Grandparental stem cells in leech segmentation: Differences in CDC42 expression are correlated with an alternating pattern of blast cell fates

Abstract

Embryonic segmentation in clitellate annelids (oligochaetes and leeches) is a cell lineage-driven process. Embryos of these worms generate a posterior growth zone consisting of 5 bilateral pairs of identified segmentation stem cells (teloblasts), each of which produces a column of segmental founder cells (blast cells). Each blast cell generates a lineage-specific clone via a stereotyped sequence of cell divisions, which are typically unequal both in terms of the relative size of the sister cells and in the progeny to which they give rise. In two of the five teloblast lineages, including the ventralmost, primary neurogenic (N) lineage, the blast cells adopt two different fates, designated nf and ns, in exact alternation within the blast cell column; this is termed a grandparental stem cell lineage. To lay groundwork for investigating unequal divisions in the leech Helobdella, we have surveyed the H. robusta genome for genes encoding orthologs of the Rho family GTPases, including the rho, rac and cdc42 sub-families, which are known to be involved in multiple processes involving cell polarization in other systems. We find that, in contrast to most other known systems the Helobdella genome contains two cdc42 orthologs, one of which is expressed at higher levels in the ns blast cells than in nf blast cells. We also demonstrate that the asymmetric divisions of the primary nf and ns blast cells are regulated by the polarized distribution of the activated form of the Cdc42 protein, rather than by the overall level of expression. Our results provide the first molecular insights into the mechanisms of the grandparental stem cell lineages, a novel, yet evolutionarily ancient stem cell division pattern. Our results also provide an example in which asymmetries in the distribution of Cdc42 activity, rather than in the overall levels of Cdc42 protein, are important regulating unequal divisions in animal cells.

Growth patterns in Onychophora (velvet worms): lack of a localised posterior proliferation zone

“…Among annelids, anti-BrdU immunolabelling revealed a distinct posterior proliferation zone in post-metamorphic stages of polychaetes, including the echiurans [15,16,54]. A similar localised region containing stem-like cells or teloblasts also occurs in clitellate embryos [13,14,18]. To obtain comparative data from Onychophora, we applied the anti-BrdU immunolabelling in elongating embryos of the velvet worm species Euperipatoides rowelli .…”

“…While it seems clear that such a localised proliferation zone is present in embryos, larvae, or juveniles of annelids, including clitellates and polychaetes [11-18], the situation is less clear for chordates. In vertebrate embryos, a higher proliferative activity, as compared to the pre-somitic mesoderm region, consistent with the presence of stem cells has been observed in the tailbud [19-24].…”

“…Traditionally, the term "posterior growth zone" has been used to describe a localised and highly proliferative terminal body region, which has been dubbed the "proliferating area" or "zone of proliferation" [ 9 - 11 ]. While it seems clear that such a localised proliferation zone is present in embryos, larvae, or juveniles of annelids, including clitellates and polychaetes [ 11 - 18 ], the situation is less clear for chordates. In vertebrate embryos, a higher proliferative activity, as compared to the pre-somitic mesoderm region, consistent with the presence of stem cells has been observed in the tailbud [ 19 - 24 ].…”

What is a segment?

“…Ablation experiments indicate that ns and nf are not an equivalence group [ 42 ]. Additionally, molecular segment polarity is found in the progeny, as the activated form of the cell cycle protein Cdc42 is expressed in higher levels in ns versus nf [ 43 ]. These data suggest that N, and by extension, Q, produce segments with intrinsic segmental polarity.…”

Developmental biology of the leech Helobdella

“…Ablation experiments suggest that the blast cells are committed to distinct f and s fates several hours before their first mitoses, and other studies suggest that there may be subtle differences in the cyokinetic processes by which nf and ns blast cells arise from the teloblast (Bissen and Weisblat, 1987), suggestive of a flip-flop mechanism. At the molecular level, it appears that the ns blast cells express higher levels of a cdc42 homolog than do nf cells (Zhang et al , 2009), but these differences were not evident in the cells most proximal to the teloblasts, so we regard this question as still open.…”