Abteilung für Evolutionsgenetik, Institut für Genetik, Universität zu Köln, Weyertal 121, 50931 Köln, Germany
* Author for correspondence (e-mail: angelika.stollewerk{at}uni-koeln.de)
Accepted 18 February 2003
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SUMMARY |
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Key words: Proneural genes, Neurogenic genes, Neurogenesis, Myriapod, Glomeris marginata
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INTRODUCTION |
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It has been shown recently that neurogenesis in the spider Cupiennius
salei (chelicerate) shares several features with Drosophila, but
that there are also several important differences. Similar to the generation
of neuroblasts in Drosophila, invagination sites arise sequentially
and at stereotyped positions in regions that are prefigured by proneural genes
(Stollewerk et al., 2001),
while neurogenic genes restrict the proportion of cells that adopt the neural
fate at each wave of neural precursor formation
(Stollewerk, 2002
). However,
in contrast to Drosophila, groups of cells, rather than single cells,
adopt the neural fate at a given time. In addition, neural stem cells,
comparable to Drosophila neuroblasts, could not be detected in the
ventral neuroectoderm of the spider. Furthermore, there is no decision between
epidermal and neural fate in the ventral neuroectoderm of the spider as in
Drosophila; instead all cells of the neurogenic region enter the
neural pathway.
In all four myriapod groups (Diplopoda, Chilopoda, Symphyla and Pauropoda),
the general development of the ventral neuroectoderm follows the same pattern.
Ventral to the limb buds, thickenings form as a result of cell proliferation.
When the embryo begins to bend about a transverse fold in the middle of the
trunk, these thickenings flatten (Anderson,
1973). After completion of ventral flexure, the middle part of the
hemisegment sinks into the embryo forming a groove
(Dohle, 1964
). Cell
proliferation takes place within this groove pushing newly formed cells
towards the basal side and leading to the formation of stacks of cells that
project out as rays from the edges of the groove. This structure is called the
`ventral organ'. During the course of neurogenesis the ventral organs are
gradually incorporated into the embryo while epidermal cells overgrow the
ventral nerve cord (Dohle,
1964
). Neurogenesis has been analysed in a variety of
representatives of all myriapod groups, but failed to reveal stem cell-like
neural precursors with morphological characteristics of insect or crustacean
neuroblasts (Heymons, 1901
;
Tiegs, 1940
;
Tiegs, 1947
;
Dohle, 1964
;
Whitington et al., 1991
).
Furthermore, Whitington and co-workers
(Whitington et al., 1991
)
showed that in the centipede Ethmostigmus rubripes the earliest
central axon pathways do not arise from segmentally repeated neurons as in
insects but by the posterior growth of axons originating from neurons located
in the brain. In addition, the axonal projections and the cell body positions
of the segmental neurons clearly diverge from the pattern described in insects
and crustaceans (Whitington et al.,
1991
).
Here, we have analysed the pattern of early neurogenesis in the myriapod
Glomeris marginata and compared it to the recently obtained data for
the spider Cupiennius salei
(Stollewerk et al., 2001;
Stollewerk, 2002
).
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MATERIALS AND METHODS |
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Archispirostreptus sp.
Adult Archispirostreptus were obtained from the Aquazoo in
Düsseldorf, Germany. Ten adults were kept at 28°C in large terraria
filled to a depth of at least 20 cm with earth. Females laid a clutch of eggs
into the earth, approximately every 3 weeks, which were collected, staged,
cleaned with bleach (under 5%) and frozen at 80°C for RNA
extraction.
Dechorionization and fixation
Glomeris eggs were removed from their egg chambers by submerging
them in water. They were transferred to a 2 ml Eppendorf tube and washed
several times in water. They were then dechorionated by leaving them in bleach
(under 5%) for 2 minutes and rinsing several times with water. Embryos were
frozen at 80°C for RNA extraction or fixed in 1 ml heptane, 50
µl formaldehyde (37%) for later use. Embryos for antibody stainings were
fixed for 20 minutes on a wheel and then washed several times with 100%
ethanol and stored at 20°C. For in situ hybridizations, embryos
were fixed for 4 hours on a wheel and washed with 100% methanol before storage
at 20°C. After storage, at least overnight, the embryos could be
devitelinized with tweezers for further staining.
PCR cloning
GmASH, GmNotch, GmDelta, AsAsh, and AsDelta were
initially found by RT-PCR on cDNA synthesized from RNA extracted from 7- to
12-day Glomeris, or 2- to 3-week Archispirostreptus embryos,
respectively. Degenerate primers for the respective genes were used as
described by Stollewerk et al. (Stollewerk
et al., 2001; Stollewerk,
2002
). The obtained PCR fragments were cloned into the pZero
vector (Stratagene) and sequenced. Larger fragments were obtained by rapid
amplification of cDNA ends (Marathon cDNA Amplification Kit, Clonetech;
GeneRacer Kit, Invitrogene). Sequence reactions were performed on plasmid
preparations with BIG DYE and run on an Abi Prism automatic sequencer. The
sequences obtained were deposited with GenBank (Accession Numbers:
GmDelta, AJ36341; GmNotch, AJ36342; AsDelta,
AJ36343; AsNotch, AJ36344; AsAsh, AJ36345; GmAsh,
AJ36346; TcASH, AJ36347).
Sequence analysis
Sequences were analysed and aligned with related amino acid sequences taken
from the NIH Blast database in Bioedit. Trees were constructed using the PAUP
NJ minimum evolution algorithm with 1000 bootstrap replicates. Positions where
an amino acid insertion was present in only one sequence were removed, as was
the variable part of the loop for the Ash alignment. Since the portion of the
Ash sequences that could be aligned is very short (the BHLH domain), the
presence or absence of a loop was used as an extra character (32/57
informative characters). For Delta, the DSL domain and EGF repeats 1 and 2
were aligned (70/109 informative characters), for Notch the 5' sequences
up to EGF repeat 12 were aligned (266/311 informative characters).
In situ hybridization
Whole-mount in situ hybridizations were performed as described for
Danio rerio, with the modification that 20x SSC pH 5.5 was used
instead of 20x SSC pH 7.4, to reduce the background
(Bierkamp and Campos-Ortega,
1993).
Histology and stainings
Phalloidin-rhodamine staining of Glomeris embryos was performed as
has been described for flies (Stollewerk,
2000). Immunocytochemistry was performed as described previously
(Stollewerk et al., 2001
). The
anti-phospho-histone 3 (PH3) antibody was provided by F. Sprenger (Institute
for Genetics, Cologne). Histology was performed as described previously
(Stollewerk et al., 1996
).
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RESULTS |
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Formation of invagination sites in the ventral neuroectoderm
To analyse the morphology of the ventral neuorectoderm in Glomeris
we stained embryos at stage 4 with phalloidin-rhodamine, a dye that stains the
actin filaments, and investigated the cell shapes in the confocal
laser-scanning microscope (LSM). At this stage a thickening of the
neuroectoderm is already visible (see above) and the extension of the ventral
neuroectoderm is clearly demarcated medially by the ventral midline and
laterally by the limb buds. We made flat preparations of embryos stained with
phalloidin-rhodamine and scanned them from apex to the base using the LSM.
Similar to the situation in the spider, we detected dots of high
phalloidin-rhodamine staining in apical optical sections of the neuroectoderm
of the head segments and the first five leg segments
(Fig. 1, see also
Fig. 3). More basal optical
sections of the same regions (at a depth of 11-21 µm from the apical
surface of the embryo) revealed that groups of basally enlarged cells are
located underneath the strongly stained dots, indicating that these dots mark
the sites of invagination of neuroectodermal cells
(Fig. 1).
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Proliferating cells are associated with invagination sites
The thickening of the ventral neuroectoderm is a result of cell
proliferation (Anderson, 1973).
To see whether there is a connection between cell proliferation and formation
of invagination sites, we double stained embryos with the mitotic marker
anti-phospho-histone 3, to analyse the pattern of cell divisions, and
phalloidin-rhodamine, to visualize the invagination sites.
In contrast to the spider, in which cell proliferation does not coincide
with the formation of invagination sites
(Stollewerk et al., 2001), in
Glomeris mitotic cells are associated with invagination sites and
seem to prefigure the regions where invagination sites arise in the ventral
neuroectoderm (Fig. 4). During
formation of the first invagination sites at least one mitotic cell abuts the
invaginating cell group (Fig.
4D-F), while groups of cells and individual cells could be
detected in the regions where invagination sites form hours later
(Fig. 4A-C). Most cell
divisions occur in the apical cell layer (data not shown), as in the spider.
It was not possible from these experiments to determine whether the cells of a
particular invagination group are clonally related.
|
Rapid amplification of the 5'and 3' ends (RACE) of GmASH resulted in a 1000 bp fragment with an 804 bp open reading frame (ORF), Archispirostreptus 5' and 3' RACE led to a 1200 bp fragment with an ORF of 864 bp. Both sequences have a single start codon with a short conserved motif also found in the CsASH genes, as well as upstream and downstream stop codons. The deduced amino acids of full-length GmASH and AsASH sequences showed a similarity of 61%, with 86% identical amino acids in the bHLH domains. The deduced amino acid sequence of GmASH is 83% identitical to Homo sapiens Achaete-Scute Complex homolog-like 1, while the Archispirostreptus sequence is 81% identical to the Gallus gallus transcriptional regulator CASH over the region of the bHLH domain. Outside of this domain, it is only possible to align a short conserved domain at the end of the protein. The alignment of the bHLH domains with other ASH proteins showed that, in contrast to insects, the millipede sequences, like their spider and vertebrate homologues, have a highly reduced loop (Fig. 5A). A tree was constructed from an alignment of the bHLH domains of nine insect, five vertebrate, two Cupiennius salei and the myriapod sequences (Fig. 6A). The node joining the myriapod, spider and vertebrate sequences has very high bootstrap support (94), while that joining the insects has low support.
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For Delta, an Archispirostreptus sequence covering the DSL domain and EGF repeats 1 and 2 with 81% similarity to the CsDelta1 protein was isolated. The complete GmDelta sequence shares 57% identical amino acids with CsDelta1, while the fragment similar to AsDelta has a similarity of only 62% with CsDelta1. An alignment of the DSL domain shows that these sequences are highly conserved (Fig. 5B). The DSL domain and EGF repeats 1 and 2 from two insect species, five vertebrates, the two myriapods, the spider Cupiennius salei and one ascidian sequence (Ciona savigny) were aligned to create the tree shown in Fig. 6B. Here, the insects and the vertebrates form two clear groups, while the myriapods and the spider form another. All three of these groups have relatively high bootstrap support: insects, 86%; vertebrates, 100%; spider and myriapods, 74%.
The obtained Glomeris Notch sequence, which shares 68% of its amino acids with the Boophilus microplus (chelicerate) Notch homologue, was aligned with seven Notch homologues to create the tree shown in Fig. 6C. The high sequence similarities between the Glomeris and the Boophilus proteins are reflected by the tree, where the node joining the Chelicerates with the vertebrates and Glomeris has a bootstrap support of 100% (Fig. 6C). The insect sequences, in contrast, are joined by a node with less than 50% support.
Expression pattern of GmASH during neurogenesis
GmASH transcripts were first detected before formation of the limb
buds at stage 1. At this time no invagination sites are visible in the ventral
neuroectoderm (Fig. 7A). The
gene is expressed in neuroectodermal cells in the middle of each hemisegment
in the head and the first two leg segments at heterogeneous levels
(Fig. 7F). Groups of cells
express high levels of the gene, while there is a weak uniform expression in
the remaining regions (Fig.
7F). At stage 2 invagination sites arise in the expression domains
of GmASH (Fig. 7B). At
this time transcripts can be detected anterior, posterior and in between the
first invagination sites (Fig.
7G, Fig. 8A). Again
the next invagination sites to arise are generated in the regions of
GmASH expression (Fig.
7C). Although the gene is simultaneously expressed in the head
segments and the first two leg segments, the expression domains in the
antennal, premandibular, mandibular and maxillar segments seem to be smaller
than in the remaining segments (Fig.
8A-D). At stage 3 the expression domains of GmASH form a
semicircle around the area where invagination sites have already formed
(Fig. 7H,
Fig. 8B). This expression
pattern again prefigures the regions where invagination sites will be formed
hours later (Fig. 7D). Before
the last wave of formation, GmASH is expressed in the corresponding
regions in between and anterior-medial to the existing invagination sites. In
addition, the gene is transiently expressed in the invaginating cell groups
and in the neural precursors of the peripheral nervous system
(Fig. 7J, Fig. 8C,D).
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Expression patterns of GmDelta and GmNotch
GmDelta is first expressed during the first wave of neural
precursor formation at stage 2. Transcripts can be detected at low levels in
all ventral neuroectodermal cells, but accumulate at higher levels in the
invaginating cell groups, similar to the expression pattern of the spider
CsDelta2 gene. GmDelta is also expressed in all invagination
sites generated subsequently (Fig.
9D-F). The expression seems to be rapidly down regulated, since
transcripts cannot be detected in all invagination sites generated during
different waves (Fig.
9A,B,D,F).
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No decision between epidermal and neural fate
The cells in the ventral neuroectoderm of Drosophila have a choice
between an epidermal and a neural fate. It has been shown recently, that this
decision does not take place in the neurogenic region of the spider. Rather,
all cells of the ventral neuroectoderm enter the neural pathway
(Stollewerk, 2002).
Analyses of transverse and horizontal sections of the ventral neuroectoderm of Glomeris embryos revealed that the invaginating cell groups detach from the apical surface at stage 6. At this stage a medial thickening forms in each hemineuromere (Fig. 11C). Subsequently, the neuroectoderm thickens at the lateral border adjacent to the limb buds (Fig. 11D) and the whole ventral neuromere sinks into the embryo while the epidermis overgrows the ventral nerve cord (Fig. 11A,D). At this time, a ladder-like axonal scaffold is already visible on the basal side (Fig. 11B), suggesting that there is no decision between epidermal and neural fate during the formation of neural precursors in the ventral neuroectoderm of Glomeris, as is the situation in the spider.
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DISCUSSION |
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However, there are some special features in the millipede that are different from spider neurogenesis. After formation of the first invagination sites, the ventral neuroectoderm forms a multi-layered structure of small cells, while in the spider there is only one single cell layer. The reason for this morphological difference is that because of limited space in the ventral neuroectoderm the invagination sites are located closer together and come to lie over and above each other. In addition, the invagination sites in Glomeris consist of up to 11 cells as compared to a maximum of eight in the spider and they do not all occupy a basal position. The invaginating cells do not all have the typical bottle-like shape as in the spider, so that their cell processes cover larger apical areas, and the dots of high phalloidin staining appear bigger in the millipede.
Furthermore, although the pattern of the invagination sites is very similar in the spider and Glomeris, the relative timing of generation of individual invagination sites is different. While in the spider the first invagination sites arise in the most anterior lateral region of the hemisegments, the first invaginating cell groups of the millipede are visible in the middle of the hemisegment. The next wave of invagination sites in the spider generates invaginating cell groups in coherent medial and posterior regions abutting the former generation sites. In contrast, in Glomeris newly formed invagination sites are distributed all over the hemisegement. Furthermore, the two most lateral anterior invagination sites that occupy strikingly similar positions in the spider and the millipede (see Fig. 1A,B) are generated during the first wave of invaginations in the spider, while they are not visible until the third wave of neural precursor formation in Glomeris.
However, the generation of neural precursors at stereotyped positions seems to be an ancient feature that has been maintained throughout the evolution of arthropods. Future analysis will show if the cells of an individual invagination site give rise to an invariant pattern of neurons in spiders and millipedes similar to the progeny of an identified neuroblast in insects and crustaceans.
Generation of neural precursors is associated with cell divisions in
Glomeris
Studies of neurogenesis in different representatives of all myriapod groups
have failed to reveal stem cell-like cells with the characteristics of insect
or crustacean neuroblasts (Heymons,
1901; Tiegs, 1940
;
Tiegs, 1947
;
Dohle, 1964
;
Whitington et al., 1991
). It
is assumed that neurons are produced by a generalized proliferation of the
ventral neuroectoderm. However, Knoll
(Knoll, 1974
) proposed that
neuroblasts are present in the apical layer of the centipede Scutigera
coleoptrata generating vertical columns of neurons; a mode of neural
precursor formation that would be very similar to the crustacean pattern.
Analysis of neurogenesis in another centipede, Ethmostigmus rubripes,
led Whitington and co-workers (Whitington
et al., 1991
) to the assumption that neural precursors with the
characteristics of insect neuroblasts are absent in this species. They could
not detect sites of concentrated mitotic activity or dividing cells that are
significantly larger than the surrounding cells. Similar results have been
obtained for the spider: scattered mitotic cells are distributed over the
neuroectoderm that do not prefigure regions where neural precursors form
(Stollewerk et al., 2001
).
However, our analysis of the mitotic pattern in the ventral neuroectoderm of
Glomeris revealed that dividing cells are associated with
invaginating neural precursors. Furthermore, groups of dividing cells seem to
prefigure the regions where invagination sites arise. In contrast to the
results from Ethmostigmus the dividing cells are significantly larger
in size than the surrounding cells in the millipede. Therefore, we assume that
stem cell-like cells are present in the apical layer of the ventral
neuroectoderm in Glomeris, although this has to be confirmed by
single cell labelling and BrdU injections. Since the dividing cell groups are
located in the apical cell layer and are present before formation of the
invagination sites, they are different from insect and crustacean neuroblasts.
In Drosophila the neuroblasts do not divide until they delaminate
from the outer layer. In contrast, the crustacean neuroblasts do not
delaminate but remain in the apical layer, dividing parallel to the surface,
so that the progenies are pushed to the basal side
(Dohle and Scholtz, 1988
;
Scholtz, 1990
;
Scholtz, 1992
).
The millipede pattern of neural precursor formation is intermediate when
compared between chelicerates and insects. While in the spider neuroectodermal
cells seem to divide randomly and are recruited for the neural fate because of
their positions in the hemisegment, the presence of neural stem cells in the
millipede links cell proliferation and generation of neural precursors in the
apical cell layer. The necessity to single out epidermal and neural precursor
cells from the ventral neuroectoderm in insects and crustaceans has led to a
separation of the generation sites: epidermal cells are produced in the apical
layer, while neural cells are generated on the basal side. Furthermore, the
fact that neuroblasts are missing in almost all lower crustaceans analysed and
that their mode of neurogenesis seems to be similar to that of myriapods, i.e.
a separation of the ganglia into the interior
(Anderson, 1973) indicates that
an entirely neurogenic ventral neuroectoderm may be the ancestral state.
All cells of the neurogenic region enter the neural pathway in
Glomeris
It is known that after completion of ventral flexure, the middle part of
each hemisegment sinks into the embryo forming a groove
(Dohle, 1964). During the
course of neurogenesis this region is gradually incorporated into the embryo
while epidermal cells overgrow the ventral nerve cord. We show here that this
process does not take place until the final differentiation of the invaginated
neural precursors, since a neuropil is already visible on the basal side, when
the neuromeres sink into the embryo. This means that as in the spider, and in
contrast to the situation in the insects, there is no decision between
epidermal and neural fate in the central neurogenic region of the
millipede.
Proneural and neurogenic genes in Glomeris
As in the spider and also in the insects, the Glomeris
achaete-scute homologue is expressed before formation of neural
precursors in the ventral neuroectoderm. Like the spider CsASH1 gene,
GmASH is expressed in patches of cells in the neuroectoderm and
becomes restricted to the invaginating cell groups, while in insects proneural
gene expression is reduced to one cell of the proneural cluster. After each
wave of neural precursor formation GmASH is re-expressed in the
regions where invagination sites form, indicating that the gene is necessary
for the formation of all neural precursors. However, this has to be confirmed
by functional analysis.
During formation of neural precursors in Drosophila, the
neurogenic genes Notch and Delta appear to be uniformly
expressed in the neuroectoderm (Baker,
2000). Although it is assumed that within a proneural cluster the
cell expressing the highest amounts of Delta is selected for the
neural fate, no variation in Delta expression has yet been observed
in the ventral neuroectoderm of fly embryos. In contrast, the expression
patterns of the spider Delta genes can be correlated to the formation
of neural precursors. While CsDelta1 is exclusively expressed in
neural precursors, CsDelta2 transcripts are distributed uniformly
throughout the neuroectoderm and accumulate in nascent neurons. The only
Delta gene we have found in Glomeris is expressed similarly
to the spider CsDelta2 gene: at low levels in almost all
neuroectodermal cells and at higher levels in the invaginating cell groups.
Furthermore, although Glomeris Notch is initially expressed uniformly
in the neuroectoderm, it resolves into a heterogeneous expression during the
first wave of neural precursor formation similar to the spider
CsNotch transcripts.
To summarize, our data support the hypothesis that myriapods are closer to chelicerates than to insects. The spider and the millipede share several features that cannot be found in equivalent form in the insects: in both the spider and the millipede, about 30 groups of neural precursors invaginate from the neuroectoderm in a strikingly similar pattern. Furthermore, in contrast to the insects, there is no decision between epidermal and neural fate in the ventral neuroectoderm of both species analysed. The sequence data also suggest a closer relationship of the millipede to the spider than to the insects. However, to confirm a sister group relationship of these arthropod groups, more morphological data from different representatives of myriapods, chelicerates and outgroups will be necessary.
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ACKNOWLEDGMENTS |
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