The hes-family

Five Capitella-bHLH-O sequences and two P. dumerilii-bHLH-O genes were isolated or found by searching through the trace files of the sequenced Capitella sp. I genome. To classify the sequences within this group of genes, phylogenetic analyses were performed.

CapI-hesr1 and CapI-hesr2 were clearly placed into the group of hesr/hey genes in all three consensus trees. These are the first two isolated hesr/hey genes of a lophotrochozoan and two of the first invertebrate hesr-genes besides the Drosophila hesr-1 gene (Kokubo et al. 1999). The amino acid sequence also shows the characteristic glycine residue right at the beginning of the bHLH domain (Davis and Turner 2001).

The CapI-hes2 and CapI-hes3 sequences were placed together with the Enhancer of split genes of Drosophila as a separate invertebrate hes group independently from the vertebrate Enhancer of split group.

The definite classification of the remaining three genes, Pdu-hes1, Pdu-hes2 and CapI-hes1, is more complicated and obviously not possible with the analyses used. All other sequences of different species and bHLH-O groups used for the calculation were expected results from compared to the phylogenetic analysis done by Davis and Turner (1997). With a Bayesian analysis, the Pdu-hes1 gene is classified as an Enhancer of split gene. Pdu-hes2

and CapI-hes1 are placed at the base of the hairy genes and invertebrate Enhancer of split genes. The Neighbor-Joining tree shows the classification of the latter two sequences into the hairy family, but also CapI-hes2 is grouped into this gene family instead of into the Enhancer of split/ hes family. The comparison of the expression patterns of all five genes (CapI-hes1, 2, 3 and Pdu-hes1, 2) is consistent with the Bayesian analysis by far. These analyses were performed to classify the isolated hes sequences into the group of bHLH-O transcription factors and inside one or two of the four subfamilies. No further predictions can be made as towards the relationship of animal groups.

Pdu-hes1, CapI-hes2 and CapI-hes3 show expression in or around the presumptive chaetal sac anlagen (for CapI-hes2 and CapI-hes3, E.C Seaver, unpublished results). After being expressed very uniformly in an early stage of development, Pdu-hes1 expression matures into a pattern surrounding the presumptive chaetal sac anlagen. Later on, it is expressed in the mesoderm of the posterior growth zone comparable to CapI-hes1 (see 4.2). The expression in the head at 4d could be associated with the development of palps and their sensory cells.

Pdu-hes2, in contrast to Pdu-hes1, is not expressed around the chaetal sacs or in the terminal growth zone. The only similarity in the expression of both genes seems to be the transcript localization in the mouth, although Pdu-hes1 is expressed around the mouth and Pdu-hes2 shows expression towards and in the stomodaeum of the embryo. All expression domains of Pdu-hes2 seem to be ectodermal, including the staining of the stomodaeum which persists throughout all stages of development or the labeling of the vetral midline cells. This very weak expression of Pdu-hes2 was confirmed by several in-situ experiments and it can be detected from 36h to 60h of development. Pdu-hes2 expression in the middle of the ventral plates is similar to the reported expression of Pdu-slit and Pdu-netrin in the early differentiating midline at the same stage of development (Denes et al. 2007). The two patches of expression posterior of the mouth could coincide with the described pigmented area (Wilson 1892), but also anlagen for the anal cirri later on. Expression of Pdu-hes2 consisting of four bilateral patches in the episphere of the embryo could be part of the developing CNS or special sensory organs.

The CapI-hes1 transcript is located in the mesoderm from the beginning of its expression.

The gene is expressed in a striped like pattern in the unsegmented part of the belly plates and later on in the posterior growth zone. With its expression pattern, it clearly follows the

al. 2005). First, nine to ten segments are formed by the lateral segment precursors and then the segmentation process is continued by the terminal growth zone. It can be proposed, judging from its expression pattern in the unsegmented lateral tissue and later in the posterior growth zone, that CapI-hes1 is involved in the segmentation process of Capitella sp. I (Fig. 15G-I). Strikingly, CapI-hes2 and CapI-hes3 expression in juveniles coincide with CapI-hes1 expression in the mesoderm of the posterior growth zone even though expression of CapI-hes2 and CapI-hes3 is broader and not as restricted as CapI-hes1 expression (Fig. 26J-P). All three genes are probably involved in segment formation during juvenile development in Capitella sp. I, but on different levels during the segmentation process like proliferation and growth of segments.

The comparison of the isolated hes genes to each other and of the two polychaetes plus their expression is very complex and quite difficult. One explanation might be the existence of several additional members of the bHLH-O gene family in one of the two species or both species. We can not determine if the isolated hes /hesr genes are orthologues of each other. The possibility that even more bHLH-O genes are existent in the genome of both polychaetes, especially in P. dumerilii, is very high. In all studied model organism like Drosophila, Xenopus or chicken, there are several members of the hes/hey/hairy family (Davis and Turner 2001; Kageyama et al. 2007). For example in the Danio genome, 15 hes genes (called her-genes for hairy and E(spl) related genes) exist and for mouse 7 hes genes were isolated so far (Sieger et al. 2004). In Drosophila, the deadpan and hairy transcription factors were identified as ‚true’ hairy genes (Bier et al. 1992;

Carroll et al. 1988; Younger-Shepherd et al. 1992). Besides those sequences, other members of the bHLH-O group were also isolated in Drosophila like a hers-gene or the Enhancer of split complex (Knust et al. 1987; Kokubo et al. 1999). This does not include the hey and hesr sequences of these model organisms. All of the already studied hes/her genes have different functions and are involved in many different developmental processes (Bessho et al. 2001; Henry et al. 2002; Kageyama et al. 2007; Shankaran et al. 2007).

Therefore, it is also not too surprising, that CapI-hes1, Pdu-hes1 and Pdu-hes2 show completely different expression patterns. Functional studies could probably confirm the different functionalities of the hes genes in Capitella sp. I and P. dumerilii.