Figure 1.
Hydractinia echinata colony structure.
(A–E) Early colony growth, viewed from above. m; mouth of primary polyp. t; tentacle. s; stolon. b; newly budding polyp. (A) Developing primary polyp, one day after induction of metamorphosis, stolon and tentacles are starting to develop. (B) Polyp 2 days after induction of metamorphosis, stolons have grown longer. (C) Colony 5 days after induction of metamorphosis, new feeding polyps have budded from the stolons. (D) Colony 7 days after induction of metamorphosis, colony has continued to grow by stolon extension and budding of new polyps. (E) Colony 10 days after induction of metamorphosis consists of numerous polyps at various stages of development and an increasing stolon network. (F, G) Hermit crabs whose shells are encrusted by mature Hydractinia colonies, consisting of thousands of polyps and extensive stolon networks. (H) Schematic illustration of colony structure. Feeding polyps (gastrozooid) and sexual polyps (gonozooid) are linked by a system of gastrovascular canals called stolons. Stolons grow along the substrate budding new polyps at regular intervals. Scale bars (A) 200 µm, (B, C) 500 µm and (D, E) 1 mm.
Figure 2.
Alignment of the partial Hydratinia echinata COUP-TF coding sequence with its closest human match, COUP transcription factor 2 isoform a, accession number NP_066285.
The light grey highlighted sequence corresponds to the conserved DNA-binding domain and the dark grey to the conserved ligand binding domain of COUP-TFs.
Figure 3.
COUP-TF expression during development.
(A) Early embryo: no expression is detectable by in situ hybridisation in the first 24 hours of embryonic development. (B) A spot of expression appears in pre-planula (approximately 24 hours post-fertilisation). (C, D) Higher magnification of pre-planula expression spots. (C) A single spot of expressing cells appears at one pole of the pre-planula only. (D) Nematocytes are visible to the right of the expression site (arrow). (E) No expression is detectable in planula larvae. (F) Putative COUP-TF expressing nerve cells are detectable in primary polyps. Scale bars (A, B & E) 200 µm, (C & F) 50 µm and (D) 20 µm.
Figure 4.
COUP-TF mRNA expression during allogeneic colony rejection.
(A) Three young colonies whose stolons have grown into contact. Original primary polyps of each colony are numbered. Arrows indicate colony stolon contact sites and corresponding COUP-TF expression. (B) Close-up of contact sites between colony 1 and 2 (arrows) and COUP-TF expression. Expression is strongest in contact regions, and decreases away from the contact zone in a graded manner. The upper contacting stolon of colony 1 is clearly hyperplastic, being swollen and overgrowing its competitor. (C–E) Higher magnification views of competing stolons. Nematocyte build up (differentiation to discharge). (C) Increased expression corresponds to nematocyte differentiation. Mature nematocytes are visibly swelling the upper stolon tip (arrow). Nests of differentiating COUP-TF expressing nematoblasts are also visible. (D) A later stage contact site. The right stolon has overgrown the left one. Differentiated nematocytes of left stolon (arrow) are lining up in a battery in preparation for discharge. (E) Close-up of the lower contact site in image B. Arrow indicates large numbers of nematocytes. Scale bars (A & B) 500 µm, (C–E) 20 µm.
Figure 5.
COUP-TF expression during stolon self contact in a single primary polyp, one day after metamorphosis.
(A) The left stolon has grown into contact with the right stolon. Expression is visible at the contact site of the left stolon and at the tip of the right stolon, adjacent to the contact site (arrows). (B) Higher magnification view of contact site. White arrow indicates putative nematoblast, black arrow indicates nematocyte. Scale bars (A) 50 µm and (B) 20 µm.
Figure 6.
Proliferation, nematoblast differentiation and altered gene expression in response to Wnt activation.
(A, B) Wnt induced outgrowth. (A) Bright field view of outgrowth. (B) Wnt induced outgrowths consist of numerous proliferating cells, as shown by BrdU labelling of proliferating nuclei (green fluorescence). (C) Nematocytes (black arrows) and nematoblast nests (white arrow) are visible in outgrowth of LiCl-treated polyp. (D) Expression levels of COUP-TF and known Wnt targets Tcf, Wnt3 & Brachyury analysed by qPCR. Expression levels normalised to GAPDH. The relative quantity (RQ) of expression of each gene in control larvae were compared to expression levels in the 18.5 hour LiCl-treated pre-planula. Scale bars 50 µm.