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

Abstract

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.

Evolutionary Dynamics of the wnt Gene Family: A Lophotrochozoan Perspective

“…The parallel bands of wnt16 positive cells in the segmental ganglia of Helobdella, with wnt16b-expressing cells lying just anterior to the wnt16a-expressing cells are of particular interest because this pattern resembles the distribution of the respective ns and nf blast cell clones (Zhang and Weisblat 2005). But the gene expression patterns do not coincide with the clone distributions.…”

Evolutionary crossroads in developmental biology: annelids

“…Alternatively, the lipophilic dye DiI has been injected (Meyer et al, 2010). Further refinement and improved resolution has been achieved by injecting mRNA coding for nuclearlocalised fluorescent proteins (Zhang and Weisblat, 2005), which improves cellular resolution by labelling the nuclei rather than having the signal distributed throughout the cytoplasm. Most recently, the degradation of these injected mRNAs has been circumvented by injecting plasmids, from which nuclearlocalised fluorescent protein coding sequences can be transcribed within the injected cells and any descendants that inherit the plasmid (Gline et al, 2009).…”

“…For example, MO injections have been used both in lineage mapping (Zhang and Weisblat, 2005) and in characterising developmental gene functions (e.g. Agee et al, 2006;Song et al, 2002;Kuo and Weisblat, 2011), and dsRNA injection has recently been used to study the novel regeneration gene grimp in Enchytraeus japonensis (Takeo et al, 2010).…”

The Evolution of Early Neurogenesis

“…Early neurogenesis has been described in greatest detail in recent papers on the polychaete annelids Platynereis dumerilii (Denes et al, 2007) and Capitella sp. (Meyer and Seaver, 2009), as well as the leeches Helobdella robusta and Theromyzon rude (Stuart et al, 1989; Wedeen and Weisblat, 1991; Ramirez et al, 1995; Shankland, 1995; Shain et al, 1998; Zhang and Weisblat, 2005). In P. dumerilii , an anterior and a posterior-ventral subdomain within the ectoderm, which give rise to the brain and ventral nerve cord, respectively, express SoxB (Kerner et al, 2009).…”