Gene expression in bryozoan larvae suggest a fundamental importance of pre-patterned blastemic cells in the bryozoan life-cycle

Background Bryozoa is a clade of aquatic protostomes. The bryozoan life cycle typically comprises a larval stage, which metamorphoses into a sessile adult that proliferates by asexual budding to form colonies. The homology of bryozoan larvae with other protostome larvae is enigmatic. Bryozoan larvae exhibit blastemic tissues that contribute to build the adult during morphogenesis. However, it remains unclear if the cells of these tissues are pre-determined according to their future fate or if the cells are undifferentiated, pluripotent stem cells. Gene expression studies can help to identify molecular patterning of larval and adult tissues and enlighten the evolution of bryozoan life cycle stages. Results We investigated the spatial expression of 13 developmental genes in the larval stage of the gymnolaemate bryozoan Bugula neritina. We found most genes expressed in discrete regions in larval blastemic tissues that form definitive components of the adult body plan. Only two of the 13 genes, BnTropomyosin and BnFoxAB, were exclusively expressed in larval tissues that are discarded during metamorphosis. Conclusions Our results suggest that the larval blastemas in Bugula are pre-patterned according to their future fate in the adult. The gene expression patterns indicate that some of the bryozoan blastemas can be interpreted to correspond to homologous adult tissues of other animals. This study challenges an earlier proposed view that metazoan larvae share homologous undifferentiated "set-aside cells", and instead points to an independent origin of the bryozoan larval stage with respect to other lophotrochozoans.


Background
Bryozoa (Ectoprocta) is a monophyletic group of sessile, colonial invertebrates and includes over 6,000 species in aquatic habitats worldwide [1]. Bryozoan life history, reproduction and anatomy are so fundamentally different from other metazoan groups (for example they lack typical circulatory structures or nephridia and the nervous systems of larvae and adults are unique), that traditional morphological investigations and the fossil record failed to clarify their evolutionary history. Bryozoa belong to Lophotrochozoa [2], but their phylogenetic position within the group is still ambiguous [3][4][5][6].
Bryozoans have indirect development and their life cycle includes a sexually produced larval stage as well as asexual reproduction by cloning to give rise to colonial adult forms [12]. The most species rich bryozoan clade with over 5,000 species, the Gymnolaemata, has evolved a fascinating diversity of reproductive mechanisms and larval forms. Less than 20 species release their eggs directly into the surrounding water, where they develop into the well known cyphonautes larvae, planktotrophic (feeding) larvae with characteristic shells. Even fewer species produce shelled larvae with a non-functioning gut called pseudocyphonautes. The great majority of gymnolaemates, however, have evolved brood protection and their embryos develop into lecitotrophic (non-feeding) "coronate" larvae. Coronate larvae usually lack both a shell and a gut, but traces of a non-functioning gut were observed in some species, which was interpreted as a vestigial gut [13,14]. Despite the above outlined differences among gymnolaemate larvae, they usually share a set of morphological characters including transitory larval structures such as larval muscles and nerves (such as an apical organ), a glandulo-sensory organ (pyriform organ), internal cells, as well as blastemic cells that give rise to definitive adult tissues [14]. During a drastic metamorphosis, the transitory larval structures are discarded and only a single species is known to retain its larval gut in the adult [15]. Typically, all adult structures (for example gut, nervous system) are built de novo from the blastemic cells, which are found in various positions in the larvae and can give rise to different adult structures during metamorphosis in individual gymnolaemate species [16,17]. However, some authors propose that only ectodermal and mesodermal cells are involved in adult body plan formation from the larva, and this process mirrors the asexual budding process, which involves proliferation of the polypide (gut and lophophore) from the pluripotent body wall [12,14,18,19].
A comprehensive cell fate map does not exist for any species of bryozoan, so it is unknown if the blastemic cells in the larvae are undifferentiated, pluri-or multipotent set-aside/stem cells, or if these cells are determined for their future fate in the adult. To help to clarify the question to what extent the adult tissues are already determined in the larval stage, we investigated the spatial expression of 13 developmental genes in the newly released, coronate larvae of the gymnolaemate bryozoan Bugula neritina. This species is one of the better-studied bryozoans with respect to larval morphology and metamorphosis, although many details are still lacking. This initial set of genes was chosen according to the specific expression of genes in certain metazoan germ layers or organs as well as their putative conserved functions in the development among taxa, and represents a foundation for future molecular studies. Amongst the investigated genes, Tropomyosin is known to be a general marker of bilaterian musculature; the genes Hox4, SoxB2, SoxE and FoxB have functions in neural development; the genes FoxA, GATA456 and Cdx (Caudal) have been shown to be involved in gut patterning among taxa; expression of GATA123 and BAMBI was observed in the ectoderm of metazoans, and Wnt genes have been shown to be involved in multiple events including axial patterning (see Discussion). Genes engaged in bilaterian gut formation were investigated to determine if there is any molecular indication for a larval gut being present as anlage of the adult gut in B. neritina. We discuss the gene expression patterns of the bryozoan larval stage in the light of available data for other metazoans.

Larval anatomy
The larval anatomy of Bugula neritina has been previously studied and relevant data are reviewed here together with our own observations ( Figure 1). The larvae have a roundish body shape (length, 260 μm; width, 190 μm) and the apical disc, which leads in swimming direction, marks the apical pole ( Figure 1A, B). The apical disc comprises a central neural area (apical organ), which is surrounded by an outer epidermal blastema and an inner mesodermal blastema ( Figure 1E) [14,[20][21][22]. The apical organ is detected in semi-thin sections (not shown). The apical disc is encircled by infolded ectodermal cells, the pallial epithelium ( Figure 1A, E). Opposite of the apical disc lies the large internal sac, which constitutes nearly half of the larval interior ( Figure 1B). The internal sac is an ectodermal invagination used for attachment to the substrate during metamorphosis (see below) and comprises the neck, wall, and roof region [22]. The wall region is extensive and is folded upon itself ( Figure 1D, E). The roof cells are elongated and contain basal inclusions of electron transparent vesicles [22]. The neck cells are recognized by their dark inclusions ( Figure 1D, E). Here, we define the region of the internal sac opening as "abapical" instead of the formerly used "oral", since coronate larvae lack a mouth. The anterior side of the larva is defined by the ciliated cleft and the pyriform organ, a typical gymnolaemate larval organ ( Figure  1C, D). It is a glandular organ, used for crawling, sensing, and transitorily attaching the larva to the substrate prior to metamorphosis [14]. The pyriform organ extends between the mesodermal blastema and the internal sac ( Figure 1B, E). The larva possesses additional nerves, muscles, and yolky cells ( Figure 1D) [12,20,23]. Some of the internal (yolky) cells are probably endodermal and mesodermal [14]; however, neither their origin nor their contribution to the adult body plan is resolved. Most of the larval surface is covered by several hundred coronal cells, which constitute the larval swimming organ ( Figure 1A, D) [14].
The terminology that describes the blastemic cell layers in bryozoans is currently confusing and it will be useful to change the nomenclature for the blastemic cells in the future, when comprehensive cell lineage studies become established, which trace the embryological origin of these cells. Herein, we define the cells of the internal sac, the pallial epithelium, the epidermal blastema, and the mesodermal blastema as blastemic cells.

Development
All data outlined in this section and in Figure 2 were gathered from previous studies. Bugula neritina is a brooder and embryos are nourished by a special placenta-like system [18]. The free swimming period of the larva is short, probably between one and 30 hours before settlement [24]. Initial attachment of the larva to the substrate and first morphogenetic movements take only a few minutes and the metamorphosis to the first feeding adult is accomplished within several hours (Figure 2A-I) [18,21]. The blastemic tissue of the internal sac aids in attachment of the larva to the substrate. The neck region of the internal sac contains dark inclusions aiding in initial attachment before being discarded, while the roof region contains secretory material and forms the permanent attachment disc of the first individual of the colony (ancestrula) [16,19]. The wall region of the internal sac subsequently forms the epidermis of the free surface of the ancestrula [19]. The epidermal blastema forms the digestive tract and the lophophore of the adult bryozoan [25]. The mesodermal blastema contributes the lophophoral coelomic lining and the splanchnic peritoneum, while the origin of the somatic peritoneum is unclear [22]. The pallial epithelium forms the epithelium of the tentacle sheath of the adult. Transitory larval structures such as the apical organ, the pyriform organ, and the corona are discarded at metamorphosis.

Gene expression patterns in the bryozoan larval stage
Here we describe the spatial expression of 13 developmental genes in the larval stage of Bugula neritina according to gene families.

Tropomyosin
In the larvae of Bugula neritina, we found BnTropomyosin expression at the upper edge of the apical disc, along the pallial epithelium, surrounding the larva in an equatorial position, as well as alongside the ciliated cleft and internally around the glandular area of the pyriform organ ( Figure 3A-F). Additional expression is found in the abapical region below the internal sac ( Figure 3A, B). Overall, BnTropomyosin expression is strongest in the coronal cells at the larval surface as well as in cells just below the coronal cells, suggesting the presence of myoepithelial cells in these areas ( Figure 3D).

Hox4
We observed BnHox4 expression in the wall and roof regions of the internal sac in the larvae (Figures 4A-C and 5A, B). Expression is also found internally in the glandular cell complex of the pyriform organ, as well as in the epidermal and mesodermal blastemas ( Figure 4B).

BAMBI
Expression of BnBAMBI is found in a defined area of a trapezoid shape on the posterior side of the bryozoan larva, including cells of the epidermal and mesodermal blastemas, as well as some internal cells. In addition, cells positioned in the abapical wall region show expression, which gives a horseshoe-shaped appearance ( Figure 4J-L).

FoxA, FoxB and FoxAB
BnFoxA is expressed in a continuous ring in the epidermal and mesodermal blastemas, as well as in the wall and roof of the internal sac (Figures 6A-C and 7A, B). BnFoxB is co-expressed with BnFoxA in the wall cells of the internal sac, but also shows expression in the circular pallial epithelium ( Figure 6D

Cdx (Caudal)
Similar to BnFoxA, BnCdx is expressed as a broad ring in the apical disc in cells of the epidermal and mesodermal blastemas, the wall region of the internal sac, and weak expression in the roof cells ( Figure 6J-L).

GATA123 and GATA456
The BnGATA factor 123 shows expression in a ring of cells in the pallial epithelium (Figures 8A-C and 9A, B).  [21,22,76]. A) Competent larva. B) Five seconds (s): post settlement: Initial attachment through eversion of the internal sac. C) 20 s: The roof region of the internal sac moves towards the substratum, the apical disc retracts. D) 60 s: The apical disc re-extends, the corona starts involuting, the pellicle (excreted by the neck region) covers the larva. E) Approximately 120 s: The corona involutes, the pallial epithelium covers the apical hemisphere, the wall rotates. F) Approximately 160 s: The edge of the pallial epithelium constricts and the apical disc is compressed, bringing the pallial epithelium into contact with neck and wall of the internal sac. G) Approximately 240 s. The pallial epithelium constricts and the wall rises towards the apical region. H) 360 to 390 s. The pallial epithelium thickens, the wall covers the apical region, the apical disc and surrounding pallial epithelium are pulled inside. Coronal cells begin to autolyse. I) Several hours: transformation from the preancestrula to ancestrula: the pre-ancestrula elongates, the wall region forms the epidermis (which then secretes the cuticle and calcium carbonate). Invaginated cells of the epidermal blastema form the digestive tract and the lophophore. The splanchnic lining is formed by cells of the mesodermal blastema. The pallial epithelium has formed the tentacle sheath. co, corona (light grey); eb, epidermal blastema (purple); gl, gland cells of pyriform organ; lo, developing lophophore; mb, mesodermal blastema (cyan); nn, nerve nodule; pe, pallial epithelium; pha, pharynx; pel, pellicle, secreted by the neck region; st, stomach; ts, tentacle sheath; internal sac (is) consisting of n, neck (blue); r, roof (dark grey); and w, wall (grey). Expression is also observed in a few cells of the epidermal blastema and beneath the mesodermal blastema in the glandular area. Weak expression is present in the neck region of the internal sac ( Figure 8A, B). In contrast, BnGATA456 is expressed in a single spot on the posterior side of the apical organ in the epidermal blastema of the Bugula neritina larvae ( Figure 8D-F).

Wnt1 and Wnt4
BnWnt1 is expressed in cells of the neck region of the internal sac, appearing as a ring at the abapical pole.

Discussion
The lecitotrophic larva of Bugula neritina contains only a few truly larval organs, including the apical sense organ and associated nerves, the swimming organ (corona), the glandulo-sensory organ (pyriform organ), internal yolk cells, and muscles. These transitory larval structures are all likely to be discarded at metamorphosis [19]. Blastemic tissues that are known to form the adult during metamorphosis are the epidermal blastema, the mesodermal blastema, the pallial epithelium, and the internal sac. Of the 13 developmental genes that we investigated, 11 genes are expressed in discreet and highly reproducible regions of one or more of the blastemic tissues in the coronate larva that form the adult during metamorphosis. The results indicate that the blastemic cells are probably molecularly pre-determined in the bryozoan larva. Some of the 11 genes show additional expression in the pyriform organ, which is used by the larval stage and resorbed at metamorphosis. Only BnTropomyosin and BnFoxAB are solely expressed in transitory larval structures, the musculature and the pyriform organ (ciliated cleft), respectively (Figures 3,  10). In the following sections, we discuss the gene expression patterns of the bryozoan larval stage in respect to available data of other metazoans.

Hox4, SoxB, SoxE, BAMBI and FoxB
In the animals investigated so far, Hox genes have been shown to play a crucial role in body plan regionalization along the anterior-posterior axis and are to a great extent expressed in ectodermal and mesodermal derivates. In Acoela, expression of a central Hox gene is found in ectodermal cells in early developmental stages which later give rise to putative neural precursor cells [26]. In several polychaetes, Hox4 is expressed in larval ectoderm and developing neural structures [27][28][29]. Accordingly, in the mollusk Haliotis, Hox4 expression is in presumptive neuroectoderm and developing ganglia in the trochophore stage and later in the mantle, suggesting partial co-option of the gene for shell formation [30]. Rather similar to Hox4, SoxB genes probably have a conserved role in nervous system development in cnidarians and spiralians [31][32][33][34][35][36]. In the bryozoan Bugula, BnHox4 and BnSoxB2 are widely co-expressed in both  transitory larval structures as well as blastemic cells ( Figure 10A, C). Ectodermal expression of Hox4 and SoxB2 in Bugula is similar to their expression in other metazoans, where they are involved in neuroectodermal patterning. Prior to our study, SoxE orthologs have only been investigated in deuterostomes, and in vertebrates they change the fate of neural stem cells into glial stem cells [37][38][39]. In the bryozoan, BnSoxE is expressed in a small subset of the BnSoxB2 expressing, blastemic cells ( Figure 10E). BAMBI is an inhibitor of TGF-β signaling and expression was earlier observed in the beetle Tribolium, where it is co-expressed with BMP/Dpp in the dorsal ectoderm [40], and in deuterostomes, where BAMBI is co-expressed with Bmp2/4 orthologs [41][42][43][44]. BnBAMBI is expressed in a subset of the BnSoxB2 expressing cells, which form parts of the adult bryozoan ( Figure 10G). FoxB orthologs play a role in neural development in the cnidarian Nematostella, the ecdysozoan Drosophila, and in chordates [33,[45][46][47][48]. In Bugula, BnFoxB is exclusively expressed in future ectodermal tissues ( Figure 10K).
The above considerations reveal that the bryozoan orthologs of Hox4, SoxB2, SoxE, BAMBI, and FoxB are to a great extent expressed in limited domains of the blastemas, and the expression patterns parallel that of other animal taxa to some degree. It is feasible that some of the investigated orthologs also play a role in nervous system patterning in bryozoans, and additional studies of gene function will be helpful to evaluate conserved and novel gene functions in the bryozoan life cycle.

FoxA, Cdx and GATA456
The genes FoxA, Cdx, and GATA456 have previously been shown to be involved in bilaterian gut development. FoxA (Forkhead, HNF3) is important for the development of components of the digestive tract in protostomes as well as in deuterostomes [49][50][51][52][53][54]. In an acoel, FoxA is expressed in the endoderm surrounding the mouth, suggesting an ancestral role of FoxA in the endoderm and a later co-option in oral ectoderm in bilaterians [55]. In annelids, FoxA is expressed during gut formation [56][57][58], and in the mollusk Patella, FoxA expression is in the endoderm and in the developing foregut [59]. In the bryozoan larva, BnFoxA is expressed in different blastemas that give rise to adult tissues during metamorphosis ( Figure 10I). Expression of BnFoxA in the epidermal blastema, which is supposed to form the adult digestive tract, suggests a function in gut development similar to other metazoans. Cdx/Caudal orthologs are involved in metazoan hindgut formation [60,61]. In addition, Cdx orthologs are expressed in the brains of both an acoel and an annelid [26,62]. In the mollusk Patella, Cdx expression is observed in the posterior neuroectoderm and mesodermal cells [63]. In the bryozoan larva, BnCdx is co-expressed with BnFoxA ( Figure 10O), and it seems likely that BnCdx is partially involved in adult gut formation as in other lophotrochozoans. However, compared to other taxa, neither FoxA nor Cdx show positional pre-patterning in the bryozoan. The gene GATA456 is involved in  endodermal specification in annelids [57,58]. In the bryozoan larva, BnGATA456 is co-expressed with BnCdx and BnFoxA in a confined domain in the epidermal blastema ( Figure 10S).
Our results lead to the conclusion that the genes FoxA, Cdx, and GATA456 are probably involved in the formation of the adult digestive tract in bryozoans and other metazoans and that the epidermal blastema is pre-patterned according to its future fate. In this study, we find no sign of a vestigial larval gut in Bugula neritina, corroborating former histological investigations [22].

Genes involved in ectoderm specification GATA123
The GATA123 factor appears to be involved in ectodermal lineage specification in annelids [57,58,64], similar to what has been described for Drosophila [65]. We observed BnGATA123 expression to the greatest extent in ectodermal larval structures as well as in blastemic tissues that form the ectoderm of the adult ( Figure  10Q), consistent with the ectodermal expression of the gene in other taxa.

Genes for axial patterning Wnt
Wnt signaling might be involved in the development of the primary body axis in most Metazoa [66]. However, from this suggested ancestral role, Wnt diversified and regulates many processes in metazoan taxa. A common feature of Wnt expression is a staggered arrangement along the anterior-posterior axis with partly overlapping domains in, for example, Nematostella, leech, or Capitella [67,68]. In Bugula, Wnt1 and Wnt4 expression is in specific, partly overlapping subsets of the future adult ectoderm, suggesting that the internal sac is regionalized and its cells might be differentially involved in adult body wall patterning (see also Figure 10U, W).

Gene expression in transitory larval tissues Tropomyosin and FoxAB
Most of the 11 genes mentioned above are largely expressed in subsets of progenitors of adult tissues, with only BnTropomyosin and BnFoxAB expressed exclusively in transitory larval tissues. Tropomyosin is a general marker of bilaterian musculature. The F-actin component of the larval musculature of Bugula species was investigated in previous studies and revealed prominent longitudinal and radial central muscles positioned between the internal sac and the apical organ, as well as body wall muscles [69,70]. The BnTropomyosin expression appears to conform with body wall muscles, and corresponds closely with a previous ultrastructural investigation of the Bugula neritina larva, which showed (1) the presence of myoepithelial cells between coronal cells, (2) a collarette of myoepithelial cells that join the infolded pallial epithelium, (3) a pair of myoepithelial cells extending along the lateral sides of the ciliated cleft and (4) a collarette joining the oral margin of the corona [21] (compare with Figure 3). The lack of Tropomyosin expression in blastemic tissues indicates that the adult musculature differentiates after metamorphosis.
Our study showed that BnFoxAB expression is confined to epidermal cells along the ciliated cleft and along a part of the abapical side of the bryozoan larva ( Figure 10M). In a brachiopod larva, FoxAB expression was observed in cells of the apical pole instead (Hejnol et al., unpublished data). Since no such expression is found in the bryozoan larva and comparable gene expression data of other metazoans are currently lacking, our conclusions must be provisional, but recruitment of FoxAB to larval structures may also be found in other taxa.

Pluripotent stem cells versus pre-patterned adult tissues
It was earlier proposed that "homologous set-aside cells" exist in "homologous larvae of protostomes and deuterostomes" [71]. These set-aside cells were supposed to have a rather unlimited division capacity and remain pluripotent and undifferentiated until late embryogenesis. This idea is rooted in the hypothesis that early metazoans were similar to modern larvae, and that the bilaterian adult stage evolved by the innovation of setaside cells, distinct from the larval cells [72]. However, this hypothesis would presume a similar gene regulatory system in all modern larvae. Here, we have shown that the blastemic cells ("set-aside cells") in the bryozoan larval stage express several metazoan developmental genes. The result indicates that the fate of the blastemic cells is already determined in the bryozoan larval stage and the blastemic cells of bryozoans are probably not pluripotent stem cells or set-aside cells. Our study rather suggests similarities between developmental gene expression in the bryozoan blastemas and adult tissues of other metazoans. In conclusion, this study does not support homology of the bryozoan larval stage and other lophotrochozoan larvae. Instead, the gene expression patterns presented herein indicate that planktonic larvae might have secondarily evolved in bryozoans. The latter hypothesis needs to be evaluated by further studies of gene expression and gene function during the development of bryozoans as well as other lophotrochozoans.

Conclusions
We have shown here the gene expression of 13 developmental genes (Tropomyosin, Hox4, SoxB2, SoxE, BAMBI, Cdx, FoxA, FoxB, FoxAB, GATA123, GATA456, Wnt1, and Wnt4) in the coronate larva of the bryozoan Bugula neritina. Eleven of the 13 genes are expressed in blastemic cells, which are precursors of adult tissues. Probably only a few of the investigated genes have their main function in the larval stage, as for example, BnTropomyosin, which is expressed in larval muscle cells. This study reveals that the blastemic cells in the bryozoan larval stage are most probably pre-patterned according to their future fate in the adult and are not pluripotent, undifferentiated set-aside cells as previously stated [71]. Our results contradict the idea that larval and adult bodies are different entities, but that there is a natural transition from the embryo to the adult with an intermediate larval stage. A comparison of expression patterns among metazoans reveals molecular similarities between bryozoan blastemas and adult tissues of other metazoans. Hence, our study does not indicate homology of the bryozoan larva with other lophotrochozoan larvae, but instead suggests conserved patterns of developmental gene expression amongst lophotrochozoan and metazoan adults. This study adds important data to the fundamental discussion about the evolution of metazoan larval stages and should trigger the interest in investigating gene expression in the "set-aside cells" of the larval stage of other lophotrochozoans as, for example nemerteans. Cell-lineage studies of bryozoans and additional gene expression studies during bryozoan development will also contribute to our understanding of the evolution of metazoan life cycles.   pointed light sources, larvae were released from spawning colonies. They were immediately collected from the water surface under dissecting microscopes and prepared for further investigation.

RNA isolation and cDNA synthesis
Larvae were fixed and stored in RNAlater at 4°C. Larval mRNA was obtained using DynaBeads mRNA DIRECT Kit (Invitrogen, Carlsbad, CA, USA) according to the supplier and stored at -80°C. Complementary DNA (cDNA) synthesis was achieved using the Advantage RT-for-PCR Kit protocol (Clontech Laboratories, Mountain View, CA, USA) following the supplier's instructions. cDNA was stored at -20°C.

Gene isolation
The sequences for the genes BnTropomyosin, BnBAMBI, BnSOXB2, BnSoxE BnHox4, BnFoxB, BnFoxAB, WNT1 were gained from a public EST library of Bugula neritina [3]. Fragments for BnCdx, BnFoxA, BnWNT4 and both GATA factors were gained using degenerate primers with larval cDNA as template. Sequences of the genes from the EST library were amplified using gene specific primers and degenerate fragments were extended using rapid amplification of cDNA ends (RACE) with a SMART RACE cDNA amplification kit (Clontech Laboratories, Mountain View, CA, USA). All fragments were cloned into pGEM-T Easy vectors (Promega Corporation, Madison, WI, USA), transformed into E.coli, and clones sequenced at Macrogen, Inc. (Seoul, South Korea). Fragments obtained from B. neritina were used for probe synthesis in in situ hybridization reactions, which are described below. Primer sequences are listed in Additional file 1 Table S1. Genes were deposited at NCBI GenBank (see below).

Gene orthology assignment
Gene orthologies for all genes (except for BnBAMBI and BnTropomyosin of which the orthology was detected by alignments) were determined by phylogenetic analyses using PhyML 3.0 [73]. Alignments were conducted using MUSCLE [74] and corrected by hand. ProtTest [75] was used to determine the best-fitting model. A total of 1,000 to 3,000 bootstraps were calculated respectively, see Additional data files.

In situ hybridization protocol
Larvae of B. neritina were relaxed in 7.14% MgCl 2 , prefixed in glutaraldehyde fixative (0.3% glutaraldehyde, 3.7% formaldehyde in seawater) for two minutes, fixed in 3.7% formaldehyde for one hour at 4°C, washed five times in PTw (1x PBS = phosphate buffered saline + 0.1% Tween 20) and once in distilled water, which was replaced three times by 100% methanol. The larvae were finally stored in the latter at -20°C. All further steps follow the protocol of Hejnol et al. [55]. Following the development of the in situ probes, larvae were then transferred into 70% glycerol and whole-mounted on glass slides and expression patterns were imaged with a Nikon DXM1200 digital camera mounted on a Nikon Eclipse E1000 microscope. For detailed examination of the expression patterns, larvae were also prepared for histology.

Histology of larva after in situ hybridization
For the histological preparations, in situ hybridized larvae stored in 70% glycerol were washed three times in PBS and dehydrated in an ascending ethanol series with a final step of dehydration in 100% propylene oxide. The larvae were transferred into a 1:1 mixture of 100% propylene oxide and Low Viscosity Resin (LVR) (Agar Scientific, Stansted, UK) over night for infiltration. The animals were embedded in 100% LVR and semithin serial sections (2 μm) were performed on a Leica RM2255 microtome using a Diatome Histo Jumbo Diamond Knife (Diatome, Hatfield, PA, USA). Sections were stained with a 1% solution of basic fuchsine (p-Rosanilin) in 70% ethanol or toluidine blue and embedded in LVR on slides. Imaging was performed with the Nikon setup described in the previous section.
Abbreviations Bn: Bugula neritina; cDNA: complementary DNA; DNA: deoxyribonucleic acid; EST: expressed sequence tag; mRNA: messenger ribonucleic acid; PBS: phosphate buffered saline; PCR: polymerase chain reaction; LVR: low viscosity resin; RACE: rapid amplification of cDNA ends the Kewalo Marine Laboratory. We are grateful to Brian Nedved and Elizabeth Perotti for help with species collection and to Christiane Todt for help with the histological preparations. Furthermore, we wish to thank three anonymous reviewers for their valuable comments on the manuscript. The work was made possible with Swedish travel grants from the Adlerbertska Stipendium Stiftelse and the Helge Ax:son Johnsons Stiftelse to JF. The research was funded by NSF AToL grant (EF05-31558), NASA Exobiology and the DFG (HE5183/2-2).