Annelids occupy a very interesting phylogenetic position in the tree of life (fig.
1.1). They belong to the super-phylum of Lophotrochozoa, one of the two subdivi-sions of protostomes, which is poorly represented within “classical” developmental model organisms. Moreover, annelids have very few phylum-specific morphologi-cal characters, meaning that their body plan didn’t undergo extensive modifications after the emergence of the group (Tessmar-Raible and Arendt, 2003). Indeed, the cambrian fauna is populated by annelid trace fossils, including worms with seg-ments, parapodia and chetae, resembling living polychaetes (Conway Morris, 2006;
Conway Morris and Peel, 2008).
The new annelid phylogeny proposed recently by Struck et al. (2011) subdivides annelids in two major clades, Errantia and Sedentaria. The two names describe the prevalent lifestyle of the group members: Errantia show adaptations to a very motile, sometimes predatory lifestyle, while Sedentaria are burrowing worms, which live in a tube as filter-feeders. In both groups, morphological traits correlated with lifestyle adaptations have been identified (Struck et al., 2011).
Within annelids, Platynereis dumerilii (Annelida, Phyllodocida, Nereididae) be-came recently a molecular model organism for evolution, development and neu-robiology. Platynereis is a polychaete worm and belongs to the clade of Errantia, thus it has a broad repertoire of sensory structures and elaborated parapodial ap-pendages for crawling. The analysis of gene structure and intron positions showed that Platynereis has a slow evolving genome (Raible et al., 2005). The possibility to keep Platynereis in the lab for its entire life cycle, and the delivery of offsprings in big numbers makes Platynereis amenable for developmental and molecular studies.
1.2.1. Life cycle and early development of P. dumerilii
The life cycle of P. dumerilii is subdivided in a pelagic and a benthic phase (fig.
1.2A, Fischer and Dorresteijn 2004). The developmental stages have been recently described in great detail by Fischer et al. (2010). After fertilization, the embryo develops quickly into a trochophora larva (fig. 1.2B), characterized by an equatorial band of ciliated cells, called prototroch, an apical assembly of sensory and ciliated cells called apical organ, a through gut, a pair of larval protonephridia and a pair of larval eyes used for phototaxis (Jékely et al., 2008). The cells anterior to the
developing brain; posterior to the prototroch, the hyposphere will form the first three body segments. This phase of development is stereotypical and characterized by a fixed lineage; moreover, the larvae develop synchronously, making it possible to directly compare different individuals of the same stage.
In comparison with other annelids, P. dumerilii larvae are lecitotrophic and have a relatively short pelagic phase. After about three days of development, the larvae elongate posteriorly and develop the parapodia and become competent to settle-ment. This new larval stage, called nectochaete larva, lasts few days and is character-ized by an higher developmental plasticity, probably as an adaptation to changing environmental conditions. The larvae start feeding and settle in the benthos only after the identification of an appropriate substrate. Afterwards, the juvenile worm grows new segments and undergoes the first metamorphosis (called cephalic meta-morphosis), which consists in the transformation of the first chaetiferous segment into part of the head; this atoke worm builds himself a tube and lives in the benthos.
A second metamorphosis takes place few months later, immediately before the reproduction, and transforms the atoke worms in sexually mature epitoke worms. P.
dumerilii is sexually dimorphic, and the sexes become recognizable at this stage, with yellow females and red-whitish males. Spawning is highly synchronized according to the moon phase; the epitoke mature worms become pelagic, find each other with pheromones, and after a nuptial dance they release their gametes in the water and die.
Nereidids development has been a subject of investigation since the end of the 19th century (Wilson, 1892). More recently, the early developmental stages of P. dumerilii have been described by Dorresteijn (1990); Fischer et al. (1996); Ackermann et al.
(2005). Like other groups of lophotrochozoans, annelids have a spiralian cleavage (fig. 1.2C): during the first hours of development, the orientation of the mitotic spindles shifts at every subsequent cleavage; as a consequence, at the eight cells stage the four dorsal cells are not aligned to the four ventral cells. The first two cell divisions are unequal and define the four main embryonic quadrants, corresponding to the four blastomeres (A, B, C and D). These cells divide further generating the first set, or quartet, of micromeres (1a, 1b, 1c and 1d), located dorsally, and the first set of macromeres (1A, 1B, 1C and 1D), which retain most of the yolk content and have a ventral location. Other three sets of micromeres are generated by the subsequent three divisions of the macromeres; at this point, the spiralian phase terminates and
apical organ
Figure 1.2: Life cycle and early development of Platynereis dumerilii. A. P. dumerilii life cycle (im-age courtesy of Dr. G. Belavoine). B. The Platynereis trochophore larva (SEM photo courtesy of Dr.
H.Hausen). C. The early cleavages of Platynereis embryos. Green indicates the trochoblasts. Redrawn from Fischer and Dorresteijn (2004). For explanation, see text.
there is a transition to bilateral cleavage. The episphere develops (almost) entirely from the first set of micromeres. The cells of the apical organ (the so-called “apical rosette”) and the prototroch cells are produced during the very first divisions of the first quartet micromeres. At the 49-cell stage, the episphere is delimited by a ring of trochoblasts (which will divide to form the prototroch cells and accessory cells). In the middle, there are the four cells of the apical rosette (which will form the apical organ) and eight more cells (two from each quadrant: 1a1121−2−1d1121−2) that will give rise to the rest of the episphere. The dorsal part of the brain is a derivative of the C and D quadrants. Interestingly, the ring of trochoblasts is interrupted dorsally by the small 2d111cell, a descendent of the second quartet micromeres.
The development of P. dumerilii is fast enough to make it amenable to developmen-tal and molecular studies. The genome and the transcriptome have been sequenced recently (Arendt lab, unpublished). Single and double whole mount in situ hy-bridization (WMISH) allow the analysis of the expression domains of genes of inter-est during embryonic and larval stages (Tessmar-Raible et al., 2005); the expression patterns can be documented with a confocal microscope together with the tubu-lin staining of the axonal scaffold (reflection microscopy, Jékely and Arendt 2007).
Since development is stereotypical and synchronized at larval stages, reflection mi-croscopy has been used for image registration of expression patterns on a common reference axonal scaffold (Tomer et al., 2010). This technique, called Profiling by Image Registration (PrImR), allows to generate average expression patterns for a gene of interest; subsequently, the average expression patterns can be compared, since they are based on the same reference scaffold. This makes possible to deter-mine in silico coexpression of multiple genes with a single cell resolution (Tomer et al., 2010).
Recently, techniques for the injection of zygotes have also been established (Kegel, 2008). This made possible, for the first time, to manipulate gene function by means of injection of synthetic mRNAs and morpholino antisense oligonucleotides, but also to establish knock out lines with the zinc finger nucleases (ZFNs) technology (described in par. 4.4). Similarly, transposon-mediated transgenesis has been estab-lished (Arendt lab and several other labs, unpubestab-lished).