Figure 1.
Amphimedon queenslandica life cycle and embryonic development.
Top panels, live specimens. (A) Adult animal. (B) Swimming larva. (C) Larva undergoing settlement with anterior part flattened on substrate. (D) Postlarva 24 hours post settlement (ps). (E) A three week old juvenile. (F) Sliced brood chamber showing developing embryos of different stages. Bottom panel, benzyl alcohol/benzyl benzoate cleared whole mount developmental stages. (G) After a series of chaotic and asymmetric cell divisions there is a population of unevenly-sized and irregular macromeres and a population of tiny micromeres that are located on the surface and between the macromeres. Micromeres are too small to be seen in this micrograph. (H) A solid blastula is formed. (I) Gastrulation results in a bilayered embryo with the outer layer thicker at the future posterior pole (top); darker pigment cells are distributed throughout the outer layer. (J) Pigment cells migrate through the outer layer towards the posterior pole and coalesce to form a pigment spot. (K–L) The cells of the pigment spot reverse direction and migrate outwards to form a ring. (L, M) Swimming larva with a posterior pigment ring, inner cell mass, ciliated epithelial layer and a subepithelial middle layer. See 21 and 22 for a more detailed description of development and larval cell types. Scale bar is 100 µm on all images except (A) where it is 1 cm. ap–anterior pole, pp–posterior pole.
Figure 2.
Expression of Wnt in Amphimedon embryos.
Top panel, whole mount in situ hybridizations; bottom panel, sectioned in situ hybridisations. (A) During cleavage, Wnt is expressed in micromeres distributed throughout the early embryo. (B) At the blastula stage, Wnt-expressing cells are predominantly localised interiorly. (C, D) Asymmetric localisation of Wnt-expressing cells occurs early in gastrulation, initially inside of the embryo. (E, F) As the gastrulation progresses, Wnt-positive cells are evident in the outer layer in the posterior pole. (G, H) At the spot stage, Wnt expression overlaps with the pigment spot and extends beyond it. (I) This expression pattern is maintained during ring formation. (J) Expression of Wnt continues in the posterior pole in swimming larvae. Scale bar is 100 µm. ap, anterior pole; pp, posterior pole.
Figure 3.
Expression of TGF-β in Amphimedon embryos.
(A) TGF-β expressing micromeres are distributed uniformly during cleavage. (B) At the blastula stage, TGF-β-positive cells are more prominent in the outer layer. (C, D) During gastrulation, TGF-β expression is restricted to the outer layer, with two stronger domains at the anterior and posterior poles. (E, F) As the pigment cells migrate to the posterior pole, TGF-β expression disappears from the posterior pole leaving the area just outside the pigment spot clearly devoid of TGF-β transcripts. A weak TGF-β expression domain persists in the very center of the spot. (F, G) The anterior pole expression remains strong at the spot and ring stages. (H) As the pigment cells begin their outward migration, TGF-β expression is strong inside of the forming ring and in the outer layer except of the pigment ring itself and area just outside of it. (I, J) In the later ring, TGF-β expression clears from the center of the ring, but persists in the inner rim of the ring. (J) The embryonic expression gradually fades at late ring stages, and expression in some cells of the follicle layer on the surface of the embryo becomes more apparent. Scale bar, 100 µm.
Figure 4.
Evolution of Wnt and TGF-β gradients in early metazoans.
Metazoan tree of life showing relationships between sponges, cnidarians and bilaterians, and a closely-related sister taxon (choanoflagellates). Major evolutionary transitions in Wnt (blue) and TGF-β (red) gradients during embryonic development. Wnt and TGF-β genes arose early in metazoan evolution, after the choanoflagellate lineage diverged from the metazoan ancestor. These genes encoded ligands that originally formed gradients along one embryonic axis, as observed in modern sponges. The TGF-β gradient shifted to a perpendicular position in relation to the Wnt gradient in the lineage leading to the last common ancestor of contemporary eumetazoans, contribution to evolution of a second body axis.