How groups of cells coordinate changes in cell shape, adhesion, and motility in order to form complex tissues is one of the central questions of developmental biology. The dynamic cell rearrangements that occur during somitogenesis make this an excellent model of tissue morphogenesis. In Xenopus laevis, cells must re-orient their long axes in a synchronized manner in order to create a morphologically distinct boundary between somites. We demonstrate here that loss of integrin α5 delays the process of rotation and the accumulation of integrin β1 at early somite boundaries, and results in persistently abnormal myotome boundaries. In addition, fibronectin matrix formation is disrupted at the boundaries between adjacent somites as well as between somites and surrounding tissues. Interestingly, these effects are less severe in the far posterior somites of the developing tail. Thus, integrin α5 is required for somite rotation and boundary formation in Xenopus, though its requirement appears to vary at different positions along the anterior/posterior axis.
The lack of observable effects of the intgα5 MO on events prior to somitogenesis highlights the usefulness of the splice morpholino strategy in avoiding defects associated with genes required in early developmental stages, and allowing examination of their function in later developmental events. As noted earlier, the maternal expression of Integrin α5 results in a pool of pre-existing mRNA and protein that is unaffected by the splice-blocking intgα5 MO. As this pool diminishes, generation of new mRNA is disrupted, resulting in the creation of nonfunctional protein. This method enabled us to directly examine the role of integrin α5 during somitogenesis, and will likely prove very useful in the investigation of later developmental roles of other Xenopus genes as well.
Integrin α5 in Somite Cell Turning
Analysis of cell turning behaviors during Xenopus somitogenesis reveals that cell movements are coordinated, but individualized (Afonin et al.,2006). Cells adjacent to the notochord are the first to show changes in orientation, and throughout somite formation cell shapes are variable, suggesting that each cell is following its own “best path” during rotation (Youn and Malacinski,1981; Wilson et al.,1989; Afonin et al.,2006). In addition, protrusive activity is consistently seen to increase during the turning process, indicating that cells are adopting a migratory phenotype. These observations raise the question of what surface somitic cells are using to create the traction necessary for rotation.
One possibility is that somitic cells crawl along other cells using cadherin-based adhesive contacts. Indeed, both paraxial protocadherin (PAPC) and protocadherin in neural crest and somites (PCNS) have been implicated in somite patterning in Xenopus (Kim et al.,2000; Rangarajan et al.,2006) and in mice (Rhee et al.,2003). In addition, disruption of cadherin I in Xenopus embryos results in disorganized somites, providing evidence that cell–cell interactions are important for the cell movements involved in somitogenesis (Giacomello et al.,2002). However, as this disruption is accompanied by a loss of MyoD staining, it is possible that the phenotype is secondary to altered somite specification (Giacomello et al.,2002).
Alternatively, somitic cells could move by migrating along the extracellular matrix. Immunostaining reveals that integrin β1 is present at cell borders and at the emerging somite boundary (Fig. 5). Furthermore, fibronectin matrix is laid down at the anterior boundaries of turning somites, and lines the junction between somites and surrounding tissues (Fig. 6; Kragtorp and Miller,2006). Finally, our data showing that somite rotation is substantially delayed in intgα5 MO injected embryos demonstrates that integrin α5β1 plays a critical role in regulating rotation.
It is probable that cells utilize a combination of cell–cell and cell–matrix interactions to generate the traction necessary for cell turning. The robust fibronectin matrix that surrounds developing somites could provide traction for cells at the periphery, while cell–cell interactions might play a larger role at the interior of the somite, where fibronectin matrix is comparatively sparse. In this model, interactions of peripheral cells with fibronectin would provide a large share of the traction necessary to drive cell rotation. Loss of this traction would result in the delayed cell rotation that we observe in intgα5 MO treated embryos, while the remaining forces from cell–cell interactions could account for the eventual completion of cell rotation.
Boundary Formation in Anterior and Posterior Somites
In addition to a delay in somite rotation, we also observe a delayed accumulation of both integrin β1 and fibronectin at developing somite boundaries in intgα5 MO treated embryos. As cells eventually attain their final, anterior/posterior orientation, some irregular boundary formation is observed in the form of disjointed integrin β1 and fibronectin accumulation. We previously observed that, in anterior somites, fibronectin accumulation at the presumptive somite boundary occasionally preceded somite turning (Kragtorp and Miller,2006). This would suggest that, in anterior somites, deposition the fibronectin matrix may serve an initial instructional role in boundary formation by creating a “track” for rotating cells to follow. We hypothesize that in the absence of this instructional cue, rotating cells lose their coordination and final cell position becomes more random. At the end of rotation, end-to-end cell contacts would determine the position of boundaries, leading to fragmented somite boundaries.
We report here that formation of posterior somites is less affected by the knockdown of integrin α5. This is reminiscent of reports of zebrafish integrin α5 mutants, in which anterior but not posterior somites are affected. When combined with other deficiencies, such as fibronectin or Delta D mutants, zebrafish somite boundary formation fails all along the anteiror/posterior axis, indicating at least one level of redundancy. Indeed, loss of fibronectin commonly results in more severe phenotypes than loss of a single integrin subunit (Georges-Labouesse et al.,1996; Yang et al.,1999; Julich et al.,2005), the activity of which could be taken over by other integrins. This prompts us to hypothesize that such a redundancy is responsible for the decreased requirement for integrin α5 activity in the posterior somites of Xenopus.
There are several possible candidates for this redundancy. In mouse embryonic cells, the activity of integrin α5β1 can be replaced by integrin αvβ1 (Yang and Hynes,1996). As integrin αv is expressed ubiquitously in the early Xenopus embryo (Joos et al.,1998), it is conceivable that this subunit contributes to the partial recovery we observe. In contrast to anterior somites, we do not observe the accumulation of fibronectin prior to somite turning in more posterior somites (Kragtorp and Miller,2006). Thus, it is also possible that interactions with other extracellular matrix molecules could provide some redundancy in the development of posterior somites. Laminin accumulates around somites and at somite boundaries (Krotoski and Bronner-Fraser,1990), and integrin α7β1, which binds laminin, is expressed in the myotomes of both chicken (Kil and Bronner-Fraser,1996) and mouse (Song et al.,1992,1993). The expression pattern of integrin α7 in Xenopus has not been investigated, however, and thus its potential role in somitogenesis remains unclear. Finally, it is possible that cadherin-based cell–cell interactions could provide a redundant mechanism for posterior somite boundary formation. In Xenopus, inhibition of cadherin I in somites resulted in tadpoles with a distinctive “kinked” tail (Giacomello et al.,2002), which could point to an increased requirement for cadherin activity in tail somites. A combinatorial approach, similar to that used in zebrafish, would be useful in Xenopus in order to create a clearer picture of the redundancies operating in the complex morphogenesis of somites.
Research conducted in mouse, quail, and zebrafish all point to a conserved role for integrin α5β1 in somite boundary formation, although the severity of the defects observed in the absence of integrin α5, integrin β1, or fibronectin, varies between species (Drake et al.,1992; Yang et al.,1993,1999; Koshida et al.,2005). These species all use epithelialization to delineate the somite boundary (Stern and Keynes,1987; Ostrovsky et al.,1988; Stickney et al.,2000), a trait not shared by Xenopus. That the requirement for integrin–fibronectin interactions in somitogenesis is conserved may indicate that their role is, at least in anterior somites, instructive: physical delineation of the somite boundary by fibronectin matrix deposition provides a cue or scaffold that assists presomitic cells in carrying out the morphogenetic program specific to that species. It appears that, despite evolving very different morphogenetic movements, each species has continued to use the same basic tools to accomplish the cell rearrangements and boundary specification necessary for somite formation.