Transgenesis enables mapping of segmental ganglia in the leech Helobdella austinensis.

The analysis of how neural circuits function in individuals and change during evolution is simplified by the existence of neurons identified as homologous within and across species. Invertebrates, including leeches, have been used for these purposes in part because their nervous systems comprise a high proportion of identified neurons, but technical limitations make it challenging to assess the full extent to which assumptions of stereotypy hold true. Here, we introduce Minos plasmid-mediated transgenesis as a tool for introducing transgenes into the embryos of the leech Helobdella austinensis (Spiralia; Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae). We identified an enhancer driving pan-neuronal expression of markers, including histone2B:mCherry, which allowed us to enumerate neurons in segmental ganglia. Unexpectedly, we found that the segmental ganglia of adult transgenic H. austinensis contain fewer and more variable numbers of neurons than in previously examined leech species.

Netrin expressed by the ventral ectoderm lineage guides mesoderm migration in epibolic gastrulation of the leech.

Netrin is a remarkably conserved midline landmark, serving as a chemotactic factor that organizes the bilateral neural architecture in the post-gastrula bilaterian embryos. Netrin signal also guides cell migration in many other neural and non-neural organogenesis events in later developmental stages but has never been found to participate in gastrulation - the earliest cell migration in metazoan embryogenesis. Here, we found that the netrin signaling molecules and their receptors are expressed during gastrulation of the leech Helobdella. Intriguingly, Hau-netrin-1 was expressed in the N lineage, which gives rise in part to the ventral midline of ectoderm, at the onset of gastrulation. We demonstrated that the N lineage is required for the entrance of mesoderm into the germinal band and that misexpression of Hau-netrin-1 in early gastrulation prevented mesoderm from entering the germinal band. Together, these results suggested that Hau-netrin-1 secreted by the N lineage guides mesoderm migration during germinal band assembly. Furthermore, ectopic expression of Hau-netrin-1 after the completion of germinal band assembly disrupted the epibolic migration of the germinal bands in a later stage of gastrulation. Thus, Hau-netrin-1 is likely involved in two distinct events in sequential stages of leech gastrulation: the assembly of germinal bands in early gastrulation and their epibolic migration in mid-gastrulation. Given that the leech netrin is expressed in the precursor cells of the ventral midline during gastrulation, we propose that a heterochronic change from the midline netrin expression had taken place in the evolution of a novel mode of gastrulation in the directly developing leech embryos.

Epidermal turnover in the planarian Schmidtea mediterranea involves basal cell extrusion and intestinal digestion.

Planarian flatworms undergo continuous internal turnover, wherein old cells are replaced by the division progeny of adult pluripotent stem cells (neoblasts). How cell turnover is carried out at the organismal level remains an intriguing question in planarians and other systems. While previous studies have predominantly focused on neoblast proliferation, little is known about the processes that mediate cell loss during tissue homeostasis. Here, we use the planarian epidermis as a model to study the mechanisms of cell removal. We established a covalent dye-labeling assay and image analysis pipeline to quantify the cell turnover rate in the planarian epidermis. Our findings indicate that the ventral epidermis is highly dynamic and epidermal cells undergo internalization via basal extrusion, followed by a relocation toward the intestine and ultimately digestion by intestinal phagocytes. Overall, our study reveals a complex homeostatic process of cell clearance that may generally allow planarians to catabolize their own cells.