Secondary embryonic axis formation by transplantation of D quadrant micromeres in an oligochaete annelid

Abstract

SUMMARYAmong spiral cleaving embryos (e.g. mollusks and annelids), it has long been known that one blastomere at the four-cell stage, the D cell, and its direct descendants play an important role in axial pattern formation. Various studies have suggested that the D quadrant acts as the organizer of the embryonic axes in annelids, although this has never been demonstrated directly. Here we show that D quadrant micromeres (2d and 4d) of the oligochaete annelid Tubifex tubifex are essential for embryonic axis formation. When 2d and 4d were ablated the embryo developed into a rounded cell mass covered with an epithelial cell sheet. To examine whether 2d and 4d are sufficient for axis formation they were transplanted to an ectopic position in an otherwise intact embryo. The reconstituted embryo formed a secondary embryonic axis with a duplicated head and/or tail. Cell lineage analyses showed that neuroectoderm and mesoderm along the secondary axis were derived from the transplanted D quadrant micromeres and not from the host embryo. However, endodermal tissue along the secondary axis originated from the host embryo. Interestingly, when either 2d or 4d was transplanted separately to host embryos, the reconstituted embryos failed to form a secondary axis, suggesting that both 2d and 4d are required for secondary axis formation. Thus, the Tubifex D quadrant micromeres have the ability to organize axis formation, but they lack the ability to induce neuroectodermal tissues, a characteristic common to chordate primary embryonic organizers.

Developmental significance of D quadrant micromeres 2d and 4d in the oligochaete annelid Tubifex tubifex

“…6C; Nakamoto et al, 2011). These results from the ablation/restoration experiment reinforce the notion that the D quadrant micromeres, 2d 11 and 4d, play a pivotal role in axial pattern formation in the Tubifex embryonic development including axial patterning (Penners, 1926). 2d and 4d are the exclusive sources of ectodermal and mesodermal segmental tissues…”

“…These studies have characterized thoroughly the developmental processes of 2d and 4d (a thorough review of these studies will be presented in the following sections). As described below, Nakamoto et al, (2011) have recently succeeded in inducing secondary axis formation by transplantation of D quadrant micromeres 2d…”

“…So we decided to follow the development in operated embryos from which damaged (or dead) cells were completely removed. For this purpose, 22-cell stage embryos were taken out of cocoons and vitelline membranes on an agar bed, and 2d 11 and 4d were ablated with fine glass needles, and discarded by means of pipets; the resulting operated embryos were cultured for 9 days in the culture medium containing antibiotics (Nakamoto et al, 2011). The operated embryos developed into rounded cell masses that failed to exhibit any morphological sign of axial development (Fig.…”

“…Similarly to the protocol used during the famous Spemann-Mangold organizer experiment, we transplanted both, 2d 11 and 4d, to an ectopic position in a host embryo. Therefore, we transplanted 2d 11 and 4d (that had been co-isolated from a donor embryo at stage 8) to the ventral region of a recipient embryo (at stage 11, i.e., about 6 hr from stage 8) from which one endodermal cell had been ablated (Nakamoto et al, 2011). This allows us to use the position of the ablated endodermal cell as mold for transplanted cells.…”

“…To determine the origin of cells comprising the secondary axis, we analyzed the cell fates of transplanted D quadrant micromeres (Nakamoto et al, 2011). Either 2d 11 or 4d of donor embryos were labeled with fluorescent tracers and they were co-isolated and transplanted to the ventral region of a normal recipient embryo as described before (see Fig.…”