Abteilung fuer Evolutionsgenetik, Institut fuer Genetik, Universitaet zu Koeln, Weyertal 121, 50931 Koeln, Germany
e-mail: angelika.stollewerk{at}uni-koeln.de
Accepted 30 August 2002
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SUMMARY |
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Key words: Delta/Notch signalling, Neurogenesis, Lateral inhibition, Chelicerate, Cupiennius salei
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INTRODUCTION |
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This system functions in a similar manner in vertebrate neurogenesis
(de la Pompa et al., 1997;
Chitnis and Kintner, 1996
;
Chitnis et al., 1995
;
Haddon et al., 1998
)
suggesting that the singling out of individual cells may be an ancestral
feature of neurogenesis that is coupled to the particular way in which
Delta/Notch signalling works. However, our previous analysis of spider
neurogenesis (Stollewerk et al.,
2001
) showed that groups of cells rather than single cells are
recruited for the neural fate. This raises the question of how such
group-specific recruitment of cells might be achieved and whether Delta/Notch
signalling is involved in this process. Therefore, I cloned homologues of
Notch and Delta to study their expression and function in
spider neurogenesis.
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MATERIALS AND METHODS |
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PCR cloning
CsDelta1, CsDelta2 and CsNotch were initially found by
RT-PCR on RNA prepared from 120- to 130-hour-old embryos, using degenerate
primers directed against conserved positions of the DSL domain (Delta) and EGF
repeats (Notch) of two invertebrate and three vertebrate neurogenic genes. We
used the following primers: DL2, TWYTGYMGNCCNMGNGAYG; DL1re,
CARTARTTNARRTCYTKRTYRCA; DL2re, NWRNCCNCCCCANYYNKY; DL3re,
CANGTNCCRTGNANRCANYYNGG; N7, TGYRTNTGYGTNAAYGGNTGG; N8,
GAYTGYWSNRANAAYWTHGAYG; N7re, RTTYTGRCANGGRTKNSW; N7re2, CCNKYRWANCCNGGCATRCA.
The PCR fragments were cloned and sequenced. Larger fragments of the genes
were obtained by rapid amplification of cDNA ends (GeneRacer kit, Invitrogen).
The sequences were deposited in the EMBL/GenBank/DDBJ databases (Accession
Numbers: CsNotch, AJ507288; CsDelta1, AJ507289; CsDelta2, AJ507290).
Histology and staining
Whole-mount in situ hybridisation were performed as described
(Damen and Tautz, 1999).
Phalloidin staining of spider embryos was performed as has been described for
flies (Stollewerk, 2000
).
Immunocytochemistry was performed as described
(Stollewerk et al., 2001
).
Anti-Horseradish peroxidase antibody was purchased from Dianova (1:500) and
anti-acetylated tubulin was purchased from Sigma (1:2000). Histology was
performed as described (Stollewerk et al.,
1996
).
Double-stranded RNA interference
Preparation of double-stranded RNA, injection and further treatment of the
embryo were performed as described previously
(Schoppmeier and Damen, 2001;
Stollewerk et al., 2001
).
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RESULTS |
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The extracellular domain of the Drosophila Delta protein contains
nine EGF repeats and the highly conserved DSL domain (Delta-Serrate-Lag2), a
cystein-rich region that is required for binding to the Notch receptor
(Fehon et al., 1990) and has
been found in all Notch ligands identified so far
(Fig. 1B). Degenerate primers
directed against the DSL domain and the second EGF repeat resulted in
amplification of 504 bp and 534 bp PCR fragments that fall into two groups and
could be distinguished by differences at 25 amino acid positions. In order to
isolate the whole sequences, I performed 5' and 3'RACE using
transcript specific primers directed against the two different PCR fragments.
With this method, two Delta genes were identified in the spider:
Cupiennius salei Delta1 (CsDelta1) and Delta 2
(CsDelta2). The 2447 bp sequence obtained for CsDelta1
encodes a deduced protein of 683 amino acids. It shares the highly conserved
DSL domain (Delta-Serrate-Lag2) and eight EGF repeats with the fly sequence
(Fig. 1B). The 2115 bp
CsDelta2 sequence encodes a protein of 437 amino acids. Although it
contains the conserved DSL domain in its extracellular part, the deduced
protein sequence can only be aligned with the first five EGF repeats of other
species, while the remaining C-terminal part is not conserved. An amino acid
sequence comparison of the DSL domains of CsDelta1 and CsDelta2 with the same
region in the Drosophila protein
(Vaessin et al., 1987
)
indicates that CsDelta1 and CsDelta2 have 62% and 57% identity to
Drosophila Delta, respectively. The DSL domains of both proteins show
the highest identity to the same region of Xenopus X-Delta-1
(Fig. 1C)
(Chitnis et al., 1995
). Besides
the DSL domain, regions of greater identity to Delta proteins of other species
exist between the first five EGF repeats, while the intracellular domains of
both spider Delta proteins are highly dissimilar.
Expression patterns of CsDelta1, CsDelta2 and
CsNotch
In contrast to Drosophila, where single neuroblasts delaminate
into the embryo during neurogenesis, groups of cells invaginate from the
ventral neuroectoderm of the spider
(Stollewerk et al., 2001).
These invagination sites consist of five to nine neural precursor cells and
are generated in four subsequent waves over approximately 3 days, beginning at
130 hours [stages according to Seitz
(Seitz, 1966
)]. The proneural
gene CsASH1 is responsible for the recruitment of the neural
precursor cells and is expressed in the appropriate regions of the ventral
neuroectoderm prior to the formation of invagination sites
(Stollewerk et al., 2001
).
Expression of CsDelta1 starts at about 130 hours after egg laying in
the first five to eight groups of cells that are going to invaginate from each
hemisegment (Fig. 2A,D). The
transcripts can also be detected in all invaginating cell groups that are
generated in the subsequent waves (Fig.
2B,C,E). The cell processes of the invaginating cells detach from
the apical surface at about 200 hours in the prosoma. At that time,
CsDelta1 expression decreases, although some of the invaginating cell
groups still show a strong expression (Fig.
2D). By contrast, CsDelta2 is uniformly expressed in the
ventral neuroectoderm during formation of the invagination sites, but shows a
stronger expression in the invaginating neural precursors throughout
neurogenesis (Fig. 2F-J). Expression of CsDelta2 decreases at the same time (at about 200
hours) in the invaginating cells as CsDelta1 expression, although
transcripts are still visible in the neuroectodermal cells that remain apical
(Fig. 2H).
|
As the fragment that is missing in the second CsNotch transcript is too small for whole-mount in situ hybridisation, the specific distribution of the two transcripts could not be resolved. Therefore, all CsNotch-expressing cells were localised by whole-mount in situ hybridisation using digoxigenin-labelled riboprobes corresponding to the complete 5' region up to EGF repeat 12. Like CsDelta2, CsNotch is expressed in the whole ventral neuroectoderm at 130 hours, but shows a slightly stronger expression in the neuroectodermal regions where the first invagination sites arise (Fig. 2K,N). After formation of most of the invagination sites these domains of higher Notch expression are reduced to small regions at the lateral anterior edge of each neuromere (Fig. 2M,O). At that time CsNotch is still expressed in all neuroectodermal cells, although there is heterogeneity in the expression levels (Fig. 2O). The same distribution of CsNotch transcripts is visible at about 200 hours, but, in addition, a medial group of cells shows strong CsNotch expression (Fig. 2M). In summary, CsDelta1, CsDelta2 and CsNotch are expressed during neurogenesis in a spatiotemporal pattern, indicating that they are involved in the specification of neural precursors in the spider ventral neuroectoderm.
Functional analysis of CsDelta1, CsDelta2 and
CsNotch
To analyse the function of the spider neurogenic genes, I injected
double-stranded RNA (dsRNA) of CsDelta1, CsDelta2 and
CsNotch, respectively, to interfere with endogenous gene function
(Fire et al., 1998;
Schoppmeier and Damen, 2001
).
Injected embryos were cultivated until about 190 hours after egg laying
(Table 1). The resulting
phenotypes were analysed by staining 10% of the embryos with
phalloidin-rhodamine, a dye that stains the actin cytoskeleton, and 20% with
neural anti-Horseradish peroxidase antibodies (anti-HRP). The remaining 70%
were hybridised with the spider proneural gene CsASH1
(Stollewerk et al., 2001
).
Morphological analysis of the resulting phenotypes in the confocal
laser-scanning microscope revealed that the invagination sites are missing to
different degrees in embryos injected with dsRNA of the neurogenic genes
(Fig. 3). Embryos injected as a
control with dsRNA corresponding to a fragment of the green fluorescent
protein (GFP) exhibit the normal number of invagination sites in the ventral
neuroectoderm, as can be seen by the dots of high phalloidin-rhodamine
staining (Fig. 3A,F,K). Groups
of invaginating cells that extend their cell processes to the apical surface
are located underneath these dots
(Stollewerk et al., 2001
). The
spot-like phalloidin-rhodamine staining is due to the constricted cell
processes of these bottle-like shaped cells
(Fig. 3K).
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|
In 35% of the embryos that were injected with CsDelta1 dsRNA, the
number of invaginating cell groups is reduced in individual segments
(Fig. 3B,C). A more severe
reduction of invagination sites can be detected after injection of
CsDelta2 dsRNA: in 47% of all embryos that show a specific phenotype,
invagination sites are absent in the whole ventral neuroectoderm
(Fig. 3C). Transverse sections
show that the morphology of the ventral neuroectoderm of embryos injected with
dsRNA of any neurogenic gene is altered in the same way as in
Drosophila neurogenic mutants
(Stollewerk, 2000): the
neuroectoderm forms bulges (Fig.
3L,M) because the cells that normally invaginate occupy a space in
the apical layer. As the proliferation rate is not affected in these embryos
(Fig. 4), newly formed cells
are either pushed to the apical (Fig.
3L,M) or to the basal side
(Fig. 3O,P).
|
After injection of CsNotch dsRNA, dots of high phalloidin-rhodamine staining can be detected in the positions that correspond to invagination sites in control embryos, although they are much smaller (Fig. 3D,I). In more severely affected embryos (Fig. 3E) there is only a diffuse phalloidin-rhodamine staining visible in positions where invagination sites form in control injected embryos (Fig. 3J). However, transverse optical sections through the neuroectoderm of embryos injected with CsNotch dsRNA revealed that invaginating cells with bottle-like shapes are missing (Fig. 3O,P). This indicates that groups of cells attach to each other at the apical surface, but the process of invagination is disturbed after injection of CsNotch dsRNA.
Alterations in the distribution of proneural gene transcripts and HRP
antigen after loss of neurogenic gene function
In Drosophila neurogenic mutants the transcriptional repression of
proneural genes to single cells of the proneural clusters fails to occur
resulting in an overproduction of neuroblasts at the expense of epidermal
cells (Marin-Bermudo et al.,
1995; Brand and Campos-Ortega,
1988
). Similar to Drosophila, the spider proneural gene
CsASH1 is expressed in patches of cells before each wave of formation
of invagination sites. The expression becomes restricted to the invaginating
cells, before the gene is re-expressed in regions where the next invagination
sites will form. I analysed the expression pattern of CsASH1 in
embryos injected with dsRNA of the neurogenic genes to see whether the
restriction of CsASH1 expression to the invaginating neural
precursors is a function of the spider neurogenic genes. At about 180 hours,
CsASH1 is expressed in a medial stripe and a patch of cells in the
lateral region of the opisthosomal hemisegments. This pattern is unchanged
after injection of GFP dsRNA (Fig.
5A). In embryos injected with CsDelta2 and
CsNotch dsRNA, respectively, a strong upregulation of CsASH1
expression can be observed (Fig.
5B,C), while after injection of CsDelta1 only a minor
change in the expression pattern is visible (data not shown).
|
To determine whether the neuroectodermal cells differentiate into neurones after interference with neurogenic gene function, injected embryos were stained with neural anti-HRP antibodies. The expression of the HRP antigen is restricted to the invaginating cells during the first phase of neurogenesis (see below). A spot-like HRP staining is visible in the apical region of the ventral neuroectoderm that corresponds to the constricted cell processes of the invaginating neural precursor cells. This pattern is unchanged after injection of GFP dsRNA (Fig. 5D). After injection of dsRNA of the neurogenic genes, there is an excess of anti-HRP staining in the apical cell layer (Fig. 5E,F), although CsDelta1 injected embryos show minor alterations in the staining pattern (data not shown). In addition, in embryos injected with CsNotch dsRNA a stronger staining is visible in the regions where invagination sites normally form (Fig. 5E).
In summary, functional analysis shows that Delta/Notch signalling mediates lateral inhibition in the ventral neuroectoderm of the spider in the same manner as in Drosophila.
No decision between epidermal and neural fate in the spider ventral
neuroectoderm
In Drosophila, the neuroectodermal cells have a choice to develop
as neuroblasts or as epidermoblasts
(Campos-Ortega, 1993). The
cells that are not singled out for the neural fate, remain in the outer layer
and differentiate into epidermal cells. To analyse the fate of the
neuroectodermal cells that do not invaginate into the spider embryo, I
investigated the morphology of the ventral neuroectoderm after the process of
invagination is completed. Light and electron microscopic analyses
(Fig. 6F,G) revealed that the
ventral nerve cord was not covered with epidermis at 220 hours, when the
invaginated cells have already formed a neuropil. Instead, the epidermal cells
arise lateral and medial to the ventral neuroectoderm and overgrow the
neuromeres between 250 and 300 hours (Fig.
6D,F,H,I). Medial to the ventral neuroectoderm, epidermal cells
that have covered the space between the separated halves of the germband are
shifted over the neuromers as the split germbands move towards each other on
the ventral surface during late embryogenesis
(Fig. 6F, arrowhead). Lateral
to the neuromers, epidermal cells are generated that start to spread medially
over the neuromers at 250 hours (Fig.
6I, asterisks). The cells that do not invaginate but remain in the
apical cell layer re-express the proneural gene CsASH1 at about 220
hours (Fig. 7A). In addition,
CsDelta2 and CsNotch are expressed in all cells of the outer
layer (Fig. 7C,D), while
CsDelta1 is still expressed in a subset of the invaginated cells:
transcripts are visible in about seven axon fascicles per hemisegment
(Fig. 7B). About 20 hours
later, the cells remaining apical differentiate into neural cells as can be
shown by staining embryos with the neural marker antibodies anti-HRP and
anti-acetylated tubulin (Fig.
6A,B). Interestingly, these antigens are differentially expressed.
While the HRP antigen is expressed in the axons of invaginated cells and in
the cells remaining apical (Fig.
6A,E), acetylated tubulin can only be detected in the apical cells
(Fig. 6B,C).
|
|
These results suggest that there is no decision between an epidermal and a neural fate in the ventral neuroectoderm of the spider, as the epidermis does not arise until neurogenesis is completed. The cells that remain in the apical layer enter a second phase of neurogenesis expressing the genes that are involved in singling out neural precursors.
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DISCUSSION |
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The two identified spider Delta homologues both contain the highly
conserved DSL domain that is required for binding of Delta to Notch
(Fehon et al., 1990). In
common with the vertebrate Delta proteins, CsDelta1 only has eight EGF repeats
as compared with nine repeats in Drosophila, while CsDelta2, in
addition, lacks three EGF repeats and is significantly divergent from
Drosophila Delta and its other homologues.
Expression patterns of neurogenic genes correlate with formation of
neural precursors
During neural cell fate specification in Drosophila, Notch and its
ligand Delta appear to be evenly expressed in proneural regions, reflected in
both RNA and protein distributions (Baker,
2000). Although it has been proposed that within a proneural
cluster the cell expressing the highest amount of Delta is selected
for the neural fate, no modulation in Delta expression has yet been observed
in the ventral neuroectoderm of fly embryos. By contrast, the expression
patterns of the zebrafish delta genes can be correlated to the
formation of neural precursors. While deltaB is expressed strongly
and selectively in the neural precursors, deltaA is expressed more
diffusely, in patches of cells showing a heterogeneous expression level
(Haddon et al., 1998
). The
cells within a deltaA patch that expresses deltaA strongly
are precisely the nascent neurones that, in addition, express deltaB.
A similar expression pattern of Delta genes is visible in the ventral
neuroectoderm of the spider. While CsDelta1, like zebrafish
deltaB, is exclusively expressed in neural precursors,
CsDelta2 transcripts are distributed uniformly throughout the
neuroectoderm and accumulate in nascent neurones similar to zebrafish
deltaA. However, there is one major difference: whereas high amounts
of deltaA and deltaB transcripts can only be detected in
scattered cells in the zebrafish, groups of cells express high levels of
CsDelta1 and CsDelta2 in the spider ventral neuroectoderm.
CsNotch is expressed in all neuroectodermal cells, but shows stronger
expression in the regions where the first invagination sites form. This
expression resolves into a more uniform distribution of transcripts during the
subsequent waves of invagination.
A similar mechanism with different outcome
Loss-of-function experiments show that similar to Drosophila and
the vertebrates the neurogenic genes of the spider regulate neurogenesis
through a mechanism of lateral inhibition limiting the proportion of cells
that segregate at each wave of neural precursor formation. However, in
contrast to Drosophila and also to vertebrates, where Delta/Notch
signalling has only been shown to specify differences in single cells within a
field of initially equivalent cells, in the spider groups of cells adopt the
neural fate simultaneously (Fig.
8).
|
According to current models, lateral inhibition is thought to operate
competitively because of a feedback loop that amplifies any initial
differences between neighbouring cells: a cell expressing more Delta activates
Notch more strongly in its neighbours. Notch activation in these cells
inhibits not only their differentiation, but also expression of Delta, thereby
reducing their ability to deliver lateral inhibition
(Chitnis, 1995;
Heitzler and Simpson, 1991
;
Sternberg, 1993
;
Ghysen et al., 1993
). The
higher expression of CsDelta2 and exclusive expression of
CsDelta1 in a group of adjacent cells suggests that Notch signalling
must be inactive in the invaginating cell groups of the spider, as these cells
would otherwise inhibit each other from adopting a neural fate. The inhibitory
effect of Delta was demonstrated by ectopically expressing X-Delta-1
in the neural plate of Xenopus embryos leading to a suppression of
primary neurogenesis (Chitnis et al.,
1995
).
It has been suggested, however, that the Delta to Notch ratio within a cell
determines its ability to receive Notch signalling. Doherty and co-workers
(Doherty et al., 1996)
observed that in the Drosophila wing imaginal disc Notch signalling
is strongest between cells that express high levels of Delta and cells with
low levels of Delta. The authors suggest that signalling only occurs when
cells with a Delta/Notch ratio low enough to allow signal reception are
juxtaposed to cells expressing high levels of Delta. In addition, Heitzler and
Simpson (Heitzler and Simpson,
1991
) showed that in mosaic animals Notch mutant cells have a
stronger capacity to send inhibitory signals than their wild-type neighbours.
These data support a model in which high proneural gene expression in groups
of neuroectodermal cells leads to an enhancement of CsDelta2
expression and a simultaneous activation of CsDelta1 expression in
the spider. This, in turn, results in a shift in the Delta/Notch ratio within
these cells, making them insensitive to Notch signalling.
Despite the fact that in the spider Delta/Notch signalling leads to groups
of five to nine cells adopting the neural fate, when compared with the
selection of single neuroblasts in the fly, there is only a minor difference
in the overall number of neurones generated per ventral hemisegment in
Drosophila and Cupiennius. The reason is that the
approximately 30 insect neuroblasts delaminate into the embryo and divide
several times to give rise to about 200 neurones per hemisegment. By contrast,
in the spider, most of the neural precursors do not divide after their
invagination. As 30 to 32 invaginating cell groups per hemisegment are formed
in the spider, it can be estimated that a neuromer consists of about 220
neurones on average. Comparison of neurogenesis in different insects
(Thomas et al., 1984) and
crustaceans (Whitington and Bacon,
1997
) has revealed that a given segmental neuroblast appears to
produce similar neurones even in widely divergent species. Therefore, the
stereotyped positions of the neuroblasts and the invariant identity of their
progeny seems to be an ancient feature that has changed little at least
through the evolution of insects and higher crustaceans. However, the
structure and development of the myriapod CNS shows little in common with the
insect and crustacean ventral nerve cord
(Whitington and Bacon, 1997
).
Future analysis will show whether the probably not clonally related
invaginating cell groups of the spider have invariant cell fates and whether
segmental neurones can be homologised to nerve cells at similar positions in
insects and crustaceans.
The ventral neuroectoderm of the spider is comparable to the neural
plate of vertebrates
In the ventral neuroectoderm of Drosophila Delta/Notch signalling
is used for a decision between two cell fates: delaminating cells become
neural precursors, while cells that remain apical give rise to epidermis. This
decision does not take place in the spider neuroectoderm, rather, the cells
remaining apical enter a second phase of neurogenesis, reexpressing proneural
and neurogenic genes. Interestingly, the early and late populations of
neurones differ in their expression of neuronal antigens. Although both
populations express the HRP antigen, acetylated tubulin can only be detected
in neurones that are generated during the second phase of neurogenesis. This
differential distribution of neural markers in primary and secondary neurones
is also visible in vertebrates (Bang and
Goulding, 1996).
As all cells of the neurogenic region develop into neurones, the ventral
neuroectoderm of the spider can be compared with the neural plate of
vertebrates. Similar to vertebrates, most cell divisions occur apical, while
the neural precursors exit the cell cycle and differentiate in deeper layers
(Stollewerk et al., 2001).
During primary neurulation in vertebrates, the original ectoderm is divided
into three sets of cells: (1) the internally positioned neural tube, which
will form the brain and the spinal cord; (2) the externally positioned
epidermis of the skin; and (3) the neural crest cells. A division of the
ectoderm into at least two populations is also visible in the spider: the
ventral neuroectodermal cells and epidermal precursors located lateral and
medial to the neurogenic region.
In summary, the data show that neurogenesis in the basal arthropod
Cupiennius salei shares features with both Drosophila and
vertebrates. Similar to the generation of neuroblasts in Drosophila,
invagination sites arise sequentially and in stereotyped positions in regions
that are prefigured by the proneural gene CsASH1
(Stollewerk et al., 2001).
However, comparable with the neuroepithelial cells of the vertebrate neural
plate, all cells of the neurogenic region of the spider seem to enter the
neural pathway, while the neurogenic genes restrict the proportion of cells
that adopt the neural fate at each wave of neural precursor formation.
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ACKNOWLEDGMENTS |
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