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Calcisponges have a ParaHox gene and dynamic expression of dispersed NK homeobox genes

Abstract

Sponges are simple animals with few cell types, but their genomes paradoxically contain a wide variety of developmental transcription factors1,2,3,4, including homeobox genes belonging to the Antennapedia (ANTP) class5,6, which in bilaterians encompass Hox, ParaHox and NK genes. In the genome of the demosponge Amphimedon queenslandica, no Hox or ParaHox genes are present, but NK genes are linked in a tight cluster similar to the NK clusters of bilaterians5. It has been proposed that Hox and ParaHox genes originated from NK cluster genes after divergence of sponges from the lineage leading to cnidarians and bilaterians5,7. On the other hand, synteny analysis lends support to the notion that the absence of Hox and ParaHox genes in Amphimedon is a result of secondary loss (the ghost locus hypothesis)8. Here we analysed complete suites of ANTP-class homeoboxes in two calcareous sponges, Sycon ciliatum and Leucosolenia complicata. Our phylogenetic analyses demonstrate that these calcisponges possess orthologues of bilaterian NK genes (Hex, Hmx and Msx), a varying number of additional NK genes and one ParaHox gene, Cdx. Despite the generation of scaffolds spanning multiple genes, we find no evidence of clustering of Sycon NK genes. All Sycon ANTP-class genes are developmentally expressed, with patterns suggesting their involvement in cell type specification in embryos and adults, metamorphosis and body plan patterning. These results demonstrate that ParaHox genes predate the origin of sponges, thus confirming the ghost locus hypothesis8, and highlight the need to analyse the genomes of multiple sponge lineages to obtain a complete picture of the ancestral composition of the first animal genome.

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Figure 1: Phylogenetic tree of the ANTP-class homeodomains.
Figure 2: SciCdx synteny and ghost loci simulations.
Figure 3: Expression of S. ciliatum ANTP-class genes.

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Accession codes

Primary accessions

ArrayExpress

European Nucleotide Archive

Data deposits

Genome assembly of S. ciliatum and the coding sequences and their translations from transcriptome assemblies of S. ciliatum and L. complicata used in this study can be accessed through http://compagen.zoologie.uni-kiel.de/ and are also deposited at http://dx.doi.org/10.5061/dryad.tn0f3. RNA-seq data have been deposited in ArrayExpress under accession numbers E-MTAB-2430, E-MTAB-2431 and E-MTAB-2890, and the cloned coding sequences of S. ciliatum ANTP-class genes have been deposited in the European Nucleotide Archive under accession numbers LN609546 to LN609553.

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Acknowledgements

This study was funded by the Sars Centre core budget to M. Adamska. Sequencing was performed at the Norwegian High Throughput Sequencing Centre funded by the Norwegian Research Council. O.M.R. and D.E.K.F. acknowledge support from the BBSRC and the School of Biology, University of St Andrews. We thank B. Bergum for collecting samples in the 2011 season and R. Holdhus from the Genomics Core Facility at the University of Bergen for help with DNA shearing.

Author information

Authors and Affiliations

Authors

Contributions

S.A.V.F. carried out gene identification and cloning, analysed gene expression by in situ hybridization, and participated in phylogenetic analyses and manuscript writing. M. Adamski performed sequence assembly, annotation, quantification of gene expression and participated in sample collection, phylogenetic analyses and manuscript writing. O.M.R. performed the synteny analyses, participated in phylogenetic analyses, manuscript writing and design of the research approach for synteny and phylogenetic analyses. S.L. isolated samples for sequencing of genomes, generated Mate Pair libraries, and participated in sample collection. J.L. generated samples for sequencing of S. ciliatum metamorphosis stages. D.E.K.F. participated in design of the research approach for synteny and phylogenetic analyses and writing of the manuscript. M. Adamska conceived the study, and participated in data analysis, sample collection and writing of the manuscript.

Corresponding author

Correspondence to Maja Adamska.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Phylogenetic tree of the ANTP-class homeodomains including representative bilaterian and non-bilaterian sequences.

A neighbour-joining tree using the JTT+G (0.5) (1,000 bootstraps) model of protein evolution is displayed. A combination of three support values obtained for three phylogenetic methods is shown: black value is the bootstrap support from the neighbour-joining method, blue value is the bootstrap support from the maximum-likelihood method (LG+G 0.5), and red value is posterior probability from Bayesian analysis (LG+G 0.5). Bootstrap support values below 10% and posterior probability values below 0.5 are not shown except for associations of calcisponge sequences. The root was determined by using selected PRD-class homeodomains as an outgroup. Aqu, A. queenslandica (Porifera/demosponges); Bfl, B. floridae (Chordata); Lco, L. complicata; Mle, M. leidyi (Ctenophora); Nve, N. vectensis (Cnidaria); Sci, S. ciliatum (Porifera/calcisponges); Tad, Trichoplax adhaerens (Placozoa); Tca, T. castaneum (Arthropoda). Scale bar indicates the number of amino acid substitutions per site.

Source data

Extended Data Figure 2 Variability of the YIT/YIS homeodomain motif within the Cdx/Cad, En and Dbx families in bilaterians, cnidarians, a placozoan and sponges.

Ame, Apis mellifera; Aqu, A. queenslandica; Bfl, B. floridae; Cte, Capitella teleta; Dme, Drosophila melanogaster; Dre, Danio rerio; Edi, Eleutheria dichotoma; Gga, Gallus gallus; Hsa, Homo sapiens; Lgi, Lottia gigantea; Mmu, Mus musculus; Nv, Nereis virens; Nve, N. vectensis; Pdu, Platynereis dumerilii; Pfl, Ptychodera flava; Tad, T. adhaerens; Tca, T. castaneum; Xla, Xenopus laevis.

Extended Data Figure 3 Phylogenetic tree including ANTP-class homeodomain families represented in sponges and two additional families characterized by the presence of the YIT motif but excluding divergent A. queenslandica sequences.

Neighbour-joining (JTT, 1,000) bootstrap support values are in black, maximum-likelihood (LG+G 0.4, 1,000 replicates) bootstrap support values are in blue and Bayesian (LG+G 0.4) posterior probability values are in red. Only bootstrap support values equal to or above 500 are shown. All families except Cdx are collapsed for clarity. Ame, A. mellifera; Aqu, A. queenslandica; Bfl, B. floridae; Cte, C. teleta; Dme, D. melanogaster; Dre, D. rerio; Edi, E. dichotoma; Gga, G. gallus; Hsa, H. sapiens; Lgi, L. gigantea; Mmu, M. musculus; Nv, N. virens; Nve, N. vectensis; Pdu, P. dumerilii; Pfl, P. flava; Tad, T. adhaerens; Tca, T. castaneum; Xla, X. laevis. Scale bar indicates the number of amino acid substitutions per site.

Source data

Extended Data Figure 4 Phylogenetic tree including ANTP-class homeodomain families represented in sponges and three additional families characterized by the presence of YIT/YIS motifs but excluding some divergent A. queenslandica sequences.

Neighbour-joining (JTT, 1,000 replicates) bootstrap support values are in black, maximum-likelihood (LG+G 0.4, 1,000 replicates) bootstrap support values are in blue and Bayesian (LG+G 0.4) posterior probability values are in red. Only bootstrap support values equal to or above 500 are shown. All subfamilies except Cdx are collapsed for clarity. Ame, A. mellifera; Aqu, A. queenslandica; Bfl, B. floridae; Cte, C. teleta; Dme, D. melanogaster; Dre, D. rerio; Edi, E. dichotoma; Gga, G. gallus; Hsa, H. sapiens; Lgi, L. gigantea; Mmu, M. musculus; Nv, N. virens; Nve, N. vectensis; Pdu, P. dumerilii; Pfl, P. flava; Tad, T. adhaerens; Tca, T. castaneum; Xla, X. laevis. Scale bar indicates the number of amino acid substitutions per site.

Source data

Extended Data Figure 5 Phylogenetic tree including ANTP-class homeodomain families represented in sponges and three additional families characterized by the presence of YIT/YIS motifs.

Neighbour-joining (JTT, 1,000 replicates) bootstrap support values are in black, maximum-likelihood (LG+G 0.4, 1,000 replicates) bootstrap support values are in blue and Bayesian (LG+G 0.4) posterior probability values are in red. Only bootstrap support values equal to or above 500 are shown. All families except Cdx are collapsed for clarity. Ame, A. mellifera; Aqu, A. queenslandica; Bfl, B. floridae; Cte, C. teleta; Dme, D. melanogaster; Dre, D. rerio; Edi, E. dichotoma; Gga, G. gallus; Hsa, H. sapiens; Lgi, L. gigantea; Mmu, M. musculus; Nv, N. virens; Nve, N. vectensis; Pdu, P. dumerilii; Pfl, P. flava; Tad, T. adhaerens; Tca, T. castaneum; Xla, X. laevis. Scale bar indicates the number of amino acid substitutions per site.

Source data

Extended Data Figure 6 S. ciliatum scaffolds containing NK genes and A. queenslandica scaffold containing cluster of NK genes.

S. ciliatum NK genes are indicated in blue and A. queenslandica NK genes are indicated in green (modified with permission from ref. 6). Annotation of the neighbouring genes (genes within 50 kb from the NK gene) in S. ciliatum was performed using BLASTp searches against the RefSeq database.

Extended Data Figure 7 Additional expression patterns of ANTP-class homeobox genes in embryonic development and during metamorphosis.

ag, All of the investigated genes (except Hex, data not shown) are expressed in oocytes. The expression of SciNKA is detectable in all blastomeres of the cleavage-stage embryos, but the transcripts are concentrated in the corner-most cytoplasm, which becomes gradually partitioned to the cross cells (arrows). lq, This subcellular localization of cross-cell-enriched transcripts is also observed for SciNanos, expression of which, similarly to SciNKA, becomes ultimately restricted to cross cells and macromeres in pre-inversion-stage embryos. rv, In metamorphosing post-larvae, SciNKA is expressed in the cells of the outer layer (r), SciNKB and SciNKG in (possibly non-overlapping) fractions of cells in the inner cell mass (s, t); SciHex is weakly expressed throughout the inner cell mass (u), and SciNKC (v) and NKD (data not shown) are not detectable in the juveniles. Scale bars: 10 μm (aq); 25 μm (rv).

Extended Data Figure 8 Samples used for quantification of expression.

af, Metamorphosis in S. ciliatum, with stages based on those described previously20 with modifications. Stage I, approximately 12 h after settlement: large flat cells derived from larval macromeres envelop the inner cell mass composed of former micromeres (a). Stage II, approximately 24 h after settlement: single-axis spicules (monaxons) are produced by sclerocytes, which have differentiated from the inner cell mass cells (b). Stage III, 2–3 days after settlement: choanocytes that have differentiated from the inner cells mass cells form a single internal chamber (c). Stage IV, approximately 4 days after settlement: osculum (exhalant opening) forms at the apical end of the spherical juvenile; first tri-radial spicules become apparent (d). Stage V, approximately 10 days after settlement: the juvenile is elongated along the apical–basal axis, long straight spicules form a crown around the osculum (e). Young syconoid sponges, approximately 8 weeks after settlement (f). ae, Photographs of live specimens in culture; photographs ad are top (apical) views, cartoon representations of sections and photograph e are side views. Scale bars: 100 μm (ae); 1 mm (f). g, Details of replicates used for the analysis. Several hundred juveniles were used in each sample. h, Plot demonstrating the results of principal component analysis of the metamorphosis series and axial dissection series of non-reproductive adults calculated according to the previously described method22 and using information about the top 500 differentially expressed genes as default parameters. Metamorphosis stages and parts of sponges are colour coded, with the ovals added manually for easier visualization of similarities and differences between the samples. Progress of development, starting from freshly released larvae and until the emergence of adult, but not yet reproductive sponges, is indicated by arrows. Note similarities of samples within replicates and with neighbouring stages of the metamorphosis series, and distinctiveness of the top (apical) samples from the basal and middle samples of the adults. i, Heat-map representation of sample-to-sample distances among all samples used in this study, calculated according to the previously described method22 and based on expression of all coding genes in S. ciliatum (approximately 18,000 sequences). Note that replicates and neighbouring stages group together, as indicated by highlighting.

Extended Data Figure 9 Heat-map representation of expression profiles.

As demonstrated in Fig. 3b but with data from individual libraries presented separately.

Extended Data Table 1 Quantification of differences in expression levels between top, middle and bottom parts of non-reproductive adult specimens of S. ciliatum

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Fortunato, S., Adamski, M., Ramos, O. et al. Calcisponges have a ParaHox gene and dynamic expression of dispersed NK homeobox genes. Nature 514, 620–623 (2014). https://doi.org/10.1038/nature13881

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