Live imaging reveals the progenitors and cell dynamics of limb regeneration.

Regeneration is a complex and dynamic process, mobilizing diverse cell types and remodelling tissues over long time periods. Tracking cell fate and behaviour during regeneration in active adult animals is especially challenging. Here, we establish continuous live imaging of leg regeneration at single-cell resolution in the crustacean Parhyale hawaiensis. By live recordings encompassing the first 4-5 days after amputation, we capture the cellular events that contribute to wound closure and morphogenesis of regenerating legs with unprecedented resolution and temporal detail. Using these recordings we are able to track cell lineages, to generate fate maps of the blastema and to identify the progenitors of regenerated epidermis. We find that there are no specialized stem cells for the epidermis. Most epidermal cells in the distal part of the leg stump proliferate, acquire new positional values and contribute to new segments in the regenerating leg.

The early development of the onychopod cladoceran Bythotrephes longimanus (Crustacea, Branchiopoda).

INTRODUCTION: Within arthropods, several crustacean groups are unique in their early development due to their stereotyped cell division patterns and cell lineages. However, it is still unclear whether these cell division patterns are homologous between the various crustacean groups and whether they could indicate the ground pattern of Tetraconata (Crustacea and Hexapoda). In this study we describe the early development of the raptorial water flea Bythotrephes longimanus as a representative of the Cladocera within branchiopods. RESULTS: In B. longimanus the early cell lineage and the cell division pattern are stereotyped up to the fifth cell division cycle. As a morphological marker a nurse cell remnant (ncr) identifies the cell lineage of the smallest and division delayed blastomere up to the 16-cell stage. This marker might be indicative of the germ line. By combining histology, confocal laser scanning microscopy, and 4D microscopy, we reconstruct the early cell lineage and cell division pattern and follow transient formations of cell morphological structures in their temporal and spatial behavior up to gastrulation. CONCLUSIONS: Correspondences to the early cleavage pattern of other Cladocera suggest that the described pattern can be assumed to be ancestral for either the entire Cladocera or for the majority of the Cladocera comprising Anomopoda, Ctenopoda and Onychopoda. The comparison to the cell division patterns of other crustacean groups such as Malacostraca, Ostracoda, and Copepoda reveals similarities that allow for a discussion of a common pattern for the crustacean groups and a ground pattern for the Tetraconata.

De novo assembly and characterization of a maternal and developmental transcriptome for the emerging model crustacean Parhyale hawaiensis.

BACKGROUND: Arthropods are the most diverse animal phylum, but their genomic resources are relatively few. While the genome of the branchiopod Daphnia pulex is now available, no other large-scale crustacean genomic resources are available for comparison. In particular, genomic resources are lacking for the most tractable laboratory model of crustacean development, the amphipod Parhyale hawaiensis. Insight into shared and divergent characters of crustacean genomes will facilitate interpretation of future developmental, biomedical, and ecological research using crustacean models. RESULTS: To generate a transcriptome enriched for maternally provided and zygotically transcribed developmental genes, we created cDNA from ovaries and embryos of P. hawaiensis. Using 454 pyrosequencing, we sequenced over 1.1 billion bases of this cDNA, and assembled them de novo to create, to our knowledge, the second largest crustacean genomic resource to date. We found an unusually high proportion of C2H2 zinc finger-containing transcripts, as has also been reported for the genome of the pea aphid Acyrthosiphon pisum. Consistent with previous reports, we detected trans-spliced transcripts, but found that they did not noticeably impact transcriptome assembly. Our assembly products yielded 19,067 unique BLAST hits against nr (E-value cutoff e-10). These included over 400 predicted transcripts with significant similarity to D. pulex sequences but not to sequences of any other animal. Annotation of several hundred genes revealed P. hawaiensis homologues of genes involved in development, gametogenesis, and a majority of the members of six major conserved metazoan signaling pathways. CONCLUSIONS: The amphipod P. hawaiensis has higher transcript complexity than known insect transcriptomes, and trans-splicing does not appear to be a major contributor to this complexity. We discuss the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models such as D. pulex and P. hawaiensis, as well as the need for development of further substantial crustacean genomic resources.

Patterns of cell lineage, movement, and migration from germ layer specification to gastrulation in the amphipod crustacean Parhyale hawaiensis.

The acquisition of specific cell fates throughout embryonic development is one of the core problems in developmental and evolutionary biology. In the amphipod Parhyale hawaiensis all three germ layers and the germ line are determined by the eight-cell stage. Despite this early fate determination, multiple cell types can be replaced following ablation of their founder cells, showing that this embryo also has significant regulative properties. Here we present a cellular-level resolution lineage analysis for P. hawaiensis embryos between fertilization and gastrulation, including analysis of cleavage patterns, division times, and clonal behaviors. We compare these cellular behaviors in wild type embryos with those in embryos where specific founder cells have been ablated, or where zygotic transcription has been inhibited. We observe that when germ line, endoderm or mesoderm founder cells are ablated, the remaining cells do not alter their cleavage or migration behaviors before the onset of gastrulation. In the absence of zygotic transcription, ingression movements proceed normally, but epibolic movements are disrupted. This indicates that the embryo's regulative response to germ layer founder loss, in the form of altered cell behavior, is realized in the ~32h between gastrulation and early germ band elongation, and is likely to require zygotic reprogramming rather than alternative deployment of maternally supplied determinants. Combining these data with the observations of previous studies, we propose a framework to elucidate the molecular mechanisms that regulate the determinative and regulative properties of the P. hawaiensis embryo.

Cleavage and gastrulation of the dendrobranchiate shrimp Penaeus monodon (Crustacea, Malacostraca, Decapoda).

The cleavage pattern of the black tiger shrimp Penaeus monodon was analyzed from the first division until gastrulation. Observations were based on microscopy combined with the use of fluorescent dyes, histological techniques, and computer based three-dimensional reconstructions. Early cleavage is holoblastic and follows a stereotypic pattern, which largely corresponds to what is known from other dendrobranchiate decapods. However, for the first time in this group, we report the presence of an intracellular structure throughout early development. This intracellular body (icb) marks the lineage of one of the two enlarged and division-delayed mesendoderm cells that initiate gastrulation. The identity of the icb as a natural marker and putative determinant of the germ line and its implications on the establishment of the body axes are discussed. The icb as a landmark reveals that the same stereotypic cell division pattern can lead to different fates of individual cells. Hence, the results of this study permit an additional approach to study the relation between cell lineage pattern and the identity of cell lineages.

The post-embryonic development of Remipedia (Crustacea)--additional results and new insights.

The post-embryonic development of a species of the enigmatic crustacean group Remipedia is described in detail for the first time under various aspects. Applying a molecular approach, we can clearly prove the species identity of the larvae as belonging to Pleomothra apletocheles. We document the cellular level of several larval stages and the differentiation of segments, limbs, and the general body morphology applying the techniques of confocal laser scanning microscopy and scanning electron microscopy. In addition, we document the swimming behavior and the peculiar movements of the naupliar appendages. A comparison of our results with published data on other Crustacea and their larval development tentatively supports ideas about phylogenetic affinities of the Remipedia to the Malacostraca.

Stages and other aspects of the embryology of the parthenogenetic Marmorkrebs (Decapoda, Reptantia, Astacida).

The early development of the parthenogenetic Marmorkrebs (marbled crayfish) is described with respect to external morphology, cell lineage, and segment formation. Due to its parthenogenetic reproduction mode, the question arises whether or not the marbled crayfish is a suitable model organism for developmental approaches. To address this question, we describe several aspects of the embryonic development until hatching. We establish ten stages based on characteristic external changes in the living eggs such as blastoderm formation, gastrulation process, formation and differentiation of the naupliar and post-naupliar segments, limb bud differentiation, and eye differentiation. The study of the post-naupliar cell division patterns, segment formation, and engrailed expression reveals distinct similarities to that of other freshwater crayfish. On this basis, we evaluate the possibility of a generalization of ontogenetic processes in the Marmorkrebs for either freshwater crayfish or other crustacean developmental systems.