4. Discussion
4.1 Spatial and temporal relationship of the Capitella sp. I gene expression patterns of
CapI-fringe
For Capitella sp. I, embryos younger than St. 4 were not examined. Notch and CapI-Delta have very similar and overlapping expression patterns in larval as well as in juveniles stages. In larvae, both genes are initially expressed in the ectoderm of the presumptive segmented tissue. CapI-Notch has a much broader expression while CapI-Delta is expressed in a discrete cells or small clusters of cells in the belly plates of the larvae. CapI-Notch also shows limited expression in the mesoderm beneath the ectodermal transcripts in the bilateral unsegmented and segmented tissue. From St. 6 on, CapI-Notch and CapI-Delta are expressed in a two row pattern of segmentally iterated patches of cells at the position of all future chaetal sacs. At this stage of development, CapI-Notch is also expressed in a broader area compared with its ligand CapI-Delta, and the two rows of expression are also detectable. Additionally, both genes are expressed in the CNS and in two patches lateral to the mouth. In later stages, both genes are expressed in the mesoderm of the posterior growth zone and to a lesser extent in the ectoderm.
The juvenile expression of both genes can be detected in the nascent segment, the adjacent segment anterior of the posterior growth zone (E.C. Seaver, unpublished results). The juvenile growth zone in Capitella sp. I was defined as a subterminal zone plus the last formed or nascent segment. CapI-Notch and CapI-Delta are mainly expressed in the mesoderm and to a lesser extent in the ectoderm.
The similar expression patterns of CapI-Notch and CapI-Delta across developmental stages and distinct tissues in Capitella sp. I are consistent with these genes acting together within a ligand-receptor signaling network (Fehon et al. 1990; Heitzler and Simpson 1993;
Rebay et al. 1991). The overlapping expression patterns of both genes in the presumptive chaetal sac anlagen before the formation of chaetae also suggest an involvement of the signaling pathway in the chaetogenesis of Capitella sp. I.
CapI-fringe expression coincides with the broad transcript localization of CapI-Notch throughout the stages it is expressed. All three characteristic expression domains, the CNS, the belly plates and the foregut, can be detected in both genes. The refinement of CapI-Notch expression and especially of CapI-Delta expression later on into two rows of dots at
transcript. This can be explained with the proposed function of fringe in other organisms.
As described previously (see Introduction), fringe seems to be a regulator of the Notch signaling pathway (Moloney et al. 2000). Depending on the ligand, it has a positive or a negative effect on the signaling process (Panin et al. 1997). Especially the border formation in diverse tissues is dependent on the influence of fringe and the Notch pathway (Irvine and Rauskolb 2001; Rauskolb et al. 1999). The overlapping expression patterns of CapI-Notch and CapI-fringe are typical for the functionality of fringe. Thus, it can inhibit other Notch ligands like Serrate or potentiate the binding of Delta to Notch (Haltiwanger 2002). Both ligand and receptor are chemically modified by fringe. This would also explain the overlapping expression of CapI-Delta and CapI-fringe. The fringe gene modulating the Notch signaling was only isolated from Capitella sp. I, but not from P.
dumerilii.
The persistent broad expression of CapI-fringe without the described later more restricted pattern can be caused for example by a negative regulation of a second CapI-Notch ligand by CapI-fringe (Panin et al. 2002), like a Serrate homologue. CapI-fringe expression in St.
8 to 9 is missing in this analysis. It would be interesting to observe this expression pattern in older larvae of Capitella sp. I to see a possible transcript location in the terminal growth zone besides the CapI-Notch and CapI-Delta expression. This would suggest an influence of CapI-fringe in the later Notch signaling pathway too.
In early stage larvae, CapI-hes1 has a very restricted expression pattern in contrast to CapI-Notch, CapI-Delta and CapI-fringe. The transcript is always associated with the unsegmented mesodermal part of the belly plates and in older larval stages is localized to the terminal growth zone with CapI-Delta and CapI-Notch. In juveniles, CapI-hes1 is expressed in the subterminal region of the mesodermal posterior growth zone and to a small extent in the nascent segment (Fig. 26G-I). Thus, CapI-hes1 expression coincides with the Delta and Notch expression of Capitella sp. I to a really small extent in the nascent segment and the subterminal region even though the latter two genes are expressed mainly anterior to hes1. The overlapping expression domains suggest that CapI-Notch and CapI-hes1 could be expressed in the same cells. This might imply a possible activation of hes1 by Notch in Capitella sp. I. during late larval and juvenile development.
The expression pattern of CapI-hes1 does not support any involvement in the formation of chaetae in comparison to CapI-Notch and CapI-Delta. Hes/Enhancer of split genes are often downstream targets of the Notch signaling pathway (Bessho et al. 2001; Iso et al.
2003; Kageyama et al. 2007; Takke and Campos-Ortega 1999). Our data for the juvenile expression of CapI-Notch and CapI-hes1 are consistent with this. In contrast to the later stages of Capitella sp. I development, the earlier embryos and larvae do not show a significant overlap or co-expression of CapI-Notch and CapI-hes1. CapI-Notch is only expressed weakly in the mesoderm of the belly plates which might be sufficient to activate and regulate CapI-hes1. Another possiblity is, that CapI-hes1 is a Notch-independent transcription factor in this stage of development and is not being regulated by this signaling pathway. Notch-independent activation of hes genes has been well documented as was already shown for developmental processes of various organisms (Bae et al. 2005; Hirata et al. 2001). Moreover, it can not be excluded, that a second or even more Notch genes exist in Capitella sp. I. We can assume a coexpression of the second CapI-Notch gene with CapI-hes1 and thus it might also be involved in the regulation of CapI-hes1. An overlap of expression with CapI-hes2 and CapI-hes3 is also possible.
CapI-hes2 as well as CapI-hes3 show prominent overlap of expression with CapI-Notch and CapI-Delta during St. 4/5 until St. 9 as well as during development of juveniles consistent with the conservation of the canoncial Notch pathway. Both hes genes could represent downstream targets for CapI-Notch, especially CapI-hes2 with its prominent expression in the future chaetal sacs. Other overlapping expression domains such as the foregut, the developing brain and later on the posterior growth zone also account for this downstream signaling.