Abteilung fuer Evolutionsgenetik, Institut fuer Genetik, Universitaet zu Koeln, Weyertal 121, 50931 Koeln, Germany
Accepted May 8, 2001
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SUMMARY |
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Key words: Neurogenesis, Proneural genes, Invagination, Mitosis, Chelicerate, Cupiennius salei
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INTRODUCTION |
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The comparative analysis of developmental processes is a powerful method to unravel phylogenetic relationships. By comparing the expression patterns of genes involved in segmentation and segment identity in holometabolous (Sander, 1988) and hemimetabolous (Akam et al., 1988; Patel et al., 1989a; Patel et al., 1989b) insects as well as crustaceans (Abhzanov and Kaufman, 1999; Abhzanov and Kaufman, 2000) and chelicerates (Damen et al., 2000; Abzhanov et al., 1999; Damen and Tautz, 1998; Damen et al., 1998; Telford and Thomas, 1998), new insights into the evolutionary relationships of the different arthropod taxa have been gained. In addition, the morphological comparison of neurogenesis in insects and crustaceans has supported the molecular evidence of a sister group relationship between these two classes: both in insects and crustaceans basally located ganglion mother cells arise by unequal divisions of neural stem cells (Pasakony, 1994; Scholz, 1990; Scholz, 1992; Dohle and Scholz, 1988; Harzsch and Dawirs, 1996). In myriapods, however, which were formerly assumed to be closely related but more basal to the insects, no neuroblasts can be detected (Whitington et al., 1991; Weygoldt, 1985; Anderson, 1973).
Within the arthropods, neurogenesis is best analysed in locusts (Bate, 1976; Bate and Grunewald, 1981) and Drosophila melanogaster (Hartenstein and Campos-Ortega, 1984). The ventral neuroectoderm consists of a sheet of ectodermal cells, of which about 25 cells per hemisegment delaminate into the embryo as neural precursor cells, called neuroblasts. Delamination takes place in five discrete pulses. The remaining cells of the neurogenic region generate the ventral epidermis. The neuroblasts form in a highly stereotyped temporal and spatial pattern (Hartenstein et al., 1987; Doe et al., 1988; Jiménez and Campos-Ortega, 1990; Campos-Ortega and Haenlin, 1992; Goodman and Doe, 1993). Based on the analysis of mutants, several Drosophila genes have been identified that regulate the decision between epidermal and neural fate (Campos-Ortega, 1993; Ghysen et al., 1993; Muskavich, 1994). The competence to take on neural fate depends on the proneural genes (Cabrera et al., 1987; Romani et al., 1989; Jiménez and Campos-Ortega, 1990; Cubas et al., 1991; Martin-Bermudo et al., 1991; Skeath and Carroll, 1992). In embryos homozygous for loss-of-function mutations in the proneural genes, neuroblasts are missing (Jiménez and Campos-Ortega, 1990), whereas ectopic expression of proneural genes gives rise to additional neural precursor cells (Campuzano et al., 1986; Brand et al., 1993; Hinz et al., 1994). The proneural genes achaete, scute and lethal of scute, together with asense, form the so-called achaete-scute complex (ASC). These genes code for related proteins that contain a basic helix-loop-helix (bHLH) domain characteristic for a family of transcriptional regulators (Murre et al., 1989; Murre et al., 1994). In groups of cells that express these proneural genes the proneural clusters proneural gene expression becomes restricted to one cell of the cluster, the future neuroblast, by the activity of the neurogenic genes. This process is called lateral inhibition and is mediated by the neurogenic genes Notch and Delta. Mutations that affect the process of lateral inhibition lead to an overproduction of neurones a neurogenic phenotype (Lehmann et al., 1981; Lehmann et al., 1983).
Much less is known about neurogenesis in the remaining arthropods. As has been mentioned above, neurone precursor cells that have many of the characteristics of insect neuroblasts could be detected in crustaceans, although it is not clear whether they are homologous to the insect neuroblasts. In contrast to insects, crustacean neuroblasts do not delaminate from the surface layer of neuroectodermal cells before or during their divisions, and they are not associated with the specialized sheath cells found in insects (Doe and Goodman, 1985a; Doe and Goodman, 1985b; Doe and Goodman, 1985c; Scholtz, 1992). In addition, at least some crustacean neuroblasts can give rise to epidermal cells after they begin to bud off ganglion mother cells (Dohle and Scholz, 1988).
The comparison of neurogenesis in myriapods and insects has led to the assumption that this developmental process is less conserved in arthropods. The formation of the ventral ganglia in myriapods is associated with the formation of ventral organs; these are shallow pits that develop within the ectoderm external to the ganglia and, in some cases, are subsequently incorporated as cavities into the ganglion (Whitington and Bacon, 1997). Furthermore, the earliest axon pathways of myriapod embryos arise by the posteriorly directed growth of axons that originate from neurones located in the brain, rather than from segmental neurones, as in insects (Whitington et al., 1991).
Apart from a few classical accounts, neural development in the chelicerates has received little attention. In xiphosurans, and most scorpions and arachnids, neurogenesis occurs by a generalized inward proliferation of neuroectodermal cells to produce paired segmental thickenings (Anderson, 1973). Neuroblasts have been described for three chelicerate species, but it is possible that the data were partly misinterpreted, owing to technical limitations at the time (Winter, 1980; Mathew, 1956; Yoshikura, 1955). The neurogenesis of Cupiennius salei (Chelicerata, Arachnida, Aranea, Ctenidae) has not been analysed until now.
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MATERIALS AND METHODS |
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PCR cloning
CsASH1 and CsASH2 were initially found by RT-PCR on RNA prepared from germband embryos, using degenerate primers directed against conserved positions in the basic region and second helix of the bHLH domain of five invertebrate and three vertebrate proneural genes. We used the following primers: ASCUL1, MGNAA-YGMNMGNGARMGNAA; ASCUL2, GMNMGNGARMGNAA-NMGNGT; ASCUL3, MGNGTNRANYWNGTNAA; ASCULre1, ACYTTNSWNADYTTYTT; ASCULre2, ARNGTNTCYACYTT-NSWNADYTTYTT. ASCUL2 and ASCULre2 were used for a nested PCR on an 1 µl aliquot of the initial PCR. The obtained PCR fragments were cloned and sequenced. Larger fragments for both genes covering the complete ORF were obtained by rapid amplification of cDNA ends (Marathon cDNA amplification kit, Clontech). The sequences obtained were deposited in the EMBL/GenBank/DDBJ databases (Accession Numbers, AJ309490 and AJ309491).
In situ hybridization
Whole-mount in situ hybridizations were performed as described (Damen and Tautz, 1999a; Damen and Tautz, 1999b).
Phalloidin staining of embryos
Phalloidin staining of spider embryos was performed as has been described for flies (Stollewerk, 2000).
YOYO staining of embryos
YOYO-1 was purchased by Molecular Probes, the Netherlands. Before staining the embryos were incubated for 2 hours in 10 µl RNaseA (Roche, Mannheim, Germany). After several washes in phosphate-buffered saline (PBS), the embryos were incubated in 1 µl YOYO per ml PBS for 1 hour.
Antibody staining
Immunohistochemistry was performed as described previously (Klämbt et al., 1991; Mitchison and Sedat, 1983). Anti-Phospho-Histone 3 (PH3) antibody was provided by F. Sprenger (Institut for Genetics, Cologne). Anti-Horseradish peroxidase antibody was purchased from Dianova, Hamburg.
Double-stranded RNA interference
Preparation of double-stranded RNA, injection and further treatment of the embryos were performed as described previously (Schoppmeier and Damen, 2001; DOI10.1007/s004270000121) with one modification: double-stranded RNA corresponding to the nucleotide positions 299 to 911 of the ORF of the pEGFP gene (provided by M. Gajewski; Institute for Genetics, Cologne) was used for control injections.
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RESULTS |
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Formation of invagination sites
Cupiennius embryos were stained with phalloidin-rhodamine, a dye that stains the actin cytoskeleton. Because the actin filaments stained by this agent are found mainly in the cortex of the cells, this technique can be used to investigate cell shapes in the confocal laser-scanning microscope (LSM). We made flat preparations of the stained embryos and scanned them from apical to basal using the LSM. At this time point, the ventral neuroectoderm of the spider consists of one single cell layer. In all embryos analysed, dots of high phalloidin staining could be detected in apical optical sections of the cephalic lobe, in all segments of the prosoma and in the first six opisthosomal segments between the edge of the limb buds and the ventral furrow (Figs 1B, 2A). Analysis of the ventral neuroectoderm at a higher magnification revealed that groups of five to nine basally enlarged cells were located underneath the strongly stained dots (Fig. 2B, arrows). Transverse sections show that these cells have a bottle-like shape (Fig. 2C,D). The cell nuclei are located basally (Fig. 2D, asterisks), whereas the bundled cell processes extend to the apical surface (Fig. 2D, arrow). These observations suggest that the apical dots of high phalloidin staining result largely from the constricted apical surfaces of these cell groups. Based on the morphology and the position of the cell groups, it can be concluded that these cells are invaginating neuroectodermal cells. Analysis of the precise arrangement of the sites of invagination in slightly older embryos revealed that they occupied the same anteroposterior and mediolateral positions within each hemisegment of all embryos analysed (Fig. 3, arrows). At that stage, there are seven rows of invagination sites, which consist of four to five invagination sites each.
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As proneural genes have not been found in arthropods other than insects, we tried to identify these genes in the spider using PCR and degenerate oligonucleotide primers that correspond to sequences conserved between the Drosophila ASC genes achaete (ac), scute (sc) and lethal of scute (l(1)sc), five invertebrate (Villares and Cabrera, 1987; Martin-Bermudo et al., 1993; Takano, 1998; Galant et al., 1998; Krause et al., 1997; Grens et al., 1995) and three vertebrate ASC homologues (Allende and Weinberg, 1994; Jasoni et al., 1994; Tanaka et al., 1999). PCR of cDNA from embryonic stages of Cupiennius yielded several weak bands of the expected size (114 to 138 bp). Amplification of these PCR products using nested PCR resulted in a 120 bp band that was cloned into pZero. Forty-eight clones with appropriately sized inserts were randomly selected and sequenced. 12 of the 48 clones had inserts, each of which contained seven conserved amino acids of the first helix and the loop region of the achaete-scute family at the deduced amino acid level, while others were unrelated. The PCR fragments fall into two groups that could be distinguished by differences at five amino acid positions. In order to isolate the whole sequences, we performed 5' and 3'RACE (rapid amplification of cDNA ends) using transcript specific primers that were directed against the two different PCR fragments. With this method, we identified two proneural genes in the spider: Cupiennius salei achaete-scute homolog 1 (CsASH1) and 2 (CsASH2).
The 840 bp sequence obtained for CsASH1 encodes a deduced protein of 197 amino acids. Comparison of the amino acid sequence of the bHLH domain shows that CsASH1 has 82% identity to the chicken ac-sc homolog CASH1 and about 70% identity to the Drosophila ASC members ac, sc and l(1)sc. (Fig. 8A). The 1056 bp sequence obtained for CsASH2 encodes a deduced protein of 203 amino acids which is 82% identical to the frog ac-sc homologue XASH1 over the region encoding the bHLH domain and about 70% identical to the gene products of the Drosophila ASC members ac, sc and l(1)sc over the same region (Fig. 8B). In common with the vertebrate ac-sc homologue proteins, the loop region of the bHLH domain of both identified genes is shorter than equivalent regions of the Drosophila ASC proteins. Both genes are clearly ac-sc homologues, as comparison of their bHLH domains with that of other families of bHLH proteins shows a much lower degree of amino acid sequence identity (data not shown). Outside of the bHLH domain CsASH1and CsASH2 diverge from all other ac-sc homologues. Comparison of the identified genes with each other revealed that there are differences at nine amino acid positions over the region of the bHLH domain, which corresponds to an identity of 82%. Apart of the first three amino acids following the translation initiation sites (MASL) the deduced amino acid sequences of CsASH1 (EMBL, AJ309490) and CsASH2 (EMBL, AJ309491) are distinct from each other outside the bHLH region.
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CsASH2 expression during neurogenesis
CsASH2-expressing cells were detected in the same way as CsASH1 transcripts, by whole-mount in situ hybridisation using digoxigenin (DIG)-labelled riboprobes. CsASH2 is expressed later in neurogenesis than CsASH1. Transcripts are first detected at 130 hours in two lateral and one median cell cluster in the cephalic lobe (Fig. 10A, arrows). At that time, the first invagination sites can already be seen in the cephalic lobe and the prosomal and first opisthosomal hemisegments (see Fig. 4B). In the ventral neuroectoderm CsASH2-expressing cells are not labelled until 175 hours. Expression can be detected only in the invaginating neuroectodermal cells (Fig. 10F,J,M). In the cephalic lobe, CsASH2 is also expressed in invaginating cells (Fig. 10B, arrow) and in addition in one median cell cluster (Fig. 10B, arrowhead). Expression has decreased in the two lateral cell clusters of the cephalic lobe labelled before. CsASH2 is strongly expressed in all 22 to 25 invaginating cell groups of each prosomal hemisegment (Fig. 10F,M,N). In the first to fifth opisthosomal hemisegments, which have developed 16 to 17 invagination sites at that time (Fig. 10P), five to eight invaginating cell groups in the anterior most lateral region show a strong expression of CsASH2 (Fig. 10O), whereas the remaining invaginating cells express the gene only weakly at that time. About 10 hours later, the gene is expressed strongly in all invaginating cells of the opisthosomal segments (data not shown). At 190 hours, CsASH2 expression has already decreased in the prosomal and the first to second opisthosomal segments, while expression can still be seen in the remaining opisthosomal segments. There is also a decrease of expression in the invaginating cells of the cephalic lobe, but now two lateral cell clusters show CsASH2 expression (Fig. 10C, arrows). At 220 hours, additional regions of CsASH2 expression can be detected in the cephalic lobe, whereas in the ventral neuroectoderm the expression has disappeared in all segments with the exception of the last one (Fig. 10H,L, arrow).
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These data show that CsASH2 is transiently expressed in neuroectodermal cells during the process of invagination. In addition, the gene is partially expressed in patches of cells in the cephalic lobe, similar to CsASH1, but not in overlapping regions (compare Fig. 9A-E with Fig. 10A-D).
Analysis of CsASH1 function during neurogenesis
Recently, the use of double-stranded RNA (dsRNA) to interfere with gene function in Caenorhabditis elegans (Fire et al., 1998) has been successfully extended to Drosophila (Kennerdell and Carthew, 1998; Misquitta and Paterson, 1999) and other insects (Brown et al., 1999; Hughes and Kaufman, 2000). The so-called RNA-mediated interference (RNAi) technique has also been successfully established for the spider Cupiennius salei (Schoppmeier and Damen, 2001). To analyse the function of CsASH1 during neurogenesis, we injected dsRNA corresponding to the basic domain and the first helix of the bHLH domain, and the complete upstream part of the gene. Injected spider embryos were cultivated (see Materials and Methods) until about 220 hours after egg laying. The resulting phenotypes were investigated by staining 10% of the embryos with phalloidin-rhodamine and the remaining 90% with HRP antibody. 60.5% of the embryos which had no developmental arrest after injection showed a specific phenotype (Table 1). Although embryos injected as a control with dsRNA corresponding to a portion of the green fluorescent protein (GFP) exhibit the normal number of invagination sites in the ventral neuroectoderm (Figs 12A, Fig. 13A,D), embryos injected with CsASH1 dsRNA are missing the invagination sites to different degrees. 42% of the affected embryos have no invagination sites either in the prosoma or the opisthosoma (Fig. 13E), 37% show a reduction of invagination sites in individual segments or over the whole region of the neuroectoderm (Figs 12B, Fig. 13B,F). 11.5% of the embryos show asymmetric effects: only one half of the germ band is affected (Fig. 13C). In 9% of the embryos that show a specific phenotype, no invagination sites can be detected at all (data not shown). Analyses of transverse optical sections in the LSM revealed that in regions of the ventral neuroectoderm where no invagination sites can be detected the morphology of the neuroectodermal cells is disturbed (Fig. 12D). The typical columnar shape of neuroectodermal cells is lost; instead, the cells have a rounded appearance (Fig. 12D). In segments where only a reduced number of invagination sites develop, the process of invagination seems to be blocked (Fig. 12E). Although in control injected embryos a basal layer of neuroectodermal cells that have already invaginated can be detected (Fig. 12C, bracket), there seems to be no invagination in the affected segments, as no second cell layer is visible (Fig. 12E).
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Analysis of CsASH2 function during neurogenesis
To analyse the function of CsASH2 during neurogenesis, we injected dsRNA corresponding to the first helix, the loop and the second helix of the bHLH domain, and the complete downstream part of the gene. The embryos were treated and analysed in the same way as has been described above for CsASH1 dsRNA injection. Injection of CsASH2 dsRNA did not affect the formation of the invagination sites nor the process of invagination itself (Fig. 14B). Rather, the differentiation of the invaginated neural precursor cells seems to be disturbed, as in the affected embryos, a reduction of the neuropil could be detected in individual or several ventral neuromeres (Fig. 14E,F). In the affected neuromeres the axon fascicle, which connects the individual neuropils is missing (Fig. 14C,D, arrowhead). In 8% of the embryos that have a specific phenotype the neuropil is either completely absent in the prosoma or in the opisthosoma. 10% of the embryos that have reduced neuropils show an asymmetric effect, as only one half of the germ band is affected. Despite the fact that the anti-HRP staining can be detected earlier during neurogenesis than CsASH2, HRP staining is reduced or partially absent in the affected embryos (Fig. 15B,D).
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DISCUSSION |
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Although the formation of invagination sites in the spider occurs sequentially, comparable with the generation of neuroblasts in Drosophila, the process of invagination itself seems then to be restricted to a short period of time. Invagination sites in the spider are generated over a period of 3 days. The fact that all 30 to 32 invagination sites are visible simultaneously until shortly before the end of inversion implies that at least some of the cells of the invaginating cell groups are still connected to the apical surface. In line with these observations, transverse optical sections reveal that basal cell layers can not be detected until the third day of neurogenesis (see Fig. 12C, bracket). Only 1 day later, when the invagination sites disappear, a thick layer of invaginated neural cells is visible basally (see Fig. 4G). This observation suggests that the recruitment of neuroectodermal cells for the neural fate during early neurogenesis is separated from the final differentiation of those cells by several days. This contrasts with Drosophila, where each population of neuroblasts delaminates from the ventral neuroectoderm before the next wave of neuroblasts arises. In addition, the delaminated neuroblasts take up their final function by dividing to produce ganglion mother cells within a few minutes of segregation.
Mitotic divisions in the ventral neuroectoderm
As described above, earlier morphological studies have in general suggested that the process of invagination is tightly connected to cell proliferation. In addition, the authors claim that the invaginated cells continue to divide when the innermost cells are differentiating into neurones and growing axons (Barth, 1985). We addressed this issue by staining Cupiennius embryos with the anti-PH3 antibody, which labels mitotic cells. Analyses of the mitotic divisions in the ventral neuroectoderm revealed that during most of neurogenesis, only single cells divide. Clusters of proliferating cells cannot be detected until the fourth wave of formation of invagination sites. At that time point, groups of mitotic cells are located in regions where new invagination sites arise (see Fig. 7F, arrow). However, proliferating cell groups can also be detected in regions of the hemisegments where all invagination sites have already formed. These data show that in contrast to earlier studies on other arachnids there is no obvious connection between cell proliferation and invagination in Cupiennius.
Furthermore, our results do not support earlier observations in other arachnids, which show that the neuroectodermal cells keep dividing after invagination. Analyses of transverse optical sections revealed that, throughout neurogenesis, almost all cell divisions occur in the apical layer of the ventral neuroectoderm. Mitotic divisions could not be detected within the groups of invaginating cells and only in rare cases were divisions seen in basally located cells that had already invaginated. These observations suggest that the basal cells are neuronal cells that differentiate further and start growing axons immediately after invagination. The fact that the neuronal anti-HRP antibody stains the invagination sites very early during neurogenesis confirms the assumption that the invaginating cells are neuronal cells that already express neuronal antigens.
Another interesting aspect of the analyses of mitotic divisions is that there seem to be no neural precursor cells with the characteristics of insect neuroblasts in the ventral neuroectoderm of the spider. The apically located proliferating cells in the ventral neuroectoderm of the spider divide perpendicular to the apical surface. This means that the daughter cells remain in the same cell layer and do not automatically shift basally after division. The fact that there is also almost no cell division in the basal layers implies that neural precursor cells that divide in a stem cell-like mode are not present in the spider. However, it is possible that at least in one case neuroblasts that divide in a stem cell-like mode are present in a chelicerate. In the pantopod Callipallene emaciata Winter (Winter, 1980) describes spindle-shaped cells that invaginate and divide to produce daughter cells that are arranged in radial rows. As the relationship of the pantopods to the remaining chelicerates is still being discussed, we do not know how these data fit into our findings.
As was mentioned above, neuroblasts that share many of the characteristics of insect neuroblasts could be detected in crustaceans. They divide asymmetrically with a spindle perpendicular to the surface. The larger basal daughter cell divides again with the same spindle orientation. Thus a column of ganglion mother cells is formed. In contrast to insects, crustacean neuroblasts do not delaminate from the apical neuroectodermal cell layer before or during their divisions. In addition, at least some crustacean neuroblasts can give rise to epidermal cells after they begun to bud off ganglion mother cells (Dohle and Scholz, 1988).
Considering this, one can speculate that the generation of neurones in the spider is the ancestral form of neurogenesis: cells divide in the apical layer of the ventral neuroectoderm and are recruited for the neural fate at a certain point in time. An invagination site forms and eventually these cells leave the neuroectodermal layer. It can be assumed that the myriapods have a similar mode of neurogenesis, as the absence of neurone progenitor cells with the characteristics of insect neuroblasts was also confirmed in myriapods by the analysis of mitotic activity in the ventral neuroectoderm (Whitington et al., 1991). As was mentioned above, in myriapods ventral ganglia are formed by the incorporation of so called ventral organs (Dohle, 1964). In the malacostracan crustaceans, specialized neural precursor cells have evolved that are capable of dividing asymmetrically. These cells remain in the neuroectodermal layer because they also produce epidermal cells. In insects, the neuroblasts have to leave the neuroectodermal layer because these cells only produce neural cells at this point, while specialized cells, the epidermoblasts, occupy the apical position in the ventral neuroectoderm.
Proneural genes in the spider
This is the first time that proneural genes have been identified in arthropods other than insects. Similar to Drosophila, both identified genes are exclusively expressed in the developing nervous system. CsASH1 transcripts can be detected in the ventral neuroectoderm before formation of the first invagination sites. A cluster of cells located in the anterior most lateral region of each hemisegment expresses the gene at this time. In this region the first five to eight invagination sites will form hours later. The medially and posteriorly extending expression domain of CsASH1 exactly prefigures the region where the next nine to 12 invagination sites will arise. After formation of the invagination sites in that region, CsASH1 expression is switched off and the gene is re-expressed in a lateral domain and a medial stripe in each hemisegment. Again, these expression domains prefigure the regions where the next five to eight invagination sites will form. Subsequently, expression decreases and the gene is re-expressed in a medial cluster of cells at the posterior end of each hemisegment. This expression domain covers the region that has not expressed the gene until now, but it does not cover the region where the last seven to ten invagination sites will form. Two new rows of invagination sites are added during the last wave of formation of invagination sites. However, it can be assumed that invagination sites that have formed in the medial domain are shifted to their final position owing to the compaction of the neuromeres between this stage and the end of inversion (see Fig. 9N,O,S,T). In addition, CsASH1 has a transient expression in the invaginating cell groups. These data show that CsASH1 is expressed in all regions of the ventral neuroectoderm where invagination sites arise. The expression pattern of CsASH1 can therefore be compared with the expression of three genes of the ASC in Drosophila: achaete, scute and lethal of scute. These three proneural genes are expressed before delamination of the neuroblasts in partially overlapping clusters of cells. As in the spider, proneural gene expression decreases after formation of one population of neuroblasts and the genes show transient expression in the neuroblasts. Subsequently, the genes are re-expressed in regions of the ventral neuroectoderm where the next neuroblasts will form. In addition, comparable with Drosophila, where the proneural genes are responsible for cell shape changes in the ventral neuroectoderm before delamination of the neuroblasts (Stollewerk, 2000), the consequence of proneural gene expression in the spider seems to be a cell shape change to the bottle-like form of the invaginating cells. However, in contrast to Drosophila, where only one cell of a proneural cluster delaminates and becomes a neuroblast, all CsASH1-expressing cells enter the neural pathway.
Analysis of CsASH1 function during neurogenesis by injection of dsRNA corresponding to this gene revealed that CsASH1 is indeed responsible for the formation of the invagination sites. The fact that it was possible to obtain embryos that do not have any invagination sites suggests that CsASH1 is the only gene that is involved in the recruitment of neural cells. This is in contrast to Drosophila, where several genes are required for the specification of the neural precursor cells.
The second proneural gene of the spider, CsASH2, is expressed later during neurogenesis in the invaginating cells that have already changed their morphology to a bottle-like shape. Strong expression of the gene can be detected only during a very short time window, when most of the invagination sites have already formed in the prosoma. However, the expression pattern in the opisthosoma suggests that the transcription rate of the gene is to low to be detected in younger stages with our in situ hybridisation methods. At 175 hours, 16 to 17 invagination site have formed in the first opisthosomal hemisegments. Five to eight of these invagination sites show a strong CsASH2 expression, while the remaining invaginating cells are only weakly stained. This expression pattern suggests that the gene is expressed immediately after formation of the invagination sites but at very low levels. The expression pattern of CsASH2 is not similar to that of the proneural genes achaete, scute and lethal of scute in Drosophila, rather it can be compared with the fourth member of the ASC, asense, which is not expressed until after the neuroblasts have segregated from the ventral neuroectoderm. asense seems to be required for the differentiation and maintenance of the specified cell fate (Alonso and Cabrera, 1988; Gonzalez et al., 1989). The observations that the neuronal HRP staining is lost and the neuropil is reduced to different degrees in embryos that have been injected with CsASH2 dsRNA, despite the fact that the invaginating cell groups are present, suggests that CsASH2 has a similar function in neurogenesis in the spider as asense, although there is only a very low degree of amino acid identity. Similarly, the vertebrate ac-sc homologues, XASH1, MASH1 and CASH1 are expressed relatively late in neurogenesis, being detectable only after neural progenitors have been specified (Lo et al., 1991; Ferreiro et al., 1992; Jasoni et al., 1994).
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ACKNOWLEDGMENTS |
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