The origin of vertebrates and the appearance of their novelties is still a debated issue. According to the “New Head” hypothesis, key innovations for vertebrate success were the neural crest and the neurogenic placodes (Northcutt and Gans, 1983). Thus, it is of interest to analyze if these crucial components originated de novo in the vertebrate lineage, or if they had precursors in the common ancestor of all chordates. In addition to vertebrates, the chordates include the cephalochordates and the tunicates, with the latter now considered most closely related to vertebrates (Delsuc et al., 2008). Sequenced tunicate genomes are very compact compared with those of vertebrates (Satou et al., 2008), and their study helps reconstruct the genetic situation of the common ancestor (Lemaire, 2011). Therefore, the tunicates can be useful living models for the study of the evolution of structures which have evolved high complexity in vertebrates (Shimeld and Holland, 2000).
In vertebrates, the cranial placodes are patches of thickened embryonic ectoderm that give rise to many sense organs and ganglia of the vertebrate head. The network of regulatory links between transcription factors involved in vertebrate placode development is still incompletely characterized, but a set of genes marks a contiguous region of non-neural ectoderm early in embryogenesis, the so-called preplacodal domain that gives rise to anterior (adenohypophyseal, olfactory, and lens) and posterior (otic, epibranchial, and lateral line) placodes. Of interest, a few of these genes, such as those of the Eya, Six1/2, and Six4/5 gene families, continue to be expressed in all placodes and their derivatives (reviewed in Schlosser, 2010). Orthologues of these transcription factors were found to have an expression domain adjacent to the anterior neural plate border and/or in sensory cells differentiating from non-neural ectoderm in both tunicates and in the cephalochordate amphioxus (Bassham and Postlethwait, 2005; Mazet et al., 2005; Kozmik et al., 2007), and more recently also at the anterior-most rim neuroectoderm of an insect (Posnien et al., 2011). However, the entire molecular pathway characterizing the cranial placodes can be found only in vertebrates (Holland and Holland, 2001; Holland, 2005; Schlosser, 2005, 2007). Intriguingly, Six1/2, Eya, and FoxI are similarly expressed during gill slit formation in all investigated deuterostomes developing these structures (Sahly et al., 1999; Solomon et al., 2003; Bessarab et al., 2004; Kozmik et al., 2007; Schlosser, 2007; Gillis et al., 2012).
In addition to molecular evidence, developmental and structural data support the idea that tunicates possess embryonic areas comparable to some cranial placodes of vertebrates (reviewed in Graham and Shimeld, 2013). In Ciona intestinalis embryos there are two thickened ectodermal domains, an anterior stomodeal and a posterior atrial, from which sensory organs derive (Manni et al., 2004). Each domain expresses a subset of placodal genes, such as Pax, Six, Eya, and FoxI and is proposed to have homology to vertebrate olfactory-adenohypophyseal and otic-lateral line placodes, respectively (Mazet et al., 2005).
Tunicates are a very diverse group and include both solitary and colonial species. Colonial species can form similar zooids through the different developmental pathways found in sexual and asexual reproduction, allowing comparison of these processes in the same organism. The colonial tunicate Botryllus schlosseri is a model species for this kind of study (Manni and Burighel, 2006); moreover, it is phylogenetically distant from C. intestinalis (Tatian et al., 2011), so comparison between the two species helps to reconstruct the hypothetical basal tunicate state. Three asexual (blastogenic) generations coexist in the colony: the adults, their buds (primary buds), which in turn produce budlets (secondary buds) (Fig. 1A). The bud rudiment appears as a thickened disc on the zooid wall. It initially organizes in a double vesicle, with the inner epithelium derived from the parental peribranchial leaflet and the outer one from the parental epidermis. The inner vesicle gives rise to the main organs, such as the branchial and peribranchial chambers, the gut, and the nervous system. Other organs, such as the heart and the gonads, derive from mesenchymal cells that arrive in the bud by means of blood circulation (Fig. 1B) (Manni et al., 2007).
Bud and embryo develop following different mechanisms with a completely different starting point; the zygote in embryogenesis and specific pluripotent somatic territories of the parental zooid in blastogenesis. However, for some structures, such as the nervous system and the branchial stigmata, developmental similarities have been found (Manni et al., 1999, 2002). For example, the nervous system of the larva differentiates from the neural plate, whereas in blastozooids, it derives from a territory of the inner bud vesicle, however in both it shows a placodal-like derivation with several ultrastructural similarities (Burighel et al., 1998; Manni et al., 1999). In addition, a molecular study has shown expression of the Pitx gene, which is normally involved in vertebrate adenohypophysial placode development, in nervous system formation during both developmental pathways (Tiozzo et al., 2005).
We here characterize B. schlosseri orthologues of Six1/2, Six3/6, Eya, and FoxI, and report their spatiotemporal expression patterns during both embryogenesis and blastogenesis. Our results show that these genes are expressed both in larva and bud during branchial fissure formation, and in two domains along the anterior–posterior axis. We hypothesize that the latter are placodal homologue territories that can be recognized during not only sexual but also asexual development of tunicates, and discuss this in the context of both placode ancestry and the co-option of gene networks.