility is that kali is not homozygous lethal.
Sample preparation and microscopy
Third instar larvae and adult brains were fixed, washed, antibody stained,
mounted, imaged and processed as previously described
(Lee and Luo, 1999;
Lee et al., 2000b
;
Ng et al., 2002
).
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RESULTS AND DISCUSSION |
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Three types of clones were expected, based on the neuroblast division
pattern in the MB lineage: neuroblast, two-cell and single-cell clones
(Fig. 1C). We observed all
three types of MB clones in this screen. As previously reported, Nb clones
predominated (Lee and Luo,
1999), most probably because perdurance of GAL80 protein in
two-cell or single-cell clones weakens GAL4-induced marker expression. The
majority of mutations were identified because of their phenotypes in
neuroblast clones.
Genes affecting cell number, membrane protein distribution and cell
size
The most frequently observed phenotype was a reduction of MB neurons in
neuroblast clones. Neuroblast clones generated in newly hatched larvae and
examined in wandering third instar larvae typically contain 150-200 neurons
(Fig. 2A). Frequently (about 1
in 10 mutagenized lines), we detected neuroblast clones with 50 or fewer
cells. This phenotype could be due to homozygous loss of housekeeping genes
that are required for cell division, cell survival or basic metabolic
functions in neuroblasts. Owing to the sheer number of such mutations and our
interest in more specific aspects of neuronal morphogenesis, we did not pursue
mutations whose only detectable phenotype was reduced neuroblast clone
size.
|
Three mutants were initially identified because, in addition to a severe
reduction in neuroblast clone size, MB neurons displayed little or no axonal
and dendritic projections (Table
1; Fig. 2B). Closer
examination revealed that these mutants have axonal and dendritic projections
that appear normal, but the membrane-targeted mCD8-GFP marker, which normally
labels neuronal cell body and axonal/dendritic processes equally well
(Lee and Luo, 1999), is now
concentrated in cell bodies and was distributed very weakly in axons and
dendrites. All three mutations failed to complement deficiencies that span
48E-49A and each other. Based on their nearly identical clonal phenotypes and
the lethal complementation data, we conclude that they represent three alleles
of the same gene, which we named weak processes (wkp). Wkp
is likely required for efficient transport of membrane protein into axons and
dendrites.
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We also found a number of mutants (Table 1; Fig. 2C,D) that exhibit abnormal concentration of the mCD8-GFP marker in intracellular structures (Fig. 2C,D, insets), probably reflecting defects in secretory pathways. However, the axonal and dendritic staining was stronger than in clones homozygous for wkp.
A third class of mutants exhibit abnormally large cell size in labeled
neuroblast clones in addition to a reduction in cell number
(Table 1;
Fig. 2E). Two of these mutants
failed to complement deficiencies within 49C1-50D2 and each other, and are
likely to be mutations in the same gene, which we have named amazon
(amz). Complementation tests suggest the remaining mutants may be
either single hits or else homozygous viable. Mutations of this class might
cause an increase in cell size by affecting cytokinesis, as is the case with
RhoA mutations (Lee et al.,
2000b); however, none of these mutations failed to complement
RhoA. Alternatively, they could affect genes that regulate cell size
(Conlon and Raff, 1999
).
roadblock mutations affect axonal transport, neuroblast
proliferation and dendritic branching
We identified three mutants that exhibit a `spotty axon' phenotype:
neuroblast or single-cell clones homozygous for these mutations have periodic
swellings along their axons or near the axon terminals
(Fig. 3A). These defects are
reminiscent of mutations that affect axonal transport, such as
microtubule-based motor protein kinesins
(Goldstein and Yang, 2000). We
have previously shown that MB neurons homozygous mutant for genes encoding
dynein heavy chain (Dhc64C) exhibit similar axon swellings
(Liu et al., 2000
). Similar
phenotypes were also seen in MB neurons homozygous mutant for Lis1
(Liu et al., 2000
), the
Drosophila homolog of human LIS1 that has been shown to be
associated with the dynein complex
(Faulkner et al., 2000
;
Smith et al., 2000
).
Haplo-insufficiency of human LIS1 causes lissencephaly or smooth
brain (reviewed by Reiner,
2000
). Dhc64C and Lis1 mutations also produce
defects in neuroblast proliferation (Liu
et al., 2000
). Interestingly, one of the new spotty axon mutants,
7A11, also has similar phenotypes. Additionally, neuroblast clones
have reduced cell number compared with wild type (
50 in third instar
larva, Fig. 3A). When examined
in adults, neuroblast clones homozygous for 7A11 are composed largely
of
neurons, the first-born class of MB neurons
(Fig. 1A). They also have few
'/ß' neurons as revealed by double labeling with
FasII, which labels
/ß neurons strongly and
neurons
weakly, and does not label
'/ß' neurons
(Lee et al., 1999
)
(Fig. 3B). These observations
suggest that the reduced size of neuroblast clones is probably caused by a
cessation of neuroblast cell division, such that only early born neurons are
generated (see Liu et al.,
2000
).
|
Deficiency mapping for 7A11 uncovered two lethal mutations at
46E1-F2 and 54C1-4. Of the 29 lethal complementation groups from a previous
saturation mutagenesis in the 46C-F region
(Goldstein et al., 2001), two
alleles in group W failed to complement 7A11. However, when
recombined with FRTG13, they did not exhibit a clonal phenotype in MB
neurons. Within 54C1-4, we found roadblock (robl), which
encodes a dynein-light chain shown to be required for axonal transport and
mitosis (Bowman et al., 1999
).
Given the phenotypic similarity between 7A11 and
Lis1/Dynein heavy chain, we tested known robl
mutants (Bowman et al., 1999
)
and found that two loss-of-function alleles
(roblB and roblZ)
failed to complement 7A11. MARCM clones for both alleles also showed
reduced cell number and spotty axons at the larval and adult stages
(Fig. 3D,E). Both alleles have
stronger phenotypes than 7A11 in that neuroblast clone size is
reduced further and the severity of axonal swellings is greater. We therefore
concluded that the 7A11 phenotypes we observe are due to a
hypomorphic mutation in the robl gene, and renamed our mutation
roblMB.
In addition to affecting neuroblast proliferation and axonal transport,
Lis1 and Dynein heavy chain mutants also cause defects in dendritic growth and
branching of MB neurons (Liu et al.,
2000). To examine whether robl also affects dendritic
morphogenesis, we quantified dendritic length and branching points in
single-cell clones homozygous for roblZ in adult.
Compared with wild type, we found a twofold reduction in both total dendritic
length [roblz: 54±11 µm, n=4;
wild type: 106±11 µm, n=6; P=0.0061
(t-test)] and branching points [roblz:
6.0±0.9, n=4; wild type: 12±1.7, n=6;
P=0.0187 (t-test)], demonstrating that, like Lis1
and Dhc64C (Liu et al.,
2000
), robl is cell-autonomously required for dendritic
branching and growth.
Although dynein heavy chain and Lis1 exhibit very similar phenotypes,
consistent with their forming a complex involved in dynein-mediated function
(Reiner, 2000), we have
previously found that there was a subtle difference in the axonal transport
phenotype. Although the axon swellings are concentrated at the distal ends of
axon branches in Dhc64C mutant MB neurons, consistent with dynein
mediating retrograde axonal transport, swellings in Lis1 mutant
neurons occur along the entire axon (Liu
et al., 2000
). Axon swellings in single-cell clones of
robl mutants occur preferentially at the distal ends of axon branches
(Fig. 3G, compared with 3F).
These observations suggest that the function of robl is more central
to the function of cytoplasmic dynein, whereas Lis1 may play
additional/different functions.
Future phenotypic analysis of two other mutants that exhibit spotty axon phenotypes (Table 1) combined with identification of the corresponding genes may contribute to our understanding of mechanisms of axonal transport, and may elucidate additional components of the Lis1 pathway, which is important for the patterning of the human cerebral cortex.
short stop mutations disrupt neuronal polarity
A number of mutants were identified based on their phenotypes in axonal and
dendritic morphogenesis (Table
1). Three mutations share similar phenotypes: MB axon staining
becomes progressively weaker further from the cell bodies, suggesting defects
in axonal extension. In addition, there are abnormal processes projecting out
from the dendritic fields of MB neurons (the MB calyx)
(Fig. 4A). These phenotypes are
reminiscent of the first characterized mutation with the MARCM method in MB
neurons, short stop (shot)
(Lee and Luo, 1999). Indeed
all three mutations failed to complement a deficiency that uncovers
shot; they also failed to complement each other and
shot3. Based on these genetic criteria and their
phenotypic similarity, we conclude that we have identified three new alleles
of shot, which we named shot6-8.
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Adult neuroblast shot3 clones also displayed
abnormal processes projecting out from the calyx
(Fig. 4B). They appear to
follow a curved route towards the antennal lobe, mimicking the trajectory of
the inner antennal cerebral tract (iACT), which contains the axons of a large
subset of projection neurons, the major input to the MB dendrites
(Stocker et al., 1990;
Jefferis et al., 2001
).
In addition to these overextension phenotypes, neuroblast clones homozygous
for shot3
(Fig. 4B), as well as for the
alleles we identified (data not shown) exhibit significantly reduced cell
number. Most or all neurons are neurons, as their axons project to the
lobe, suggesting a defect in the continuous generation of new neurons
from the neuroblast.
Originally identified as a mutation in which embryonic motoneurons fail to
reach their targets (Van Vactor et al.,
1993), shot has subsequently been found to affect CNS and
PNS axon growth (Kolodziej et al.,
1995
; Lee et al.,
2000a
) and dendritic morphogenesis
(Gao et al., 1999
;
Prokop et al., 1998
) in
Drosophila embryos. It also is required for morphogenesis of other
embryonic and imaginal epithelial and mesodermal tissues
(Gregory and Brown, 1998
;
Lee and Kolodziej, 2002a
;
Strumpf and Volk, 1998
).
shot is allelic to kakapo, which encodes a large
cytoskeletal linker protein similar to vertebrate plakin
(Gregory and Brown, 1998
;
Lee et al., 2000a
;
Strumpf and Volk, 1998
).
Recent structure-function analysis suggests that the actin and
microtubule-binding domains must be present on the same molecule for Shot to
function in axon extension (Lee and
Kolodziej, 2002b
).
We originally characterized shot phenotypes in larval MB neuronal
morphogenesis based on defects in axon fasciculation and misguidance, as many
`axons' project out of the calyx, rather than following the peduncles
(Lee and Luo, 1999). In light
of the finding that shot also affects dendritic development in the
embryonic PNS and CNS (Gao et al.,
1999
; Prokop et al.,
1998
), we re-examined the processes projecting from the calyx. A
fusion protein made of a microtubule motor Nod and ß-galactosidase
(Nod-ßgal) is highly enriched in MB dendrites and their tips but largely
absent from axons (Lee et al.,
2000b
) (Fig. 4D).
We found that the processes projecting out of the calyx stained strongly for
Nod-ßgal (Fig. 4C).
Strikingly, however, in shot neuroblast clones, axons that follow the
normal route through the peduncle are also strongly labeled for Nod-ßgal
(Fig. 4C). Sometimes the
Nod-ßgal fusion protein is present in both the dorsal and medial lobes
all the way to their terminals (Fig.
4C,E), despite the fact that total axon staining become
progressively weaker as they are further from the cell bodies (reflecting axon
growth defects). This is in contrast to wild type, where Nod-ßgal
staining rapidly diminishes along the axonal peduncle
(Fig. 4D,E)
(Lee et al., 2000b
). These
observations suggest that neuronal polarity, as measured by the microtubule
polarity in axons and dendrites, is perturbed in shot mutants.
In neurons, dendrites normally collect input and axons send output. As
such, there are a number of distinctions between these two neuronal processes
structurally, functionally and developmentally
(Craig and Banker, 1994). In
particular, differences in microtubule polarity have been suggested as a
hallmark between axons and dendrites. In a number of vertebrate neurons
studied so far, microtubules in axons uniformly orient with their plus end
pointing distally. By contrast, microtubules in dendrites have both
plus-end-distal and plus-end-proximal orientations, the latter population
gradually diminishing further away from the cell body
(Baas, 1999
;
Craig and Banker, 1994
).
Although direct evidence of similar microtubule polarity in invertebrate
neurons is lacking, our studies provide two lines of indirect evidence that
microtubule distribution in MB neurons is similar to mammalian neurons. First,
Nod, a likely minus-end-directed microtubule motor
(Clark et al., 1997
), when
fused with ß-gal is highly enriched in MB dendrites but largely absent in
MB axons (Lee et al., 2000b
)
(Fig. 4D). This observation is
consistent with the notion that in MB dendrites microtubules are
bi-directional, so a minus-end-directed reporter can enter efficiently into
the most distal parts of the dendrites, but cannot be efficiently transported
along the axons if microtubules are oriented only plus-end distally. Second,
dynein is known to be a minus-end-directed microtubule motor. We observed
that, in Dhc64C mutants (Liu et
al., 2000
) and now in robl mutants
(Fig. 3), axon swellings are
preferentially located at the distal tips of MB axons. This is consistent with
the notion that in MB axons microtubules are distributed with their plus end
pointing distally.
Little is known about the establishment of microtubule polarity differences
in axons and dendrites during development. Recent experiments in hippocampal
cultured neurons have indicated the importance of polarized microtubule
distribution in neuronal polarity development and maintenance. A kinesin
superfamily member, CHO1/MKLP1, is distributed in dendrites and has the
ability to transport minus-end distal microtubules in the dendrites.
Disruption of CHO1/MKLP1 by antisense oligonucleotides resulted in failure of
dendritic differentiation in young hippocampal neurons
(Sharp et al., 1997), and
conversion of dendritic processes into axon-like processes in mature neurons
(Yu et al., 2000
).
shot mutants appear to have the opposite phenotype: conveying
specific dendritic properties to axonal compartments. It will be very
interesting in the future to determine how this microtubule/actin cytoskeletal
linker protein regulates neuronal polarity in conjunction with proteins such
as CHO1/MKLP1.
fmi mutants overextend processes from the dendritic
field
We isolated a mutant, 39B17, in which many processes extend beyond
the typical MB dendritic field, often as far as the axon lobes. We quantified
the number of short and long (defined as less or more than one calyx diameter,
respectively) overextended processes in 39B17 and for wild type
(Table 2). Neuroblast clones
homozygous for 39B17 have a marked increase of long over-extended
processes. Overextended processes from the calyx are also evident in adult,
projecting along similar tracks as the iACT as in the case of shot
mutant neurons (data not shown; see below). Mutant neuroblast clones also have
fewer cells than wild type, which becomes more obvious in adult clones. These
clones contain neither nor
' dorsal lobes, indicating an
arrest of neuroblast proliferation before the generation of
'/ß' neurons (data not shown; see below).
|
Deficiency mapping uncovered a lethal mutation in the 47A1-47D2 region for
39B17. We tested our mutation for complementation against a mutant
allele of flamingo (fmi), also known as starry
night, which encodes a seven transmembrane cadherin
(Usui et al., 1999;
Chae et al., 1999
).
Loss-of-function mutations of fmi exhibit defects in planar polarity
(Usui et al., 1999
;
Chae et al., 1999
) and
excessive dendritic outgrowth and misguidance in embryonic sensory neurons
(Gao et al., 1999
;
Sweeney et al., 2002
).
39B17 failed to complement fmiE59, which
has a stop codon early in the extracellular domain and is believed to be a
null allele (Usui et al.,
1999
). Two additional lines of evidence demonstrate that the
overextension phenotype in 39B17 is due to a mutation in
fmi. First, MARCM clones of fmiE59 also
exhibited phenotypes of process overextension and reduction of neuroblast
clone size similar to that of 39B17
(Fig. 4D; Table 2). Second, using MARCM,
we created MB clones homozygous mutant for
fmi39B17 or fmiE59
in which a full-length fmi cDNA was also expressed under the control
of UAS; this UAS-fmi expression was able to rescue the process
overextension and cell number reduction phenotypes in third instar larvae and
adults (Fig. 5E,F; Table 2). Thus, our mutation is
an allele of flamingo and we named this allele
fmiMB.
|
To determine the nature of the overextended processes, we constructed flies carrying fmiMB/E59 and UAS-Nod-ßgal, and made clones using the MARCM system. Nod-ßgal staining is observed in a subset of overextended processes (Fig. 5C; data not shown), suggesting that a portion of the overextended processes are dendrites. The remaining overextensions are either misguided axons in the dendritic field or dendrites in which Nod-ßgal transport was inefficient.
Fmi has recently been shown to regulate dendritic extension in embryonic
and larval sensory neurons (Gao et al.,
1999; Gao et al.,
2000
; Sweeney et al.,
2002
). fmi mutant sensory neurons extend their dorsal
dendrites beyond their normal territory. Although dorsal dendrites from
homologous neurons appear to repel each other at the dorsal midline in wild
type, they do not do so in fmi mutants
(Gao et al., 2000
). Our data
extend these previous findings into dendrites of CNS neurons and suggest a
general function for fmi in regulating dendritic extension.
Fmi overexpression results in axon retraction
While neuroblast clones expressing wild-type Fmi do not exhibit any
phenotypes and could indeed rescue the fmi mutant phenotypes
(Fig. 5E,F), we found that
whole MB overexpression of Fmi using GAL4-OK107 results in loss of the dorsal
branches of axons when examined in adult
(Fig. 6B, compare with
Fig. 6A). FasII staining, which
allows us to distinguish the three classes of MB neurons (see above), suggests
that ß, ß' and lobes are present when Fmi is expressed
in all MB neurons. Coupled with the lack of cell loss, we suspect that either
the
and
' axons fail to extend dorsally, or they extend
and retract, as is the case for MB neurons expressing double-stranded RNA
corresponding to Drosophila p190 RhoGAP
(Billuart et al., 2001
).
|
To distinguish between these two possibilities, we performed a
developmental study and found a progressive worsening of the phenotype. High
level expression of Fmi in all MB neurons does not result in any detectable
phenotypes in wandering third instar larvae
(Fig. 6C). At 18 hours after
puparium formation, wild-type MB neurons undergo pruning whereas
'/ß' neurons retain their larval branches including
the dorsal
' lobe (Fig.
1A). All MBs overexpressing Fmi retain at least a portion of the
dorsal lobes, with 63% more than half the length of the normal dorsal lobe and
25% full length, indicating that at least 25% and perhaps all
'/ß' axons extend normally
(Fig. 6D,G). Over the next
12-24 hours, dorsal lobes become progressively shorter until they are not
detectable at 48 hours after puparium formation
(Fig. 6E-G). Although failure
of dorsal lobe extension could in theory also contribute to the phenotypes,
these developmental studies indicate that dorsal lobe phenotypes mainly result
from axon retraction.
These phenotypes are qualitatively similar to (albeit stronger than)
inhibition of p190 RhoGAP (Billuart et al.,
2001), which we have previously shown to be caused by activation
of RhoA, Drok and phosphorylation of myosin regulatory light change encoded by
spaghetti squash (sqh)
(Billuart et al., 2001
). We
tested whether Fmi may signal through the RhoA/Drok/Sqh pathway. Despite
considerable efforts, however, our biochemical studies and genetic interaction
experiments failed to provide such a link (A. P., E. K. S. and L. L.,
unpublished).
The opposite phenotypes (overextension versus retraction) observed in fmi mutant and Fmi overexpression neurons suggest a general role for Fmi as a negative regulator of neuronal process extension.
Two novel genes that affect axonal and dendritic morphogenesis
One mutant, 13B44, exhibited remarkable phenotypic similarities to
fmi in many respects. Neuroblast clones homozygous for 13B44
consistently extend their processes out of the calyx and follow the typical
arc-like projection (Fig. 7A)
found in fmi neuroblast clones
(Fig. 5A;
Table 2). Nod-ß-gal
staining indicates that this fusion protein is present in the proximal part of
some but not all of the overextending processes
(Fig. 7B). In addition, we
observed a mild reduction of cell number in larval neuroblast clones
(Fig. 7A); adult neuroblast
clones consist of mainly early born neurons, with a few
'/ß' neurons (Fig.
7B).
|
We recovered a second allele (see Materials and Methods) that exhibited identical clonal phenotypes as the original 13B44 allele (Fig. 7A; Table 2). No deficiency uncovered either allele. We named this new gene heron (hrn) for its phenotypic similarity with flamingo (stan FlyBase).
Another mutant, 41A13, exhibited a 100% penetrant dendritic
overextension phenotype. Unlike flamingo or hrn mutants,
these overextended dendrites project in all directions and are always strongly
positive for Nod-ßgal (Fig.
7C,D), highly reminiscent of clonal phenotypes for the small
GTPase RhoA (Lee et al.,
2000b). A complementation screen identified a number of mutations
that failed to complement kali1 but that did not
reproduce the kali1 clonal phenotype.
Future identification of the molecular identity of hrn and kali, as well as studies of their mechanisms of action including their relationship with Flamingo and RhoA, will further our understanding of the mechanisms that regulate dendritic extension and dendritic field formation.
Pleiotropy of gene function in neuronal morphogenesis
In this study, we screened labeled MB clones in 4600 mutagenized lines on
chromosome 2R. The majority of mutations we identified are single alleles
(Table 1), indicating that the
screen is not saturated. However, we did identify multiple alleles for several
genes, suggesting that we have sampled a significant proportion of the
20% of the fly genes located on this chromosome arm.
The ability to visualize mutant cells in a mosaic animal using a method such as MARCM provides great sensitivity in detecting morphological phenotypes. The complex and stereotypical morphogenetic programs of MB neurons further allow us to study different aspects of neuronal morphogenesis. The nature of our mosaic screen allows for identification of pleiotropic genes important for a specific biological process. Interestingly, almost all genes we identified perform multiple functions. All mutations we identified, for example, have reduced cell number in homozygous neuroblast clones examined in adult. Although some could be due to multiple mutations generated on the same chromosome arm, in many cases this is unlikely as the same phenotype is also seen in different alleles in distinct genetic backgrounds, and in one case (flamingo) all phenotypes can be rescued by supplying the wild-type transgene. These observations imply that many genes used in the morphological development of MB neurons (and likely other neurons) are used in multiple developmental processes rather than each gene having one specific, dedicated function.
Although mosaic analysis allowed us to identify genes with pleiotropic functions, at the same time the pleiotropy posed a limitation. If the candidate gene is required for and presumably expressed in neural precursors, homozygous mutant clones will inherit the wild-type protein from their heterozygous precursors so they will not lose the gene activity immediately. This perdurance of wild-type protein could prevent identification of genes that function in the early stages of neuronal morphogenesis, such as in the establishment of neuronal polarity or initial axon outgrowth. Perdurance of gene activity could also explain why most of our mutations were isolated based on their defects in neuroblast clones, which presumably dilute the inherited proteins much more rapidly than do single-cell clones.
Hence, it is possible that the nature of our screen precludes identification of proteins that are expressed in neural precursors and are required for early stages of neuronal development. A priori we expected that some genes required for early neuronal differentiation would be turned on only in postmitotic neurons, negating perdurance issues. Given the scale of our screen and the fact that all the mutations we identified appear to play a role in neuroblast proliferation or survival, it seems likely that most of the genes necessary for differentiation in postmitotic neurons also function in neural precursors. Some of these genes in fact play multiple functions in postmitotic neurons (Table 1). These observations make it essential to couple mosaic analysis such as MARCM with careful phenotypic analysis to unravel the complex process of neuronal morphogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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