The origin of many of the defining features of animal body plans, such as symmetry, nervous system, and the mesoderm, remains shrouded in mystery because of major uncertainty regarding the emergence order of the early branching taxa: the sponge groups, ctenophores, placozoans, cnidarians, and bilaterians. The "phylogenomic" approach [1] has recently provided a robust picture for intrabilaterian relationships [2, 3] but not yet for more early branching metazoan clades. We have assembled a comprehensive 128 gene data set including newly generated sequence data from ctenophores, cnidarians, and all four main sponge groups. The resulting phylogeny yields two significant conclusions reviving old views that have been challenged in the molecular era: (1) that the sponges (Porifera) are monophyletic and not paraphyletic as repeatedly proposed [4-9], thus undermining the idea that ancestral metazoans had a sponge-like body plan; (2) that the most likely position for the ctenophores is together with the cnidarians in a "coelenterate" clade. The Porifera and the Placozoa branch basally with respect to a moderately supported "eumetazoan" clade containing the three taxa with nervous system and muscle cells (Cnidaria, Ctenophora, and Bilateria). This new phylogeny provides a stimulating framework for exploring the important changes that shaped the body plans of the early diverging phyla.

Striated muscles are present in bilaterian animals (e.g. vertebrates, insects, annelids) and some non-bilaterian eumetazoans (i.e. cnidarians and ctenophores). The striking ultrastructural similarity of striated muscles between these animal groups is thought to reflect a common evolutionary origin1, 2. Here we show that a muscle protein core set, including a Myosin type II Heavy Chain motor protein characteristic of striated muscles in vertebrates (MyHC-st), was already present in unicellular organisms before the origin of multicellular animals. Furthermore, myhc-st and myhc-non-muscle (myhc-nm) orthologues are expressed differentially in two sponges, compatible with the functional diversification of myhc paralogues before the origin of true muscles and the subsequent deployment of MyHC-st in fast-contracting smooth and striated muscle. Cnidarians and ctenophores possess myhc-st orthologues but lack crucial components of bilaterian striated muscles, such as troponin complex and titin genes, suggesting the convergent evolution of striated muscles. Consistently, jellyfish orthologues of a shared set of bilaterian z-disc proteins are not associated with striated muscles, but are instead expressed elsewhere or ubiquitously. The independent evolution of eumetazoan striated muscles through the addition of novel proteins to a pre-existing, ancestral contractile apparatus may serve as a paradigm for the evolution of complex animal cell types.

The germ cell lineage in Xenopus is specified by the inheritance of germ plasm, which originates within a distinct "mitochondrial cloud" (MC) in previtellogenic oocytes. Germ plasm contains localized RNAs implicated in germ cell development, including Xcat2 and Xdazl. To understand the mechanism of the early pathway through which RNAs localize to the MC, we applied live confocal imaging and photobleaching analysis to oocytes microinjected with fluorescent Xcat2 and Xdazl RNA constructs. These RNAs dispersed evenly throughout the cytoplasm through diffusion and then became progressively immobilized and formed aggregates in the MC. Entrapment in the MC was not prevented by microtubule disruption and did not require localization to germinal granules. Immobilized RNA constructs codistributed and showed coordinated movement with densely packed endoplasmic reticulum (ER) concentrated in the MC, as revealed with Dil16(3) labeling and immunofluorescence analysis. Vg1RBP/Vera protein, which has been implicated in linking late pathway RNAs to vegetal ER, was shown to bind specifically both wild-type Xcat2 3' untranslated region and localization-defective constructs. We found endogenous Vg1RBP/Vera and Vg1RBP/Vera-green fluorescent protein to be largely excluded from the MC but subsequently to codistribute with Xcat2 and ER at the vegetal cortex. We conclude that germ line RNAs localize into the MC through a diffusion/entrapment mechanism involving Vg1RBP/Vera-independent association with ER.

Regionalised activation of canonical Wnt signalling via β-catenin stabilisation is a key early step in embryonic patterning in many metazoans, including the basally diverging cnidarians, but the upstream maternal cues appear surprisingly variable. In Clytia, regionalised β-catenin stabilisation defining a presumptive 'oral' territory is determined by two maternally coded Frizzled family Wnt receptors of opposite localisation and function. We have identified a maternally coded ligand, CheWnt3, the RNA of which is localised to the animal cortex (future oral side) of the egg. Antisense morpholino oligonucleotide experiments showed that CheWnt3 is required maternally for regionalised oral β-catenin stabilisation in the early embryo, being only the second clear example of a maternally required Wnt ligand after Xenopus Xwnt11. In line with the determinant role of the maternally localised Frizzleds, CheWnt3 overexpression by RNA injection initially had little effect on establishing the oral domain. Subsequently, however, overexpression had dramatic consequences for axis development, causing progressive expansion of β-catenin stabilisation to yield spherical 'oralised' larvae. Upregulation of both CheFz1 and CheFz3 RNAs in CheWnt3 morpholino embryos indicated that CheWnt3 participates in an active axial patterning system involving reciprocal downregulation of the receptors to maintain oral and aboral territories. Localised introduction of CheWnt3 RNA induced ectopic oral poles in CheWnt3 morpholino embryos, demonstrating its importance in directing oral fate. These findings suggest that the complete ligand-dependent Wnt signalling cascade is involved in axial patterning in ancestral eumetazoans. In Clytia, two variant Frizzled receptors and one Wnt ligand produced from localised RNAs cooperate to initiate regionalised Wnt pathway activation.

In phylogenetically diverse animals, including the basally diverging cnidarians, “determinants” localised within the egg are responsible for directing development of the embryonic body plan. Many such determinants are known to regulate the Wnt signalling pathway, leading to regionalised stabilisation of the transcriptional coregulator β-catenin; however, the only strong molecular candidate for a Wnt-activating determinant identified to date is the ligand Wnt11 in Xenopus. We have identified embryonic “oral–aboral” axis determinants in the cnidarian Clytia hemisphaerica in the form of RNAs encoding two Frizzled family Wnt receptors, localised at opposite poles of the egg. Morpholino-mediated inhibition of translation showed that CheFz1, localised at the animal pole, activates the canonical Wnt pathway, promotes oral fates including gastrulation, and may also mediate global polarity in the ectoderm. CheFz3, whose RNA is localised at the egg vegetal cortex, was found to oppose CheFz1 function and to define an aboral territory. Active downregulation mechanisms maintained the reciprocal localisation domains of the two RNAs during early development. Importantly, ectopic expression of either CheFz1 or CheFz3 was able to redirect axis development. These findings identify Frizzled RNAs as axis determinants in Clytia, and have implications for the evolution of embryonic patterning mechanisms, notably that diverse Wnt pathway regulators have been adopted to initiate asymmetric Wnt pathway activation.

Jellyfish (medusae) are a distinctive life-cycle stage of medusozoan cnidarians. They are major marine predators, with integrated neurosensory, muscular and organ systems. The genetic foundations of this complex form are largely unknown. We report the draft genome of the hydrozoan jellyfish Clytia hemisphaerica and use multiple transcriptomes to determine gene use across life-cycle stages. Medusa, planula larva and polyp are each characterized by distinct transcriptome signatures reflecting abrupt life-cycle transitions and all deploy a mixture of phylogenetically old and new genes. Medusa-specific transcription factors, including many with bilaterian orthologues, associate with diverse neurosensory structures. Compared to Clytia, the polyp-only hydrozoan Hydra has lost many of the medusa-expressed transcription factors, despite similar overall rates of gene content evolution and sequence evolution. Absence of expression and gene loss among Clytia orthologues of genes patterning the anthozoan aboral pole, secondary axis and endomesoderm support simplification of planulae and polyps in Hydrozoa, including loss of bilateral symmetry. Consequently, although the polyp and planula are generally considered the ancestral cnidarian forms, in Clytia the medusa maximally deploys the ancestral cnidarian-bilaterian transcription factor gene complement.

The separation of the germ line from the soma is a classic concept in animal biology, and depending on species is thought to involve fate determination either by maternally localized germ plasm ("preformation" or "maternal inheritance") or by inductive signaling (classically termed "epigenesis" or "zygotic induction"). The latter mechanism is generally considered to operate in non-bilaterian organisms such as cnidarians and sponges, in which germ cell fate is determined at adult stages from multipotent stem cells. We have found in the hydrozoan cnidarian Clytia hemisphaerica that the multipotent "interstitial" cells (i-cells) in larvae and adult medusae, from which germ cells derive, express a set of conserved germ cell markers: Vasa, Nanos1, Piwi and PL10. In situ hybridization analyses unexpectedly revealed maternal mRNAs for all these genes highly concentrated in a germ plasm-like region at the egg animal pole and inherited by the i-cell lineage, strongly suggesting i-cell fate determination by inheritance of animal-localized factors. On the other hand, experimental tests showed that i-cells can form by epigenetic mechanisms in Clytia, since larvae derived from both animal and vegetal blastomeres separated during cleavage stages developed equivalent i-cell populations. Thus Clytia embryos appear to have maternal germ plasm inherited by i-cells but also the potential to form these cells by zygotic induction. Reassessment of available data indicates that maternally localized germ plasm molecular components were plausibly present in the common cnidarian/bilaterian ancestor, but that their role may not have been strictly deterministic.

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