* Department of Zoology and Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom
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Abstract |
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The lethal mutation l(2)CA4 causes specific
defects in local growth of neuronal processes. We uncovered four alleles of l(2)CA4 and mapped it to bands
50A-C on the polytene chromosomes and found it to be
allelic to kakapo (Prout et al. 1997. Genetics. 146:275- 285). In embryos carrying our kakapo mutant alleles,
motorneurons form correct nerve branches, showing
that long distance growth of neuronal processes is unaffected. However, neuromuscular junctions (NMJs) fail
to form normal local arbors on their target muscles and
are significantly reduced in size. In agreement with this finding, antibodies against kakapo (Gregory and
Brown. 1998. J. Cell Biol. 143:1271-1282) detect a specific epitope at all or most Drosophila NMJs. Within
the central nervous system of kakapo mutant embryos,
neuronal dendrites of the RP3 motorneuron form at
correct positions, but are significantly reduced in size.
At the subcellular level we demonstrate two phenotypes potentially responsible for the defects in neuronal
branching: first, transmembrane proteins, which can
play important roles in neuronal growth regulation, are
incorrectly localized along neuronal processes. Second,
microtubules play an important role in neuronal
growth, and kakapo appears to be required for their organization in certain ectodermal cells: On the one hand,
kakapo mutant embryos exhibit impaired microtubule organization within epidermal cells leading to detachment of muscles from the cuticle. On the other, a specific type of sensory neuron (scolopidial neurons)
shows defects in microtubule organization and detaches
from its support cells.
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Introduction |
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THE ability to form synaptic contacts is a fundamental property of developing neurons. To build a
neural network, neurons send out processes in a
regulated manner, initially by long-distance growth into
defined target areas, followed by recognition and contact
of target cells within that region. This final growth phase is
characterized by local sprouting of neurites in the target areas and terminal arborization on the surface of target
cells, and appears to require mechanisms different from
those underlying long distance growth (Caroni, 1997). Although it is well established that the development of neuronal circuits depends on a sequence of precisely regulated
growth events, the underlying mechanisms are still poorly
understood.
Neurite growth is carried out by specialized structures,
growth cones, that are formed either at the distal end of
the neurite or by budding off from already established axons. Growth cones respond to diffusible and contact-mediated signals in their environment, which can be attractive
or repulsive, pulling and pushing the growth cone along its
path (Tessier-Lavigne and Goodman, 1996). Such extrinsic information can be used not only for guidance of growth cones, but also to change and regulate their growth
behavior. For example, in vertebrates, agrin released from
muscles downregulates long-distance growth of incoming
growth cones and upregulates their ability to form terminal branches and to differentiate synapses (Ruegg and
Bixby, 1998
). Furthermore, neuronal growth is regulated by intrinsic properties of the growing neuron. For example, in crayfish different motorneuronal terminals arborize
in the absence of target muscles in culture, and the degree
to which they arborize is neuron-specific and reminiscent
of their growth behavior in vivo (Arcaro and Lnenicka,
1995
). Similar intrinsic determination of the size of neuronal terminals has been demonstrated in vivo for sensory
neurons of crickets or Drosophila (Murphey and Lemere, 1984
; Canal et al., 1998
). Also in vertebrates, graft experiments suggest that certain growth properties, such as the
length to which axons extend, can be crucially dependent
on intrinsic cues (Caroni, 1997
). Thus, neuronal growth is
regulated by a combination of extrinsic signals and intrinsic properties of the growing neuron.
Migrating growth cones extend filopodia which are
filled with actin bundles, and distinct changes in the actin
cytoskeleton cause newly assembling microtubules to accumulate at the base of these filopodia, consolidating a
new part of the axon or dendrite (Bentley and O'Connor,
1994; Smith, 1994
). The molecular machinery which intrinsically regulates these events comprises (a) the cytoskeletal components tubulin and actin, (b) components associating
with these cytoskeletal components, e.g., microtubule-
associated proteins, (c) transmembrane molecules involved in adhesion and signaling, (d) components of second messenger pathways, e.g., Gap-43 and Cap-23, Cdc42,
Rac, Rho, and (e) proteins involved in anterograde and
retrograde transport such as nonmuscle myosin or the
product of the Drosophila glued gene (Landmesser et al.,
1990
; Avila et al., 1994
; Nobes and Hall, 1995
; Caroni,
1997
; Reddy et al., 1997
; Suter and Forscher, 1998
). Insights into the function of some of these components give
first explanations for how neuronal growth can be regulated and subdivided into different growth phases. For example, repressing tau function (a microtubule-associated protein) suppresses the formation of axons (Caceres et al.,
1992
) whereas MAP2 (another microtubule-associated
protein) or CAP-23 and GAP-43 proteins appear to function specifically in local sprouting events but not in long-distance growth (Caceres et al., 1991
; Dinsmore and
Solomon, 1991
; Caroni, 1997
).
Here we report the isolation and phenotypic characterization of a paralytic mutation in Drosophila, l(2)CA4,
which affects local neuronal growth. l(2)CA4 turned out to
be allelic to kakapo (kak; Prout et al., 1997). The kak mutation affects terminal branch formation of embryonic motorneurons on muscle surfaces and local sprouting of their
dendrites in the central nervous system (CNS).1 However,
long-distance growth of axons appears unaffected in kak
mutant embryos. We demonstrate that kak is required for
(a) the restricted localization of membrane proteins along
axons and (b) for the organization of the microtubule cytoskeleton in scolopidial sensory neurons and epidermal
cells. Loss of these types of function could account for kak
mutant phenotypes in local neuronal growth. The phenotypes reported here are in good agreement with the finding that kak encodes a potential actin binding cytoskeletal
element (Gregory and Brown, 1998
; Strumpf and Volk,
1998
).
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Materials and Methods |
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Fly Stocks and Genetic Mapping of kak
The kakapo (kak) alleles kak91K, kakel3, kakHG25, and kakSF20 were discovered as second-site lethals on chromosomes isolated from four independent ethylmethane sulfonate (EMS) mutagenesis experiments which were designed to recover new lethal and visible mutations in the Adh region.
kakel3 was found on the elbow3 chromosome (Ashburner et al., 1980),
kakSF20 on wingblisterSF20 (Ashburner et al., 1980
), kak91k on l(2)35Fc91k
(Ashburner, M., and J. Roote, unpublished data), and kakHG25 was isolated in a screen for new alleles of wingblister in which mutagenized chromosomes were screened over wingblisterSF20 (Ashburner, M., and J. Roote, unpublished data). The gene responsible for this unmapped lethality was designated l(2)CA4 and now named kakapo.
Meiotic mapping using the multiple-marked chromosome al dp b pr c
px sp located kak 17.5 map U to the right of pr, 20 map U to the left of px,
and 1.5 map U on either side of c (data not shown) i.e., on chromosome
arm 2R, within bands 50-53 of the polytene chromosomes. This location
was confirmed and refined when it was discovered that the kak alleles
were lethal with Df(2R)CX1 [Df(2R)49D1; 50D1] and Df(2R)MK1
[Df(2R)50B3-5; 50D1-4; Strumpf and Volk, 1998] but not Df(2R)50C-38
[Df(2R)50C; 50D] or Df(2R)50C-101 [Df(2R)50C; 50D; Preston et al.,
1996
], Df(2R)vg-B [Df(2R)49D3-4; 49F15-50A3], Df(2R)vg-C [Df(2R)49A4-13; 49E7-F1] or Df(2R)vg-D [Df(2R)49C1-2; 49E2-6]. The haplo-lethal
deletion segregant from the transposition Tp(2;3)6r35 [Tp(2;3)50A1-15;
50E1-50F9; 84D1-84D14; Eberl et al., 1989
], i.e., Df(2R)6r35 [Df(2R)50A1-15; 50E1-50F9], does not complement kak and shows the typical kak mutant neuromuscular and muscle phenotypes in embryos when heterozygous with kak alleles. The duplication segregant from Tp(2;3)6r35, Dp(2;
3)6r35 [Dp(2;3)50A1-15; 50E1-50F9; 84D1-14] is homozygous lethal but,
in heterozygosis, completely rescues the lethality and phenotype of kak
transheterozygotes, e.g., kakel3/kakSF20; Dp(2;3)6r35 flies are viable and
phenotypically wild-type. Taken together, these data place the kak locus
in the interval 50A to 50C.
Immunohistochemical Methods
Antibody stainings were carried out using standard techniques (Prokop et al.,
1996), at stage 16 on whole mounts and at stage 17 on embryos dissected
flat with the help of histoacryl glue (Braun, Melsungen, Germany). At
stages 16 and 17, mutant embryos were identified with the help of CyO
balancer chromosomes expressing lacZ, at stage 17 also by paralysis and
the typical muscle detachment phenotype. FasIIeb112 mutant embryos were
identified by lack of anti-FasII staining. Embryos carrying four copies of
kak were collected from a y;Dp(2;3)6r35/TM6,y+ strain and identified by
their yellow mouthhooks.
As antibody probes we used anti-FasII 1D4 2F3 (mouse monoclonal;
1:20) (Van Vactor et al., 1993), anti-Fas III (mouse monoclonal; 1:4)
(Halpern et al., 1991
); anti-cysteine string protein (mouse polyclonal; 1:10)
(Zinsmaier et al., 1994
), anti-synaptotagmin (rabbit polyclonal; 1:1,000)
(Littleton et al., 1993
), anti-
-adaptin (rabbit polyclonal; 1:200) (González-Gaitán and Jäckle, 1997
), and 22C10 (mouse monoclonal; 1:10)
(Fujita et al., 1982
). For stainings with anti-kakapo antibody (rabbit polyclonal; 1:20) (Gregory and Brown, 1998
) flat dissected embryos were fixed
for 2 min in 0.25% glutaraldehyde and mildly blocked for 10 min in 10%
calf serum in phosphate-buffered saline containing 0.1% Triton-X 100.
Stage 16 embryos were either transferred to araldite and sucked as
whole mounts into borosilicate capillaries (Hilgenberg, Malsfeld, Germany) (Prokop and Technau, 1993), or they were transferred to 70% glycerol and dissected flat thereafter. Stage 17 flat preparations were dehydrated and covered with araldite, cut off the glass with a razor blade
splinter, and then the mount was embedded under a coverslip. Images
were scanned directly from the microscope via a video camera (Kontron
Elektronik ProgRes 3012; Eching/Munich, Germany). For clarity, different focal planes were combined into one picture using Photoshop 4.0 software (Adobe Systems, Mountain View, CA). The significance of measurements was tested with a nonparametric Mann-Whitney-U test using
StatView software. Neuropile and fascicle areas (see Fig. 5, A and B) were
measured on scanned drawings using the Histogram function within the
Photoshop 4.0 software.
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DiI Labeling and Analysis
Stage 16 embryos were dissected on poly-L-lysine-coated coverslips and
stage 17 embryos were dissected flat on a layer of Sylgard (Dow Corning
Corp., Midland, MI) using Histoacryl glue (Braun, Melsungen, Germany)
(Broadie and Bate, 1993). DiI labeling was carried out as described elsewhere (Landgraf et al., 1997
). In brief, embryos were treated with 0.2 mg/
ml collagenase IV (Sigma Chemical Co., St. Louis, MO) in saline for 1.5 min, rinsed with saline, fixed with 3.7% formaldehyde in saline for 2.5 min, and then rinsed with saline once more. DiI (Molecular Probes, Eugene, OR) was dissolved in vegetable oil and backfilled into sharpened
glass capillaries which were then bevelled. A small droplet of DiI was deposited on the cleft between muscle VL3/4 (muscle nomenclature according to Bate, 1993
) and left to diffuse overnight at 4°C. Labeled neurons
were then either photoconverted or were scanned at 0.5-µm steps on a
Bio-Rad 1024 confocal microscope (Hercules, CA). Confocal images were
projected and analyzed using NIH image (Bethesda, MD). Tracings of
photoconverted preparations were made using a Zeiss Axiophot (Carl
Zeiss Inc., Thornwood, NY) attached to a video monitor and data
analyzed with a two-sample, unpaired t test using Minitab software (Coventry, UK).
Electrophysiology
Whole-cell recordings of muscles VL3/4 in wild-type and kak mutant embryos at late stage 17 were made using standard patch-clamp techniques
and solutions (Broadie and Bate, 1993). Muscles were voltage-clamped at
60 mV. Signals were amplified using an Axopatch-1D amplifier (Axon
Instruments, Foster City, CA), filtered at 2 or 10 KHz and analyzed using
Axotape software (Axon Instruments). Series resistance was 16-22
MOhm, electrode resistance was ~5 MOhm, and capacitance was 18-27 pF.
Electron Microscopy
Ultrastructural analyses were carried out as described previously (Prokop
et al., 1996, 1998
). In brief, embryos were injected with 5% glutaraldehyde
in 0.05 M phosphate buffer, pH 7.2, the injected specimens were cut open
at their tips with a razor blade splinter, postfixed for 30-60 min in 2.5%
glutaraldehyde in 0.05 M phosphate buffer, briefly washed in 0.05 M phosphate buffer, fixed for 1 h in aqueous 1% osmium solution, briefly washed
in dH2O, treated en bloc with an aqueous 2% solution of uranyl acetate for 30 min, dehydrated, and then transferred to araldite. Serial sections of
30-50 nm (silver-grey) thickness were transferred to formvar-covered carbon-coated slot grids, poststained with lead citrate for 5-10 min, and then
examined on a JEOL 200CX (Peabody, MA) or Hitachi H600 (Tokyo, Japan). Transverse serial thin sections were taken ~10-15 µm behind the
anterior border of the denticle belts, which can be visualized in semithin
sections with the light microscope.
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Results |
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The kak Mutant Alleles Affect Size and Shape of Neuromuscular Junctions, but Neuromuscular Synapses Can Form
We uncovered four lethal alleles of the gene l(2)CA4 as
second-site lethals from four independent EMS mutageneses and mapped them genetically to the cytological location 50A-C on the right arm of chromosome 2 (Materials
and Methods). In subsequent complementation tests our
l(2)CA4 alleles failed to complement the lethal phenotype of kakapoV168 and kakapol(2)k03405 mutant fly strains (Gregory and Brown, 1998), demonstrating that l(2)CA4 belongs to the kakapo (kak) complementation group (Prout et al., 1997
). All four alleles (kak91k, kakel3, kakHG25, and
kakSF20) are embryonic lethals, they fail to complement
each other, and show a paralytic phenotype when homozygous, transheterozygous or hemizygous over deficiencies
(Materials and Methods). Paralysis might be caused by
dysfunction or developmental defects in the nervous system or in the musculature. In kakapo mutant embryos we
find defects in both tissues: muscles detach from the epidermis in all alleles (see later), and we consistently find a
reduction in the size of motorneuronal terminals on muscles and of neuronal branches in the CNS at late stage 17.
We first describe the neuromuscular junction (NMJ)
phenotype. In wild-type embryos at stage 17, motorneuronal terminals have branches on their target muscles with
varicosities (boutons) of up to 1 µm in diameter (Broadie
and Bate, 1993; Yoshihara et al., 1997
). We visualized boutons with antibodies raised against synaptotagmin, cysteine string protein, or
-adaptin, proteins involved in fusion or recycling of synaptic vesicles (Fig. 1, A, C, and E)
(Littleton et al., 1993
; Zinsmaier et al., 1994
; González-Gaitán and Jäckle, 1997
). In kak mutant embryos NMJs in
all locations occupy far less surface of their respective
muscles, their branches are reduced in length, and boutons
appear reduced in number and size (tested allelic combinations: SF20/SF20, el3/el3, 91k/91k, SF20/el3, SF20/91k, 91k/HG25, SF20/Df(2R)6r35, SF20/Df(2R)MK1, el3/Df(2R)-
6r35, el3/Df(2R)CX1, 91k/Df(2R)MK1, HG25/Df(2R)-
MK1). Whereas some allelic combinations exhibit an
almost complete absence of NMJs (Fig. 1 F), other combinations show less severe phenotypes (Fig. 1, B and D), but
their phenotype is nevertheless significant (e.g., relation of
NMJ length to muscle length on muscles VL3 and 4 in
central abdominal segments is 44 ± 8% in controls, n = 18, and 28 ± 10% in SF20/91k, SF20/el3, and el3/Df(2R)-
6r35, n = 38; P = 0.0001). Although NMJs are severely reduced in kak mutant embryos, presynaptic marker expression is mainly restricted to neuromuscular sites (for
example Fig. 1 F) and can hardly be found in ectopic locations (in contrast to other classes of mutant embryos;
Prokop et al., 1996
). This reduced and restricted appearance of synaptic markers in kak mutant embryos hints at a
requirement for kak within the presynaptic terminal (see
Discussion).
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Ultrastructural analyses of kak mutant embryos reveal
that presynaptic boutons can form normal cell junctions
with the muscle, interspersed by morphologically normal
synapses (Fig. 1 H; for details of wild-type NMJs see Fig. 1
G) (Broadie et al., 1995; Prokop et al., 1996
). However, we
found examples where synapses were indicated by structured material in the neuromuscular cleft, but typical presynaptic specializations (T-bars) were missing (Fig. 1 J). If
T-bars were found, they were restricted to neuromuscular sites, corroborating our light microscopic findings. Furthermore, neuromuscular contacts and synapses were
found less frequently compared with controls, which is in
agreement with the reduction of NMJs observed at the
light microscopic level. To test whether transmission occurs at kak mutant NMJs we carried out patch recordings on kakSF20/kakel3 mutant muscles. These recordings revealed excitatory junctional currents, clearly indicating
that neuromuscular transmission occurs (Broadie and Bate,
1993
). In four cases we stained the NMJs with antibodies
raised against cysteine string protein subsequent to recording (Fig. 1 D) and confirmed that in all cases the NMJ
was clearly misshapen and reduced in size. Occurrence of
neuromuscular transmission is furthermore demonstrated
by the presence of strong muscle contractions in kak mutant embryos observed under polarized light in vivo.
Taken together, ultrastructural, electrophysiological and in vivo observations suggest that NMJs, although abnormal in shape, are functional in kak mutant embryos. This suggests that kak might be required specifically for growth and shaping of branches at motorneuronal terminals.
kak Function Appears To Be Required for Local, but Not Long-distance Growth
The reduction of NMJ size could be the result of a general
inhibition or delay of axonal growth. To test this idea we
used the axonal markers anti-Fasciclin II (Fas II) and Fasciclin III (Fas III) to analyze the peripheral branching pattern of motor axons during stage 16, when these axons
have just reached their target muscles in the wild type (Fig.
2) (Halpern et al., 1991; Van Vactor et al., 1993
): peripheral nerves can form correctly in kak mutant embryos
(tested combinations: SF20/el3, 91k/HG25, SF20/Df(2R)-
MK1). Only the short SNb-branch has a tendency to stall
at the entry point into the ventral muscle field, as observed by SNb-specific Fas III staining in kak91k/kakHG25 and
kakSF20/Df(2R)MK1 mutant embryos at early stage 16 (data not shown). However, as the longest nerves (SNa
and ISN) reach their target areas in kak mutant embryos
there is no indication of general impairment of neuronal
growth (Fig. 2 B). At stage 17 the peripheral nerve pattern
has become distorted in kak mutant embryos due to muscle detachment (see below). However, in interpretable
cases, anti-Fas II stainings reveal a normal pattern of
nerve branches (Fig. 2 D).
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Thus, in kak mutant embryos motorneurons appear capable of navigating along correct paths to their target muscles and maintaining these contacts thereafter. This suggests that our kak mutant alleles affect NMJ formation
during the differentiation phase, when muscle-attached growth cones reshape into the branches and boutons of
mature NMJs (Broadie and Bate, 1993; Yoshihara et al.,
1997
).
kak Protein Is Expressed at NMJs
To investigate whether kak function might be required directly within the nerve terminal, we used an anti-kak antiserum (Gregory and Brown, 1998). Our staining procedure (Materials and Methods) failed to detect strong
staining at NMJs in wild type or hemizygous embryos,
however, in Dp(2;3)6r35 embryos NMJs are labeled more reliably and strongly (Fig. 3, compare A and C with B).
Dp(2;3)6r35 embryos carry four copies of kak due to a duplication of a chromosomal region involving kak (Materials and Methods), suggesting that the enhanced immunoreactivity at the NMJ is specific for kak. Also at late
larval stages anti-kak antibodies label spots of up to 2 µm
at the NMJ, which by size appear to be presynaptic boutons (Fig. 3 D).
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kak Mutations Affect Dendritic Growth of RP3 Motorneurons in the CNS
Local neuronal growth is not restricted to branch formation at the NMJ but also occurs within the CNS during the
development of dendritic branches at stage 16/17. We
tested whether this growth might also be affected in kak
mutant embryos. We labeled dendrites retrogradely by applying DiI to the NMJ of RP3 motorneurons on muscles
VL3/4 (Landgraf et al., 1997). In wild-type embryos, RP3 sends an axon contralaterally through the dorsally located
anterior root of the intersegmental nerve. On the ipsilateral side a second projection leaves the soma of RP3, projecting along a similar path as the contralateral process,
but remaining confined to the neuropile. Both projections
have numerous local arborizations (Fig. 4, C and E)
(Landgraf et al., 1997
; Sink and Whitington, 1991
). In
kakSF20/kakel3 and in kakSF20/kak91k mutant embryos the ipsilateral local arborizations are almost normal, but the
contralateral arborizations are severely reduced and often
form swellings or blobs (Fig. 4, D and F). We quantified the spread of the dendritic arborization by measuring either the longest distance of dendrites from the midline in
the mediolateral axis, or the maximal spread of dendrites
in the anterior-posterior axis. With both methods only the
spread of the contralateral dendritic arbor is significantly
reduced in kak mutant embryos whereas the spread of the
ipsilateral side is similar to wild type (Fig. 4 G). The failure
of RP3 to elaborate its contralateral dendrites is apparent
from late stage 16 (Fig. 4, compare A with B), suggesting kak function to be required for the process of outgrowth
rather than maintenance of dendrites. As in kak mutant
embryos, the contralateral arborizations of RP3 form
within the correct region of the neuropile (kak: 37-76% of
the mediolateral neuropile diameter; controls: 24-91%)
they might reach their correct target areas, as similarly observed for their neuromuscular projections.
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Consistent with the findings for the RP3 dendrites, the whole neuropile is reduced in size in kak mutant embryos compared with wild type, but appears normal in its organization. In transverse sections of anti-Fas II-labeled nerve cords at stage 17 the neuropile area is reduced to 73% (P = 0.0084) in kakSF20/kakel3 mutant embryos, but the number and position of Fas II-positive longitudinal fascicles is normal (for details see Fig. 5, A and B). However, measurements of midline-associated fascicles (Fig. 5, A and B, M1 and M2) reveal a significant reduction to 50% of wild-type size (n = 17 for kak and 22 for wild type; P = 0.0001). Taken together, our findings suggest that neurons project correctly, but fail to elaborate part of their local branches, leading to smaller NMJs in the periphery and smaller dendrites and thus reduced neuropile volume centrally.
kak Function Is Required To Regulate the Localization of Membrane Proteins within Neuronal Processes
The phenotypes shown so far strongly suggest a specific requirement for kak function in specific local growth events. In the following we describe further kak mutant phenotypes, i.e., mislocalization of axonal proteins and disorganization of the cytoskeleton, both of which are potential causes underlying the specific defects in neuronal branch formation.
First, we observed a mislocalization of proteins along
neuronal processes. For example, Fas II, which encodes a
transmembrane protein of the immunoglobulin superfamily (Goodman and Doe, 1993), is expressed at low levels in
the nerve roots and stops at the entry point into the neuropile of stage 16 and 17 control embryos (Fig. 5, C and E).
Fas II expression in kakSF20/kakel3 and kak91k/kakHG25 mutant nerve cords exhibits no obvious differences at stage 16 (data not shown), but by stage 17 Fas II expression in all
nerve roots is strongly upregulated and the anterior root
of the intersegmental nerve extends to the dorsal part of
the neuropile (Fig. 5, B, D, and F). This phenotype at late
stage 17 was confirmed using other allelic combinations of
kak (el3/Df(2R)CX1, SF20/SF20, SF20/91k; 91k/HG25)
(data not shown). Thus, it appears as if Fas II fails to localize properly along neuronal processes. In contrast, 22C10 immunoreactivity, which detects membrane-associated or
transmembrane proteins (Angaut-Petit et al., 1998), appears to be distributed normally in kak mutant nerve roots
(Fig. 5, compare G with H), but we found a mislocalization
phenotype in another type of neuron, the dorsal bipolar
neuron of the peripheral nervous system. The dorsal bipolar neurons have longitudinal projections that span the entire length of the segment (Bodmer and Jan, 1987
), but
only the proximal regions of these processes are labeled by
22C10 antibodies in the wild type (Fig. 5 J). However, in
kakSF20/kakel3, kakSF20/kak91k, and kak91k/kakHG25 mutant
embryos the entire length of these lateral bipolar projections is 22C10-positive (Fig. 5, K). Thus, kak function is required for the correct localization of (membrane) proteins
within neuronal processes, and the mislocalization of such
proteins is a potential cause for defects in local branching
in the neuropile or at the NMJ (Suter and Forscher, 1998
)
(see Discussion).
The Microtubule Cytoskeleton Is Affected in kak Mutant Embryos
Neuronal growth requires a dynamic cytoskeleton (Bentley and O'Connor, 1994; Suter and Forscher, 1998
). Some
kak mutant phenotypes suggest that kak function might be
required for cytoskeletal organization. This is most obvious for muscle attachments to the epidermis. At stage 16 we could not detect any obvious defect in the muscle pattern of kak mutant embryos (SF20/el3 and 91k/HG25)
(data not shown). However, at stage 17 the same alleles of
kak cause severe detachment of muscles from the cuticle,
but many muscles remain attached to each other (Fig. 6 F).
Muscle attachments are formed by hemiadherens junctions, the adhesion of which depend on PS integrins (Fig.
6, D and arrowheads in B) (Tepass and Hartenstein, 1994
;
Prokop et al., 1998
). In late stage 17 kak mutant embryos the extracellular adhesion of hemiadherens junctions is intact (Fig. 6, C and arrowheads in A) and, accordingly,
PS
integrin is expressed at the muscle tips (data not shown).
However, we observe a striking phenotype on the intracellular face of hemiadherens junctions, only on the epidermal side. Normally the intracellular face of epidermal
hemiadherens junctions contains a thick layer of electron-dense material, which connects to the stress-resisting microtubules (Fig. 6 D, bent white arrow). In kak mutant epidermal cells the layer of dense material is thinner (Fig. 6
C, bent white arrow) and microtubules, although present
within the cell (Fig. 6, open arrows), seem not to be attached to the remaining layer of dense material. As a result, epidermal cells rupture (Fig. 6, A and F, black arrows), and the retracting muscles take with them the
epidermal cell fraction around the hemiadherens junction. This phenotype is similar to BPAG1 mutant mice where
intermediate filaments, the major stress-resisting cytoskeletal elements in epidermal cells of vertebrates, fail to adhere to hemidesmosomes (Guo et al., 1995
).
Epidermal cells at sites of muscle attachments contain
1-tubulin, which is also strongly expressed throughout
the central nervous system and in the scolopidia, which are
part of the peripheral nervous system (also called chordotonal organs; Buttgereit et al., 1991
). As growth defects in
the nervous system are restricted to small dendrites and
neuromuscular side branches, potential defects in their cytoskeleton are expected to be subtle, and so far we have
not been able to pinpoint specific defects at the ultrastructural level. However, analysis of the more prominent scolopidia yielded interesting results. In the wild type, scolopidial sensory neurons form a long process stretched
between cap and sheath cells and the soma bulges out
asymmetrically on one side (Hartenstein, 1988
; Carlson et
al., 1997
) (Fig. 7 A). The neuronal processes contain a typical ciliary apparatus with a cilium, basal body, and rootlet.
The rootlets are surrounded by a circle of microtubules,
especially in the distal parts of the processes. This circle of
microtubules is mostly absent or very poorly developed in
kak mutant embryos (Fig. 7, compare G with H), and the
prominent dendrites appear collapsed in 22C10-labeled
specimens (Fig. 7 B, white arrowhead and white bent arrow). The cilia contain a ring of nine microtubule doublets
and are each located in a lymph-filled capsule formed by
scolopale and cap cells (Fig. 7 E). The cilia are anchored
with their apical ends in extracellular matrix at the tip of
the capsules (Fig. 7 C). In kak mutant embryos the cilia
look normal (data not shown), however, they fail to anchor at the capsule tips and are retracted (Fig. 7 F). In
some mutant embryos we found grey inclusions within the
extracellular matrix at the capsule tips, which might be
remnants of the cilia (Fig. 7 D). This could suggest an intracellular detachment of the cilia similar to that seen in
the epidermis, leaving behind pieces of fractured membrane.
|
Taken together, we could demonstrate defects in the
cytoskeleton of two ectodermal tissues, suggesting that
growth defects in the nervous system might have a similar
cause. The synaptic phenotype, as well as the localization
of kak-immunoreactivity at the NMJ, suggest that kak is
required within the neuron (see Discussion). This interpretation is in good agreement with sequence data suggesting that kak codes for a cytoskeletal element with homologies to the actin-binding domain of plectin and
BPAG1, the coiled-coil region of dystrophin, and parts of
the GAS2 protein (Gregory and Brown, 1998; Strumpf
and Volk, 1998
).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
kak Mutations Specifically Affect Local Neuronal Growth
Growth of neuronal processes can be subdivided into (a)
long-distance growth into target areas and (b) local growth
within target areas. Here we describe the phenotypes of
four independently isolated alleles of kakapo (kakSF20,
kakel3, kak91k, and kakHG25), all of which show specific defects in local neuronal growth. In kak mutant embryos
growth cones project into the correct target areas in the
muscle field and in the neuropile but, subsequently, subsets of dendrites or NMJ branches fail to develop. Local
dendritic growth in the neuropile is likely to be carried out
by secondary growth cones budding off primary axons de
novo (Tessier-Lavigne and Goodman, 1996). Branch formation at the NMJ takes place at about the same time as
dendrite formation but occurs in a slightly different manner, in that the primary growth cone has already occupied
space on the muscle but refines its shape into branches and
boutons (Broadie and Bate, 1993
; Yoshihara et al., 1997
). The kak mutant phenotype strongly suggests that the final
formation of both kinds of structures requires common
molecular mechanisms.
The transition from long-distance growth to local branch
formation appears to be a regulated process. It seems to be
facilitated or accelerated by the function of the late
bloomer gene, as loss of late bloomer function causes delay
of NMJ differentiation after correct arrival of the growth
cone (Kopczynski et al., 1996). One of the genes downstream in this pathway could be kak, since loss of kak function seems to cause growth defects after the growth cone
arrives at the muscle. Loss of kak function leads to smaller NMJ branches and boutons, and the number of synapses
and T-bars appears reduced. Also in mbc, mef2 or twist
mutant embryos NMJs are reduced or absent (caused indirectly by defects of the target muscles; Prokop et al., 1996
),
but the respective motorneurons still form varicosities and
T-bars in fairly normal amounts, and T-bars which can't be
placed apposed to muscle surfaces get localized at extrajunctional sites. In contrast, presynaptic specializations (presynaptic markers and T-bars) (Fig. 1) remain restricted
to neuromuscular sites in kak mutant embryos, in spite of
the fact that NMJs are severely reduced. We conclude that
our kak mutant alleles might cause structural defects
within the presynaptic terminals independently of target
muscles. This interpretation is in agreement with cloning
data demonstrating that kak encodes a cytoskeletal element with actin binding properties and a coiled coil region
(Gregory and Brown, 1998
; Strumpf and Volk, 1998
). As
kak is localized at the motorneuron terminal (Fig. 3) it
might play a direct role in changes of the terminals' cytoskeleton which are required for growth and synapse formation.
kak Function Is Required for the Regulation of the Microtubule Cytoskeleton and Localization of Membrane Proteins
In addition to the growth defects in motorneurons, kak
mutant embryos exhibit defects of the microtubule cytoskeleton in epidermal cells at muscle attachments and in
scolopidial sensory neurons. The clearest phenotype is at
muscle attachments, where the dense material associated
with hemiadherens junctions on the basal side of the epidermis is reduced in thickness and fails to anchor microtubules. Thus, kak is required to mediate the link between
the intracellular cytoskeleton and membrane-associated
proteins. Thinning of the dense material at kak mutant
hemiadherens junctions suggests that kak protein might be
an integral part of the membrane-associated cytoskeleton,
which would be in accordance with immunocytochemical localization of kakapo at the basal surface of muscle-attached epidermis cells (Gregory and Brown, 1998;
Strumpf and Volk, 1998
). If this interpretation is correct,
kak could bind to the actin network underlying the membrane and link (indirectly) to microtubules, thus mediating
attachment of microtubules to the membrane.
However, kak may not be restricted to specialized membrane areas like hemiadherens junctions. For example,
dystrophin (in part homologous to kak) is concentrated at
specialized junctions like synapses, but also found at nonspecialized membrane surfaces (Cartaud et al., 1992). If
kak were similarly spread over neuronal surfaces it could
regulate the localization of Fas II or 22C10 antigens (Fig.
5) by linking them to the underlying actin cytoskeleton. Loss of this kind of kak function might also cause the irregular appearance of the cell surfaces of dorsal bipolar
dendrite neurons (Fig. 5 K) or the soma of RP3 (Fig. 4, D
and F). Kak may even be localized in the cytoplasm, like
its partial homologue BPAG1, which is localized in membrane-associated dense material of epidermal hemidesmosomes, but in the cytoplasm of neurons (in another splice version; Fuchs and Cleveland, 1998
). Similarly, the organization of microtubules in scolopidial sensory neurons (Fig.
7 H) might require kak function within the cytoplasm
where kak could link microtubules to the actin network or
to the ciliary rootlet. Alternatively the defect of microtubules in scolopidial neurons could be caused secondarily
due to loss of kak-mediated anchoring at the dendrite tip,
comparable to the phenotype at epidermal hemiadherens
junctions. The latter possibility is supported by the finding
that kak is localized at the dendrite tip of scolopidial neurons (Gregory and Brown, 1998
). Interestingly, our staining procedure failed to detect kak staining in the epidermis or scolopidial organs at stage 16, 17 or in the late larva.
This might hint at different splice versions of kak or at a
different molecular context.
Possible Causes for Defects in Local Sprouting
We have demonstrated two different defects at the subcellular level in kak mutant embryos. First, the localization of axonal proteins is affected and, secondly, there are defects in the microtubule organization of some cell types. Both defects may be the underlying cause for the observed reduction in local growth of dendrites and at NMJs.
It has been shown that branching of motorneuronal terminals and axonal defasciculation require a reduction of
neuronal cell adhesion molecule (N-CAM)-mediated interaxonal adhesion in vertebrates (Landmesser et al.,
1990) and, in agreement with this, overexpression of Fas
II, the Drosophila homologue of N-CAM, antagonizes
nerve branching (Lin et al., 1994
). Hence, we reasoned
that the inhibition of dendrite and branch formation might
be due to the observed mislocalization of Fas II to axonal
areas where dendrites and terminal branches are usually
forming. However, combining kak with the Fas IIeb112 null
allele (Grenningloh et al., 1991
) did not show any obvious suppression of the neuromuscular phenotype (data not
shown). Thus, mislocalization of Fas II alone does not explain the growth defects, but its involvement might be obscured by mislocalization of other redundant CAMs of
similar function (Speicher et al., 1998
). Mislocalization of
membrane proteins might be the consequence of their lack
of a kak-mediated linkage to the membrane-associated cytoskeleton (see above). Conversely, loss of such a physical
link could cause disruption of growth regulation, as transmembrane proteins have been shown to instruct the assembly of the actin cytoskeleton in neuronal growth cones
(Thompson et al., 1996
; Suter and Forscher, 1998
).
Neuronal growth defects in kak mutant embryos might
be caused directly by defects in cytoskeleton assembly. Microtubules are essential for axonal growth and are regulated in a complex way. For example, low concentrations
of taxol do not interfere with growth cone advance in general, but render growth cones unable to turn when they
come into contact with a repellent signal (Challacombe et al.,
1997). The assembly of microtubules during growth is preceded by formation of the actin cytoskeleton. Accordingly,
growth cone turning can be blocked upon low application
of cytochalasin-B, indicating cooperation between the actin and tubulin cytoskeleton in this specific growth event
(Challacombe et al., 1996
). The fine regulation of microtubules has been shown to require MAPs (Avila et al., 1994
).
Similarly, the fine regulation of actin could require actin-associated proteins, and kak might be one of them. This
might explain why loss of kak function suppresses only a
specific subset of neuronal growth events, i.e., local growth
at NMJs and of contralateral RP3 dendrites but not long distance growth or ipsilateral RP3 arbors. The specific
growth defects in kak mutant embryos might be due to
subcellular-specific compartmentalization of kak or local
posttranslational modifications, as has similarly been demonstrated for MAPs (Avila et al., 1994
). Alternatively, unaffected branches may contain redundant cytoskeletal molecules that the affected branches lack. Possible molecular differences might reflect a general difference between
affected and unaffected branches. For example, affected
branches might represent preferentially presynaptic output branches (certainly true for NMJs) and unaffected
branches may represent postsynaptic or input branches.
Alternatively, the qualitative differences might consist in
the origin of the branches: arborizations derived from an
axon (NMJ, contralateral RP3 dendrites) may require kak function, but not those derived from somatic extensions
(ipsilateral RP3 dendrites).
![]() |
Footnotes |
---|
Address correspondence to A. Prokop, Institut für Genetik, Zellbiologie, Universität Mainz, Becherweg 32, D-55128 Mainz, Germany. Tel.: (49) 6131-394328 or 393293. Fax: (49) 6131-395845. E-mail: prokop{at}goofy.zdv.uni-mainz.de
Received for publication 24 April 1998 and in revised form 14 September 1998.
M. Bate was funded by a grant from the Wellcome Trust (052032/Z/
97/Z), A. Prokop by a research fellowship from the Lloyd's of London
Tercentenery Foundation (LTF/GL/FEL95), and J. Uhler by a grant from
the Overseas Research Students Awards Scheme.
We are grateful to M. Ashburner (University of Cambridge, Cambridge, UK), in whose laboratory the mutageneses were carried out, to H. Bellen (Baylor College of Medicine, Howard Hughes Medical Institute [HHMI], Houston, TX), M. González-Gaitán (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany), C.S. Goodman (University of California, HHMI, Berkeley, CA), and K. Zinsmaier (University of Pennsylvania School of Medicine, Philadelphia, PA) for providing antibodies, to C. Schuster (Max Planck Institute) for providing flies, to N. Brown, S. Gregory (both from University of Cambridge, Wellcome Institute), D. Strumpf, and T. Volk (both from Weizmann Institute, Rehovot, Israel) for helpful exchange of information and fly stocks and generously sending anti-kakapo antisera. We would like to thank R. Baines, M. Landgraf, and N. Sánchez-Soriano (all three from University of Cambridge) for critical comments on the manuscript and S.D. Carlson for comments on Figure 7. J. Uhler would like to thank A. Sossick and A. Brand (both from University of Cambridge, Wellcome Institute) for help and advice on confocal microscopy. A. Prokop is grateful to R. Baines for teaching and advising on the patch-clamp technique and to K. Broadie (University of Utah, Salt Lake City, UT) for additional advice, to G. Technau in whose laboratory part of the work was carried out, and to E. Sehn and G. Eisenbeis (all three from University of Mainz, Mainz, Germany) for advice and help on the Hitachi microscope.
![]() |
Abbreviations used in this paper |
---|
CNS, central nervous system; Fas, fasciclin; kak, kakapo gene; MAP, microtubule-associated protein; NMJ, neuromuscular junction.
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