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     

Generating lineage-specific markers to study Drosophila development

To generate cell- and tissue-specific expression patterns of the reporter gene lacZ in Drosophila, we have generated and characterized 1,426 independent insertion strains using four different P-element constructs. These four transposons carry a lacZ gene driven either by the weak promoter of the P-element transposase gene or by partial promoters from the even-skipped, fushi-tarazu, or engrailed genes. The tissue-specific patterns of β-galactosidase expression that we are able to generate depend on the promoter utilized. We describe in detail 13 strains that can be used to follow specific cell lineages and demonstrate their utility in analyzing the phenotypes of developmental mutants. Insertion strains generated with P-elements that carry various sequences upstream of the lacZ gene exhibit an increased variety of expression patterns that can be used to study Drosophila development.     

Expression of hunchback during trunk segmentation in the branchiopod crustacean Artemia franciscana

Comparative studies have shown that some aspects of segmentation are widely conserved among arthropods. Yet, it is still unclear whether the molecular prepatterns that are required for segmentation in Drosophila are likely to be similarly conserved in other arthropod groups. Homologues of the Drosophila gap genes, like hunchback, show regionally restricted expression patterns during the early phases of segmentation in diverse insects, but their expression patterns in other arthropod groups are not yet known. Here, we report the cloning of a hunchback orthologue from the crustacean Artemia franciscana and its expression during the formation of trunk segments. Artemia hunchback is expressed in a series of segmental stripes that correspond to individual thoracic/trunk, genital, and postgenital segments. However, this expression is not associated with the segmenting ectoderm but is restricted to mesodermal cells that associate with the ectoderm in a regular metameric pattern. All cells in the early segmental mesoderm appear to express hunchback. Later, mesodermal expression fades, and a complex expression pattern appears in the central nervous system (CNS), which is comparable to hunchback expression in the CNS of insects. No regionally restricted expression, reminiscent of gap gene expression, is observed during trunk segmentation. These patterns suggest that the expression patterns of hunchback in the mesoderm and in the CNS are likely to be ancient and conserved among crustaceans and insects. In contrast, we find no evidence for a conserved role of hunchback in axial patterning in the trunk ectoderm.     

Developmental control of cell division in leech embryos

During embryogenesis, cell division must be spatially and temporally regulated with respect to other developmental processes. Leech embryos undergo a series of unequal and asynchronous cleavages to produce individually recognizable cells whose lineages, developmental fates and cell cycle properties have been characterized. Thus, leech embryos provide an opportunity to examine the regulation of cell division at the level of individual well-characterized cells within a community of different types of cells. Isolation of leech homologues of some of the highly conserved regulators of the cell division cycle, and characterization of their patterns of maternal and zygotic expression, indicate that the cell divisions of early leech embryos are regulated by cell type-specific mechanisms. These studies with leech embryos contribute to the emerging appreciation of the diverse mechanisms by which animals regulate cell division during early development.     

The leech hunchback protein is expressed in the epithelium and CNS but not in the segmental precursor lineages

We are interested in identifying the regulatory genes involved in segmental pattern formation in annelids. The Drosophila segmentation gene hunchback (hb) is critical for the proper anteroposterior development of the fly embryo, but its function outside the diptera is currently unknown. Here, the protein expression pattern of Leech Zinc Finger II (LZF2), a leech orthologue of hb is characterized. In early embryogenesis, LZF2 protein is expressed in a subset of micromeres and is later expressed in the micromere-derived epithelium of the provisional epithelium and prostomium. LZF2 protein is detected in the ventral nerve cord during organogenesis, first in interganglionic muscle cells and later in subsets of neurons in each neuromere of the CNS. The location of immunoreactive cells during development and the similarity of the expression pattern of LZF2 to the expression of the Caenhorhabditis elegans hb homologue hbl-1 suggests that LZF2 plays a role in the morphogenetic movements of leech gastrulation and later in CNS specification but not in anteroposterior pattern formation. Received: 28 May 1999 / Accepted: 21 December 1999     

Leech segmentation: A molecular perspective

A variety of leech homeobox genes have been identified by homology with genes that are known to bring about the regionalization and segmentation of the anteroposterior body axis in other organisms. Embryonic expression patterns suggest a number of interphyletic similarities in the way that these genes are utilized. However, several interesting differences have also been observed. In particular, transplantation experiments in the leech embryo have shown that axially aligned patterns of homeobox gene expression are not specified by a global pattern of positional cues. Rather, the leech independently establishes anteroposterior patterns of gene expression in each of five discrete stem cell lineages, and these patterns are brought into their final alignment through a process of morphogenetic assembly.     

JAK-STAT signalling is required throughout telotrophic oogenesis and short-germ embryogenesis of the beetle Tribolium

In Drosophila, the JAK-STAT signalling pathway regulates a broad array of developmental functions including segmentation and oogenesis. Here we analysed the functions of Tribolium JAK-STAT signalling factors and of Suppressor Of Cytokine Signalling (SOCS) orthologues, which are known to function as negative regulators of JAK-STAT signalling, during telotrophic oogenesis and short-germ embryogenesis. The beetle Tribolium features telotrophic ovaries, which differ fundamentally from the polytrophic ovary of Drosophila. While we found the requirement for JAK-STAT signalling in specifying the interfollicular stalk to be principally conserved, we demonstrate that these genes also have early and presumably telotrophic specific functions. Moreover, we show that the SOCS genes crucially contribute to telotrophic Tribolium oogenesis, as their inactivation by RNAi results in compound follicles. During short-germ embryogenesis, JAK-STAT signalling is required in the maintenance of segment primordia, indicating that this signalling cascade acts in the framework of the segment-polarity network. In addition, we demonstrate that JAK-STAT signalling crucially contributes to early anterior patterning. We posit that this signalling cascade is involved in achieving accurate levels of expression of individual pair-rule and gap gene domains in early embryonic patterning.     

Intermediate filament genes as differentiation markers in the leech <Emphasis Type="Italic">Helobdella</Emphasis>

The intermediate filament (IF) cytoskeleton is a general feature of differentiated cells. Its molecular components, IF proteins, constitute a large family including the evolutionarily conserved nuclear lamins and the more diverse collection of cytoplasmic intermediate filament (CIF) proteins. In vertebrates, genes encoding CIFs exhibit cell/tissue type-specific expression profiles and are thus useful as differentiation markers. The expression of invertebrate CIFs, however, is not well documented. Here, we report a whole-genome survey of IF genes and their developmental expression patterns in the leech Helobdella, a lophotrochozoan model for developmental biology research. We found that, as in vertebrates, each of the leech CIF genes is expressed in a specific set of cell/tissue types. This allows us to detect earliest points of differentiation for multiple cell types in leech development and to use CIFs as molecular markers for studying cell fate specification in leech embryos. In addition, to determine the feasibility of using CIFs as universal metazoan differentiation markers, we examined phylogenetic relationships of IF genes from various species. Our results suggest that CIFs, and thus their cell/tissue-specific expression patterns, have expanded several times independently during metazoan evolution. Moreover, comparing the expression patterns of CIF orthologs between two leech species suggests that rapid evolutionary changes in the cell or tissue specificity of CIFs have occurred among leeches. Hence, CIFs are not suitable for identifying cell or tissue homology except among very closely related species, but they are nevertheless useful species-specific differentiation markers.     

Drosophila segmentation genes and blastoderm cell identities

The formation of the segmentation pattern in Drosophila embryos provides an excellent model for investigating the process of pattern formation in multicellular organisms. Several genes required in an embryo for normal segmentation have been analyzed by classical and molecular genetic and morphological techniques. A detailed consideration of these results suggests that these segmentation genes are combinatorially involved in translating the positional identities of individual cells at an early stage in Drosophila development.     

Epithelial differentiation in Drosophila

Our understanding of epithelial development in Drosophila has been greatly improved in recent years. Two key regulators of epithelial polarity, Crumbs and DE-cadherin, have been studied at the genetic and molecular levels and a number of additional genes are being analyzed that contribute to the differentiation of epithelial cell structure. Epithelial architecture has a profound influence on morphogenetic movements, patterning and cell-type determination. The combination of embryological and genetic/molecular tools in Drosophila will help us to elucidate the complex events that determine epithelial cell structure and how they relate to morphogenesis and other developmental processes.     

Molecular characterization and embryonic expression of innexins in the leech Hirudo medicinalis

Gap junctions are direct intercellular channels that permit the passage of ions and small signaling molecules. The temporal and spatial regulation of gap junctional communication is, thus, one mechanism by which cell interactions, and hence cell properties and cell fate, may be regulated during development. The nervous system of the leech, Hirudo medicinalis, is a particularly advantageous system in which to study developmental mechanisms involving gap junctions because interactions between identified cells may be studied in vivo in both the embryo and the adult. As in most invertebrates, gap junctions in the leech are composed of innexin proteins, which are distantly related to the vertebrate pannexins and are encoded by a multi-gene family. We have cloned ten novel leech innexins and describe the expression of these, plus two other previously reported members of this gene family, in the leech embryo between embryonic days 6 and 12, a period during which the main features of the central nervous system are established. Four innexins are expressed in neurons and two in glia, while several innexins are expressed in the excretory, circulatory, and reproductive organs. Of particular interest is Hm-inx6, whose expression appears to be restricted to the characterized S cell and two other neurons putatively identified as presynaptic to this cell. Two other innexins also show highly restricted expressions in neurons and may be developmentally regulated. Electronic Supplementary Material Supplementary material is available for this article at .     

Pair-rule and gap gene mutants in the flour beetle Tribolium castaneum

 Early pattern formation in the Drosophila embryo occurs in a syncytial blastoderm where communication between nuclei is unimpeded by cell walls. During the development of other insects, similar gene expression patterns are generated in a cellular environment. In Tribolium, for instance, pair-rule stripes are transiently expressed near the posterior end of the growing germ band. To elucidate how pattern formation in such a situation deviates from that of Drosophila, functional data about the genes involved are essential. In a genetic screen for Tribolium mutants affecting the larval cuticle pattern, we isolated 4 mutants (from a total of 30) which disrupt segmentation in the thorax and abdomen. Two of these mutants display clear pair-rule phenotypes. This demonstrates that not only the expression, but also the function of pair-rule genes in this short-germ insect is in principle similar to Drosophila. The other two mutants appear to identify gap genes. They provide the first evidence for the involvement of gap genes in abdominal segmentation of short-germ embryos. However, significant differences between the phenotypes of these mutants and those of known Drosophila gap mutants exist which indicates that evolutionary changes occurred in either the regulation or action of these genes. Received: 8 May 1998 / Accepted: 17 June 1998     

The tardigrade Hypsibius dujardini, a new model for studying the evolution of development

Studying development in diverse taxa can address a central issue in evolutionary biology: how morphological diversity arises through the evolution of developmental mechanisms. Two of the best-studied developmental model organisms, the arthropod Drosophila and the nematode Caenorhabditis elegans, have been found to belong to a single protostome superclade, the Ecdysozoa. This finding suggests that a closely related ecdysozoan phylum could serve as a valuable model for studying how developmental mechanisms evolve in ways that can produce diverse body plans. Tardigrades, also called water bears, make up a phylum of microscopic ecdysozoan animals. Tardigrades share many characteristics with C. elegans and Drosophila that could make them useful laboratory models, but long-term culturing of tardigrades historically has been a challenge, and there have been few studies of tardigrade development. Here, we show that the tardigrade Hypsibius dujardini can be cultured continuously for decades and can be cryopreserved. We report that H. dujardini has a compact genome, a little smaller than that of C. elegans or Drosophila, and that sequence evolution has occurred at a typical rate. H. dujardini has a short generation time, 13–14 days at room temperature. We have found that the embryos of H. dujardini have a stereotyped cleavage pattern with asymmetric cell divisions, nuclear migrations, and cell migrations occurring in reproducible patterns. We present a cell lineage of the early embryo and an embryonic staging series. We expect that these data can serve as a platform for using H. dujardini as a model for studying the evolution of developmental mechanisms.     

Roles of the sister chromatid cohesion apparatus in gene expression,development, and human syndromes

The sister chromatid cohesion apparatus mediates physical pairing of duplicated chromosomes. This pairing is essential for appropriate distribution of chromosomes into the daughter cells upon cell division. Recent evidence shows that the cohesion apparatus, which is a significant structural component of chromosomes during interphase, also affects gene expression and development. The Cornelia de Lange (CdLS) and Roberts/SC phocomelia (RBS/SC) genetic syndromes in humans are caused by mutations affecting components of the cohesion apparatus. Studies in Drosophila suggest that effects on gene expression are most likely responsible for developmental alterations in CdLS. Effects on chromatid cohesion are apparent in RBS/SC syndrome, but data from yeast and Drosophila point to the likelihood that changes in expression of genes located in heterochromatin could contribute to the developmental deficits.