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First published online April 25, 2008
doi: 10.1242/10.1242/dev.020784

Development 135, 1761-1769 (2008)
Published by The Company of Biologists 2008
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FGF signalling controls formation of the apical sensory organ in the cnidarian Nematostella vectensis

Fabian Rentzsch1,*, Jens H. Fritzenwanker1, Corinna B. Scholz2 and Ulrich Technau1,3,*

1 Sars Centre for Marine Molecular Biology, University of Bergen, N-5008 Bergen, Norway.
2 Miltenyi Biotec, Friedrich-Ebert-Str. 68, 51429 Bergisch-Gladbach, Germany.
3 Faculty of Life Sciences, University of Vienna, Althanstrasse 14, 1090 Wien, Austria.


Figure 1
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  		Fig. 1. Phylogenetic position and embryonic development of the cnidarian Nematostella. (A) Simplified phylogenetic tree according to published data (Bourlat et al., 2006Go; Delsuc et al., 2006Go), showing that Cnidaria is a sister phylum to Bilateria. The presence of apical organs is indicated by asterisks. (B-E) Overview of Nematostella embryonic development: (B) gastrula, (C) planula, (D) metamorphosis and (E) primary polyp. The blastopore/oral pole is marked by asterisks. at, apical tuft; bc, blastocoel; ect, ectoderm; end, endoderm; gc, gastric cavity; mes, mesentery; pha, pharynx; ten, tentacle. Note the short cilia covering the ectoderm in C.

 

Figure 2
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  		Fig. 2. Expression pattern of Nematostella FGFa, FGFb and FGFRa. (A) Temporal expression profile determined by RT-PCR; NvFGFa1 is not expressed maternally. ooc, unfertilised eggs; blast, blastula (12 hpf); gastr, gastrulation (24 hpf); e.plan, early planula (48 hpf); l.plan, late planula (96 hpf); pp, primary polyp (7 dpf). (B-S) Spatial expression pattern determined by in situ hybridisation. Lateral views, blastoporus/mouth to the right, apical pole and swimming direction of planula larvae to the left. (B-E) NvFGFa2, (F-I) NvFGFa1, (J-M) NvFGFRa. (B,F,J) Gastrula, (C,G,K) early planula, (D,H,L) late planula, (E,I,M) primary polyp. (N-S) Fluorescent double in situ hybridisation at late gastrula stage with indicated probes. The NvFGFa1 and NvFGFa2 expression domains are identical; the NvFGFRa expression domain is wider than that of NvFGFa2 (and NvFGFa1).

 

Figure 3
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  		Fig. 3. Opposite effects of NvFGFa1 and NvFGFa2 morpholinos on apical organ formation. (A) Autoradiograph of transcription-translation reactions in the presence of 35S-labeled methionine. Plasmids and MOs added to the reaction are indicated above the lanes. Tested MOs only inhibit translation of the corresponding transcripts. (B-D) Scanning electron microscopy of 4-day-old planulae injected with the MOs indicated. The aboral pole is marked by asterisks; arrows in B point to the apical tuft. NvFGFa1 MO leads to loss, NvFGFa2 MO to expansion of the apical organ. (E-G) Visualisation of the ciliary tuft by anti-acetylated tubulin antibody staining of 48-hpf planulae injected with MOs indicated. Lateral views, aboral pole to the left. The NvFGFa2 MO causes premature formation of an expanded apical tuft. (H,I) Animals with a differentiated apical organ treated from 72 hpf to 120 hpf with 0.1% DMSO or 20 µM SU5402/0.1% DMSO. SU5402 causes loss of the apical organ.

 

Figure 4
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  		Fig. 4. NvFGF signalling regulates patterning within the aboral region. (A-P) In situ hybridisation of 48 hpf planulae. Lateral views, aboral pole to the left; probes are indicated above the panels, injected morpholinos to the left. Knockdown of NvFGFa1 and NvFGFRa causes specific loss of the apical organ marker, knockdown of NvFGFa2 results in expansion of the apical tuft. Displayed expression patterns were obtained in: (A) 38/42, (B) 31/38, (C) 21/23, (D) 36/36, (E) 48/53, (F) 58/65, (G) 14/18, (H) 29/31, (I) 39/42, (J) 24/33, (K) 22/24, (L) 46/52, (M) 29/34, (N) 27/29, (O) 26/36 and (P) 40/42 embryos.

 

Figure 5
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  		Fig. 5. NvFGFRa signalling is required for the expression of FGF pathway components and expansion of the apical organ in NvFGFa2 morphants. (A-O) In situ hybridisation of 48-hpf planulae. (A-L) Lateral views, aboral pole to the left; injected morpholinos are indicated to the left of the panels, probes above. NvFGFa1 and NvFGFRa are required for their own transcription. (M-O) In situ hybridisation of 48-hpf planulae probed with NvFGFa1. (M) NvFGFa2 MO-injected planula; (N) planula co-injected with NvFGFa2 and NvFGFRa MOs; (O) planula injected with the NvFGFa2 MO and treated with 20 µM SU5402 from 20 hpf on.

 

Figure 6
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  		Fig. 6. MAP kinase ERK acts downstream of NvFGF signalling in apical organ formation. (A) Western blot to detect levels of phosporylated and total ERK. (B-G) In situ hybridisation of 48-hpf planulae. Lateral views, aboral pole to the left. (B-D) Embryos treated with 10 µM UO126 from 20-48 hpf; probes are indicated in the lower left corner. (E-G) In situ hybridisation with a NvFGFa1 probe; treatments are indicated in the upper right corner, inhibitor treatment was carried out from 20-48 hpf.

 

Figure 7
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  		Fig. 7. Metamorphosis, but not swimming, is affected by the manipulation of FGF signalling. (A,B) Frames from movies of individual 4-dpf planula larvae injected with the morpholino indicated. Total width of the frame is ~3 mm; time interval between first and last frame was ~1.2 seconds in A and ~1.7 seconds in B. (C) Illustration of the effect of the indicated morpholinos on metamorphosis at 12 dpf. Uninjected, n=156; control (ctr) MO, n=133; NvFGFa1 MO, n=98; NvFGFa2 MO, n=85. (D-F) Micrographs showing live animals at 12 dpf; injected morpholinos are indicated. All animals injected with the NvFGFa1 MO are still planula larvae, most of the NvFGFa2 MO-injected animals are primary polyps. (G) Effect of SU5402 treatment on metamorphosis. Late planula stage embryos (5 dpf) were continuously incubated with DMSO or 20 µM SU5402 until 9 dpf. DMSO, n=159; SU5402 treatment, n=165. (H) Schematic model of the autoregulation of FGF pathway components in Nematostella apical organ development.

 

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