Evolution of neurogenesis in arthropods

angelika stollewerk

Several alternative hypotheses have been suggested that support various phylogenetic groupings of the individual euarthropod taxa, the chelicerates, myriapods, crustaceans and insects. The Tetraconata hypothesis suggests a sister-group relationship of insects and crustaceans in contrast to the traditional monophyletic grouping of myria-pods and insects, the Tracheata or Atelocerata (see references in Stollewerk and Chipman 2006). The Mandibulata hypothesis suggests a clade consisting of insects, crustaceans and myriapods (see references in Harzsch et al. 2005). However, the relationships within this clade are being debated since this hypothesis excludes neither the Pancrustacea nor the Tracheata concept. The latest hypothesis suggests a sister-group relationship of chelicerates and myriapods. Although this theory was initially based on molecular phylogenetic analysis (Friedrich and Tautz 1995, Hwang et al. 2001, Kusche and Burmester 2001, Nardi et al. 2003, Mallatt et al. 2004, Pisani et al. 2004), recent morphological and molecular data on neurogenesis in these groups potentially support a close relationship (Stollewerk et al. 2001, 2003, Dove and Stollewerk 2003, Kadner and Stollewerk 2004, Stollewerk and Simpson 2005, Chipman and Stollewerk 2006, Stollewerk and Chipman 2006). On top of the various ideas on the relationships within the euarthropods, none of the groups except the insects is generally accepted as monophyletic.

New insights into the evolutionary relationships between the different taxa have been gained by comparing morphological features and expression patterns of genes involved in developmental processes. Besides the analyses of the expression domains of segmentation genes and Hox genes (see references in Stollewerk et al. 2001), morphological

Evolving Pathways: Key Themes in Evolutionary Developmental Biology, ed. Alessandro Minelli and Giuseppe Fusco. Published by Cambridge University Press. # Cambridge University Press 2008.

comparison of neurogenesis in insects and crustaceans has confirmed the molecular evidence of a sister-group relationship between these two groups (see references in Schachtner et al. 2005, Harzsch 2006, Strausfeld et al. 2006a,b). Furthermore, neurogenesis in myriapods is more similar to chelicerates than to insects and thus the data support a Myriochelata clade and contradict the Tracheata hypothesis (Dove and Stollewerk 2003, Kadner and Stollewerk 2004, Chipman and Stollewerk 2006, Pioro and Stollewerk 2006, Stollewerk and Chipman 2006). However, without knowing the ancestral state of neurogenesis (and any other developmental process for that matter) a correct interpretation of the molecular and morphological data is not possible. Since analysis of outgroups to the euarthropods has turned out to be difficult and time-consuming because of the microscopic size (tardigrades) or almost year-long embryogenesis (onychophorans), a thorough analysis of many representatives of each arthropod group can be used as an alternative method of reconstructing the ancestral pattern of developmental processes. It can be assumed that characters conserved in all arthropod groups are ancestral and thus were present in the last common ancestor. Characters that can only be found in subsets of arthropod groups can consecutively be analysed in outgroups to the arthropods to see if they are synapomorphies or rather reflect the ancestral pattern. With this approach, studies in difficult outgroups can be kept to a minimum since we have to focus only on specific questions using already established methods.

Neurogenesis is a perfect system to study since the complexity of this developmental process provides a pool of various characters that have to correspond in detail to be judged as homologous, reducing the risk of assessing superficial similarities as homologies. Here I re-evaluate published data on early neurogenesis in all arthropod groups to uncover ancestral and possibly derived homologies and speculate on the sequence of evolutionary changes that might have led to the different modes of neurogenesis in arthropods.

neural precursor formation: the morphological processes

Individual stem-cell-like neuroblasts are formed in the ventral neuroectoderm of insects and crustaceans

Our detailed knowledge of the generation of neurons in insects is mainly based on studies of neurogenesis in Drosophila melanogaster and Locusta

Figure 19.1 Comparison of the different modes of neurogenesis in the four euarthropod groups. Neuroectodermal cells are represented in light grey, neural precursors in multi-shaded grey, epidermal cells in middle grey. A, In insects, single neuroblasts (NBs) are recruited from the ventral neu-roectoderm. Within minutes after their specification, the neuroblasts delaminate from the neuroectoderm into the interior of the embryo and divide to give rise to ganglion mother cells (GMC). Those cells that remain apical after five waves of neuroblast formation will give rise to epidermo-blasts (middle grey) and divide in the plane of the neuroectoderm. B, In crustaceans, neuroblasts do not delaminate. After rotation of the mitotic spindle, the neuroblasts bud off ganglion mother cells into the interior of the embryo. Crustacean neuroblasts can switch from producing ganglion mother cells to generating epidermal cells (dark grey). C, In chelicerates the central region of the neuroectoderm gives rise exclusively to neural cells. Groups of mainly postmitotic neural precursors form an invagination site that persists in the neuroectoderm for several days. After formation of all invagination sites the groups eventually detach from the apical layer. D, A similar mode of neurogenesis is seen in myriapods, although in contrast to chelicerates single mitotic cells (dark grey) and groups of mitotic cells (not shown) are associated with forming invagination sites.

migratoria (Bate 1976, Bate and Grunewald 1981, Hartenstein and Campos-Ortega 1984). Individual stem cells - so-called neuroblasts -delaminate from a single-layered neuroectoderm to the interior of the embryo in five subsequent waves (Figure 19.1A, top). In this basal (interior) position, they divide asymmetrically to self-renew and to produce smaller ganglion mother cells that divide once to give rise to two neural cells (i.e. neurons or glia; Figure 19.1A). About 500 neuroblasts are generated in the ventral neuroectoderm forming a highly stereotyped temporal and spatial pattern (Hartenstein and

Campos-Ortega 1984, Goodman and Doe 1993). The cells remaining in the apical cell layer give rise to epidermal cells. The decision between epidermal and neural fate depends on direct cell-cell interactions of the ventral neuroectodermal cells (see below). This mode of neurogenesis seems to be representative for insects since consecutive studies on the flour beetle Tribolium castaneum and the silverfish Ctenolepisma longicau-data have confirmed the presence of stem-cell-like cells in the neuroectoderm which are arranged in a pattern similar to Drosophila melanogaster and Locusta migratoria (Truman and Ball 1998, Wheeler et al. 2003).

Neuroblasts have also been described in higher crustaceans (Malacostraca) and in two Branchiopoda, but their origin and position in the developing neuromeres is different from that of insects (see references in Whitington 2004, Stollewerk and Simpson 2005). In malacostracan crustaceans, amphipods excepted, neuroblasts arise from stereotyped divisions of ectoteloblasts. These are stem-cell-like cells that are located in the posterior region of the germ band anterior to the proctodeum. The asymmetric divisions of ectoteloblasts produce transverse rows of stereotyped individually identifiable cells that form the grid-like pattern of the postnaupliar germ band. These cells lack the typical morphology of neuroectodermal cells. They divide several times until the first neuroblasts can be identified by their typical mode of mitotic division. In contrast to insects, crustacean neu-roblasts do not delaminate from the outer cell layer into the embryo but remain apical and divide perpendicular to the surface so that the daughter cells are pushed into the embryo (Figure 19.1B, top). After several rounds of division a dorso-ventral column of ganglion mother cells is visible (Dohle and Scholtz 1988; Figure 19.1B, bottom). Similar to insects, the ganglion mother cells divide once to give rise to two neural cells. In contrast to insects, crustacean neuroblasts can switch from the production of ganglion mother cells to the generation of epidermal cells (Figure 19.1B, middle). Interestingly, despite these differences the neuroblast pattern is similar in crustaceans and insects. In both groups, 25 to 30 neuroblasts are arranged in seven rows in each hemisegment (Dohle and Scholtz 1988, Scholtz 1992, Ungerer 2006).

Groups of neural precursors are recruited from the ventral neuroectoderm of chelicerates and myriapods

In a few classical accounts, neuroblasts have been described in three chelicerate species, but it is possible that the data were partly misinterpreted because of technical limitations at the time (Yoshi-kura 1955, Mathew 1956, Winter 1980). Apart from these studies, the literature suggests that neurogenesis occurs by a generalised inward proliferation of neuroectodermal cells to produce paired segmental thickenings both in chelicerates and myriapods (Anderson 1973). In some arachnids, the amblypygids and araneids, each neuro-mere is supposed to be formed by a number of invaginations (Wey-goldt 1985). Groups of cells divide and form small clusters that invaginate. Recent analysis of neurogenesis in three chelicerates (spiders: Cupiennius salei, Pholcus phalangioides; Xiphosura: Limulus polyphemus) and four myriapods (diplopods: Glomeris marginata, Archispir-ostreptus sp.; chilopods: Lithobius forficatus, Strigamia maritima) showed that neural precursor formation is indeed significantly different from that of insects and crustaceans (Stollewerk et al. 2001, 2003, Dove and Stollewerk 2003, Kadner and Stollewerk 2004, Chipman and Stollewerk 2006, Pioro and Stollewerk 2006, Stollewerk and Chipman 2006). (Note the term 'neural precursor' refers to cells that are committed to the neural fate but have not yet developed into neurons and glial cells.) In the ventral neuroectoderm of cheli-cerates and myriapods, groups of precursors are specified for the neural fate (Figure 19.1C, D; Figure 19.2). The precursor groups form invagination sites at stereotyped positions which persist in the neuroectoderm for several days (Figure 19.1C, D, middle). After invagination, most of the neural precursors do not divide but directly differentiate into neurons and glia.

Figure 19.2 Comparison of the expression pattern of invaginating neural precursor groups in single hemisegments of three arthropod species. Confocal micrographs of embryos stained with phalloidin-rhodamine. Anterior is towards the top, the midline towards the left. The arrangement of invagination sites is similar in the chilopod Lithobius forficatus (A) the spider Cupiennius salei (B) and the diplopod Glomeris marginata (C). The arrows point to two lateral invagination sites that are located at similar positions in all three species.

Figure 19.2 Comparison of the expression pattern of invaginating neural precursor groups in single hemisegments of three arthropod species. Confocal micrographs of embryos stained with phalloidin-rhodamine. Anterior is towards the top, the midline towards the left. The arrangement of invagination sites is similar in the chilopod Lithobius forficatus (A) the spider Cupiennius salei (B) and the diplopod Glomeris marginata (C). The arrows point to two lateral invagination sites that are located at similar positions in all three species.

Differences in the timing of neural precursor formation in individual chelicerate and myriapod species

Although neurogenesis follows the same general pattern in chelicerates and myriapods, differences in the timing of formation of invagination sites were observed that might coincide with distinct modes of embryogenesis in the individual species. In the spider Cupiennius salei the same numbers of invaginating cell groups arise simultaneously in the proso-mal segments. Neural precursor groups are formed in four waves generating 5 to 13 invagination sites each. In the opisthosoma, invagination sites are formed in an anterior to posterior gradient, since new opistho-somal segments are generated by the posterior growth zone during the course of neurogenesis. In a similar way, in the diplopod Glomeris margin-ata, the same number of invaginating cell groups arises simultaneously in the five head segments and the first three leg segments, while the invagination sites are formed in an anterior to posterior gradient in the remaining leg segments. Similar to the spider, four waves of neural precursor group formation have been described. In the chilopod Lithobius forficatus and the diplopod Archispirostreptus sp., neurogenesis is less synchronised in the trunk segments as compared with Glomeris mar-ginata. The formation of neural precursors seems to occur consecutively in each trunk segment in Archispirostreptus sp., while no more than two trunk segments show the same pattern of invagination sites in Lithobius forficatus. Although embryogenesis takes about the same time in these myriapods and the spider, neurogenesis lasts only two days in Lithobius forficatus and Archispirostreptus sp., while in the spider and Glomeris mar-ginata this process takes five days to complete. The different timing of neural precursor formation might therefore be an adaptation to the acceleration of neurogenesis in these species. A distinct pattern of neu-rogenesis is also seen in the geophilomorph centipede Strigamia maritima. In contrast to Lithobius forficatus and Glomeris marginata, Strigamia maritima undergoes epimorphic development. Myriapods showing this kind of development generate all segments during embryogenesis, whereas in Lithobius forficatus and Glomeris marginata further segments are added during posthatching larval stages. In Strigamia maritima, about 50 segments are generated during embryogenesis and differentiate in quick succession. Expression studies and morphological analyses showed that each trunk segment exhibits a different differentiation state along the anterior-posterior axis during neurogenesis. In contrast to the other myriapods and the spider, neural precursor groups arise one-by-one in each hemisegment, rather than in several waves. This distinct mode of neural precursor formation might be an adaptation to the independent initiation of neurogenesis in each segment.

However, despite these differences the final pattern of invagination sites is strikingly similar in all chelicerate and myriapod species analysed. In each species about 30 invagination sites per hemisegment are arranged in a regular pattern of seven rows consisting of four to six invagination sites each.

Differences in the number and morphology of invaginating neural precursors

While the pattern of invagination sites is conserved between different chelicerate and myriapod species, the morphology of the invagination sites and the number of neural precursors forming an invagination group is not consistent. In the diplopod Glomeris marginata and the chilopod Strigamia maritima up to 12 cells contribute to an individual invagination site, while in the spider Cupiennius salei and in the chilopod Lithobius forficatus the invaginating neural precursor groups consist of only five to nine cells. Furthermore, in Strigamia maritima, the cell processes of the neural precursors are not attached to the apical surface but to a single cell of the precursor group. Initially the invagination groups form three rows per hemisegment which are rearranged to a final pattern of seven rows during the convergent extension movements that lead to an extension of the germ band. It can be speculated that the specific morphology of the invagination groups is necessary for the rearrangement of the neural precursor groups from three rows to seven rows during these medio-lateral movements in Strigamia maritima.

Neural stem cells comparable to insect and crustacean neuroblasts are missing in chelicerates and myriapods

Analysis of the mitotic pattern in the spider Cupiennius salei and the myriapod Glomeris marginata suggests that the neural precursors of the invaginating cell groups are not comparable to insect and crustacean neuroblasts. In contrast to the literature that suggests a connection between cell proliferation and invagination (see above), dividing cell groups or single mitotic cells, which prefigure regions where invagination sites arise, could not be detected in the neuroectoderm of the spider Cupiennius salei. In addition, most mitotic divisions occur in the apical cell layer. The neuroectodermal cells divide in the plane of the apical surface so that the daughter cells remain in the surface layer and are not pushed into the embryo. Since there are no cell divisions in the invaginating cell groups, it can be concluded that mainly postmitotic neuroectodermal cells are recruited for the neural fate. These results contrast with those for insects, since neuroblasts do not divide until they delaminate into the embryo and produce ganglion mother cells by asymmetric cell divisions (Hartenstein and Campos-Ortega 1984). They are also in contrast to crustaceans, since crustacean neuroblasts divide asymmetrically similar to insects, although without delamina-tion. The absence of asymmetric cell divisions in the spider was confirmed by analysing the expression pattern of the neural cell fate determinant Prospero (Weller and Tautz 2003). In Drosophila, Prospero is asymmetrically distributed into ganglion mother cells during neuroblast division (Doe et al. 1991), while in the spider Prospero is equally distributed to both daughter cells in the few neural precursors that divide after invagination.

Studies of neurogenesis in different representatives of all myria-pod groups have in most cases failed to reveal neural stem-cell-like cells with the characteristics of insect and crustacean neuroblasts (Heymons 1901, Tiegs 1940, 1947, Dohle 1964, Whitington et al. 1991). Knoll (1974) proposed that neuroblasts are present in the ventral neuroectoderm of the chilopod Scutigera coleoptrata generating vertical columns of neurons - a mode of neural precursor formation similar to the crustacean pattern. However, the cells that Knoll identified as neuroblasts are only insignificantly larger than the neural cells in the basal cell layers. Analysis of the mitotic pattern in the ventral neuroectoderm of Glomeris marginata revealed that single dividing cells are associated with invaginating neural precursors. Furthermore, groups of dividing cells seem to prefigure the regions where invagination sites arise. The single dividing cells are significantly larger in size than the surrounding cells. This pattern can be interpreted in two ways. The large dividing cells might be neural stem cells that divide asymmetrically in the plane of the neuroectoderm to produce a group of neural precursor cells that subsequently invaginates. On the other hand, the presence of groups of dividing cells suggests that a single cell divides giving rise to two daughter cells which divide again and so forth, until a group of about 12 cells is generated. However, it would be possible to distinguish between these two scenarios by dye labelling of individual mitotic cells. If the progeny of these cells give rise to clones of about 12 cells which subsequently invaginate, we can assume that neural stem cells are present in the ventral neuroectoderm of Glomeris marginata.

The ventral neuroectoderm of chelicerates and myriapods is comparable to the neural plate of vertebrates

Neurogenesis in chelicerates and myriapods shows an additional distinct feature compared with insects and crustaceans. In chelicerates and myriapods, the central region of the ventral neuroectoderm generates exclusively neural cells, while in the remaining arthropods both neural and epidermal cells arise from the ventral neurogenic region. This mode of neurogenesis is actually more similar to vertebrates. During primary neurulation in vertebrates the ectoderm becomes divided into the internally positioned neural plate, which will form the brain and the spinal cord and the externally positioned region from which the epidermis of the skin will arise. A similar division of the ectoderm into a medial neurogenic region and lateral epidermal precursors is visible in the ventral neuroectoderm of chelicerates and myriapods. In addition, most cell divisions occur in the apical neuroec-toderm, while the neural precursors exit the cell cycle and differentiate in deeper cell layers. This mode of neurogenesis is also more similar to vertebrates than to insects and crustaceans. These data suggest that neurogenesis in chelicerates and myriapods reflects the ancestral pattern, while the formation of the nervous system in insects and crustaceans is derived.

The morphological processes of neural precursor formation

To summarise, several characters have been described in chelicerates and myriapods that cannot be found in equivalent form in the remaining arthropods. (1) Groups of neural precursors invaginate from the ventral neuroectoderm of chelicerates and myriapods, while single neuroblasts are specified in crustaceans and insects. (2) In contrast to insects and crustaceans, mainly postmitotic neural precursors are recruited for the neural fate. (3) The central region of the ventral neu-roectoderm in chelicerates and myriapods generates exclusively neural cells, while in insects and crustaceans both neural and epidermal cells arise from the ventral neurogenic region. Despite these differences, the pattern of neural precursor groups/neuroblasts is strikingly similar in all arthropod groups. In all species analysed, about 30 neuroblasts/ neural precursor groups per hemisegment are arranged in seven transverse rows with four to six neural precursor groups/neuroblasts each indicating that this pattern is a conserved character of neurogenesis in arthropods.

neural precursor formation: conserved genes

Proneural and neurogenic genes are essential for the specification of neuroblasts

In Drosophila, early neurogenesis is controlled by proneural genes that encode transcription factors with a basic domain necessary for DNA binding and two helices that allow for the formation of heterodimers with other basic helix-loop-helix (bHLH) proteins (see references in Pioro and Stollewerk 2006). The proneural genes belong to two major subfamilies, the achaete-scute group and the atonal group. In the ventral neuroectoderm of Drosophila, members of the Achaete-Scute Complex (AS-C; achaete, scute and lethal of scute) are expressed in a stereotyped, partially overlapping pattern and are necessary for neuroblast formation. In loss of function mutants fewer neuroblasts are generated. Proneural proteins can only bind DNA as heterodimers with the ubiquitously expressed bHLH protein Daughterless. By recruiting proneural proteins to autoregulatory enhancer elements, Daughterless assists in an up-regulation of proneural gene expression in the precursor cell which is essential for activation of the neural programme.

Despite their function in selecting neural precursors, proneural genes specify neuronal subtype identity indicating that these genes activate both a common neural programme and neuronal subtype-specific target genes. In the Drosophila PNS, achaete and scute specify external sensory organ identity, while atonal mainly specifies chordotonal organ identity. Prior to delamination of the neuroblasts, the proneural genes are expressed in clusters of cells in the ventral neuroectoderm (Figure 19.3A). Because of the activity of a second group of genes, the neurogenic genes, the expression of the proneural genes becomes restricted to a single cell of the cluster, the future neuroblast. This process is called lateral inhibition and is mediated by the transmembrane proteins Notch and Delta. Binding of the ligand Delta to the Notch receptor eventually leads to the activation of the Enhancer of split gene complex. The gene products of this complex repress proneural gene expression. Since production of the ligand Delta is positively regulated by the proneural genes, activation of the Notch signalling pathway leads to a down-regulation of Delta. Because of this feedback loop that takes place between the cells of a proneural cluster, a slightly elevated level of proneural gene expression in one cell of the cluster, the future neuroblast, leads to a repression of proneural gene expression in the neighbouring cells. Mutations affecting the process of lateral inhibition lead to an overproduction of neurons - a neurogenic phenotype.

Figure 19.3 Expression of proneural genes in the insect Drosophila melano-gaster and the spider Cupiennius salei. Flat preparations of embryos stained with a DIG-labelled Drosophila melanogaster achaete probe (A) and a DIG-labelled Cupiennius salei ASH1 probe (B). The midline is towards the left. A, In Drosophila melanogaster, the proneural gene achaete is first expressed in groups of cells (arrows). Because of lateral inhibition, expression becomes up-regulated in single cells (arrowheads) and down-regulated in the remaining cells of the proneural cluster. B, In the spider Cupiennius salei, CsASH1 is expressed in fields of cells at the beginning of neurogenesis (arrows). In contrast to Drosophila, proneural gene expression becomes restricted to groups of neural precursors (arrowheads).

Figure 19.3 Expression of proneural genes in the insect Drosophila melano-gaster and the spider Cupiennius salei. Flat preparations of embryos stained with a DIG-labelled Drosophila melanogaster achaete probe (A) and a DIG-labelled Cupiennius salei ASH1 probe (B). The midline is towards the left. A, In Drosophila melanogaster, the proneural gene achaete is first expressed in groups of cells (arrows). Because of lateral inhibition, expression becomes up-regulated in single cells (arrowheads) and down-regulated in the remaining cells of the proneural cluster. B, In the spider Cupiennius salei, CsASH1 is expressed in fields of cells at the beginning of neurogenesis (arrows). In contrast to Drosophila, proneural gene expression becomes restricted to groups of neural precursors (arrowheads).

Within the insect group, proneural genes have been identified in several dipterans, a butterfly and the flour beetle Tribolium castaneum (Galant et al. 1998, Wülbeck and Simpson 2000, 2002, Pistillo et al. 2002, Skaer et al. 2002, Wheeler et al. 2003). A proneural function of the achaete-scute homologues in these dipteran species has been suggested by analysis of their expression in the peripheral nervous system. The butterfly achaete-scute homologue shows a restricted expression in small proneural clusters in the embryonic ventral neuroectoderm that cannot account for the formation of all neural precursors. However, the expression of the gene is down-regulated to single cells indicating a similar mode of neurogenesis to that in the remaining insects. The Tribolium achaete-scute homologue (TcASH) is expressed in proneural clusters in the ventral neuroectoderm and becomes restricted to single neuroblasts, in a pattern similar to Drosophila. Recently, two achaete-scute homologues have been identified in the branchiopod crustacean, Triops longicaudatus. Tl-ASH1 seems to be expressed like a pro-neural gene, while Tl-ASH2 is exclusively expressed in neuroblasts.

The function of achaete-scute homologues in neural precursor specification seems to be conserved in chelicerates and myriapods

Two achaete-scute homologues have been identified in the spider Cupien-nius salei (Stollewerk et al. 2001), both of which are exclusively expressed in the developing central and peripheral nervous system. CsASH1 shows a proneural expression pattern (Figure 19.3B). Transcripts prefigure the regions where invagination sites arise at each wave of neural precursor formation. However, in contrast to insect and crustacean proneural genes, expression of CsASH1 becomes restricted not to single cells but to groups of neural precursors. The second achaete-scute homologue CsASH2 is exclusively expressed in the invaginating neural precursor groups. The expression pattern of CsASH2 can be compared with that of the fourth member of the Drosophila Achaete-Scute Complex, asense, which is exclusively expressed in neuroblasts. Analyses of CsASH1 and CsASH2 function during neurogenesis by RNA mediated interference revealed that CsASH1 is required for the recruitment of all neural precursors, while CsASH2 has a later function during differentiation.

One achaete-scute homologue each has been identified in the diplo-pod Glomeris marginata and the chilopod Lithobius forficatus. Both homologues show a proneural mode of expression and accumulate at higher levels in the neural precursors that are going to invaginate.

Furthermore, a member of the Atonal family has been identified in Glomeris marginata (Pioro and Stollewerk 2006). Gm Atonal, like its Drosophila homologue, is expressed exclusively in the PNS. However, in contrast to Drosophila, Gm Atonal seems to be expressed in external sensory organs. The expression domains at the tip of the appendages correspond to the positions of developing chemosensory organs (cone sensilla). Gm Atonal is not expressed in the sensory precursors of the body wall, lateral to the limb buds, while GmASH transcripts accumulate in this area. Similarly, the spider achaete-scute homologues CsASH1 and CsASH2 are partially expressed in non-overlapping domains in the developing PNS. These data suggest that the proneural genes of chelicerates and myriapods are involved in specification of neuronal subtype identity, similar to the Drosophila homologues.

In addition the identification of a daughterless homologue in Glo-meris marginata suggests conserved interactions of the genetic network involved in neural precursor specification (Pioro and Stollewerk 2006). Similar to Drosophila, the heterodimerisation partner of the proneural genes is expressed ubiquitously during development, which might account for a function in a large number of developmental processes. However, in contrast to Drosophila, a higher accumulation of daughterless transcripts seems to correlate with regions where neural precursor groups form in the central and peripheral nervous system of Glomeris marginata. It has been speculated that up-regulation of daughterless in a proneural field might refine the precise position of proneural clusters in concert with the proneural genes (Pioro and Stollewerk 2006). The heterogenous expression of daughterless in the central and peripheral nervous system of Glomeris marginata supports this model.

Neurogenic genes mediate lateral inhibition in chelicerates and myriapods

Although groups of neural precursors, rather than single neuroblasts, are recruited for the neural fate from the ventral neuroectoderm of che-licerates and myriapods, the regular pattern and sequential generation of the invagination sites suggested that neurogenic genes might restrict the proportion of cells that arise at each wave of neural precursor formation. One Notch and two Delta homologues were identified in the spider Cupiennius salei (Stollewerk 2002). While CsDelta1 is exclusively expressed in the invaginating cell groups, CsDelta2 is expressed in all ventral neuroectodermal cells but shows a higher expression in the inva-ginating cells. Like CsDelta2, CsNotch transcripts are distributed over the entire ventral neuroectoderm, although there is heterogeneity in the expression level. CsNotch transcripts seem to accumulate at higher levels in the invagination sites after their formation suggesting a dual function of Notch in neural precursor formation and invagination. A similar pattern of expression of the single Delta and Notch homologues is seen in the ventral neuroectoderm of the myriapods Glomeris marginata and Lithobius forficatus (Dove and Stollewerk 2003, Kadner and Stollewerk

2004). Functional analysis by RNA mediated interference confirmed that the neurogenic genes of the spider mediate lateral inhibition. This is an interesting result, since the current model for singling out neural precursors from a group of initially equivalent cells via the Delta/Notch signalling pathway only applies to single cells rather than to groups of cells. The data suggest that the genetic interactions of components of the Notch signalling pathway must have changed during evolution to permit generation of single neuroblasts in insects and crustaceans on the one hand, but recruitment of groups of neural precursors in chelice-rates and myriapods on the other hand. In this context, the expression pattern of the single Delta homologue in Strigamia maritima shows an interesting expression pattern (Chipman and Stollewerk 2006, Stollewerk and Chipman 2006). StmDelta seems to be expressed at higher levels in single cells of the neural precursor groups. However, it is also possible that Delta transcripts accumulate around individual cells, since the cell processes of all cells of an invagination group are attached to a single cell of the group (see above). In any case, the data suggest that individual cells of the precursor group are distinct. Although the whole precursor group will eventually invaginate and give rise to neural cells, Delta/Notch signalling might generate single cells with distinct properties within the precursor groups. These cells might have an important function during convergent extension movements in keeping individual cell groups together (see above). Therefore, neurogenesis in Strigamia maritima might represent an intermediate state between recruitment of groups of neural precursors in the remaining myriapods and chelice-rates and singling out of individual neuroblasts in insects and crustaceans.

In the ventral neuroectoderm of Drosophila melanogaster, the decision between epidermal and neural fate depends on Delta/Notch signalling. Cells that eventually remain apical give rise to epidermis, while delaminating cells become neural precursors. Although this process has not been analysed in insects other than Drosophila, it can be assumed that Notch signalling is used in a similar way within this arthropod group, since the mode of neurogenesis is similar in all insects analysed. While in insects neuroblasts are singled out by cell-cell interactions between initially equivalent cells, neuroblasts of malacostracan crustaceans arise from stereotyped cell lineages. In addition, neuroblasts can switch from the production of ganglion mother cells to the production of epidermal precursors, indicating that the choice between two cell fates occurs within a single cell lineage rather than groups of equivalent cells as in Drosophila. This raises the question of whether

Notch signalling is required at all for the decision between epidermal versus neural fate in crustaceans. However, neurogenic genes have not been identified in crustaceans up to now. In chelicerates and myriapods, the ventral neurogenic region gives rise exclusively to neural cells (see above). The epidermal cells are derived from lateral regions of the neu-roectoderm and overgrow the neurogenic region only after formation of all neural precursors. Therefore, Notch signalling is merely involved in the timing of neural precursor formation in the neurogenic region of chelicerates and myriapods, rather than in the decision between epidermal and neural fate.

Pan-neural genes switch on a common neural programme

Once neural precursors are selected, a group of genes referred to as panneural genes, such as hunchback, deadpan and snail, is expressed in most or all neuroblasts in Drosophila. These genes are either involved in asymmetric cell division or are part of a common neural programme and promote neural differentiation (see references in Stollewerk et al. 2003). In the Drosophila ventral neuroectoderm, two members of the Snail zinc finger family, snail and worniu, have a pan-neural mode of expression. Together with the third Snail family gene, escargot, they have partially redundant functions in the formation of the CNS and the mesoderm. Triple mutants show severe defects in the development of both the mesoderm and the nervous system. In these mutants, the neural determinants Prospero and Numb are no longer asymmetrically segregated into GMCs upon neuroblast division and the generation of GMCs is disrupted.

One snail homologue each has been identified in the myriapod Glo-meris marginata and the spider Cupiennius salei which are both expressed in most or all neural precursor cells of the CNS, similar to Drosophila (Stollewerk et al. 2003, Pioro and Stollewerk 2006). In contrast to the spider, Glomeris marginata snail transcripts can be detected in the whole neuroectoderm at the beginning of neurogenesis and accumulate in groups of cells prior to formation of invagination sites. This expression is comparable to Drosophila where snail is expressed similar to the AS-C genes in proneural clusters in the ventral neuroectoderm. During specification of the neuroblasts, snail transcripts become restricted to all, or most, neural precursor cells. While the spider homologue is also expressed in the PNS, similar to Drosophila, GmSnail expression is restricted to the CNS. Additionally, GmSnail shows a strong expression in the ventral midline, which has been observed neither in Drosophila nor in the spider. It has been suggested that Gm snail is involved in cell shape changes in the ventral midline, since in Drosophila Snail induces cell shape changes in the wing imaginal disc and during ventral furrow formation. In addition, Snail might have a similar function in the ventral neuroectoderm of chelicerates and myriapods. In the Drosophila neuroectoderm, the snail genes are necessary for the asymmetric distribution of the cell fate determinants Prospero and Numb to ganglion mother cells. Since most of the neural precursors in chelicerates and myriapods do not proliferate after their specification and Prospero is not asymmetrically distributed to daughter cells (at least in the spider), Snail must have a different function in the invaginating neural precursors. The spider and millipede snail homologues might be involved in the maintenance of the cell shape changes that occur during formation of the invagination sites in the ventral neuroectoderm.

Within the arthropods, the expression pattern of the pan-neural protein Prospero has only been analysed in Drosophila and in the spider Cupiennius salei. In Drosophila Prospero is asymmetrically localised to the basal membrane of the neuroblasts. During mitosis Prospero is exclusively distributed into one daughter cell, the ganglion mother cell, where it translocates from the cytoplasm into the nucleus. It has been shown that Prospero inhibits expression of multiple cell cycle regulatory genes in ganglion mother cells entering their final mitotic division. In the spider Prospero is expressed in the nuclei of neural precursors which are located basally within the invagination groups (Weller and Tautz 2003). Most of these precursors do not divide after invagination but differentiate into neurons and glial cells. These data suggest a conserved role of Prospero in neural cell fate determination in the spider and the fly.

Generation of neural precursor diversity

It has been shown in the insect Drosophila melanogaster that once the neural precursors are selected they divide in a unique and invariant pattern generating a stereotyped sequential series of ganglion mother cells (GMC) (Doe 1992). Each GMC divides once to give rise to two neural cells. Neural precursor diversity in Drosophila is achieved by both spatial and temporal patterning mechanisms. During neurogenesis segment polarity and dorso-ventral patterning genes subdivide the ventral neuroectoderm into a grid-like structure (reviewed by Skeath 1999). Each proneural cluster thus expresses a unique set of genes giving rise to neuroblasts with spatial heterogeneity. The spatial cues change over time so that the identities of neuroblasts also correlate with their time of formation (Berger et al. 2001). After delamination from the ventral neuroectoderm, neuroblasts become independent of spatial patterning cues. Subsequently, temporal patterning mechanisms generate additional diversity among the cell lineages of individual neuroblasts (see references in Chipman and Stollewerk 2006). Temporal identity in neuroblasts is regulated by sequential expression of Hunchback, Krüppel, Pdm and Castor. The temporal expression profile is maintained in the progeny of the neuroblasts leading to expression of transcription factors in mutually exclusive cell layers in the ventral neuromeres. Hunchback is expressed in early-born neurons that are located in the deepest layer, while Krüppel is expressed at low levels in the Hunchback layer and in a distinct layer between Hunchback and Pdm. Castor transcripts accumulate in the late-born superficial layer neurons.

There are few comparative studies of the events that generate neural precursor diversity, following the recruitment of neural precursors, during early development of the ventral nerve cord in the different arthropod groups, and those studies are incomplete (Stollewerk and Simpson 2005). However, from the limited data available, the expression of the segment polarity genes does appear to have been conserved in arthropods. These genes are expressed during neurogenesis suggesting an additional (or even primary) function in neural precursor identity. Studies on the segment polarity gene engrailed have indeed revealed that this gene is specifically expressed in neuroblasts/neural precursor groups of rows 1, 6 and 7 in all arthropod groups (for references see Stollewerk and Chipman 2006). Within the arthropods, the expression pattern and function of the dorso-ventral patterning genes ventral nerve cord defective, intermediate nerve cord defective and muscle segment homeodo-main have only been studied in Drosophila melanogaster and Tribolium cas-taneum (Skeath 1999, Wheeler et al. 2005). The overall expression of these genes in three longitudinal columns seems to be conserved, although slight differences in the spatiotemporal pattern were observed between the species.

However, it is obvious that spatial information from segment polarity genes and dorso-ventral patterning genes alone cannot account for the high complexity of cell types in the nervous system of arthropods. In Drosophila temporal identity genes like hunchback, Krüppel, Pdm and castor generate diversity within individual neuroblast lineages. But temporal identity mechanisms of the sort used by Dros-ophila cannot operate in a similar way in the remaining arthropod groups. In malacostracan crustaceans, neuroblasts do not delaminate and thus cannot escape the spatial cues of the neuroectoderm to initiate an independent temporal program. Furthermore, in chelicerates and myriapods stem-cell-like neuroblasts are absent and neural precursors are mainly postmitotic after invagination. Although this process has not been analysed in crustaceans, recent studies in chelicerates and myriapods suggest that the time-dependent expression of neural identity genes such as hunchback and Krüppel within the ventral neuroectoderm might generate additional diversity of neural precursor groups (Stollewerk et al. 2003, Chipman and Stollewerk 2006).

To summarise, although the components of the genetic network involved in specification of neural precursors are conserved in arthropods, the function of some of the genes might have changed, leading to different outcomes in the individual groups.

the ancestral pattern of neurogenesis in arthropods

The presented data indicate that some morphological and molecular aspects of neurogenesis are conserved in all arthropods and thus might have been present in their last common ancestor.

The ground pattern ofneurogenesis in arthropods seems to be the successive formation of about 30 neuroblasts/groups of neural precursors that are arranged in seven rows in each hemisegment. The genetic network that controls the specification and identity of neural precursors is conserved, although the function of the genes is adapted to the specific modes of neurogenesis in the individual arthropod groups. One possible reason for the stereotyped arrangement of neural precursors is the connection of neural precursor identity with spatial cues that confer anterior-posterior and dorso-ventral identities within a segment. The neural precursor pattern might have been constrained along with the general patterning mechanisms. Hence, we would expect a similar pattern of neural precursors in all animals that show expression of segment polarity and dorso-ventral patterning genes comparable to the euarthropods. However, this theory has to be tested in the future.

How has neurogenesis evolved in arthropods? It is tempting to speculate that the ancestral pattern of neurogenesis is the formation of groups of neural precursors, since this mode of neurogenesis is present in the basal arthropod groups. Neurogenesis in myriapods might be the crosslink between formation of groups of neural precursors and single neuroblasts. In contrast to chelicerates, neural precursor formation in myriapods seems to be associated with a proliferation pattern that indicates a clonal relationship of the precursor groups. Furthermore, although in Strigamia maritima groups of cells are specified for the neural fate, one cell of the group is different since all the remaining cells of the group are attached to this cell during the convergent extension movements that lead to an elongation of the germ band. The next step in evolution could have been the appearance of single neuroblasts in the lineage leading to insects and crustaceans. However, another possibility is that the modifications in the mode of neurogenesis in the individual arthropod groups do not reflect the actual sequence of evolution but are merely adaptations to the specific modes of embryogenesis in the individual species.

Indeed, differences in neurogenesis seem to coincide with different modes of embryogenesis in arthropods. For example, the different timing and order of neural precursor formation in Strigamia maritima and Archispirostreptus sp. as compared with the spider and the remaining myriapods might be an adaptation to the acceleration of neurogenesis relative to segment formation in these species. Similarly, differences in the number of neural precursors in a group and the initial arrangement of invagination sites seem to coincide with distinct morphologies of the neuroectoderm. The neuroectoderm of the diplopods and the geophilo-morph centipede analysed seems to consist of many more cells than that of the spider and Lithobius forficatus. Correspondingly, up to 12 cells contribute to an individual invagination site in the diplopods and Strigamia maritima, while in the spider and Lithobius forficatus only five to nine cells were counted. Furthermore, in Strigamia, neural precursor groups are initially arranged in three rows. The invagination sites become rearranged to a pattern similar to the remaining myriapods and the spider during an expansion of the germ band along the longitudinal axis. This process does not take place during neural precursor formation in the spider and the remaining myriapod species analysed, and thus the arrangement of the invagination sites remains the same throughout neurogenesis.

To summarise, ifwe disregard the adjustments to the specific morphologies ofembryogenesis in the individual species, the ground pattern of neurogenesis in arthropods seems to be the successive formation of about 30 neuroblasts/groups of neural precursors that are arranged in seven rows in each hemisegment. However, analyses of neurogenesis in outgroups to the euarthropods are necessary to confirm this assumption and to show whether the formation of groups of neural precursors, rather than the generation of individual neuroblasts, is the plesio-morphic state for this phylum.

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