Comparative Genomics and Transcriptomics in Placozoa

We have been sequencing and are currently analyzing several genomes and transcriptomes of placozoan species. We expect valuable insights into the phylogeny, genetic diversity and mechanisms of selection of an entire phylum. Placozoa have become an emerging model system for evo-devo research because of their simple bauplan and a basal position in the tree of life. We propose that placozoans are also well suited to become a model phylum in marine comparative genomics and ocean climate studies because: 1. they are abundant throughout the oceans in benthic communities 2. different lineages differ in their geographical distribution and associated tolerance for environmental factors 3. their compact genome is comparatively time and cost efficiently to sequence 4. assumed passive dispersal along oceanic currents allows to study effects of migration and local adaptation.

genome projects placozoa

Placozoan genome or transcriptome sequencing projects in our group projected onto a 16S phylogenetic tree: A finished sequencing project of a placozoan 16S haplotype is marked with a green star. A sequencing project in progress is indicated by a yellow star. The coloration of the placozoan clades follows that of Eitel et al., 2013.

The Enigmatic Phylum Placozoa

The first placozoan species, Trichoplax adhaerens, was discovered by F.E. Schulze in 1883 [1]⁠ in aquarium samples. More members (species, yet undescribed) of this phylum have been found in shallow tropical, sub-tropical and temperate waters [2,3]⁠. They are irregular disc-shaped benthic animals of only a few millimeters which crawl over hard substrates. Placozoans exhibit the simplest morphology of all living metazoans, harboring only six somatic cell types [4,5]⁠ that are arranged in an upper epithelium, a lower epithelium and an intermediate layer (for review see [6]⁠). This simple bauplan and a supposed basal position of placozoans in the tree of life has led to many speculations regarding the origin of Metazoa [7]⁠. Consequently the Trichoplax mitochondrial [8]⁠ and nuclear [9]⁠ genomes have been among the first sequenced non-bilaterian genomes and support the phylum's basal position [10]⁠.

placo panel

(A) Schematic cross section of a placozoan. Placozoans possess two epithelia, a lower epithelium (le) facing the substrate and an upper epithelium (ue) facing the water. In between lie several layers of syncytial fiber cells (fc). Marginal cells (mc) are thought to represent pluripotent stem cells. The “shiny spheres” (ss) are lipid filled droplets in the upper epithelium probably used as a chemical defense mechanism (from Eitel et al., 2013). (B) Live placozoan on a rock grazing on algae. (C) Placozoan on a microscope slide.

A Case to Sequence the Phylum Placozoa

The fact that T. adhaerens has remained the only described species can mainly be attributed to the uniform morphology of the phylum and to the difficulty of observing placozoans in the field. This view changed when genetic studies by Voigt et al. and Eitel et al. [3,11,12]⁠ revealed substantial diversity within the phylum. Moreover, different lineages show remarkable differences in their geographical range and tolerance for environmental factors. For example, the cosmopolitan haplotype H2 has been found in tropical to temperate waters in all oceans, while the haplotype H3 seems to be restricted to the Caribbean. Currently the Placozoa consists of around 25 lineages, presently defined by their 16S haplotypes [3,12]⁠. While their genetic distances as well as physiological differences leave no doubt that they represent valid species, attempts for species descriptions have only been partially successful, again, mainly due to the simple morphology of placozoans [13]⁠.
To resolve the tabula rasa Placozoa and to assess the phylum’s genetic diversity, the genomes and transcriptomes of the major lineages are currently sequenced, a process which is greatly facilitated by the small (100Mb) genome size of placozoans. In the future we plan to establish a genotyping-by-sequencing approach [14] to study global population dynamics of placozoans.

References

[1] Schulze FE. Trichoplax adhaerens, nov. gen., nov. spec. Zool Anz 1883;6:92–7.
[2] Pearse VB, Voigt O. Field biology of placozoans (Trichoplax): distribution, diversity, biotic interactions. Integr Comp Biol 2007;47:677–92. doi:10.1093/icb/icm015.
[3] Eitel M, Osigus H-J, DeSalle R, Schierwater B. Global Diversity of the Placozoa. PLoS One 2013;8:e57131. doi:10.1371/journal.pone.0057131.
[4] Jakob W, Sagasser S, Dellaporta S, Holland P, Kuhn K, Schierwater B. The Trox-2 Hox/ParaHox gene of Trichoplax (Placozoa) marks an epithelial boundary. Dev Genes Evol 2004;214:170–5. doi:10.1007/s00427-004-0390-8.
[5] Smith CL, Varoqueaux F, Kittelmann M, Azzam RN, Cooper B, Winters CA, et al. Novel cell types, neurosecretory cells, and body plan of the early-diverging metazoan Trichoplax adhaerens. Curr Biol 2014;24:1565–72. doi:10.1016/j.cub.2014.05.046.
[6] Schierwater B. My favorite animal,Trichoplax adhaerens. BioEssays 2005;27:1294–302. doi:10.1002/bies.20320.
[7] DeSalle R, Schierwater B. Key transitions in animal evolution. Integr Comp Biol 2007;47:667–9. doi:10.1093/icb/icm042.
[8] Dellaporta SL, Xu A, Sagasser S, Jakob W, Moreno M a, Buss LW, et al. Mitochondrial genome of Trichoplax adhaerens supports placozoa as the basal lower metazoan phylum. Proc Natl Acad Sci U S A 2006;103:8751–6. doi:10.1073/pnas.0602076103.
[9] Srivastava M, Begovic E, Chapman J, Putnam NH, Hellsten U, Kawashima T, et al. The Trichoplax genome and the nature of placozoans. Nature 2008;454:955–60. doi:10.1038/nature07191.
[10] Schierwater B, Eitel M, Jakob W, Osigus H-J, Hadrys H, Dellaporta SL, et al. Concatenated Analysis Sheds Light on Early Metazoan Evolution and Fuels a Modern “Urmetazoon” Hypothesis. PLoS Biol 2009;7:e1000020. doi:10.1371/journal.pbio.1000020.
[11] Voigt O, Collins AG, Pearse VB, Pearse JS, Ender A, Hadrys H, et al. Placozoa -- no longer a phylum of one. Curr Biol 2004;14:R944-5. doi:10.1016/j.cub.2004.10.036.
[12] Eitel M, Schierwater B. The phylogeography of the Placozoa suggests a taxon-rich phylum in tropical and subtropical waters. Mol Ecol 2010;19:2315–27. doi:10.1111/j.1365-294X.2010.04617.x.
[13] Guidi L, Eitel M, Cesarini E, Schierwater B, Balsamo M. Ultrastructural analyses support different morphological lineages in the phylum placozoa Grell, 1971. J Morphol 2011;272:371–8. doi:10.1002/jmor.10922.
[14] Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES, et al. A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species. PLoS One 2011;6:e19379. doi:10.1371/journal.pone.0019379.

 

The tumor suppressor p53 plays a crucial role in apoptosis and cell cycle regulation and ensures a cell’s integrity [1, 2]. Endogenous p53 levels are regulated by the ubiquitin ligase Mdm2 [3]. Both p53 and Mdm2 have been shown to be conserved throughout the animal kingdom and orthologs of both genes have been found in the simplest animal, Trichoplax adhaerens [4, 5, 6]. Our first studies have shown that the interplay is crucial for the animals’ health and chemical interruption of the p53/Mdm2 interaction leads to abnormal phenotypes.

Roscovitine 15 800  Roscovitine 17 800

Artificially induced cancer and tumor-like growth in Trichoplax adhaerens.

We also seek to better understand the interplay between Myc and Max in Trichoplax. Myc is a proto-oncogene and its malfunction leads to an enhanced formation of tumors [7, 8] As a transcription factor belonging to a family of bHLH-Zip proteins, Myc interacts with Max and several other proteins to activate or repress downstream target genes [9, 10]. Myc and Max are conserved within the Metazoa [11, 12] and are of fundamental importance for cellular processes. Within the diploblasts, research on these transcription factors has mainly been conducted on the freshwater polyp Hydra [13, 14]. To elucidate their functions we use functional genetics combined with biochemical and biophysical approaches.

Alpha tubulin and DAPI staining in Trichoplax adhaerens.
The bar marks 50 µm.

References:
[1] Levine AJ, Oren M (2009) The first 30 years of p53: growing ever more
complex. Nat Rev Cancer 9(10):749–758

[2] Vousden KH, Prives C (2009) Blinded by the light: the growing complexity
of p53. Cell 137(3):413–431

[3] Manfredi JJ (2010) The Mdm2-p53 relationship evolves: Mdm2 swings both ways as an oncogene and a tumor suppressor. Genes Dev
24(15):1580–1589

[4] Lane DP, Cheok CF, Brown C, Madhumalar A, Ghadessy FJ, Verma C
(2010) Mdm2 and p53 are highly conserved from placozoans to man. Cell Cycle 9(3):540–547

[5] Srivastava M, Begovic E, Chapman J, Putnam NH, Hellsten U,
Kawashima T, Kuo A, Mitros T, Salamov A, Carpenter ML,
Signorovitch AY, Moreno MA, Kamm K, Grimwood J, Schmutz
J, Shapiro H, Grigoriev IV, Buss LW, Schierwater B, Dellaporta SL,
Rokhsar DS (2008) The Trichoplax genome and the nature of
placozoans. Nature 454(7207):955–960

[6] Chevallerie vd, K, Rolfes, S, Schierwater, B (2014) Inhibitors of the p53-Mdm2 interaction increase programmed cell death and produce abnormal phenotypes in the placozoon Trichoplax adhaerens (F.E. Schulze). Dev Genes Evol 224:79–85

[7] Meyer N, Penn LZ (2008) Reflecting on 25 years with MYC. Nat Rev Cancer 8, 976-990

[8] Sheiness D, Fanshier L, Bishop JM (1978) Identification of nucleotide sequences which may encode the oncogenic capacity of avian retrovirus MC29. J Virol 28, 600-610

[9] Grandori C, Eisenman RN (1997) Myc target genes. Trends Biochem Sci 22, 177-181

[10] Blackwell TK, Kretzner L, Blackwood EM, Eisenman RN, Weintraub H (1990) Sequence-specific DNA binding by the c-Myc protein. Science 250, 1149-1151

[11] Sebe-Pedros A, de Mendoza A, Lang BF, Degnan BM, Ruiz-Trillo I (2011) Unexpected repertoire of metazoan transcription factors in the unicellular holozoan Capsaspora owczarzaki. Mol Biol Evol 28, 1241-1254

[12] King N, Westbrook MJ, Young SL, Kuo A, Abedin M, Chapman J, Fairclough S, Hellsten U, Isogai Y, Letunic I, et al. (2008) The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans. Nature 451, 783-788

[13] Hartl M, Mitterstiller AM, Valovka T, Breuker K, Hobmayer B, Bister K (2010) Stem cell-specific activation of an ancestral myc protooncogene with conserved basic functions in the early metazoan Hydra. Proc Natl Acad Sci U S A. 2010 Mar 2; 107(9): 4051–4056
[14] Hartl M, Glasauer S, Valovka T, Breuker K, Hobmayer B, Bister K (2014) Hydra myc2, a unique pre-bilaterian member of the myc gene family, is activated in cell proliferation and gametogenesis. Biology Open (2014) 000, 1–11

 

  1. Cnidarian EvoDevo research

Our second focus in diploblast research is on the evolution and development of cnidarians, because, like placozoans, they represent a key transition in animal complexity - from a sac-like bauplan with an oral-aboral axis and only two germ layers to the oral-aboral axis and three germ layers in triploblasts. Our model system is the hydrozoan Eleutheria dichotoma which was the first cnidarian representative in which Hox-like genes were identified [1,2]⁠. Subsequent research on Eleutheria homeobox genes gave valuable insights about the evolution of the hox system and the developmental role of homeobox genes in cnidaria [3–5]⁠. In the future we plan to sequence the estimated 500 Mb genome of Eleutheria dichotoma because it would ideally complement the cnidarian genomes already sequenced or planned to sequence: Eleutheria not only represents a typical cnidarian in having both polyp and medusa life cycle stages, but it also has some distinguishing features like internal fertilization, a brood pouch where planula develop, a crawling medusa and the possession of ocelli.

eleu panel 800

(A1) Eleutheria dichotoma polyp (A2) Polyp budding a medusa at its base (B) Medusa of Eleutheria with bifurcated tentacles: lower (walking) branch with adhesive disk, upper branch with cnidocyst knob (C) Expression pattern of the Hox-like gene Cnox-3 around the manubrium of the medusa (C from Kamm et al., 2006).

In collaboration with Professor David Miller from the James Cook University, Townsville, Australia, we are also interested in the developmental signaling pathway of fibroblast growth factors in Cnidaria. In animals the FGF and FGF-related signaling pathway is important for cell-cell communication during development and it is now clear that these important genes emerged early during metazoan evolution [6]⁠. Recent findings about FGF signalling in Nematostella vectensis [7] and Hydra vulgaris [8]⁠ gave first clues about the evolution and function of FGF genes in cnidarians. While Hydra represents a highly diverged hydrozoan representative, molecular and functional data from Eleutheria and other cnidarians will certainly improve our knowledge about the FGF signaling pathway in early metazoan evolution.

 

[1]       Schierwater B, Murtha M, Dick M, Ruddle FH, Buss LW. Homeoboxes in cnidarians. J Exp Zool 1991;260:413–6. doi:10.1002/jez.1402600316.

[2]       Kuhn K, Streit B, Schierwater B. Homeobox Genes in the CnidarianEleutheria dichotoma:Evolutionary Implications for the Origin ofAntennapedia-Class (HOM/Hox) Genes. Mol Phylogenet Evol 1996;6:30–8. doi:10.1006/mpev.1996.0055.

[3]       Kamm K, Schierwater B, Jakob W, Dellaporta SL, Miller DJ. Axial patterning and diversification in the cnidaria predate the Hox system. Curr Biol 2006;16:920–6. doi:10.1016/j.cub.2006.03.036.

[4]       Jakob W, Schierwater B. Changing hydrozoan bauplans by silencing Hox-like genes. PLoS One 2007;2:e694. doi:10.1371/journal.pone.0000694.

[5]       Kamm K, Schierwater B. Ancient linkage of a POU class 6 and an anterior hox-like gene in cnidaria: implications for the evolution of homeobox genes. J Exp Zool Part B Mol Dev Evol 2007;308B:777–84. doi:10.1002/jez.b.21196.

[6]       Bertrand S, Iwema T, Escriva H. FGF signaling emerged concomitantly with the origin of Eumetazoans. Mol Biol Evol 2014;31:310–8. doi:10.1093/molbev/mst222.

[7]       Rentzsch F, Fritzenwanker JH, Scholz CB, Technau U. FGF signalling controls formation of the apical sensory organ in the cnidarian Nematostella vectensis. Development 2008;135:1761–9. doi:10.1242/dev.020784.

[8]       Lange E, Bertrand S, Holz O, Rebscher N, Hassel M. Dynamic expression of a Hydra FGF at boundaries and termini. Dev Genes Evol 2014;224:235–44. doi:10.1007/s00427-014-0480-1.

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