Sinigaglia C et al. (2015), Molecular characterization of the apical organ ...

ECB-ART-43754

Dev Biol 2015 Feb 01;3981:120-33. doi: 10.1016/j.ydbio.2014.11.019.

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Molecular characterization of the apical organ of the anthozoan Nematostella vectensis.

Sinigaglia C , Busengdal H , Lerner A , Oliveri P , Rentzsch F .

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Apical organs are sensory structures present in many marine invertebrate larvae where they are considered to be involved in their settlement, metamorphosis and locomotion. In bilaterians they are characterised by a tuft of long cilia and receptor cells and they are associated with groups of neurons, but their relatively low morphological complexity and dispersed phylogenetic distribution have left their evolutionary relationship unresolved. Moreover, since apical organs are not present in the standard model organisms, their development and function are not well understood. To provide a foundation for a better understanding of this structure we have characterised the molecular composition of the apical organ of the sea anemone Nematostella vectensis. In a microarray-based comparison of the gene expression profiles of planulae with either a wildtype or an experimentally expanded apical organ, we identified 78 evolutionarily conserved genes, which are predominantly or specifically expressed in the apical organ of Nematostella. This gene set comprises signalling molecules, transcription factors, structural and metabolic genes. The majority of these genes, including several conserved, but previously uncharacterized ones, are potentially involved in different aspects of the development or function of the long cilia of the apical organ. To demonstrate the utility of this gene set for comparative analyses, we further analysed the expression of a subset of previously uncharacterized putative orthologs in sea urchin larvae and detected expression for twelve out of eighteen of them in the apical domain. Our study provides a molecular characterization of the apical organ of Nematostella and represents an informative tool for future studies addressing the development, function and evolutionary history of apical organ cells.

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Species referenced: Echinodermata
Genes referenced: cep89 fgf LOC100887844 LOC115919910 LOC581706 LOC583082 LOC764881 pole tubgcp2

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	Fig. S1. The AO gene set is enriched for ciliary genes. The percentage of BLAST hits against the custom ciliary database (Supplementary File 1) is shown for the AO dataset (red square) and for 10 random Nematostella datasets. The values of the random datasets are lower than the value for the AO dataset.
	Fig. S2-1. Expression patterns ofNematostellaapical organ genes. In situ hybridizations at gastrula and planula stages for the genes identified in this study. All images are lateral views with aboral pole to the left. The insets are: (D) aboral view, (DA, AB, VA) higher magnifications. Note: some pictures are also shown in the main figures. Scale bar=100 µm
	Fig. S2-2.
	Fig. S2-3.
	Fig. S2-4.
	Fig. S3-1. Expression patterns of putative sea urchin apical organ genes. In situ hybridizations for the sea urchin genes most similar to the indicated Nematostella genes (by reciprocal BLAST). For each gene from left to right: lateral views of mid-gastrula, late gastrula (with apical pole to the top) and pluteus (oral side indicated by asterisk) stages, respectively. For SPU_007284 an apical pole view is shown in the middle.
	Fig. S3-3.
	Fig. 1. Nematostella vectensis as a model for the identification of apical organ genes (A) Apical organs are found in the developing stages of diverse invertebrates: Cnidaria, Lophotrochozoa, Echinodermata and Hemichordata (red asterisks). (B) N. vectensis is a representative of the Anthozoa, the sister group to all other cnidarians. Anthozoa is the only class of Cnidaria where apical organs with long cilia have been described. Embryonic development comprehends a swimming stage, the planula larva, which bears a tuft of long cilia at the aboral pole. The apical organ disappears after about one week of development, when the larva settles and develops the tentacles. (C) Experimental design for the microarray analysis. Apical organs (red tuft in the drawing) were manipulated by injecting antisense morpholinos directed against two FGF ligands with opposite functions: NvFGFa1 MO produces larvae lacking an apical organ, while NvFGFa2 MO leads to larvae with an expanded organ. The samples were preserved until total RNA was extracted from the different conditions (including the control wild type). The transcription profiles of the three phenotypes were compared in a microarray analysis.
	Fig. 2. Examples of new apical organ genes related to cilia development and function A BLAST search against a custom dataset of cilia-related genes allowed the identification of 52 putative ciliary genes. These included genes that are related to general aspects of ciliogenesis (A–E), like a β-tubulin gene (A), and genes which might give a clue about the nature of the apical organ cilia, being related to provision of energy (F and G), transduction of signals (H and I) and cell–cell interactions (J and K). Interesting is also a number of conserved but uncharacterized genes, here identified with the putative orthologous human gene (L–N). The embryos displayed are all at planula stage, the aboral pole is to the left. Each gene is identified by the assigned ID and a name, either attributed by the genome annotation, or obtained through a BLAST search. Scale bar=100 µm.
	Fig. 3. Selected non-ciliary apical organ genes The genes which did not produce any hit against the custom dataset of cilia-related genes are considered as “non-ciliary”. (A-D) Two genes involved in the Wnt signalling pathway were recovered, the Wnt receptor NvFz5/8 (A and B) and the secreted protein NvSRFP1 (C and D). Both genes are expressed in a broader aboral domain at gastrula stage (A and C), then restrict to the most aboral pole of the planula. Aboral endodermal expression is also visible at this stage. (E and F) NvFGF1E is expressed in a relatively small aboral domain at gastrula stage and in a subset of apical organ cells at planula stage. (G and H) A gene orthologous to the E. coli TauD gene was also found. The gene is involved in the catabolism of taurine, an amino acid that has been implicated in metamorphosis. NvTauD is expressed in a ring within the apical organ domain (see aboral view of the planula, in E), demonstrating the existence of different sub-domains within the apical organ domain. The aboral pole is to the left, each gene is identified by the assigned ID and a name, either attributed by the genome annotation, or obtained through a BLAST search. Scale bar=100 µm.
	Fig. 4. Apical organ genes with additional cell-type specific expression (A) The O-linked-mannose beta-1,2-N-acetylglucosaminyltransferase gene NvPOMGnT1-like (ao51) is expressed in scattered cells in the ectoderm and in the pharynx. (B) NvCellulase positive cells (ao132) are enriched in a broad domain in the aboral ectoderm and in the pharynx, scattered ectodermal cells are also present. (C) The uncharacterized gene identified by the ID number 239479 (ao81) is expressed in few ectodermal cells in the aboral half of the larvae. (D) The coiled-coil domain containing gene NvCCDC81 (ao155) is detected in individual cells throughout the entire ectoderm (picture focuses on the surface). The embryos displayed are all at planula stage, the aboral pole is to the left. Scale bar=100 µm.
	Fig. 5. Expression patterns displayed by AO homologous genes in the purple sea urchin. Different stages are displayed, to exemplify the expression dynamics: genes could be either restricted to the apical organ (e.g. A–D) or be broadly expressed in the early stages and then restricted to the apical organ (E–H, K, L), or display additional domains like in the ciliary bands (I and J, arrow indicates ciliary band). Pictures are lateral views (except B), oriented with the blastopore at the bottom. Each gene is identified with the name obtained from the sea urchin database at www.spbase.org; the corresponding Nematostella gene is also reported.

References [+] :

Altenburger, Neuromuscular development in Novocrania anomala: evidence for the presence of serotonin and a spiralian-like apical organ in lecithotrophic brachiopod larvae. 2010, Pubmed