1 Zoology Department, Stockholm University, S-106 91 Stockholm, Sweden
2 Institute of Genetics, University of Mainz, Saarstrasse 21, D-55122 Mainz,
Germany
3 European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg,
Germany
4 Center of Molecular Biology Severo Ochoa, Universidad Autónoma de
Madrid, Cantoblanco, 28049 Madrid, Spain
* Author for correspondence (e-mail: rcantera{at}zoologi.su.se)
Accepted 13 September 2002
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SUMMARY |
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Key words: Drosophila, spalt, Neurodegeneration, Cell adhesion, Neuronal differentiation, Nervous system development
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INTRODUCTION |
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To investigate this question, we have studied the CNS in mutant embryos, initially with the aid of transmission electron microscopy (TEM). Tissues fixed at 60% of embryonic development had a strong neurodegenerative phenotype but, surprisingly, those fixed at a slightly later stage (80% of development) looked almost normal. A simple explanation to this finding would be that the phenotype defined by electron microscopy reverted in the few hours between the two stages. To test such a provoking possibility, we studied the dynamics of the phenotype with a combination of techniques applied either in situ, in vitro or after transplantation of single mutant precursors into wild-type tissue. The results indicate that during embryonic CNS development sal is important for cell adhesion and the physical integrity of neuronal axons and membranes, probably during a short developmental period around 60% of embryonic development.
A related phenotype is that mutations in this gene affect the expression of particular adhesion and cytoskeletal proteins in the CNS. Although many of the morphological effects are transient, specific branching projection defects of certain neurones persist. Thus, the absence of Sal compromises the capacity of some central neurones to differentiate correctly. We discuss the concept of genetic redundancy in the context of this result.
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MATERIALS AND METHODS |
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Transmission electron microscopy
Embryos were manually dechorionated, mounted in halocarbon oil to avoid
dessication, and their genotype (GFP-expression) and stage determined upon
examination at 40x on a Zeiss Axiovert microscope at room temperature.
At least six embryos of each genotype and stage were processed. Heterozygotes
(GFP positive) and wild type (w1118) were also compared. A
minimum of 100 histological sections were prepared from each embryo according
to standard procedures (2 µm thickness and stained with 0.1% boracic
Toluidine Blue) and carefully analysed, covering representative regions of
brain and nerve cord. Three embryos from each sample were also analysed with a
transmission electron microscope (JEOL 100CX) operated at 60 kV. As
GFP-negative mutant embryos develop at slower and somewhat erratic rates,
individual fixation of carefully staged mutant embryos was used to obtain
samples of similar developmental age. Upon reaching the desired stage, as
defined by morphological and temporal criteria, the embryo was quickly
pre-fixed by shaking in a cold mixture of heptane and aldehydes under 10
minutes, devitellinized by hand, post-fixed in dialdehyde solution for 1 hour,
and for another hour in 1% osmium tetroxide. The dialdehyde fixative was
freshly prepared in 0.1 M sodium cacodylate buffer pH 7.3, and contained 4%
paraformaldehyde, 3% glutaraldehyde and 2% tannic acid
(Afzelius, 1992). Ultrathin
sections (silver), contrasted by serial incubation in lead citrate, uranyl
acetate and lead citrate (Daddow, 1982), were mounted directly onto 300 mesh
copper grids.
Immunocytochemistry, laser confocal microscopy and image
analysis
Transheterozygous Df(2L)32FP-5;sal445 mutant embryos of
two ages (stage early 16 and 17) were processed for whole-mount
immunocytochemistry according to standard techniques
(Patel, 1994) with a
double-staining protocol in which one of the two primary antibodies always was
mouse or rabbit anti-ß-galactosidase for genotype assignment. The other
antibody probes were specific for N2-Armadillo
(Peifer and Wieschaus, 1990
;
Loureiro and Peifer, 1998
),
DN-Cadherin (Iwai et al.,
1997
), Faint Sausage (Lekven
et al., 1998
), Fasciclin 2
(Schuster et al., 1996
),
Fasciclin 3 (Patel et al.,
1987
), Neurexin IV (Baumgartner
et al., 1996
), Neuroglian
(Bieber et al., 1989
),
Neurotactin (Speicher et al.,
1998
), Notch (Johansen et al.,
1989
), Tubulin (Sigma BioSciences) and Futsch/22C10 (Developmental
Studies Hybridoma Bank). Stained embryos were mounted in PBS-buffered
glycerine and studied with laser confocal microscopy on a Zeiss LSM 510. For
general surveys either XY-sections or Z-series covering selected CNS areas
were produced. For quantification of fluorescence intensity, single optical
sections scanned with exactly the same parameters and under the same session
were compared, always on exactly the same anteroposterior and dorsoventral
location in the thoracic neuromeres. Care was taken to choose a CNS area
relevant for each marker (for example, longitudinal tracts for Fasciclin 2,
but ventral cell body cortex for Notch). Histograms of pixel intensity for
each channel were prepared directly with the Histogram tool of LSM 510, or,
after import into Photoshop, with the Histogram tool of the Adobe software.
Each staining was made at least twice, each time with embryos collected from a
different cross.
TUNEL staining
For the detection and quantification of apoptosis at a single cell level a
fluorescein in situ cell death detection kit was used (Roche; standard
labelling protocol for tissues). Staining was applied to flat preparations of
embryos between stages 14 and 17. Z-series covering 5-6 abdominal neuromeres
per embryo (n=14 for sal mutants or wild-type controls) were
captured under a fluorescent microscope (Zeiss Axiophot) equipped with a
digital camera (Sony MC-3255). Focal planes were combined using Photoshop
(Adobe) and total numbers of TUNEL-positive cells were counted.
Dil labelling
To trace the cell lineages of individual neural precursors in sal
mutant background, we applied the DiI labelling technique as described in
detail elsewhere (Bossing and Technau,
1994; Bossing et al.,
1996
). Individual neuroectodermal precursor cells were labelled
with DiI at stage 7 and allowed to further develop until stage 17. Clones
generated by the labelled precursors were uncovered upon photoconversion of
the dye in flat preparations and documented with a video camera (Sony
MC-3255), Photoshop and Illustrator (Adobe).
Cell transplantations
Transplantations were performed as described previously
(Prokop and Technau, 1993).
Cells were removed from the neuroectoderm of horseradish peroxidase (HRP)
labelled donors at stage 7 and individually transplanted into the
neuroectoderm of wild-type hosts at the same stage. Hosts were allowed to
develop until stage 17 when the clones derived from the transplanted
precursors were uncovered and documented (see above) upon staining for HRP in
flat preparations. Donor strain was kept over a GFP balancer, and the genotype
of donor embryos was identified by GFP fluorescence.
Cell culture and time-lapse video analysis
Primary cultures of single precursor cells, time-lapse recordings and
staining procedures of clones in vitro were performed as described elsewhere
(Lüer and Technau, 1992).
Cells were taken from the neuroectoderm of stage 7 donors and grown in culture
for up to 4 days. As the donor strain was kept over a green balancer, the
genotype of donor embryos was identified by GFP fluorescence. Clones were
stained with anti-HRP (Dianova), anti-Repo
(Halter et al., 1995
) and
anti-tubulin (Sigma) antibodies.
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RESULTS |
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sal mutant embryos develop a strong but reversible
neurodegenerative phenotype
A strong and fully penetrant phenotype was detected in the CNS of
Df(2L)32FP-5;sal445 mutant embryos by TEM at early stage
16 (60% of embryonic development). Neuronal and glial cells in the brain
and nerve cord were seen to be loosely attached or widely separated by a
dramatic enlargement of the extracellular space
(Fig. 1A). At this stage
neuronal cell bodies are tightly packed in wild-type tissue
(Fig. 1C). Membrane `whorls',
autophagosomes, and other membranous profiles typical of neurodegenerative
processes were observed in the cytoplasm of neuronal and glial cells. The
lacunar spaces between cell bodies and neuronal fibres in the neuropil
contained large amounts of extracellular membranous material, mostly in the
form of vacuoles of a wide size range, the largest approaching the size of
whole cell bodies (Fig. 1A,E). These vacuoles either seemed empty or contained smaller vacuoles, but not
organelles or cytoplasmic remnants characteristic of cellular debris resulting
from cell death. We did not observe an increase in apoptotic profiles as
compared with wild-type tissue either with TEM or TUNEL staining (see
Materials and Methods, data not shown). Similar membranous formations result
from mutations in spongecake (Min
and Benzer, 1997
). Axonal calibre was reduced in sal null
mutants (compare insets in Fig.
1E,G) and filopodia emanating from growth cones were often
clumped. The phenotype was observed in all sal mutant embryos, but
not in heterozygous or wild-type embryos examined as controls. The phenotype
was not observed in peripheral nerves.
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Surprisingly, the CNS appeared to recover rapidly from the degenerative process, as embryos fixed a few hours later, by late stage 16 or stage 17 (between 80 and 90% of embryonic development), exhibited an almost normal organisation (see Fig. 1B for cell bodies, and 1F for neuropil; compare with wild-type morphology in Fig. 1D,H, respectively). At this stage, most of the extracellular membranous material had disappeared and the neuronal cell bodies in the mutant were almost as tightly packed as in the wild type.
If the recovery depends on the activity of another protein, with the capacity to compensate for the loss of Sal, simultaneous deletion of this protein should substantially diminish the capacity of the tissue to recover and perhaps make the phenotype irreversible. A potential candidate for this hypothetical redundant function could be the paralogous protein Salr. To test this hypothesis, we examined embryos lacking Sal and Salr due to a small deficiency. However, these homozygous Df(2L)32FP-5; Df(2L)32FP-5 mutants exhibited the same phenotype caused by the lack of Sal alone and the phenotype reversed within the same developmental interval (data not shown).
sal mutant embryos have abnormal levels of cell adhesion
proteins
A possible interpretation of the phenotype defined above would be that
components of cell adhesion are seriously compromised in the CNS of
sal embryos during early stage 16. To test this hypothesis we used
specific antibodies and laser confocal microscopy to survey the expression of
molecules known to be important for cell adhesion in embryonic CNS at early
stage 16. All the markers were detectably expressed in
Df(2L)32FP-5;sal445 mutant embryos at both stages, and
their spatial patterns of expression in the CNS were normal, showing that
sal is not essential for any of these proteins to be expressed.
However, the quantification of fluorescence intensity revealed that most
markers were present in abnormally high or low levels. Representative examples
are shown in Fig. 2A for seven
of the analysed markers (see Materials and Methods for explanation of
quantification of fluorescence levels using the laser confocal microscope's
software). In transheterozygous Df(2L)32FP-5;sal445
mutants at early stage 16, when the strong TEM phenotype is manifest, we
measured lower fluorescence levels for Armadillo, N-Cadherin, Neuroglian,
Fasciclin 2 and Fasciclin 3; higher fluorescence levels for Notch; and levels
similar to wild type for Neurotactin, Neurexin IV and Faint Sausage
(Fig. 2B and data not shown).
Comparison between wild-type, heterozygous and null sal mutant
embryos revealed a stepwise decrease in the fluorescence levels for Armadillo
and N-Cadherin (Fig. 2C),
indicating that the effect of the mutation is dominant.
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We next measured the fluorescence levels at the stage when the TEM phenotype is reverted (stage 17). The wild-type fluorescence for the three markers studied in this regard (Armadillo, Fasciclin 2, Neuroglian) changed between early stage 16 and stage 17, indicating that during this short developmental interval the levels of cell adhesion proteins are regulated (Fig. 3). Relative to these new wild-type levels, the three proteins that were not affected during the expression of the TEM phenotype (Neurotactin, Neurexin IV and Faint Sausage) remained normal in the mutant (not shown). The levels of Notch switched from abnormally high to slightly lower than normal. All other markers still exhibited lower-than-normal fluorescence levels, with the exception of N-Cadherin, which exhibited a partial recovery (data not shown).
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Taken together, these data lead to the conclusions that the expression of sal is necessary to maintain correct dynamic levels of several adhesion molecules in the CNS and that sal exerts this function in a persistent and dominant fashion.
sal is necessary for normal neuronal growth and
differentiation in vitro
To gain a more detailed understanding of the dynamics of the sal
phenotype, we used cell cultures derived from single neuronal precursors
isolated either from the neuroectoderm or the midline region of mutant
embryos. Unlike the wild type, mutant cells were extremely fragile and
sometimes disintegrated upon suction into the microcapillary. Moreover, the
cells had a rounded morphology and showed obvious difficulties to establish
and maintain a normal attachment to the substrate. Upon inspection of
time-lapse recordings, we even found examples of cells that attached and lost
contact with the substrate repeatedly. Wild-type neuroectodermal precursors,
however, strongly adhered to the bottom of the culture chamber and adopted a
more flattened morphology (see also
Lüer and Technau, 1992).
Proliferation of the sal mutant-derived cells did not appear to be
affected. However, their progenies exhibited a clearly slower rate of branch
growth and differentiation (Fig.
4A, compare with wild type in 4B). Even after several days of
culture, most mutant clones still had a poor branching when compared with
wild-type clones (Fig. 4C,
compare with wild type in 4D). The fibres growing from sal-derived
cells were often very thin (Fig.
4A), confirming the original TEM observations, and sometimes
displayed abnormal fasciculation. Mutant-derived clones were often surrounded
by debris, probably representing material shed from living cells
(Fig. 4C).
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Staining with antibodies specific for the glial nuclear protein Repo
(Halter et al., 1995) showed
that this glial marker is expressed in some cells derived from
sal-mutant explants (Fig.
4E). An unusual morphology frequently found in
sal-derived clones was an anastomosing network of distal branches
intercalated with wider, flattened structures (see arrowheads in
Fig. 4E). Time-lapse analysis
of these anastomosing structures revealed that they were very dynamic, with
new branches and connections being made and dismantled over intervals of some
minutes (Fig. 5A-D).
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Our TEM data showed that the enlarged extracellular space of the mutant CNS tissue appeared to be packed with vacuoles. Time-lapse recording of cultured cells was used to document directly the loss of membrane material that could be the origin of these vacuoles. We detected blistering along sal mutant-derived fibres, which could represent an early step in the formation of vacuoles. However, these structures were observed only rarely and were always reabsorbed (Fig. 5E-H). We were thus not able to directly document the loss of membrane or cell material from mutant cells in culture.
The neuronal cytoskeleton is modified in sal mutant
embryos
The results obtained from time-lapse recordings suggested that the cells
derived from sal mutant CNS have a deficient cytoskeleton. To
investigate this possibility we stained for tubulin in vitro and found two
major differences with wild-type neurones. In mutant neurones, tubulin did not
reach into the growth cone (Fig.
6A) and patches of poor fluorescence were also detected along the
axon (Fig. 6B), suggesting the
existence of interruptions along the core of axonal microtubules. In wild-type
neurones, anti-tubulin fluorescence extends homogeneously along the entire
axons and reaches almost the distal border of the growth cone
(Fig. 6C). Laser confocal
microscopy of embryos stained with three cytoskeletal markers (F-actin,
tubulin and the tubulin-associated protein Futsch) revealed additional
differences. The three markers were correctly expressed across brain and nerve
cord, with the typical accumulation along major axonal tracts
(Fig. 6D), but the fluorescence
levels were abnormally higher for F-actin, and lower for tubulin and its
associated protein Futsch (Fig.
6E).
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sal is necessary for correct neuronal differentiation in
situ
The data gathered from our cell cultures showed on a single cell level that
neurons derived from sal mutant neuroectodermal precursors
differentiate poorly in vitro. Next, we tested to what extent the lack of Sal
affects the differentiation of individual cell lineages in the developing CNS
tissue. In Drosophila each neural precursor (neuroblast) produces a
stereotyped combination of cells identifiable by cell body position and the
pattern of axonal projections. Extensive data obtained from lineage analysis
in wild type make it possible to identify each neuroblast lineage on the basis
of its neuroanatomy (Bossing and Technau,
1994; Bossing et al.,
1996
; Schmidt et al.,
1997
). We decided to exploit this knowledge to investigate the
capacity of sal mutant neuroblasts to produce normal cell clones in
situ. Single neuroectodermal and midline precursors were labelled with DiI and
cell lineages derived from these precursors were analysed at late stage
17.
The clones obtained in sal null embryos can be classified into three categories according to their degree of neuroanatomical abnormality. Some clones differentiated into morphologies showing no obvious similarities to identified wild-type lineages (n=2; Fig. 7A), others exhibited abnormally projecting axons but were as a whole identifiable as particular wild-type lineages (n=7; Fig. 7B) and others, finally, were almost indistinguishable from their wild-type counterparts (n=7; Fig. 7C). Interestingly, some of the clones derived from labelled midline precursors also developed abnormalities, although sal expression has not been detected in these precursors (Fig. 7D and data not shown). At high magnification, spherical thickenings were found along the axons, which resemble the blistering observed in the time-lapse studies (Fig. 7E).
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The function of sal during embryonic CNS development is
probably cell autonomous
Although sal is expressed only in a subset of neurones, the
embryonic sal mutant CNS exhibit a general disorganisation,
indicating that the surrounding cells cannot overcome the lack of Sal. To test
whether the phenotype is expressed cell autonomously, we used a
transplantation assay. HRP-labelled neuroectodermal precursors (stage 7) were
taken from sal mutant donors and singly transplanted into wild-type
embryos, and their lineages were documented at stage 17. Again we could detect
clones falling into the three categories listed above: non-identifiable
lineages (n=8; Fig.
7F), partially abnormal morphologies (n=3;
Fig. 7G) and apparently normal
morphologies (n=17; Fig.
7H). Clones derived from midline precursors also showed
abnormalities (Fig. 7I). These
data together with the in vitro observations provide support for a cell
autonomous function of Sal in the developing CNS. However, we can not exclude
that non-autonomous aspects of Sal function also exist, e.g. considering that
abnormalities occur in midline clones even though the expression of
sal has not been detected in the midline.
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DISCUSSION |
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At least five adhesion proteins seemed to be under-represented in
sal null mutants and three cytoskeletal proteins were also
quantitatively mis-expressed. At least some of the molecular phenotypes are
also exhibited by sal heterozygotes to a lower level, indicating that
sal acts in a dose-dependent fashion. Armadillo and N-Cadherin are
expressed ubiquitously and the same values were obtained for each individual
when different CNS areas were framed for the measurement of fluorescence
levels, indicating that the differences between wild-type, heterozygous and
sal null embryos do not represent changes in specific neuronal
subpopulations but the state of the entire CNS. A likely explanation of these
molecular phenotypes is that Sal, which acts as a transcription factor,
regulates quantitatively transcription of genes that are directly or
indirectly involved in cell adhesion and the cytoskeleton, thus offering a
link to the observed cellular defects. It must be kept in mind, however, that
some of the target genes can have functions other than cell adhesion. For
example, Armadillo protein not only firmly attaches to the cell membrane but
can also enter the nucleus to influence the transcription of genes involved in
cell fate decisions (reviewed by Cavallo et
al., 1997).
Adhesion-related abnormalities in attachment and branching were also exhibited by sal mutants at the cellular level. Both in tissue culture and in vivo, whether in situ or following transplantation into wild-type embryos, certain neuronal clones exhibited abnormal branching pattern. This suggests that these cellular phenotypes could be cell autonomously expressed. However, this does not seem to be the case for mutant CNS midline precursors, which showed abnormalities, although they normally do not express sal detectably. Defects were also seen in lineages derived from heterozygous precursors (data not shown), again indicating sal dominance. Importantly, both molecular and lineage defects were persistent. In these two respects, dominancy and persistency, these defects differ from the prominent ultrastructural phenotype, which is both recessive and transient. As the recessive ultrastructural defects undergo reversal when the molecular phenotypes that we have studied persist, we conclude that the proper organisation of the developing central nervous tissue is stabilised by genetic redundancy. The reversal of the ultrastructural defects suggest sequential redundancy. However, the fact that the ultrastructural defects are only seen in sal null mutants (contrasting with the co-dominance of molecular phenotypes) suggest coincident redundancy. The putative genes that are redundant with sal are as yet unknown.
It is generally assumed that many loss-of-function mutations for a given
cell adhesion protein do not result in overt phenotypes throughout the CNS.
Mutations in Armadillo, Fasciclin 2, Fasciclin 3, Neurotactin, Neurexin IV or
Neuroglian have phenotypes restricted to de-fasciculation of particular axons,
partial disorganisation of major tracts and nerve roots, or other `localised'
adhesion defects (Schuster et al.,
1996; Patel et al.,
1987
; Peifer and Wieschaus,
1990
; Speicher et al.,
1998
; Baumgartner et al.,
1996
) (reviewed by Goodman,
1996
). The current interpretation is that normal adhesive
properties in a complex tissue as the CNS depends on the combination of
several molecules, some of which can possibly compensate for the absence of
others. Examination of double mutants supports this hypothesis
(Speicher et al., 1998
), as
stronger phenotypes are detected after simultaneous deletion of two cell
adhesion proteins.
The rapid recovery of sal CNS during the course of stage 16 could
be explained by the robustness inherent to a system in which adhesion is
mediated by a combination of proteins and the possible compensatory effect
mediated by upregulation of other members of the system. But we will also like
to propose an alternative view. The ultrastructural recovery may as well
reflect the normal dynamics of combinations of adhesion proteins expressed
successively along embryonic development. From this point of view, the rapid
recovery from the adhesion phenotype will reflect the normal transition
between two particular combinations of adhesion proteins expressed at early or
late stage 16. For this to be valid, the expression levels of several adhesion
proteins must change along this interval during normal development.
Interestingly, our data do support this possibility, as the fluorescence
levels for Armadillo, Fasciclin 2 and Neuroglian did change between stages 16
and 17 in wild-type CNS. Whether sal is required for the regulation
of a combination of cell adhesion and cytoskeletal proteins at a particular
developmental stage could be tested by deleting the expression of Sal
exclusively in CNS tissue within short developmental intervals. This approach
could now be possible using techniques based on combinations of the GAL4-UAS
system and RNA interference (Piccin et
al., 2001).
The fragility of the precursors during their manipulation with
micro-capillaries and the difficulties they exhibited to settle down and
maintain themselves adhered to the substrate clearly demonstrates, in
combination with the previous data, that sal expression is necessary
for the development of neural cells with normal adhesion capacity. Time-lapse
recordings clearly showed that the axons of these cells often lose adhesion
from the substrate and exhibited an unusual behaviour: They showed a
`vibrating' movement suggestive of increased tension, instead of the relaxed
appearance of a well-attached wild-type axon. These two observations, and the
previously commented fragility during micro-manipulation lead us to suspect an
abnormal composition of the cytoskeleton. Staining for tubulin and actin
confirmed this assumption. Cell adhesion and the cytoskeleton are functionally
related, making difficult to clarify without further experiments which of
these two cellular features is the original cause of the phenotype.
Alternatively, sal could be necessary for the correct maintenance of
both features. It is worth noting that loss of function in sal
interferes with the migration of respiratory tubes
(Kühnlein and Schuh,
1996), a phenotype that could also be explained by cytoskeletal
defects.
An intriguing feature of the phenotype revealed by electron microscopy is
the accumulation of `vacuoles' in the enlarged extracellular space separating
the loosely attached CNS cells. Vacuolation is a common feature in mammalian
neuropathies and has also been recorded for mutations causing
neurodegeneration in Drosophila
(Coombe and Heisenberg, 1986;
Min and Benzer, 1997
;
Wittmann et al., 2001
). We
were able to detect in vitro the formation of two types of structures that
could represent the correlate of the membranous material detected with TEM.
First, mutant clones were associated with more abundant extracellular debris
than wild-type clones, which could represent loss of membrane material but
probably also represent cell death if mutant cells are less tolerant to the
culture conditions. Second, we detected blistering along the axons. However,
these structures were seen only rarely and were reabsorbed, so we were not
able to document directly in our time-lapse experiments any event of material
loss through this mechanism. We also detected a possible morphological
correlate to this axonal blistering in neurones labelled with DiI during our
lineage studies. Axonal swellings are often correlated with defective axonal
transport. In Drosophila they are caused by mutations in kinesin
heavy chain, Dynein heavy chain (reviewed by
Goldstein and Yang, 2000
), and
Lis1 (Liu et al., 2000
). From
this point of view, it is interesting to notice that neurones derived from
sal mutant tissue exhibited both axonal blisterings and
discontinuities in the tubulin staining along the axon.
Staining of nervous tissue with 22C10 and detailed clonal analysis also demonstrated the persistence of irregularities in neuronal projections at the end of embryonic development. Smaller departures from normal neuroanatomy of nerve tracts and cell body location where also detected in larval tissues (data not shown). These might be due to effects on axonal growth and path-finding during stage 16, which are not reversible once axons are formed, or to lack of maintenance of fasciculation patterns at later stages.
In conclusion, we show that embryonic Drosophila CNS possess a
hitherto unknown capacity to recover from a strong adhesion phenotype, and
that the zinc-finger nuclear protein Sal is necessary for the integrity of
central nervous tissue, most probably acting in morphogenetic pathways that
directly or indirectly control cell adhesion, the cytoskeleton and membrane
integrity. Sal has a strictly nuclear localisation and is thought to function
as a transcription factor (Kühnlein
et al., 1994). An interesting question to be addressed will hence
be whether the activity of genes, which mediates normal dynamics of neural
cell adhesion and cytoskeleton, is under transcriptional control of Sal. Based
on the phenotype reported here, the genes that codify Fasciclin 2, Fasciclin
3, Armadillo, N-Cadherin and Neuroglian should be regarded as probable
candidates.
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ACKNOWLEDGMENTS |
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