Germ-layer commitment and axis formation in sea anemone embryonic cell aggregates

Abstract

Robust morphogenetic events are pivotal for animal embryogenesis. However, comparison of the modes of development of different members of a phylum suggests that the spectrum of developmental trajectories accessible for a species might be far broader than can be concluded from the observation of normal development. Here, by using a combination of microsurgery and transgenic reporter gene expression, we show that, facing a new developmental context, the aggregates of dissociated embryonic cells of the sea anemone Nematostella vectensis take an alternative developmental trajectory. The self-organizing aggregates rely on Wnt signals produced by the cells of the original blastopore lip organizer to form body axes but employ morphogenetic events typical for normal development of distantly related cnidarians to re-establish the germ layers. The reaggregated cells show enormous plasticity including the capacity of the ectodermal cells to convert into endoderm. Our results suggest that new developmental trajectories may evolve relatively easily when highly plastic embryonic cells face new constraints.

Keywords: body axes; embryonic cell aggregates; germ layers; self-organization.

Fig. 1.

The course of aggregate development. (A) Scheme of the dissociation–reaggregation experiment. (B–M) Successive stages of aggregate development analyzed by confocal and scanning electron microscopy. Directly after centrifugation, no epithelium is observed (B and C). Ectoderm epithelialization begins by 12 hpd (D and E: note a stretch of epithelialized ectoderm along the dotted line between white arrowheads in D) and is complete by 24 hpd (F: longitudinal optical section; G: transverse optical section). First signs of mouth formation become visible (F and H). Endoderm starts to form an epithelial layer by 48 hpd (I) and completes the process by 3 dpd (J: note also a well-developed pharynx). Mouth, hypostome, and tentacles form over the next several days (K–M). Black box (K, dashed line) masks the original scale bar. (N–T) Larger aggregates form multiple heads. (N) Double in situ hybridization with the oral marker FoxA (red) and aboral marker FGFa1 (blue) shows that the number of heads/number of aboral poles ratio is 3/1. SEM shows that the number of heads per aggregate and tentacles per head can vary (O–S). Head structures can form in close proximity to each other, as visualized by SEM at the polyp stage and by in situ hybridization with an oral marker Brachyury at an earlier stage (S and T). (B–G, I, and J) Red: nuclei; green: F-actin. Asterisks, mouth; dpd, days post dissociation; ecto, ectoderm; endo, endoderm; hpd, hours post dissociation; mpd, minutes post dissociation. (Scale bars: 100 μm.)

Fig. 2.

Differences in capacities of gastrula cells for axis formation and cell-fate specification in the aggregates. (A–E) Aggregates made of oral halves of gastrulae develop into polyps. (F–J) Aggregates made of aboral halves of gastrulae develop into ciliated balls. (J) Confocal imaging shows that, outside, they have an ectodermal epithelial layer and that their inside is filled with numerous small cells. (K–N) In aggregates made of oral halves of wild-type gastrulae and aboral halves of gastrulae ubiquitously expressing lifeact-mOrange2, glowing cells are dispersed throughout the aggregate and can be observed both in aboral and oral positions of the polyp (yellow arrows in N). (O–R) In aggregates made of ectoderm of wild-type gastrulae and endoderm of gastrulae ubiquitously expressing lifeact-mOrange2, fluorescent cells migrate into the endoderm. (S–V) In aggregates made of aboral ectoderm of wild-type gastrulae and endoderm of gastrulae ubiquitously expressing lifeact-mOrange2, fluorescent cells migrate into the endoderm although the organizer cells are missing. (W–Z) In aggregates made of only endodermal cells, the cells become mesenchymal and migrate out of the aggregate. (A′–E′) Immediately after centrifugation, mCherry is not expressed in aggregates made of aboral ectoderm of endoRed gastrulae and blastopore lip ectoderm of the wild-type gastrulae (B′). Endodermal promoter-driven mCherry expression starts to be detectable in the internal cells of the aggregate from 28 hpd on (yellow arrows in C′). Glowing cells are then observed in the endoderm of the forming polyps (E′). (F′–J′) In aggregates made of oral halves of endoRed gastrulae, mCherry is continuously expressed in the endodermal cells. Sample size >30 in every experiment. dpd, days post dissociation; eR, endoRed; hpd, hours post dissociation; mOr, lifeact-mOrange2; mpd, minutes post dissociation; wt, wild type. Black bars on gastrulae denote the position of the cut. (Scale bars: J, 15 µm; all others, 100 μm.)

Fig. 3.

The summary of the fate of cells during normal development (A) and in aggregates (B).

Fig. 4.

The role of Wnt/β-catenin and BMP signaling during aggregate development. (A–C) Axis formation and endoderm segregation is rescued in 15 of 17 aggregates made from aboral halves of gastrulae, which were coinjected into a single blastomere at the eight-cell stage with plasmids coding for untagged Wnt1 and Wnt3 driven by the EF1α promoter and fluorescent tracer (glowing cells in B). (D–F) BMP2/4 knockdown results in the lack of morphologically distinct body axes in the aggregates. n = 32. (Scale bars: 100 µm.)