Gangliogenesis in leech: morphogenetic processes leading to segmentation in the central nervous system

 Using intracellular lineage tracers to study the main neurogenic lineage (N lineage) of the glossiphoniid leech embryo, we have characterized events leading from continuous columns of segmental founder cells (nf and ns primary blast cells) to discrete, segmentally iterated ganglia. The separation between prospective ganglia was first evident as a fissure between the posterior boundary of nf- and the anterior boundary of ns-derived progeny. We also identified the sublineages of nf-derived cells that contribute parallel stripes of cells to each segment. These stripes of cells project ventrolaterally from the dorsolateral margin of each nascent ganglion to the ventral body wall. The position and orientation of the stripes suggests that they play a role in forming the posterior segmental nerve; they are not coincident with the ganglionic boundary, and they form well after the separation of ganglionic primordia. Previous work has shown that cells in the anterior stripe express the leech engrailed-class gene. Thus, in contrast to the role of cells expressing engrailed in Drosophila, the stripes of N-derived cells expressing an engrailed-class gene in leech do not seem to play a direct role in segmentation or segment polarity. Received: 10 October 1997 / Accepted: 12 December 1997     

Early axonogenesis in the embryo of a primitive insect,the silverfish Ctenolepisma longicaudata

The pattern of axon growth from the population of neurons that pioneers the major axon pathways in the central nervous system is highly conserved in winged insects. This study sought to determine whether the same pattern of axon growth is shared by an apterygotic insect, the silverfish. We have found that homologues to at least nine early differentiating winged insect neurons are present in the silverfish. The axon trajectories and the sequence of axon outgrowth from these neurons are very similar in silverfish and winged insects, suggesting that the pterygotic and apterygotic insects share a common developmental Bauplan for the construction of the central nervous system. Some of these neurons do show differences in several aspects of axon growth, including the relative timing of axonogenesis, the polarity of axon growth and the pattern of axon fasciculation. In addition, a major, early-appearing fascicle in the posterior commissure of the silverfish is pioneered by a neuron which does not appear to have an equivalent in the winged insects. These differences are similar in character to, albeit more pronounced than, differences previously reported between two winged insects, the fruitfly Drosophila and the grasshopper. Some of the features of early central axon growth, that set the silverfish embryo apart from the winged insects, are shared by crustacean embryos, providing support for the claim that insects and crustaceans share a common developmental Bauplan for the construction of central axonal pathways.     

Evolution of the pair rule gene network: Insights from a centipede

Comparative studies have examined the expression and function of homologues of the Drosophila melanogaster pair rule and segment polarity genes in a range of arthropods. The segment polarity gene homologues have a conserved role in the specification of the parasegment boundary, but the degree of conservation of the upstream patterning genes has proved more variable. Using genomic resources we identify a complete set of pair rule gene homologues from the centipede Strigamia maritima, and document a detailed time series of expression during trunk segmentation. We find supportive evidence for a conserved hierarchical organisation of the pair rule genes, with a division into early- and late-activated genes which parallels the functional division into primary and secondary pair rule genes described in insects. We confirm that the relative expression of sloppy-paired and paired with respect to wingless and engrailed at the parasegment boundary is conserved between myriapods and insects; suggesting that functional interactions between these genes might be an ancient feature of arthropod segment patterning. However, we find that the relative expression of a number of the primary pair rule genes is divergent between myriapods and insects. This corroborates suggestions that the evolution of upper tiers in the segmentation gene network is more flexible. Finally, we find that the expression of the Strigamia pair rule genes in periodic patterns is restricted to the ectoderm. This suggests that any direct role of these genes in segmentation is restricted to this germ layer, and that mesoderm segmentation is either dependent on the ectoderm, or occurs through an independent mechanism.     

Gene expression during postembryonic segmentation in the centipede <Emphasis Type="Italic">Lithobius peregrinus</Emphasis> (Chilopoda,Lithobiomorpha)

Postembryonic segmentation (anamorphosis) is widespread among arthropods, but only partially known as for its developmental mechanics and control. Studies on developmental genetics of segmentation in anamorphic arthropods are mostly limited to the germ band stage, during early phases of embryonic development. This work presents the first data on the postembryonic expression of a segmentation gene in a myriapod. Using real-time PCR, we analyzed engrailed expression patterns during the anamorphic stages of the centipede Lithobius peregrinus. A variation pattern in en RNA level during anamorphosis suggests that gene expression is precisely modulated during this period of development and that engrailed is mainly expressed in the posterior part of the body, in the newly differentiating segments of each stage. As anamorphosis is possibly the primitive segmentation mode in arthropods, the postembryonic en expression pattern documented here provides evidence for a conservation of en role in ontogeny, across the embryonic/postembryonic boundary, as well as in phylogeny, across the same boundary, but in the opposite direction, from primitive postembryonic expression to the more derived expression in clades with exclusively embryonic segmentation.     

Neurogenesis in the chilopod <Emphasis Type="Italic">Lithobius forficatus</Emphasis> suggests more similarities to chelicerates than to insects

In a recent comparative study on neurogenesis in the diplopod Glomeris marginata we have shown that the millipede and the spider share several features that cannot be found in homologous form in insects and crustaceans. The most distinctive difference is that groups of neural precursors are singled out from the neuroectoderm of the spider and the diplopod, rather than individual cells (i.e. neuroblasts) as in insects or crustacean. This observation constitutes the first morphological indication for a close myriapod/chelicerate relationship that has otherwise only been suggested by molecular phylogenetic analysis. To see whether the pattern of neurogenesis described for the diplopod is representative for myriapods, we analysed neurogenesis in the basal chilopod Lithobius forficatus. We show here that groups of cells invaginate from the chilopod neuroectoderm at strikingly similar positions as the invaginating cell groups of the diplopod and the spider. Furthermore, the expression patterns of the proneural and neurogenic genes reveal more similarities to the chelicerate and the diplopod than to insects. Thus, chelicerates and myriapods share the developmental mechanism for neurogenesis, either because they are true sister groups, or because this reflects the ancestral state of neurogenesis in arthropods.Edited by P. Simpson     

Embryonic expression of the single Tribolium engrailed homolog

We have cloned and sequenced the single Tribolium homolog of the Drosophila engrailed gene. The predicted protein contains a homeobox and several domains conserved among all engrailed genes identified to date. In addition it contains several features specific to the invected homologs of Bombyx and Drosophila, indicating that these features most likely were present in the ancestral gene in the common ancestor of holometabolous insects. We used the cross-reacting monoclonal antibody, 4D9, to follow the expression of the Engrailed protein during segmentation in Tribolium embryos. As in other insects, Engrailed accumulates in the nuclei of cells along the posterior margin of each segment. The first Engrailed stripe appears as the embryonic rudiment condenses. Then as the rudiment elongates into a germ band, Engrailed stripes appear in an anterior to posterior progression, just prior to morphological evidence of the formation of each segment. As in Drosophila (a long germ insect), expression of engrailed in Tribolium (classified as a short germ insect) is preceeded by the expression of several homologous segmentation genes, suggesting that similar genetic regulatory mechanisms are shared by diverse developmental types. © 1994 Wiley-Liss, Inc.     

Neurogenesis in myriapods and chelicerates and its importance for understanding arthropod relationships

Several alternative hypotheses on the relationships betweenthe major arthropod groups are still being discussed. We reexaminehere the chelicerate/myriapod relationship by comparing previouslypublished morphological data on neurogenesis in the euarthropodgroups and presenting data on an additional myriapod (Strigamiamaritima). Although there are differences in the formation ofneural precursors, most euarthropod species analyzed generateabout 30 single neural precursors (insects/crustaceans) or precursorgroups (chelicerates/myriapods) per hemisegment that are arrangedin a regular pattern. The genetic network involved in recruitmentand specification of neural precursors seems to be conservedamong euarthropods. Furthermore, we show here that neural precursoridentity seems to be achieved in a similar way. Besides theseconserved features we found 2 characters that distinguish insects/crustaceansfrom myriapods/chelicerates. First, in insects and crustaceansthe neuroectoderm gives rise to epidermal and neural cells,whereas in chelicerates and myriapods the central area of theneuroectoderm exclusively generates neural cells. Second, neuralcells arise by stem-cell-like divisions of neuroblasts in insectsand crustaceans, whereas groups of mainly postmitotic neuralprecursors are recruited for the neural fate in cheliceratesand myriapods. We discuss whether these characteristics representa sympleisiomorphy of myriapods and chelicerates that has beenlost in the more derived Pancrustacea or whether these characteristicsare a synapomorphy of myriapods and chelicerates, providingthe first morphological support for the Myriochelata group.     

<Emphasis Type="Italic">Engrailed</Emphasis> is expressed in larval development and in the radial nervous system of <Emphasis Type="Italic">Patiriella</Emphasis> sea stars

We documented expression of the pan-metazoan neurogenic gene engrailed in larval and juvenile Patiriella sea stars to determine if this gene patterns bilateral and radial echinoderm nervous systems. Engrailed homologues, containing conserved En protein domains, were cloned from the radial nerve cord. During development, engrailed was expressed in ectodermal (nervous system) and mesodermal (coeloms) derivatives. In larvae, engrailed was expressed in cells lining the larval and future adult coeloms. Engrailed was not expressed in the larval nervous system. As adult-specific developmental programs were switched on during metamorphosis, engrailed was expressed in the central nervous system and peripheral nervous system (PNS), paralleling the pattern of neuropeptide immunolocalisation. Engrailed was first seen in the developing nerve ring and appeared to be up-regulated as the nervous system developed. Expression of engrailed in the nerve plexus of the tube feet, the lobes of the hydrocoel along the adult arm axis, is similar to the reiterated pattern of expression seen in other animals. Engrailed expression in developing nervous tissue reflects its conserved role in neurogenesis, but its broad expression in the adult nervous system of Patiriella differs from the localised expression seen in other bilaterians. The role of engrailed in patterning repeated PNS structures indicates that it may be important in patterning the fivefold organisation of the ambulacrae, a defining feature of the Echinodermata.     

Regionalization and segmentation of the leech

Regionalization and segmentation of the leech body plan have been examined by numerous approaches over the years. A wealth of knowledge has accumulated regarding the normally invariant cell lineages of the leech and the degree of developmental plasticity that is possible in each cell line in early development and in neurogenesis. Homologues of genes that control regionalization and segmentation in Drosophila have been cloned from the leech and the expression patterns reveal conserved features with those in Drosophila and other organisms. Possible developmental functions of the en-class proteins in spatial and temporal modes of segment formation are discussed in light of leech and Drosophila development. Annelida and Arthropoda cell lineages of engrailed-class gene expression are compared in leech blast cell clones and crustacean parasegments. In addition, future directions for molecular analysis of segmentation of the leech are summarized. © 1995 John Wiley & Sons, Inc.     

Effects of engrailed in the genital disc of Drosophila melanogaster

engrailed has been postulated to be the “selector gene” involved in the establishment of the anterior-posterior compartment border in several imaginal discs and in at least the first two abdominal segments of Drosophila melanogaster. Our study of the effects of different mutant engrailed genotypes on genital disc development provided the following major results: All three terminal primordia (female and male genitalia, and analia) were affected. Different heteroallelic combinations showed different expressivities, and the three terminal primordia were differently affected by the same mutant genotype. The engrailed genotypes deleted specific elements of the adult terminalia without causing associated pattern duplications. The reduced morphology of the male engrailed genital disc was analogous to the pattern deletions observed in the adult terminalia. That the engrailed phenotype is stable was demonstrated by culturing in vivo intact and fragmented engrailed genital discs. Cell death was found in a significant number of mature male en²/en³ genital discs. The results are discussed in terms of the segmental organization of the genital disc and in terms of the “selector gene” function postulated for the engrailed locus. The interpretation that each terminal primordium has an anterior and a posterior compartment is presented and it is assumed that in the genital disc engrailed transforms posterior cells into anterior cells that do not develop, thereby causing the deficiency pattern of the engrailed phenotype.     

Segmentation in the crustacean Artemia: engrailed staining studied with an antibody raised against the Artemia protein

We have studied the process of post-embryonic segmentation in the anostracan crustacean Artemia franciscana using a specific antibody raised against the engrailed protein of this organism. Three cephalic segments are specified during embryonic development, before larval hatching, whilst trunk (thoracic) segmentation begins after the first stage free-swimming nauplius larva has emerged from the dormant cyst. Thus, cephalic and trunk segmentation seem to be at least in part independent and superimposed processes. Trunk stripes of engrailed expression are added one at a time as segments are generated from the posterior growth zone. The first detectable decision in engrailed expression is the establishment of a line of engrailed-expressing cells, interpretable as delineating the parasegmental boundary. The subsequent widening of engrailed stripes is not correlated with cell lineage events but is probably mediated by the combination of inheritance of the active state and recruitment of new cells into the engrailed-expressing stripe.     

Neurogenesis in the water flea Daphnia magna (Crustacea, Branchiopoda) suggests different mechanisms of neuroblast formation in insects and crustaceans

Within euarthropods, the morphological and molecular mechanisms of early nervous system development have been analysed in insects and several representatives of chelicerates and myriapods, while data on crustaceans are fragmentary. Neural stem cells (neuroblasts) generate the nervous system in insects and in higher crustaceans (malacostracans); in the remaining euarthropod groups, the chelicerates (e.g. spiders) and myriapods (e.g. millipedes), neuroblasts are missing. In the latter taxa, groups of neural precursors segregate from the neuroectoderm and directly differentiate into neurons and glial cells. In all euarthropod groups, achaete–scute homologues are required for neuroblast/neural precursor group formation. In the insects Drosophila melanogaster and Tribolium castaneum achaete–scute homologues are initially expressed in clusters of cells (proneural clusters) in the neuroepithelium but expression becomes restricted to the future neuroblast. Subsequently genes such as snail and prospero are expressed in the neuroblasts which are required for asymmetric division and differentiation. In contrast to insects, malacostracan neuroblasts do not segregate into the embryo but remain in the outer neuroepithelium, similar to vertebrate neural stem cells. It has been suggested that neuroblasts are present in another crustacean group, the branchiopods, and that they also remain in the neuroepithelium. This raises the questions how the molecular mechanisms of neuroblast selection have been modified during crustacean and insect evolution and if the segregation or the maintenance of neuroblasts in the neuroepithelium represents the ancestral state. Here we take advantage of the recently published Daphnia pulex (branchiopod) genome and identify genes in Daphnia magna that are known to be required for the selection and asymmetric division of neuroblasts in the fruit fly D. melanogaster. We unambiguously identify neuroblasts in D. magna by molecular marker gene expression and division pattern. We show for the first time that branchiopod neuroblasts divide in the same pattern as insect and malacostracan neuroblasts. Furthermore, in contrast to D. melanogaster, neuroblasts are not selected from proneural clusters in the branchiopod. Snail rather than ASH is the first gene to be expressed in the nascent neuroblasts suggesting that ASH is not required for the selection of neuroblasts as in D. melanogaster. The prolonged expression of ASH in D. magna furthermore suggests that it is involved in the maintenance of the neuroblasts in the neuroepithelium. Based on these and additional data from various representatives of arthropods we conclude that the selection of neural precursors from proneural clusters as well as the segregation of neural precursors represents the ancestral state of neurogenesis in arthropods. We discuss that the derived characters of malacostracans and branchiopods – the absence of neuroblast segregation and proneural clusters – might be used to support or reject the possible groupings of paraphyletic crustaceans.     

Initial axon growth rate from embryonic sensory neurons is correlated with birth date

Axon growth rate from different populations of sensory neurons is correlated with the distance they have to grow to reach their targets in development: neurons with more distant targets extend axons at intrinsically faster rates. With growth of the embryo, later‐born neurons within each population have further to extend their axons to reach their targets than early‐born neurons. Here we examined whether the axon growth rate is related to birth date by studying the axon growth from neurons that differentiate in vitro from precursor cells isolated throughout the period of neurogenesis. We first showed that neurons that differentiated in vitro from different precursor cell populations exhibited differences in axon growth rate related to in vivo target distance. We then examined the axon growth rate from neurons that differentiate from the same precursor population at different stages throughout the period of neurogenesis. We studied the epibranchial placode precursors that give rise to nodose ganglion neurons in the chicken embryo. We observed a highly significant, threefold difference in axon growth rate from neurons that differentiate from precursor cells cultured early and late during the period of neurogenesis. Our findings suggest that intrinsic differences in axon growth rate are correlated with the neuronal birth date.     

Embryonic expression of the engrailed homologue of Rhynchosciara americana

The segment polarity gene engrailed is involved in the determination of segment posterior identity in Drosophila. engrailed has been largely used for comparative developmental studies due to its evolutionary conservation from nematodes to humans. By in situ hybridization of an engrailed cDNA probe from Drosophila to polytene chromosomes of fourth instar larvae of Rhynchosciara americana we have shown that engrailed-like sequences must be localized in band 6 of chromosome A in this species. The pattern of engrailed protein expression during R. americana embryo development is diffuse at first evolving into a nuclear striped pattern after quite a length of time. In addition, our results suggest a possible developmentally regulated molecular modification of engrailed protein in R. americana embryos.     

Gene expression suggests conserved mechanisms patterning the heads of insects and myriapods

Segmentation, i.e. the subdivision of the body into serially homologous units, is one of the hallmarks of the arthropods. Arthropod segmentation is best understood in the fly Drosophila melanogaster. But different from the situation in most arthropods in this species all segments are formed from the early blastoderm (so called long-germ developmental mode). In most other arthropods only the anterior segments are formed in a similar way (so called short-germ developmental mode). Posterior segments are added one at a time or in pairs of two from a posterior segment addition zone. The segmentation mechanisms are not universally conserved among arthropods and only little is known about the genetic patterning of the anterior segments. Here we present the expression patterns of the insect head patterning gene orthologs hunchback (hb), orthodenticle (otd), buttonhead-like (btdl), collier (col), cap-n-collar (cnc) and crocodile (croc), and the trunk gap gene Krüppel (Kr) in the myriapod Glomeris marginata. Conserved expression of these genes in insects and a myriapod suggests that the anterior segmentation system may be conserved in at least these two classes of arthropods. This finding implies that the anterior patterning mechanism already existed in the last common ancestor of insects and myriapods.     

Two engrailed-related genes in the cockroach: cloning, phylogenetic analysis, expression and isolation of splice variants

engrailed-related genes have been isolated in numerous taxa. Within the insects, some species have a single engrailed-related gene whilst others have two copies, raising the question of when and how often gene duplications have occurred. Here we report the cloning, in the cockroach Periplaneta americana, of two engrailed-related genes Pa-en1 and Pa-en2. By comparing conserved domains and by carrying out a phylogenetic analysis, we conclude that these two genes are likely to be the product of a recent duplication in the cockroach lineage. Pa-en1 and Pa-en2 are co-expressed during early embryogenesis and their segmental pattern of expression appears in an anterior-posterior progression. We have also isolated potential splice variants of Pa-en2 which lack some regulatory domains. The roles these splice variants may play in regulating developmental processes are discussed. Received: 6 August 1999 / Accepted: 4 April 2000     

Segmental expression of Pax3/7 and Engrailed homologs in tardigrade development

How morphological diversity arises through evolution of gene sequence is a major question in biology. In Drosophila, the genetic basis for body patterning and morphological segmentation has been studied intensively. It is clear that some of the genes in the Drosophila segmentation program are functioning similarly in certain other taxa, although many questions remain about when these gene functions arose and which taxa use these genes similarly to establish diverse body plans. Tardigrades are an outgroup to arthropods in the Ecdysozoa and, as such, can provide insight into how gene functions have evolved among the arthropods and their close relatives. We developed immunostaining methods for tardigrade embryos, and we used cross-reactive antibodies to investigate the expression of homologs of the pair-rule gene paired (Pax3/7) and the segment polarity gene engrailed in the tardigrade Hypsibius dujardini. We find that in H. dujardini embryos, Pax3/7 protein localizes not in a pair-rule pattern but in a segmentally iterated pattern, after the segments are established, in regions of the embryo where neurons later arise. Engrailed protein localizes in the posterior ectoderm of each segment before ectodermal segmentation is apparent. Together with previous results from others, our data support the conclusions that the pair-rule function of Pax3/7 is specific to the arthropods, that some of the ancient functions of Pax3/7 and Engrailed in ancestral bilaterians may have been in neurogenesis, and that Engrailed may have a function in establishing morphological boundaries between segments that is conserved at least among the Panarthropoda. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

EphA receptors direct the differentiation of mammalian neural precursor cells through a mitogen-activated protein kinase-dependent pathway

Ephrins are cell surface-associated ligands for Eph receptor tyrosine kinases and are implicated in repulsive axon guidance and cell migration. EphA2, 3, and 4 receptors and one of their cognate ligands, ephrin-A2, are expressed by cells in the subventricular zone and ganglionic eminence of the embryonic day 14.5 telencephalon and by neural precursor cells in vitro. Activation of EphA receptors in dissociated neural precursor cells in vitro facilitates the commitment to neuronal fates. The majority of ephrin-A1-induced neurons is immunoreactive for tyrosine hydroxylase. Blocking the signal by the extracellular domain of EphA in forebrain slices results in a decrease in neurogenesis. Extracellular signal-regulated kinase is activated by the ligand binding to EphA receptors and is involved in the neurogenesis through EphA receptors. Rap1, but not Ras, is activated in response to ephrin-A1. Our results identify EphA receptors as positive regulators of the mitogen-activated protein kinase pathway that exerts neurogenesis of neural precursor cells from the developing central nervous system.     

The involvement of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">wingless</Emphasis> during segmentation in the onychophoran <Emphasis Type="Italic">Euperipatoides kanangrensis</Emphasis> (Peripatopsidae: Onychophora) (Reid 1996)

As the putative sister group to the arthropods, onychophorans can provide insight into ancestral developmental mechanisms in the panarthropod clade. Here, we examine the expression during segmentation of orthologues of wingless (Wnt1) and engrailed, two genes that play a key role in defining segment boundaries in Drosophila and that appear to play a role in segmentation in many other arthropods. Both are expressed in segmentally reiterated stripes in all forming segments except the first (brain) segment, which only shows an engrailed stripe. Engrailed is expressed before segments are morphologically visible and is expressed in both mesoderm and ectoderm. Segmental wingless expression is not detectable until after mesodermal somites are clearly distinct. Early engrailed expression lies in and extends to both sides of the furrow that first demarcates segments in the ectoderm, but is largely restricted to the posterior part of somites. Wingless expression lies immediately anterior to engrailed expression, as it does in many arthropods, but there is no precise cellular boundary between the two expression domains analogous to the overt parasegment boundary seen in Drosophila. Engrailed stripes extend along the posterior part of each limb bud, including the antenna, while wingless is restricted to the distal tip of the limbs and the neurectoderm basal to the limbs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Formation and specification of neurons during the development of the leech central nervous system

In the leech embryo, neurogenesis takes place within the context of a stereotyped cell lineage. The prospective germ layers are formed during the early cleavage divisions by the reorganization and segregation of circumscribed domains within the cytoplasm of the fertilized egg. The majority of central neurons arise from the ectoderm, and central neuroblasts are distributed throughout both the length and width of each ectodermal hemisegment. Much of the segmental ganglion arises from medial neuroblasts, but there are also lateral ectodermal neuroblasts and mesodermal neuroblasts that migrate into the nascent ganglion from peripheral sites of origin. Some of these migratory cells are committed to neurogenesis prior to reaching their central destination. In addition, the leech embryo exhibits a secondary phase of neurogenesis that is restricted to the two sex segment ganglia. Secondary neurogenesis requires that a mitogenic or trophic signal be conveyed from the peripherally located male sex organ to a particular set of centrally located neuroblasts, apparently via already differentiated central neurons that innervate the sex organ. The differential specification of neuronal phenotypes within the leech central nervous system occurs in multiple steps. Some aspects of a neuron's identity are already specified at the time of its terminal cell division and would seem to involve the lineal inheritance of developmental commitments made by one of the neuron's progenitors. This lineage-based identity can then be modified by interactions between the postmitotic neuron and other neurons or non-neuronal target cells encountered during its terminal differentiation. © 1995 John Wiley & Sons, Inc.