1 Department of Genetics and the Rappaport Family Institute for Research in the
Medical Sciences, Faculty of Medicine, Technion-Israel Institute of
Technology, Haifa 31096, Israel
2 Unit of Electron Microscopy, Faculty of Medicine, Technion-Israel Institute of
Technology, Haifa 31096, Israel
* Author for correspondence (e-mail: adis{at}tx.technion.ac.il)
Accepted 28 February 2003
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SUMMARY |
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VVL is also expressed in developing external sensory organs in the embryo and in the adult. In the embryo, loss of VVL function results in increased apoptosis in specific es organs. Analysis of vvl mutant clones in adults revealed a requirement for VVL in the control of cell number within the bristle lineage.
Key words: vvl, ut, PNS, Chordotonal, POU-domain, Drosophila
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INTRODUCTION |
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In the embryonic PNS, abdominal segments A1-A7 share an identical pattern,
consisting of a dorsal, a lateral and two ventral neuronal clusters. There are
eight ch organs in each of these segments. The pentascolopidial ch organ
(lch5) and the v'ch1 organ are located in the lateral cluster, and vchA and
vchB reside in the ventral cluster (Fig.
3D) (Campos-Ortega and
Hartenstein, 1985; Ghysen et
al., 1986
). In Drosophila, sensory organs are generally
born in their final position. However, the precursors of the lch5 are born in
the dorsal region (Bier et al.,
1990
), yet by an unknown mechanism, so far termed lch5 migration,
the mature organs assume a lateral position. Genetic screens identified
mutations that perturb this migration and result in a `dorsal ch' phenotype
(Salzberg et al., 1994
;
Salzberg et al., 1997
;
Kania et al., 1995
). This
phenotype is also characterized by an incorrect orientation of the affected
neurons. When the lch5 neurons are located in a normal lateral position, their
dendrites point dorsally; when they are abnormally located within the dorsal
PNS cluster, their dendrites point ventrally or posteriorly. The correlation
between the position of the neurons along the dorsoventral axis and their
orientation suggests that descending to the lateral cluster is accompanied by
counter clockwise rotation of the lch5 neurons
(Salzberg et al., 1994
). The
non-neuronal cells of the lch5 organs have not been characterized with respect
to the lch5 migration process.
|
We show that ut is allelic to ventral veinless
(vvl), also known as drifter. VVL, a POU-domain
transcription factor, has been shown to participate in the development of the
embryonic tracheal system and CNS, and in wing formation in the adult
(Anderson et al., 1995;
de Celis et al., 1995
). We
find that VVL is also required for normal development of both embryonic and
adult PNS. In vvl mutant embryos the lch5 organs fail to rotate and
to stretch ventrally, the two steps required for achieving their lateral
localization, as we suggest in a new model. The expression pattern of VVL and
the results of rescue experiments suggest that VVL functions either
non-autonomously or both autonomously and non-autonomously in this process. In
addition, in the embryo, VVL is expressed in the developing es organs, and in
its absence increased apoptosis is observed in these lineages. In the adult,
VVL expression is observed in all cells of the developing bristles at early
stages, and becomes restricted mainly to the socket cells at later stages of
development. vvl mutant clones exhibit defects in bristle
development, which are characterized by excessive numbers of cells and
abnormal differentiation.
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MATERIALS AND METHODS |
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Sequencing
The coding sequence of vvl was amplified by PCR from genomic DNA
of heterozygous utH599, utH76, and
utM638 flies and controls. The PCR products were cleaned
using the Qiaquick gel extraction kit (Qiagen, Valencia, CA) and sequenced
using the Big Dye terminator cycle sequencing kit (PE Applied Biosystems,
Foster City, CA) and capillary electrophoresis on an ABI PRISM 310 automated
sequencer. The presence of mutations identified in genomic DNA was verified by
repeating PCR and sequencing on extracts from single homozygous embryos,
following the protocol for PCR from single flies
(Gloor et al., 1993).
Immunohistochemistry and TUNEL
Staining of whole-mount embryos was performed using standard techniques
(Patel, 1994) with minor
modifications. For the labeling of pupal tissues, pupae were aged at 25°C,
dissected in PBS and fixed in 4% formaldehyde for 15-30 minutes. After several
washes in PBT (PBS, 0.1% Tween 20), staining was performed as for embryos,
except that incubation with antibodies was performed in PBT plus 5% normal
goat serum. Primary antibodies were: mAb 22C10 (1/20)
(Fujita et al., 1982
); rat
anti-VVL (1/300) (Llimargas and Casanova,
1997
); mAb 21A6 (1/10; obtained from S. Benzer); mAb MR1A
anti-Prospero (PROS) (1/5) (Spana and Doe,
1995
); rabbit anti-REPO (1/200)
(Halter et al., 1995
); rabbit
anti-a85E (1/50) (Matthews et al.,
1990
); mouse and rabbit anti-ß-galactosidase (ß-Gal)
(1/1000; Promega and Cappel, respectively); rat anti-Suppressor of Hairless
(Su(H)) (1/1000-1/2000) (Gho et al.,
1996
); rabbit anti-DPax2 (1:200)
(Fu and Noll, 1997
); mouse
anti-ELAV and rat anti-ELAV (1/20 and 1/10, respectively)
(O'Neill et al., 1994
) as well
as mAb8D12 anti-REPO (1/10), mAb BP102 (1/20)
(Seeger et al., 1993
) and mAb
2B10 anti-Cut (CT) (1/20) were obtained from the Developmental Studies
Hybridoma Bank at the University of Iowa. Secondary antibodies for fluorescent
staining were Cy3, FITC or Cy5-conjugated anti-mouse/rabbit/rat (Jackson).
Secondary antibodies for non-fluorescent staining were biotinylated
anti-mouse/rabbit/rat detected with Vecta-Stain Elite ABC-HRP kit (Vector
Laboratories). Stained embryos and pupal tissues were viewed using bright
field and confocal microscopy (Zeiss Axioskop and Radiance 2000, BioRad).
Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) reaction was carried out following fluorescent staining and proteinase K treatment, using the In Situ Cell Death Detection Kit, TMR red (Roche Molecular Biochemicals).
Cuticle preparations and scanning electron microscopy
Anesthetized adult flies were boiled in a 10% NaOH solution for 10 minutes.
The cuticles were washed in water, dissected and mounted in Hoyer's. The
preparations were examined on a Zeiss Axioskop. For electron microscopy adult
flies were prepared following the HMDS method
(Wolff, 2000). The flies were
examined in a Jeol T-300 scanning electron microscope, operated at 25 kV.
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RESULTS |
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To map the ut gene further, complementation tests were performed
between the ut mutant strains and chromosomal deficiencies in the
65A-E region. Two deficiency chromosomes failed to complement the ut
mutant chromosomes: Df(3L)ZN47 (64C2-64C10; 65C1-65D4) and
Df(3L)v65c (64E1; 65C1-65D6). The Df(3L)CH12 (65A11; 65C2)
chromosome did complement the ut mutations. These results indicated
that the ut locus resided either in the 64E1;65A11 or 65C2;65D4
genomic interval. One of the candidate genes in these genomic regions is
ventral veinless (vvl), which maps to 65C5
(FlyBase, 1999). Two
observations strongly suggested that ut and vvl could be
allelic. First, a cross between ut alleles and the
In(3LR)282 chromosome, which has a breakpoint in the 65C-D region,
yielded adult progeny that lacked the L4 wing veins (not shown). vvl
is known to be required for wing vein formation
(de Celis et al., 1995
).
Second, vvl mutant embryos exhibit a collapse of the anterior
segments of the ventral nerve cord (VNC), a phenotype we observed also in
ut mutant embryos (not shown). Complementation tests between the
embryonic lethal allele vvlGA3 and ut alleles
demonstrated that ut and vvl were indeed allelic.
To characterize the nature of the ut mutations, we amplified and sequenced the coding region of vvl from the utH599, utH76 and utM638 strains. The coding sequence of vvl from utH599 flies revealed a point mutation converting tryptophan in position 138 to a stop codon. In utH76 flies we identified a mutation changing tryptophan in position 351 into a stop codon. We were not able to identify any mutation in the coding sequence of the utM638 allele, suggesting a possible mutation interfering with regulatory elements of the gene.
VVL is required for the correct positioning of lch5 organs
The most prominent defect in the PNS of vvl homozygous embryos is
the frequent abnormal positioning of lch5 neurons
(Salzberg et al., 1994) (this
work). In wild-type embryos, these neurons are invariably located in the
lateral cluster of abdominal segments A1-A7
(Fig. 1A). In 60-70% of the
abdominal segments of utH599 or vvlGA3
mutant embryos (n=138 and n=115 respectively), the lch5
neurons were situated in a more dorsal position than normally
(Fig. 1B,C). As described
previously (Salzberg et al.,
1994
), the orientation of the neurons differed depending on their
location along the dorsoventral axis. When the lch5 neurons were situated high
within the dorsal cluster, their dendrites pointed ventrally, similar to the
dch3 in thoracic segments T2 and T3 (not shown). If the lch5 neurons were
located just above or below the level of the dorsal bipolar dendrite (dbd)
neuron, their dendrites pointed in a ventroposterior or a dorsoposterior
direction, respectively (Fig.
1B,C). Only when these neurons were located near or within the
lateral cluster, their dendrites pointed dorsally as in a normal embryo. The
number of the lch5 neurons was not affected in these mutants. Of the three
additional ch neurons present in each abdominal segment the v'ch1 neuron was
invariably present in its correct position in vvl mutant embryos
(Fig. 1B,C). The vchA and vchB
neurons were always present too; however, in about 40% of the segments, either
one or both vch neurons were abnormally oriented, more often the vchA neuron
(not shown).
|
To verify this hypothesis we examined whether the dorsoventral location of the ligament cells, relative to the other cells of the organ, changes in the course of lch5 development in normal embryos. We double-stained ato-lacZ embryos, in which lacZ is expressed in all cells of the ch lineage, with anti-ß-GAL and anti-REPO. Indeed, in stage 12 embryos the REPO-expressing ligament cells were often observed in a dorsal position within the organ. During stage 13, these cells acquired the most ventral position within the organ (Fig. 1H-J). These data suggest that the ligament cells migrate ventrally, possibly pulling the whole organ with them. We therefore conclude that the concept of lch5 neuronal migration is inaccurate, and refer to this process as lch5 lateral localization.
Where is VVL function required for lch5 lateral localization?
Lateral localization of the developing lch5 organs is probably directed by
factors expressed within these organs and factors expressed in the surrounding
environment. In embryos, VVL expression has been previously detected in the
developing tracheae, midline glia, oenocytes and ectoderm
(Anderson et al., 1995). This
expression pattern suggests a non-autonomous function of VVL in lch5
localization. However, the ectodermal expression of VVL, which begins at stage
12, may mask concomitant expression in the developing PNS.
In order to determine whether VVL is expressed in the developing lch5 lineage, we double labeled ato-lacZ embryos with anti-VVL and anti-ß-GAL. We found that VVL is not expressed in the lch5 precursors in stage 11 (Fig. 2A). During stages 12-13, very low levels of VVL could be detected in some of the lacZ-expressing cells (Fig. 2B,C). Because, at these stages, all the cells of the developing lch5 organs are clustered together, we could not identify the VVL-expressing cells unambiguously. However, double labeling with anti-REPO and anti-VVL demonstrated that the ligament cells did not express VVL. From stage 14 onwards, a slightly stronger VVL expression was clearly detected in the lch5 neurons (Fig. 2D-F).
|
VVL is required for the normal development of embryonic es
organs
Characterization of VVL expression in the embryonic PNS revealed it was
expressed in developing es organs in stage 12 and older embryos
(Fig. 3A-C). In accordance with
this expression pattern, vvl mutant embryos stained with mAb 22C10
exhibited a mild loss of es neurons. Of the 15 es neurons normally present in
each of the abdominal segments A1-A7 (Fig.
3D) [nomenclature according to Ghysen et al. and Bodmer et al.
(Ghysen et al., 1986;
Bodmer et al., 1989
)], desC,
desD, lesB, lesC, v'esA and vesC were often missing. All other es neurons
seemed largely unaffected by the lack of VVL. The absence of these neurons was
verified by anti-ELAV staining, which revealed accordingly reduced numbers of
neuronal nuclei in all four neuronal clusters
(Fig. 3E,F).
Such reduction in the number of es neurons could be the result of cell fate transformations within the lineage, or a failure of the entire organ to form. To distinguish between the two possibilities, we stained mutant embryos for several es organ cell type-specific markers. Sheath cell nuclei were detected by anti-PROS, and outer support cells were detected using anti-Su(H) and the A1-2-29 enhancer trap. All of these markers showed a decreased number of cells when compared with wild-type embryos (not shown). For example, in the two dorsalmost es organs, desC and desD, the neurons were missing in 53-69% of the segments (n=45, desD and desC respectively). Sheath cells were missing in 24% (desD) or 57% (desC) of the segments (n=70). The two outer support cells were both missing in 18% (desD) or 61% (desC) of the segments (n=94), and only rarely there was only one cell present (not shown). Staining with anti-CT, which labels all cells of the es organ, demonstrated that it is not the whole organ that fails to form, rather, only some of the cells are lost (Fig. 3G,H). These results indicate that VVL is not required for precursor formation or for cell fate decisions within the es lineage.
Certain POU-domain containing proteins have been implicated in
differentiation and survival of PNS cells
(McEvilly et al., 1996;
Xiang et al., 1998
;
Gan et al., 1999
). In order to
find whether VVL has a similar role in embryonic es organ development, we
performed TUNEL analysis on vvl mutant embryos and examined the
number of apoptotic nuclei in the PNS. We focused on the lateral cluster, in
which one or two of the three es organs are often affected. In stage 14
utH599/+ embryos, very few apoptotic nuclei are present in
the PNS of each abdominal segment. In the lateral cluster, typically one or
two apoptotic nuclei are evident (Fig.
3I). In utH599 homozygous embryos, a marked
increase in the number of apoptotic nuclei in the lateral cluster is observed
(Fig. 3J), indicating that lack
of VVL expression results in enhanced death of cells in the developing PNS. To
verify that the apoptotic nuclei belong to PNS cells, we performed TUNEL
analysis on vvlGA3 embryos, which carry one copy of the
A101 transgene, stained with anti-ß-GAL. This analysis confirmed
that at least some of the increased apoptosis in vvl mutant embryos
takes place in the PNS (Fig.
3K).
VVL is expressed in developing es organs of the adult
Although all four alleles of vvl used in this work are embryonic
lethal when homozygous, the
utM638/vvlGA3 and
utM638/utH76 allelic combinations
resulted in a low percentage of pharate adults, presenting bristle defects
(see below). This suggested that VVL functions in adult es organ development
as well. We therefore looked for VVL expression in the developing adult PNS.
Staining pupal heads and nota revealed that VVL is indeed expressed in the
developing es organs. At 16 hours after puparium formation (APF), many of the
es organ precursors have already divided once to generate the two daughter
cells pIIa and pIIb. VVL expression was detected both in the precursors and in
the two daughter cells (Fig.
4A). At 24 hours APF, when the division of pIIa and pIIb was
completed, uniform expression of VVL could be observed in all four
post-mitotic cells (Fig. 4B).
In later stages this expression gradually decreased in three of the four cells
(Fig. 4C), and by 42 hours
APF, strong VVL expression was detected in one large nucleus, which according
to its position within the cluster belonged to the socket cell. A much weaker
expression was occasionally observed in the adjacent large nucleus of the
shaft cell (Fig. 4D). The
identity of the VVL expressing nuclei was verified by double labeling with
anti-VVL and with either anti-Su(H), which labels socket cells
(Gho et al., 1996
)
(Fig. 4D), or
anti-Drosophila Pax2, which labels shaft cell nuclei
(Kavaler et al., 1999
) (data
not shown). Staining pupal retinas revealed that VVL was also expressed in the
developing interommatidial bristles (Fig.
4E-G).
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In the eyes, the phenotype was somewhat different and extremely consistent. All affected bristles had dramatically shortened shafts, but the number of shaft and socket cells appeared normal (Fig. 4M,N).
Loss of VVL results in supernumerary cells in adult es organs
If the increased numbers of shaft and socket cells were the result of cell
fate transformations within the es lineage, then a reduced number of the
internal cells, namely the neuron and sheath cell, is expected. However, no
such reduction was observed and the number of internal cells was either normal
or increased. Mutant clones stained with mAb 22C10, which labels the neuron
and shaft cells, revealed that the number of neurons was mostly unchanged,
whereas the number of shaft cells was often increased. Rarely, two neurons
were observed in a single developing organ. Staining with anti-PROS
demonstrated an occasional increase in the number of sheath cells to two or
three per organ (not shown). These results suggest that no cell fate
transformations occur in the mutant bristle lineages, rather extra cell
divisions generate supernumerary cells. This conclusion was corroborated by
anti-CT staining which demonstrated many clusters with five or more
CT-expressing nuclei, instead of only four in normal developing bristles
(Fig. 4O,P). This conclusion,
however, does not apply to interommatidial bristles, as we did not observe an
increased number of cells in these organs.
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DISCUSSION |
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By visualizing all the cells of lch5 organs located in a variety of positions between the dorsal and lateral clusters, we were able to determine that the polarity of the neurons reflects the polarity of the whole organ. When the neurons point ventrally, the ligament cells are the most dorsal cells of the organ and the cap cells are the most ventral, whereas for the dendrites to point dorsally, the ligament cells must be ventral and the cap cells dorsal. In normal stage 12 embryos, the ligament cells can be detected at the dorsal part of the organ, whereas in older embryos they migrate ventrally to become the ventralmost cells of the organ. Based on these observations, we propose a two-step model for the lateral localization of lch5 organs (Fig. 5A). In the first step, rotation of the organ takes place. The organ rotates around the attachment cells, which anchor it to the ectoderm and thus function as a pivot. The rotation results in both bringing the organ to its correct orientation and placing it in a more ventral position, closer to the lateral cluster. This step occurs during stage 12 and perhaps early stage 13. Once in their correct orientation the lch5 organs go through the second step, which involves ventral stretching into their final shape and position, as seen in stage 15 or older embryos. This model is further supported by the fact that when the thoracic dch3 are forced to descend to the lateral cluster by overexpressing abd-A, their orientation is reversed and the ligament cells are found at their ventral edge (Fig. 5B).
|
The role of VVL in lch5 lateral positioning
Mutations in three loci, abd-A, hth and sal, have been
shown previously to perturb the lateral localization of lch5 organs
(Heuer and Kaufman, 1992;
Kurant et al., 1998
;
Rusten et al., 2001
).
Mutations in these three loci result in both abnormal localization and
abnormal number of lch5 organs. However, decisions of organ number and organ
localization are not always coupled. For example, mutations in the EGFR
pathway gene rhomboid and the EGFR pathway antagonist argos,
affect the number of lch5 organs but only rarely affect their position
(Bier et al., 1990
;
Okabe et al., 1996
) (A.I and
A.S., unpublished). vvl is the first gene that affects the
localization of the lch5 organs without affecting their number. The abnormal
localization of lch5 organs in vvl mutant embryos is similar to the
abnormal localization of these organs in hth and abd-A
mutant embryos, suggesting these genes are required in the same developmental
pathway. However, epistasis experiments did not provide evidence for direct
genetic interactions between vvl and hth or vvl and
abd-A (A.I. and A.S., unpublished).
VVL, a class III POU-domain transcription factor, and its mammalian
homologs, have been shown to be required for cell migration. Brn1 and Brn2,
the mouse homologs of VVL, have a crucial role in the migration of cortical
neurons (McEvilly et al.,
2002; Sugitani et al.,
2002
). In the CNS of Drosophila embryos, VVL is required
for the migration of midline glial cells
(Anderson et al., 1995
). In the
embryo, VVL is also required for tracheal cell migration and in its absence
the tracheal tree fails to form (Anderson
et al., 1995
; de Celis et al.,
1995
).
The mechanism by which VVL affects the lateral localization of lch5 organs
is not clear. Cell migration requires the existence of signals from the
environment and the ability of the migrating cell to receive and respond to
these signals. Previous work has demonstrated that in tracheal development,
VVL functions autonomously and it was suggested to regulate the expression of
cell surface molecules necessary for the migration of tracheal cells
(Anderson et al., 1995). In
lch5 organs, VVL expression is detected in the neurons. However, expressing
VVL under elav-Gal4 regulation in vvl mutant background
could not rescue the mutant phenotype. This result suggests that the neuronal
expression of VVL is either not required or not sufficient for lch5 lateral
localization. Driving VVL expression with ato-Gal4 could rescue the
mutant phenotype; however, in a much lower efficiency than when VVL was
expressed under arm-Gal4 regulation. The major differences between
these two drivers are that while ato-Gal4 drives strong expression in
the lch5 lineage and in a small group of ectodermal cells, arm-Gal4
induces strong expression throughout the ectoderm and only weak expression in
lch5 organs. Thus, the results of these experiments cannot determine
unambiguously where VVL is required during lch5 lateral localization, and
suggest it could function in both lch5 organs and the surrounding ectoderm, or
in the ectoderm alone. The more efficient rescue generated by
arm-Gal4 may indicate that the ectodermal expression of VVL is the
main factor with regard to lch5 positioning. In the ectoderm, VVL could be
involved in the generation of a positional cue. Although the rescue of lch5
localization was achieved by ubiquitous expression of VVL in the ectoderm, it
should be noted that the normal ectodermal expression of VVL during critical
stages of lch5 positioning (stages 12 and early 13) is not uniform. VVL is
more strongly expressed in a dorsal domain of the embryo
(Fig. 5C), from the position of
the lateral cluster dorsally. Later during stage 13, VVL expression becomes
uniform throughout the ectoderm. It is not clear yet whether this differential
expression is significant in the context of lch5 positioning. VVL has been
shown to interact with other transcription factors in the CNS and trachea
(Ma et al., 2000
;
Zelzer and Shilo, 2000
). Thus,
another possibility is that an unidentified partner of VVL confers a spatial
specificity to its activity.
Two additional cell types in the vicinity of the developing lch5 organs
express high levels of VVL: tracheal cells and oenocytes. The trachea is
probably not involved in the process of lch5 localization, as
trachealess mutants do not exhibit a `dorsal ch' phenotype (A.S.,
unpublished). The possible role of oenocytes in lch5 migration is intriguing.
Impaired lch5 localization is many times accompanied by partial or complete
loss of oenocytes, as seen in embryos mutant for abd-A
(Brodu et al., 2002),
sal (Rusten et al.,
2001
), hth and vvl (A.I. and A.S., unpublished).
However, rhomboid mutants lack oenocytes
(Elstob et al., 2001
), yet
their lateral ch organs (which consist of three, instead of five, scolopidia)
are almost always positioned properly. Thus, it seems more probable that lch5
organs and oenocytes are independently affected by the same mutations.
Apoptosis in the PNS in the absence of VVL a common theme
with mammalian POU-factors
In Drosophila, loss of es organ cells has been attributed to one
of two reasons. Either the organ completely fails to form because of
interference with the function of the proneural genes, or cell fate
transformations occur between the cells comprising these organs. However, in
vvl mutant embryos the decreased number of these cells is a result of
increased apoptosis. Any of the cells of the organ could be affected, and the
remaining cells expressed typical markers, suggesting that initial decisions
of cell fates were not impaired. It is therefore possible that VVL is required
for cell survival in the developing es lineages. Another possibility is that
VVL is required for the differentiation of these organs, and that in its
absence some of the cells fail to differentiate properly and go through
apoptosis.
In mammals, POU-domain transcription factors were shown to play significant
roles in survival of cells in the nervous system. Members of the class IV
POU-factors are known to be essential for differentiation and survival of PNS
cells (McEvilly et al., 1996;
Xiang et al., 1998
;
Gan et al., 1999
). The most
interesting of those in the context of Drosophila es organ
development is Brn3c, which is required for maturation and survival of the
inner ear hair cells (Xiang et al.,
1998
). The vertebrate inner ear hair cells are mechanosensory
organs, considered homologous to Drosophila bristles in many aspects.
The parallelism between the two types of organs was shown at the levels of
function, structure and the molecular mechanisms responsible for their
development (Adam et al., 1998
)
(reviewed by Eddison et al.,
2000
). Mice deficient for Brn3c fail to develop inner ear hair
cells and are completely deaf (McEvilly et
al., 1996
; Xiang et al.,
1998
). A mutation in the human homolog of this gene was shown to
cause progressive hearing loss (Vahava et
al., 1998
). The defects seen in the development of the hair cells
in Brn3c-null mice are limited to maturation and survival of these
organs (Xiang et al.,
1998
).
Although there is not sufficient evidence to consider a functional homology between VVL, a class III POU-factor, and the mammalian class IV POU-factors, it will be interesting to determine whether the similarity of their loss-of-function phenotypes extends further at the molecular level.
The role of VVL in adult bristle development
vvl mutant clones in adult head tissue caused defects in bristle
development, which typically resulted in supernumerary cells. One possible
explanation for an increase in the number of bristle cells is that too many
precursors were formed as a result of inefficient lateral inhibition. In such
case, we would expect the appearance of complete ectopic organs. However, the
supernumerary cells did not constitute separate organs, rather they increased
the number of cells within a single es organ. This observation suggests that
one or two extra cell divisions took place, resulting in the production of
extra cells within the lineage. Thus, it is possible that VVL is required in
these cells for exit from the cell cycle.
Many abnormalities were also observed in the structure of the external support cells of the mutant bristles, especially in the shaft. Whether the structural defects are secondary to the abnormal pattern of cell division, or they represent another independent role for VVL in the differentiation of these structures remains to be determined.
Loss of VVL function in the embryonic and adult es lineages results in what seem to be two very different phenotypes: loss of cells in the embryo as opposed to overproduction of cells in the adult. However, it is possible that both phenotypes reflect a failure of the es cells to commence differentiation. These cells may behave differently when unable to differentiate properly, depending on their developmental context.
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ACKNOWLEDGMENTS |
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