Applications of mRNA injections for analyzing cell lineage and asymmetric cell divisions during segmentation in the leech Helobdella robusta

Department of Molecular and Cell Biology , University of California, Berkeley, Berkeley, California, United States
Development (Impact Factor: 6.46). 06/2005; 132(9):2103-13. DOI: 10.1242/dev.01802
Source: PubMed


Synthetic mRNAs can be injected to achieve transient gene expression even for 'non-model' organisms in which genetic approaches are not feasible. Here, we have used this technique to express proteins that can serve as lineage tracers or reporters of cellular events in embryos of the glossiphoniid leech Helobdella robusta (phylum Annelida). As representatives of the proposed super-phylum Lophotrochozoa, glossiphoniid leeches are of interest for developmental and evolutionary comparisons. Their embryos are suitable for microinjection, but no genetic approaches are currently available. We have injected segmentation stem cells (teloblasts) with mRNAs encoding nuclear localized green fluorescent protein (nGFP) and its spectral variants, and have used tandem injections of nGFP mRNA followed by antisense morpholino oligomer (AS MO), to label single blast cell clones. These techniques permit high resolution cell lineage tracing in living embryos. We have applied them to the primary neurogenic (N) lineage, in which alternate segmental founder cells (nf and ns blast cells) contribute distinct sets of progeny to the segmental ganglia. The nf and ns blast cell clones exhibit strikingly different cell division patterns: the increase in cell number within the nf clone is roughly linear, while that in the ns clone is almost exponential. To analyze spindle dynamics in the asymmetric divisions of individual blast cells, we have injected teloblasts with mRNA encoding a tau::GFP fusion protein. Our results show that the asymmetric divisions of n blast cells result from a posterior shift of both the spindle within the cell and the midbody within the mitotic spindle, with differential regulation of these processes between nf and ns.

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    • "The six other Hes/Hey-related genes expressed in structures related to the segmentation are localized in the teloblasts of the SAZ. The mitotic behavior of Platynereis teloblasts is coordinated to a certain degree [82] and they presumably undergo asymmetric divisions as leech teloblasts do [102]. One can suppose that those genes may be involved in such cellular processes and so have a role in the specification of a segmental periodicity. "
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    ABSTRACT: Background The Hes superfamily or Hes/Hey-related genes encompass a variety of metazoan-specific bHLH genes, with somewhat fuzzy phylogenetic relationships. Hes superfamily members are involved in a variety of major developmental mechanisms in metazoans, notably in neurogenesis and segmentation processes, in which they often act as direct effector genes of the Notch signaling pathway. Results We have investigated the molecular and functional evolution of the Hes superfamily in metazoans using the lophotrochozoan Platynereis dumerilii as model. Our phylogenetic analyses of more than 200 Metazoan Hes/Hey-related genes revealed the presence of five families, three of them (Hes, Hey and Helt) being pan-metazoan. Those families were likely composed of a unique representative in the last common metazoan ancestor. The evolution of the Hes family was shaped by many independent lineage specific tandem duplication events. The expression patterns of 13 of the 15 Hes/Hey-related genes in Platynereis indicate a broad functional diversification. Nevertheless, a majority of these genes are involved in two crucial developmental processes in annelids: neurogenesis and segmentation, resembling functions highlighted in other animal models. Conclusions Combining phylogenetic and expression data, our study suggests an unusual evolutionary history for the Hes superfamily. An ancestral multifunctional annelid Hes gene may have undergone multiples rounds of duplication-degeneration-complementation processes in the lineage leading to Platynereis, each gene copies ensuring their maintenance in the genome by subfunctionalisation. Similar but independent waves of duplications are at the origin of the multiplicity of Hes genes in other metazoan lineages.
    Full-text · Article · Sep 2014 · EvoDevo
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    • "Transient labeling methods involve in vivo delivery of vital dyes, DNA constructs, synthesized mRNAs or purified proteins that fluorescently label treated embryos. Transient labeling was first applied in established models such as Drosophila, zebrafish or mouse (Köster and Fraser, 2004; Minden et al., 1989; Zernicka-Goetz et al., 1997), and is still the method of choice in other model and non-model species, including crustaceans, annelids, echinoderms, ascidians and vertebrates (Corbo et al., 1997; Damle et al., 2006; Köster and Fraser, 2004; Price et al., 2010; Teddy and Kulesa, 2004; Zernicka-Goetz et al., 1996; Zhang and Weisblat, 2005). Labeling agents can also be delivered together with DNA/RNA constructs for gene overexpression or knockdown (Link and Megason, 2008). "
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    ABSTRACT: Studies on new arthropod models such as the beetle Tribolium castaneum are shifting our knowledge of embryonic patterning and morphogenesis beyond the Drosophila paradigm. In contrast to Drosophila, Tribolium embryos exhibit the short-germ type of development and become enveloped by extensive extra-embryonic membranes, the amnion and serosa. The genetic basis of these processes has been the focus of active research. Here, we complement genetic approaches with live fluorescence imaging of Tribolium embryos to make the link between gene function and morphogenetic cell behaviors during blastoderm formation and differentiation, germband condensation and elongation, and extra-embryonic development. We first show that transient labeling methods result in strong, homogeneous and persistent expression of fluorescent markers in Tribolium embryos, labeling the chromatin, membrane, cytoskeleton or combinations thereof. We then use co-injection of fluorescent markers with dsRNA for live imaging of embryos with disrupted caudal gene function caused by RNA interference. Using these approaches, we describe and compare cell and tissue dynamics in Tribolium embryos with wild-type and altered fate maps. We find that Tribolium germband condensation is effected by cell contraction and intercalation, with the latter being dependent on the anterior-posterior patterning system. We propose that germband condensation drives initiation of amnion folding, whereas expansion of the amniotic fold and closure of the amniotic cavity are likely driven by contraction of an actomyosin cable at the boundary between the amnion and serosa. Our methodology provides a comprehensive framework for testing quantitative models of patterning, growth and morphogenetic mechanisms in Tribolium and other arthropod species.
    Full-text · Article · Aug 2013 · Development
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    • "Prostomial and other non-segmental tissues arise in part from the earlier-born micromeres from all four quadrants (Weisblat et al. 1980, 1984; Huang et al. 2002) and from the early blast cells of selected teloblast lineages contribution (Zhang and Weisblat 2005; Gline et al. 2011). During stage 9, the non-segmental head ganglia and the prominent proboscis begin to form within the prostomial domain. "
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    ABSTRACT: 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.
    Full-text · Article · Sep 2011 · Development Genes and Evolution
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