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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02605
Institute of Genetics, University of Mainz, D-55099 Mainz, Germany.
* Authors for correspondence (e-mail: urbach{at}uni-mainz.de; technau{at}unimainz.de)
Accepted 31 August 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: CNS, Brain, Neuroectoderm, Neuroblasts, Dorsoventral patterning, Cell fate, vnd, msh, ind, Glia, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Poulson,
1950). The developmental processes that underlie NB formation,
cell fate specification and pattern formation have been extensively studied in
the embryonic VNC. In early stages of embryogenesis, each segment of the
truncal neuroectoderm is subdivided by segment-polarity genes into transverse
rows along the anteroposterior axis, and by `columnar genes' (DV patterning
genes) into longitudinal columns along the dorsoventral axis. The
superimposition of their gene activities establishes a Cartesian coordinate
system of positional cues conferring specific identities to neural equivalence
groups, of which each gives rise to a single NB (reviewed by
Bhat, 1999;
Skeath, 1999;
Skeath and Thor, 2003).
Accordingly, each NB acquires a unique identity, which is reflected by the
time and position of its delamination from the neuroectoderm, by the
combination of genes it expresses, and by the production of a specific cell
lineage (e.g. Bossing et al.,
1996; Broadus et al.,
1995; Doe, 1992;
Schmidt et al., 1997). Through
the expression of proneural genes, each cell of an equivalence group (or
proneural cluster) acquires the potential to become a NB (e.g.
Skeath and Carroll, 1992).
Notch signaling ensures that only a single cell adopts a NB fate and the
others assume an epidermal fate (reviewed by
Campos-Ortega, 1995).
The truncal neuroectoderm is subdivided along the dorsoventral axis into
adjacent longitudinal columns by the expression of three genes: ventral
nervous system defective (vnd) in the ventral, intermediate
neuroblasts defective (ind) in the intermediate and muscle
segment homeobox [msh; Drop (Dr) - FlyBase] in
the dorsal neuroectodermal column
(Buescher and Chia, 1997;
Chu et al., 1998;
D'Alessio and Frasch, 1996;
Isshiki et al., 1997;
Jimenez et al., 1995;
Mellerick and Nirenberg, 1995;
Weiss et al., 1998).
Experimental data provide evidence that these homeobox genes interact in a
hierarchical cascade of transcriptional repression, which is necessary to
establish and maintain the sharp boundaries between their domains of
expression. According to that hierarchy, vnd represses ind
(and msh) in the ventral column, and ind represses
msh in the intermediate column (reviewed by
Cornell and Von Ohlen, 2000;
Skeath, 1999).
vnd, the prototype of the Nkx2-type gene family, is
expressed not only in the ventral column neuroectoderm, but also in descending
NBs, and is maintained in subsets of their daughter cells. Coincident with its
pattern of expression, vnd is required for specification of ventral
column neuroectoderm, as well as for formation and specification of ventral
NBs and their progeny (Chu et al.,
1998; Jimenez and
Campos-Ortega, 1990; McDonald
et al., 1998; Mellerick and
Nierenberg, 1995; Mellerick
and Modica, 2002; Shao et al.,
2002; Skeath et al.,
1994). Similar roles are fulfilled by ind in
intermediate, and msh in dorsal, column neuroectoderm and NBs,
although msh is dispensable for the formation of dorsal NBs
(Isshiki et al., 1997;
Weiss et al., 1998). Owing to
much higher complexity and hidden segmental organization, the mechanisms that
control pattern formation in the brain are largely unknown. The brain can be
subdivided (from posterior to anterior) into the tritocerebrum, deutocerebrum
and protocerebrum, which derive from the intercalary, antennal and ocular
(plus labral) segments, respectively (Schmitt-Ott and Technau, 1992) (reviewed
by Urbach and Technau, 2003a).
Recently, we have described the pattern of the entire population of brain NBs
(Urbach et al., 2003) and
could show that, based on the combination of genes expressed, each brain NB
acquires a unique identity (Urbach and
Technau, 2003b). Furthermore, we have presented first data on the
expression of the columnar genes in the early embryonic brain
(Urbach and Technau,
2003c).
In this study, we have investigated the function of columnar genes during early brain development with the main focus on vnd. We show that vnd is required for formation of ventral brain NBs, and that cell death contributes to the loss of brain NBs in embryos lacking vnd function. Detailed analysis of the expression of an array of molecular markers in loss- and gain-of-function backgrounds indicates that vnd is also necessary, and to some extent sufficient, for the proper specification of ventral and intermediate pNE and brain NBs. We further demonstrate that control of ind and msh expression by vnd differs among brain neuromeres and between brain and VNC. Finally, we show that the generation of normal numbers of early glial cells in the protocerebrum is independent of msh and vnd, which is in contrast to the situation in the trito-/deutocerebrum and the VNC.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), svp-lacZ (H162)
(Mlodzik et al., 1990),
vnd6 (Jimenez and
Campos-Ortega, 1990) and mshd68
(Isshiki et al., 1997). The
UAS-Gal4 system (Brand and Perrimon,
1993) was used to overexpress vnd in the neuroectoderm,
crossing UAS-vnd (Chu et al.,
1998) with the sca- Gal4 driver line
(Klaes et al., 1994).
Staging, flat preparation, and mounting of embryos
Staging of the embryos was carried out according to Campos-Ortega and
Hartenstein (Campos-Ortega and
Hartenstein, 1997). Flat preparations of the head ectoderm of
stained embryos and mounting were carried out as described previously
(Urbach et al., 2003).
Antibodies and immunohistochemistry
Embryos were dechorionated, fixed and immunostained according to previously
published protocols (Patel,
1994). The following primary antibodies were used:
rabbit-anti-Atonal [1:5000 (Jarman et al.,
1993); kindly provided by A. Jarman], rabbit anti-cleaved human
caspase 3 [1:50 (Brennecke et al.,
2003); Cell Signaling Technology], mouse-anti-Dachshund [1:250
(Mardon et al., 1994); DSHB],
rabbit-anti-Deadpan [1:300 (Bier et al.,
1992); kindly provided by H. Vaessin], anti-DIG-AP (1:1000,
Roche), rat-anti-Empty spiracles [1:1000
(Walldorf and Gehring, 1992);
kindly provided by U. Walldorf], mouse-anti-Engrailed [4D9,1:7
(Patel et al., 1989); DSHB],
rabbit-anti-Eyeless (1:1000, kindly provided by U. Walldorf),
mouse-anti-ß-Galactosidase (1:500, Promega),
rabbit-anti-ß-Galactosidase (1:2500, Cappel), rat-anti-Gooseberry-distal
[16F12 and 10E10, 1:2 (Zhang et al.,
1994); kindly provided by B. Holmgren], mouse-anti-Ladybird early
[1:2 (Jagla et al., 1997);
kindly provided by K. Jagla], rabbit-anti-Muscle segment homeobox (1:500;
kindly provided by M. P. Scott), rabbit-anti-Repo (1:500)
(Halter et al., 1995),
rat-anti-Sloppy paired [1:300 (Cadigan et
al., 1994); kindly provided by W. Gehring], rabbit-anti-Ventral
nervous system defective [1:2000 (McDonald
et al., 1998); kindly provided by C. Doe] and mouse-anti-Wingless
(1:10, DSHB). The secondary antibodies (donkey anti-mouse, goat anti-rabbit;
donkey anti-rat; Dianova) were either biotinylated or alkaline
phosphatase-conjugated and diluted 1:500.
Whole mount in situ hybridization
DIG-labeled intermediate neuroblast defective RNA probe (kindly
provided by M. P. Scott) was synthesized using HindIII linearized
pNB40-ind as a template with T7 polymerase, and DIG-labeled lethal of
scute RNA probe was synthesized using the EST clone lsc RE59335 (BDGP)
cloned in PstI linearized pFLC1 as a template with T3 polymerase
according to the manufacturers protocol (Roche). The hybridization on embryos
was performed as described previously
(Plickert et al., 1997;
Tautz and Pfeifle, 1989).
Documentation
Embryos were viewed under a Zeiss Axioplan equipped with Nomarski optics
using 40x, 63x and 100x oil immersion objectives. Pictures
were digitized with a CCD camera (Contron progress 3012) and different focal
planes were combined using Adobe Photoshop 7.0. Semi-schematic presentations
are based on camera lucida drawings.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Thus, in contrast to the situation in the trunk, Vnd expression in the
early brain is highly dynamic and becomes progressively confined to three
separate ventral domains, encompassing different numbers of NBs and their
progeny in the posterior compartments of the TC, DC and PC [for Vnd expression
at later stages, see Sprecher et al.
(Sprecher et al., 2006)].
Absence of Vnd affects formation of ventral brain NBs
To assay the presence of individual brain NBs in embryos lacking
vnd function (referred to as `vnd embryos'), we used
morphological criteria (size and position), the NB marker Deadpan (Dpn) and
Seven up-lacZ (Svp-lacZ). Between stages 9 and 11
Svp-lacZ-positive brain NBs represent stable reference points, also
allowing identification of Svp-lacZ-negative NBs surrounding them
(Urbach et al., 2003). We
focused mainly on the pattern of NBs at stage 9 (corresponding to the S2 NB
pattern in the ventral nerve cord, VNC), and at stage 11 (corresponding to the
S5 NB pattern in the VNC) (Doe,
1992) when the full complement of brain NBs is established
(Urbach et al., 2003). The
expression of all molecular markers used in this study for the identification
of individual NBs in wild-type and mutant embryos is summarized in
Fig. 2 and
Table 1. The analysis of large
samples of embryos allowed us to quantify the data and thus to determine the
variability of the phenotypes. The frequency at which each NB is found in
vnd embryos is indicated in percent in
Fig. 2B',D'.
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)].
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Taken together, a considerable proportion of ventral NBs is not formed in the TC, DC and PC of vnd embryos. The probability of being part of this fraction significantly differs among the individual NBs.
Cell death contributes to loss of ventral brain NBs in vnd mutant embryos
In order to investigate whether cell death accounts for loss of ventral
brain NBs, we performed antibody staining against activated caspase 3 (Casp3).
Casp3 activation is one of the early hallmarks of cell death. In the wild-type
pNE, cell death first becomes detectable by late stage 10, and increases
rapidly during stages 11/12 (see also
Nassif et al., 1998). A
comparable time course is observed in vnd embryos. However, apoptosis
is significantly more pronounced in the ventral pNE of the developing DC and
PC, but not TC (Fig. 4A,B).
During early/middle stage 11, Casp3 staining was found in cells of the pNE and
in brain NBs (Fig. 4C), at
relative positions corresponding to the loss of deuto- and protocerebral NBs
at later stages (compare Fig. 4C with
4D,E). We did not detect apoptotic NBs in the TC. These data
suggest that apoptosis of neuroectodermal progenitor cells, and of already
developed NBs in the DC and PC, partially accounts for the defects in the
pattern of brain NBs in vnd embryos [for apoptosis in vnd
mutant brain at later stages, see Sprecher et al.
(Sprecher et al., 2006)].
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).
The combinations and frequencies in which the various markers are expressed in
individual brain NBs of vnd embryos are summarized in
Fig. 2B,D and in
Table 1. In wild type, Msh is
expressed in the dorsal pNE of the TC and DC, but not of the PC
(Fig. 6A-D). In vnd
embryos, Msh is derepressed in the ventral pNE and residual ventral NBs in the
TC and DC, but again not detected in the PC
(Fig. 6E-H). In vnd
mutants, Ems expression becomes ectopically expanded into large parts of the
ventral antennal and intercalary pNE, and into part of the residual ventral
NBs (three to four in the DC and one in the TC;
Fig. 5A-D), resembling the
pattern of ectopic Msh. Lbe expression in the pNE of the TC (and slightly in
the DC) is expanded ventrally, although we did not detect descending ectopic
Lbe-positive NBs (Fig. 5E-H).
Dac is derepressed in part of the ocular Vnd mutant domain
(Fig. 5I-L), and we identified
Dac in two to four additional ventral NBs in the PC descending from this
ectopic Dac domain (Fig. 5L).
Furthermore, Ey is lacking in intermediate pNE (and respective NBs) of the TC,
but is ectopically expressed in the ventral pNE
(Fig. 5M-P). These data
indicate that the expression of several dorsal marker genes is expanded in
vnd mutants into the ventral pNE and deriving NBs, suggesting that
loss of Vnd at least in part leads to a ventral-to-dorsal transformation of
ventral pNE and residual ventral NBs.
To find out whether `intermediate NBs', which normally derive from the
insulated domains of ind expression (e.g. Td1, Td2, Td3 and Dd1), are
also affected in the mutant, we investigated the expression of
intermediate neuroblasts defective (ind), Ey,
Gooseberry-distal (Gsb-d), Wingless (Wg) and Svp-lacZ, markers which
are co-expressed in part of these precursors in wild type (see
Fig. 2A,C,
Table 1)
(Urbach and Technau, 2003b).
In vnd embryos, Td3 in most cases develops properly
(Fig. 2D,
Fig. 3D,
Fig. 6T). By contrast, lack of
ind is found in positions of Td1, Td2 and Dd1
(Fig. 2D, compare
Fig. 6P with 6T). Expression of
the other marker genes suggests that Td2 and Dd1 are mostly formed but become
improperly specified, whereas Td1 is not formed
(Fig. 2D,
Fig. 3D).
Together, these data indicate that Vnd is required for proper specification of ventral pNE and residual ventral, as well as for intermediate NBs.
Ectopic Vnd changes the identity of dorsal pNE and dorsal brain NBs
To test whether vnd is able to alter the fate of dorsal pNE and
NBs, we ectopically expressed Vnd (using sca-Gal4; embryos in the
following termed sca-vnd) and examined the expression of Dac, Ems,
En, Ey, Lbe, Msh and Wg, which are specifically expressed in dorsal and/or
ventral parts of the pNE and brain NBs. In sca-vnd embryos, these
markers are largely suppressed or completely absent in the dorsal pNE and NBs
of the TC (Ems, Ey, Lbe, Msh and Wg), DC (Ems, En, Ey, Lbe, Msh, and Wg) and
PC (Dac, Ems, En, Ey, and Wg) (Fig.
5Q-X, Fig. 6I-L and
data not shown). The effects of ectopic Vnd on the level of individual NBs are
summarized in Table 1.
Susceptibility to ectopic Vnd significantly differs among dorsal NBs in a
cell-specific manner. For example, Lbe is frequently missing in the dorsal Dd7
(40%), and always in the dorsal Td4 (0%). The two ventral Lbe-positive NBs in
the PC, Ppv3 and Pcv8, are maintained (100%), and, in addition, we usually
identified ectopic Lbe in one to six adjacent NBs in the dorsal PC (compare
Fig. 5F with 5T). This
indicates that ectopic Vnd suppresses Lbe in the dorsal TC and DC, but confers
ventral fates to a subset of dorsal NBs in the PC.
Taken together, in the vnd gain-of-function situation, we recognized a wide-ranging loss of specific marker gene expression and a partial dorsal-to-ventral transformation of dorsal pNE and corresponding brain NBs.
Regulation of ind and msh by Vnd in the pNE and brain NBs differs from the trunk in a segment-specific manner
In wild type, the expression pattern of ind and msh
differs between head and trunk and among head segments. ind is
expressed in three separate pNE patches, of which the intercalary and antennal
ind spot are located at intermediate position between the dorsal Msh
and ventral Vnd domain. The domains of Msh and Vnd share a common border at
sites lacking an intervening ind domain. In the ocular segment, the
ind domain is spatially separated from the Vnd domain, and Msh is not
expressed (Fig. 6A-D,M-P) (see
also Urbach and Technau,
2003c). To assay whether vnd regulates these DV
patterning genes in the pNE and brain NBs, we examined the expression of Msh
and ind in vnd loss-of-function and gain-of-function
embryos.
In vnd mutants, ind is derepressed within the ventral
part of the truncal neuroectoderm (Weiss
et al., 1998). Surprisingly, we did not observe such a
derepression of ind in the ventral intercalary and antennal segment
(compare Fig. 6M,O with 6Q,S).
Instead, expression of ind in the intercalary segment is delayed
(starting after stage 9) and found in a smaller area. Even more unexpectedly,
ind in the antennal pNE and Dd1 was never detected [0%,
n=30; `cluster1' in McDonald et
al. (McDonald et al., 1998)]. This indicates that in the antennal
segment, Vnd is necessary for activating ind, rather than for
repressing it. ind transcript in the ocular pNE often appears
slightly expanded ventrally (70%, n=30;
Fig. 6O,S).
Msh expression in vnd embryos is ectopically expanded into the
ventralmost pNE of the intercalary and antennal segment and corresponding
residual NBs, apparently owing to the lack of repression by vnd and
ind (compare Fig. 6A-D with
6E-H). We observed derepression of Msh already before the early
phase of NB formation (stage 8/9; Fig.
6E,F). This is in contrast to observations made in the trunk,
where ind (instead of msh) is derepressed in the ventral
neuroectoderm at these stages (McDonald et
al., 1998) [although Msh is found in the ventral neuroectoderm at
later stages (Chu et al.,
1998)]. Early ectopic Msh is likely to `dorsalize' the ventral pNE
and developing residual NBs. We found, that ectopic Msh also encompasses the
region of the missing antennal ind spot, suggesting that cell fate of
intermediate Dd1 (which is mostly formed) may also be affected by ectopic Msh.
By contrast, ectopic Msh is largely absent at positions of the intercalary
ind spot (compare Fig. 6H with
6T), indicating that, at least in the TC, the genetic interaction
of ind and msh (repression of msh by ind)
may be comparable with the situation in the trunk.
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In sca-vnd embryos, expression of ind is lacking in the intercalary domain in 85% of cases, and diminished in the ocular domain in 37% cases (n=50), indicating that, as in the trunk neuroectoderm, ectopic Vnd is able to suppress ind expression (compare Fig. 6M-P with 6U-X). However, the antennal ind spot is almost unaffected under these conditions (observed in 92% cases by stage 10/11; n=50). Thus, ind expression in the antennal (and residual ocular) domain is less sensitive to ectopic Vnd.
Taken together, these data indicate that interactions between vnd, ind and msh differ among brain segments and compared with the VNC (Fig. 6Y). In the brain, ind is expressed in three separate segmental domains, and Msh is not expressed in the PC. Whereas in the trunk Vnd negatively regulates ind, Vnd does not repress ind (but msh) in the ventral pNE and NBs of the DC and TC. Instead, in the deutocerebral region, Vnd is necessary for activating and/or maintaining ind expression.
Normal production of glial cells depends on Msh and Vnd in the posterior brain (TC and DC) but not in the anterior brain (PC)
In the VNC, most glial cells derive from dorsal NBs
(Schmidt et al., 1997), and
msh is implicated in proper specification of these NBs and their
progeny (Buescher and Chia,
1997; Isshiki et al.,
1997). Likewise, Msh is expressed in dorsal pNE and NBs of the TC
and DC (Fig. 6A-D). First,
glial cells in the TC and DC are closely associated with dorsal NBs, and are
thus likely to derive from these progenitors
(Urbach et al., 2003) (data
not shown). In order to find out whether msh influences glial cell
development in the brain, we counted Repo-positive cells in msh
mutant embryos at early stage 12; until this stage it is possible to relate
glial cells to their neuromere of origin. We observed that the number of glial
cells in the DC and TC is strongly reduced, but is unchanged in the PC
(Fig. 7A-D;
Table 2).
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|
). Instead, specification of dorsal NBs and their glial
progeny in the PC must depend on other factors. Although in a vnd
mutant background these factors (unlike msh) do not expand the
territory of their glial promoting function, they (like msh) remain
repressable by ectopic Vnd expression. Taken together, these data indicate that, in the posterior brain (TC and DC), production of normal numbers of glial cells depends on msh expression (as in the VNC), which is ventrally delimited by Vnd (but by Ind in the VNC). This is in clear contrast to the anterior brain (PC), where early glial development is independent from msh and vnd.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Skeath et al.,
1994; Chu et al.,
1998; McDonald et al.,
1998).
In the absence of Vnd, we found an increase in cell death, which
contributes to the loss of ventral brain NBs. Apoptosis acts at the level of
both pNE progenitor cells and NBs [for cell death occurring at later stages,
see Sprecher et al. (Sprecher et al.,
2006)]. It is not yet clear whether the reduction of ventral NBs
is solely due to cell death, or whether it also involves activity of proneural
genes. In the truncal neuroectoderm, proneural genes of the AS-C complex
promote NB formation (reviewed by
Campos-Ortega, 1995). There is
evidence that vnd interacts with proneural genes, but also that it
has additional function in promoting NB formation apart from activating
proneural genes. The latter assumption is supported by the finding that, in
vnd embryos, lethal of scute (l'sc) can still be
expressed in the ventral proneural clusters of, for example, NB5-2, although
the respective NB is missing (Skeath et
al., 1994; Chu et al.,
1998). In the pNE, genes of the AS-C complex are expressed in
large proneural domains, of which those of achaete, but especially of
l'sc (Urbach et al.,
2003; Younossi-Hartenstein et
al., 1996), seem to overlap with the vnd expression
domain, suggesting a possible genetic interaction. However, in vnd
embryos, we observe no substantial differences in the expression pattern of
l'sc transcript compared with the wild type (R.U., unpublished).
Thus, similar to the situation in the trunk, Vnd does not appear to exert
proneural function through activation of l'sc. However, our data
propose a possible interaction between vnd and the proneural gene
atonal. In vnd mutants, expression of atonal is
often missing in proneural clusters of the sensory organ precursors of the
hypopharyngeal-latero-hypopharyngeal organ (see Fig. S1 in the supplementary
material). Clearly, further investigations are required to clarify in how far
interactions between vnd and proneural genes play a role in the
formation of ventral brain NBs.
We noticed that, in vnd mutants, not only ventral, but also
intermediate brain NBs in the TC and DC show defects in their formation or
specification, comparable with the situation in the trunk
(Jimenez and Campos-Ortega,
1990; McDonald et al.,
1998; Mellerick and Modica,
2002). As intermediate brain NBs do not express vnd (but
ind), these defects appear to be non-cell-autonomous. Another, more
likely explanation is that determination occurs at the blastodermal stage,
when Vnd is transiently expressed in a much larger population of cells in the
pNE, which presumably include progenitors of intermediate NBs. A similar
proposal was made for intermediate NBs in the trunk
(McDonald et al., 1998).
Furthermore, early commitment of ventral neuroectodermal cells and
cell-autonomous expression of ventral and intermediate NB fates has been
demonstrated by heterotopic transplantations of neuroectodermal cells from
ventral to dorsal sites at the early gastrula stage
(Udolph et al., 1995).
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;
Skeath, 1999;
Skeath and Thor, 2003). Most
of these genes are also expressed in specific domains of the pNE before NBs
delaminate, although in a segment-specific manner
(Urbach and Technau, 2003b).
Our present data show that Vnd influences the expression of such site-specific
marker genes ('NB identity genes') already in the pNE, before NBs are formed.
In vnd embryos, we noticed a derepression of dorsal-specific genes in
the ventral pNE (e.g. of msh and ems in the intercalary and
antennal segment, and dac in the ocular segment) and in the
descending NBs, and conversely, a loss of ventral-specific gene expression
(e.g. lbe in the PC). The altered expression of `NB identity genes'
in vnd mutants reflects a ventral-to-dorsal transformation of ventral
pNE and residual NBs. Further evidence for such a transformation is the
production of (ectopic) glial cells by these ventral NBs, which normally is a
trait specific to dorsal NBs (see below). By contrast, in the trunk, absence
of Vnd results in a ventral-to-intermediate transformation, owing to the
derepression of ind (instead of msh in pNE), which induces
specification of intermediate NB fates
(Chu et al., 1998;
Weiss et al., 1998). Together, our data in the vnd loss- and gain-of-function backgrounds indicate that vnd is required, and is at least partially sufficient, for the induction of ventral fate in brain NBs through the activation of genes specific for the ventral pNE, and through the repression of genes specific for dorsal pNE.
Interactions between columnar genes vnd, msh and ind differ in the neuroectoderm of head and trunk
Our analysis revealed differences in the regulation of DV patterning genes
among the intercalary (IC), antennal (AN) and ocular (OC) head segments,
giving rise to the TC, DC and PC, respectively. Overexpression of vnd
leads to repression of ind within the IC, but loss of
vnd-function does not seem to cause ventral expansion of the
ind intercalary spot. Unexpectedly, ind is completely absent
in the AN of vnd mutants, suggesting that in this segment
vnd is necessary for activation and/or maintenance of ind
(rather than repression). This is supported by our previous finding that the
ind antennal spot transiently co-expresses Vnd
(Urbach and Technau, 2003c),
which is unique in the neuroectoderm, and by our present finding that in
vnd gain-of-function background the ind antennal spot is
almost unaffected. In the OC, however, ind expression is partially
repressed upon Vnd overexpression, and ventrally expanded in the absence of
Vnd, similar to the situation in truncal segments. However, because, in wild
type the ind ocular spot does not adjoin the ocular vnd
domain (Urbach and Technau,
2003c), its expansion in vnd embryos cannot be due to a
cell-autonomous effect.
Overexpression of vnd abolishes Msh almost completely in the neuroectoderm of all body segments. Yet, absence of Vnd reveals segment-specific differences in the regulation of msh. Owing to insulated ind expression in the IC and lack of ind in the AN of vnd mutants, Msh (instead of ind) is found in the ventral pNE of these segments, which is unique in the CNS anlagen, except for the mandibular segment, which exhibits equivalent expression (compare Fig. 6A,M with 6E,Q).
Among the pregnathal segments, the degree of conservation with regard to
the expression and interactions of DV patterning genes seems to be highest in
the posterior IC (TC) [ind and msh being repressed by
(ectopic) vnd, and msh by ind]. In the anterior
head, endogenous Msh expression in the dorsal pNE reaches the segmental border
between AN (DC) and OC (PC), but does not cross it
(Urbach and Technau, 2003c).
Ectopic Msh in vnd mutants does also not cross this border, which
suggests interference with regulatory factors acting in AP axis.
|
).
Requirement for further factors in DV patterning of the anterior brain
As discussed above, in the pNE, vnd is necessary for the formation
and specification of brain NB. It remains to be shown whether ind and
msh exert analogous functions. However, more than 50% of the
identified brain NBs do not express any of the three DV patterning genes
(Urbach and Technau, 2003c).
Most of these NBs derive from pNE of the preantennal head, which implies that
further factors are involved in DV patterning of the anterior pNE and brain.
Several other genes have been reported to be crucial for DV patterning in the
truncal neuroectoderm, such as the EGF-receptor homolog Egfr
(Skeath, 1998;
Udolph et al., 1998;
Yagi et al., 1998), the Sox
genes SoxNeuro and Dicheate
(Buescher et al., 2002;
Sanchez-Soriano and Russell,
2000; Zhao and Skeath,
2002), and Nk6 (Uhler
et al., 2002). For most of them it has been shown that they are
involved in formation and/or specification of truncal NBs. Egfr, both
Sox genes (J.S. and R.U., unpublished) and Nk6
(Uhler et al., 2002) are also
expressed in the pNE, before and during the phase of NB formation. However, in
Egfr mutant embryos the number and pattern of brain NBs is unaffected
(Dumstrei et al., 1998). How
far the Sox genes and Nk6 contribute to the formation and/or
specification of brain NBs awaits further investigation.
Segment-specific requirements of Msh and Vnd for the production of glial cells in the brain
Most of the glial cells in the VNC derive from dorsal NBs (neuroglioblasts
or glioblasts) (Schmidt et al.,
1997), which depend on msh for proper specification.
Accordingly, glial cells deriving from these progenitors are missing or
improperly differentiated in msh mutants
(Isshiki et al., 1997), as
well as in sca-vnd embryos
(Mellerick and Modica, 2002).
Likewise, in the TC and DC, first glial cells are closely associated with
dorsal NBs that descend from Msh-expressing pNE
(Urbach and Technau, 2003c).
In the TC, some dorsal NBs have been identified as glial progenitors, e.g. the
neuroglioblast Td4 and the glioblast Td7, which are putative serial homologs
of the truncal neuroglioblast NB5-6 and the glioblast LGB, respectively
(Urbach et al., 2003). In
absence of Vnd, we found the number of glial cells in the TC, and especially
in the DC, to be increased. This is most probably due to the segment-specific
early derepression of Msh in the ventral pNE and NBs of the TC and DC. In the
truncal segments, however, ind instead of Msh is derepressed in the
ventral NE (Chu et al., 1998;
McDonald et al., 1998), and
the number of glial cells is not significantly affected in the VNC of
vnd mutants (data not shown). Furthermore, in msh mutants,
glial development in the TC and DC is almost completely abolished, which
parallels the phenotype observed upon vnd overexpression (leading to
repression of msh in the dorsal pNE and NBs). Thus, comparable with
the situation in the VNC, Msh promotes glial fate in the TC and DC. However,
in the PC, glial development must be regulated differently (at least in its
early phase). Until stage 12 we did not detect Msh in this part of the brain,
and in msh mutants the number of glial cells in the PC is normal.
Glial cell fate in the PC is also not affected by loss of vnd,
although it remains repressable by ectopic Vnd. Therefore, as opposed to the
TC and DC, and to the VNC, normal production of early glial cells in the PC
does not depend on msh, nor indirectly on vnd or
ind.
Evolutionary conservation of DV patterning in the brain
There are striking similarities in the spatial order of expression of
vnd, ind and msh in the Drosophila neuroectoderm
and homologous genes in the neural plate and neural tube of vertebrates:
vnd homologs of the Nkx2 family are expressed in ventral
regions; the ind homologs Gsh1 and Gsh2 are
expressed in the intermediate regions; the msh homologs Msx1,
Msx2 and Msx3 are expressed in the dorsal region of the neural
tube (reviewed by Arendt and
Nübler-Jung, 1999;
Cornell and Ohlen, 2000). This
dorsoventral order of expression is conserved not only in the anlagen of the
truncal CNS but also in those that form the posterior part of the brain (in
Drosophila, TC and DC; in vertebrates, hindbrain)
(Urbach and Technau, 2003c).
Moreover, the anterior borders of the expression domains of these columnar
genes correspond in the early brains of Drosophila and mouse:
expression of vnd/Nkx2 extends most rostrally (mouse ventral
forebrain), followed by ind/Gsh1 and, finally, msh/Msx3
expression (reviewed by Urbach and
Technau, 2004). Thus, the expression of columnar genes in the
brain is, to some extent, evolutionarily conserved both along the DV axis and
along the AP axis.
|
;
Sussel et al., 1999).
Moreover, ind/Gsh2 expression is not expanded in Nkx2.1 mutants
(Corbin et al., 2003)
(reviewed by Rallu et al.,
2002), and residual basal (ventral) pallidal structures become
transformed into dorsal striatal structures
(Sussel et al., 1999). Thus,
in both Drosophila and mouse, loss of vnd/Nkx2 in the brain
leads to a transformation of ventral into dorsal structures, rather than into
intermediate structures, which has been shown to be the case in the truncal
CNS of both species (Briscoe et al.,
1999; Chu et al.,
1998; McDonald et al.,
1998). Therefore, in the developing brains of Drosophila
and vertebrates, vnd/Nkx2 is crucial for the formation and
specification of ventral brain structures, and interacts with other
dorsoventral patterning genes in a region-specific manner.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/21/4315/DC1
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ACKNOWLEDGMENTS |
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