Hox complement and gene orthology
We identified 8 Hox genes in the transcriptome of Ph. harmeri and our phylogenetic analysis allowed their assignment to particular orthology groups (Fig. 2). Those genes represent orthologues of the genes labial (lab),, proboscipedia (pb),, hox3, deformed (dfd),, lox5, antennapaedia (antp),, lox4 and post2 (Figs. 1A and 2). Moreover, in addition to the gene cdx reported by Andrikou et al.(85), we identified two other paraHox genes in the transcriptome of Ph. harmerigsx and xlox. Most of the Hox orthologues form distinct clades in our phylogenetic tree (Fig. 2). Sequences from the two orthologues (sex combs reduced (scr) and antp) do not form clades but rather grades of similar sequences (Fig. 2), which nevertheless allow exact orthology assessment. We found that the gene identified by Luo et al. as lox2 in the genome of P. australis(89) and its orthologue in Ph. harmeri do not fall into the clade containing lox2 sequences from other Spiralia, but instead they group in the grade containing antp sequences. Accordingly, sequence of those two phoronid genes lack most of the residues proposed as signature of lox2 by de Rosa et al. (Additional File 1: Fig. S1; (93)).
Embryonic and larval development of Ph. harmeri
Embryos and larvae of Ph. harmeri are relatively transparent and many aspects of their morphology can be easily observed with the light microscope using Nomarski interference contrast (Fig. 3). At 9°C the blastula stage is reached at about 6–8 hours post fertilization (hpf). Around 12 hpf a swimming blastula with a large blastocoel (bc) is formed (Fig. 3 A, A’). At 20 hpf the gastrulation process is initiated, which leads to the formation of the gastrula (Fig. 3 B, B’) that displays a distinctive blastopore (bp),, the archenteron (ar) and the anterior mesoderm (am).. Subsequently, the embryo (including the archenteron) elongates along the A-P axis and the oral hood (oh) develops anteriorly leading to the formation of the early larval stage, at approximately 40 hpf (Fig. 3C, C’). In the posterior part of the early larval gut the intestine (in) develops, which merges with the posterior ectoderm and forms an anus. Laterally to the intestine the first undifferentiated rudiments of the protonephridia are present (pr in Fig. 3C, C’). At 60 hpf the pre-tentacle larval stage is reached (Fig. 3D, D’), which possesses a tripartitioned digestive tract (with esophagus, es; stomach, st and intestine, in),, an apical organ (ao),, protonephridial rudiments (pr) and rudiments of the first three pairs of tentacles (rt).. 3 days post fertilization (dpf) larvae can be already identified as early 6-tentacle actinotrocha (Fig. 3E, E’) due to the presence of three pairs of well-defined tentacles (te).. At this stage the larval protonephridia reach their definite branching form (pn, Fig. 3E), the rudiments of posterior mesoderm are morphologically distinguishable (pm, Fig. 3E)and the posterior telotroch starts to form around anal opening (tt, Fig. 3E’). At 5 dpf (Fig. 3 F, F’) the telotroch is fully formed, while the posterior mesoderm forms rudiments of the posterior coelom compartment (metacoel), displacing the terminal organs of larval protonephridia to the more anterior position. The actinotrocha reach the 8-tentacle stage at 7 dpf (Fig. 3G, G’). At this stage the post-tentacular region of the body elongates and the metasomal sac, a rudiment of the body wall of the prospective adult worm, is formed (ms, Fig. 3G, G’). The metasomal sac at this stage appears as an ectodermal thickening located on the ventral side between tentacle bases and the telotroch. The thickening is concave and it is possible to discriminate between its opening (which will represent the anterior part of the adult trunk), and bottom (which after eversion will contribute to the posterior ampulla). The morphological details of the embryonic and larval development of Ph. harmeri are well described elsewhere(66, 72, 75–78, 80–82, 84, 85), therefore we did not examined further the embryonic and larval morphology.
Hox gene expression
We did not detect expression of any of the Hox genes in blastula and gastrula stages (Additional File 1: Fig. S2). Expression of the anterior Hox gene lab is detected for the first time during development at the late 6-tentacle actinotrocha stage (Fig. 4A g and h). The gene is expressed in the ventro-posterior ectodermal domain, between the tentacle bases and the telotroch (black arrowhead, Fig. 4A g and h) and in the paired domains of the dorso-lateral posterior mesoderm (red arrowheads, Fig. 4A g and h). Both of the expression domains persist to the 8-tentacle actinotrocha stage (Fig. 4A i and j). At this developmental stage the ectodermal domain is part of the metasomal sac, where lab is expressed in the cells of the anterior and bottom portion of the sac (Fig. 5A and A’).
The second anterior Hox gene, pb, is the earliest expressed among all Hox genes in Ph. harmeri as its expression can be already detected in the early larva stage (42 hpf) in the cells at the border of intestine and anus (red arrowheads, Fig. 4B a and b). This expression domain remains in the pre-tentacle stage (56 hpf, 4B c and d) and later it is restricted to the small lateral domains (Fig. 4B e and f), which in late 6-tentacle actinotrochae expands and the gene labels lateral, mesodermal sheet-like structures, extending along posterior ectoderm (red arrowheads Fig. 4B g and h). In 8-tentacle actinotrochae pb is expressed in two mesodermal stripes, which extends from the metasomal sac to the base of tentacles (Fig. 4B i and j, 5G) and corresponds to the telotroch flexor muscles, as described in the late actinotrocha of Ph. harmeri based on phalloidin staining(81).
Hox3 expression is detected in the late 6-tentacle actinotrochae in an ectodermal domain between the tentacle bases and telotroch (black arrowhead, Fig. 4C g and h). At the 8-tentacle actinotrocha stage hox3 is uniformly and exclusively expressed in the ectodermal cells of the metasomal sac (black arrowheads, Fig. 4C i and j; Fig 5B and B’).
Dfd expression initiates only at the 8-tentacle actinotrocha stage (Fig. 4D i and j), where the gene is expressed in a small, anterior subset of the metasomal sac (Fig. 5C and C’).
Transcripts of the gene lox5 are detected first in the early 6-tentacle actinotrocha in posterior cells of the developing telotroch (green arrowhead, Fig. 4E e and f). Later on, lox5 remains expressed in the telotroch, expanding its expression domain to the entire structure (green arrowheads, Fig. 4E g–j). Two additional expression domains of lox5 also appear: the metasomal sac rudiment (black arrowhead, Fig. 4E h), which later encompass the entire metasomal sac (black arrowheads Fig. 4E j and inset between i and j; Fig 5D and D’), and an asymmetric domain in the left ventro-lateral posterior mesoderm, located between metasomal sac, midgut and left body wall (red arrowheads Fig. 4 i and j and inset between g and h; Fig. 5H, I).
Expression of antp is not detected until the 8-tentacle actinotrocha stage. Transcripts of the gene are found in ectodermal cells around the opening of the metasomal sac (black arrowheads, Fig. 4F i and j; Fig. 5E and E’), which in a dorso-ventral view look like a ring on the ventral body surface between the tentacles base and the telotroch (Fig. 4F i).
Similarly, lox4 expression is not detected until the 8-tentacle actinotrocha stage, where the gene is exclusively labeling the ring of the endodermal cells at the junction between stomach and intestine (blue arrowheads, Fig. 4G i and j; Fig.5J).
The only posterior Hox gene, post2, is expressed from the early 6-tentacle actinotrocha (3dpf) in the telotroch (green arrowheads, Fig. 4H e and f), initially in the posterior portion of the organ but later on the expression domain is uniformly surrounding the anus (green arrowheads, Fig. 4H g–j). However, compared to lox5 expression (which is also demarcating the telotroch), post2 labels only the inner ring of epidermal cells of the organ (compare Fig. 4E g–j and 4H g–j) and not the entire structure. At the 8-tentacle actinotrocha stage the gene post2 is additionally expressed in the small posterior portion of the metasomal sac (black arrowhead, Fig. 4H j; Fig 5F and F’).
In addition to the investigation of Hox genes we tested expression of several head-specific genes in the early larva (42 hpf) and advanced 8-tentacle actinotrocha. The genes, which expression we investigated, were foxG (also known as brain factor–1 or BF–1), foxQ2, six3/6, otx and pax4/6, all commonly considered as head markers (38, 45, 89, 94–98). One of the two foxG paralogues (see methods section for details), which expression we managed to detect, foxGa,is expressed in the early larva in the epidermal cells, from which the tentacular crown will develop (Fig. 6A–C). In 8-tentacle stage the gene expression was not detected (data not shown). We managed to clone one of two foxQ2 paralogues, FoxQ2b, and detected its exclusive expression in the apical organ of both early and 8-tentacle stage larvae (Fig. 6D–G). six3/6 is expressed in the early larvae in the apical organ, hood mesoderm, postoral ectoderm and in the anterior portion of the digestive tract (Fig. 6H and I) as previously reported(85). In 8-tentacle actinotrocha six3/6 is expressed in the apical organ, hood muscles, some cells associated with dorso-anterior part of digestive system and in the stripe of mesodermal cells along ventral side of anterior digestive system (Fig. 6J–M). In early larvae otx is expressed in the apical organ, ventral preoral ectoderm, anterior portion of the digestive tract and in two spots in the ventro-posterior ectoderm, which lay in the prospective tentacular crown territory (Fig. 6N–P), following the expression pattern described before(85). In the 8-tentacle stage otx is expressed in the apical organ, rim of the oral hood, anterior portion of the digestive tract and in the small spots close to the tips of each tentacle (Fig. 6Q–U). pax4/6 is expressed in the early larvae in the two stripes of cells which extend along ventral side of the larva, from mouth to about half of the body length (Fig. 6V, W) and which correspond with the position to the tentacular neurite bundles (78). In 8-tentacle stage pax4/6 expression is detected along frontal side of each larval tentacle (Fig. 6X and Y), which also corresponds to the tentacular innervation(78). In general the head specific genes are broadly expressed in both developmental stages in the body structures anterior to the Hox-expressing territory (Fig. 6Z).