The Migratory Behavior of the Presomitic Mesoderm Cells

Somites are formed one-by-one from the posterior region of the paraxial mesoderm, the region also known as the presomitic mesoderm. The morphogenetic movements that account for the extension, segmentation, and narrowing of this tissue have been studied using live imaging mainly in the chicken and zebrafish, due to its accessibility, among other technical reasons. Nevertheless, the early studies of Wilson and colleagues performed in Xenopus embryos where cell explants of paraxial mesoderm were filmed during full somitogenesis, described similar cell behaviors to what Thorogood and Wood described 2 years before in fish (Thorogood and Wood 1987; Wilson et al. 1989). Convergent extension movements, including first radial intercalation, followed by mediolateral intercalation and finally cell-shape changes that occurred in a sequential fashion that was repeated from posterior-to-anterior PSM over time. Wilson and coauthors found that only the most posterior region of the PSM contributed to tissue elongation and that neither cell-shape changes nor cell division were the driving force of axis extension in Xenopus embryos but solely cell rearrangements (Wilson et al. 1989). Staining the membranes of small groups of cells at different positions along the PSM by iontophoretic injection of the fluorescent lipophilic dye Dil in chicken eggs allowed Kulesa and Fraser (2002), using time-lapse imaging, to discover that cells from the posterior PSM move more and exchange neighbors in a more dynamic way, compared to the anterior PSM. This is similar to what Wilson and colleagues found in the frog. When Benazeraf and colleagues tracked the orientation of these cell movements they found a decreasing pos- terior-to-anterior gradient of cell motility with an opposed gradient of cell density, where all PSM cells move toward the caudal end of the embryo and only anterior cells show convergent movements directed to the midline (Benazeraf et al. 2010). Nevertheless, given that embryo elongation by itself causes tissue deformation, the authors cleverly subtracted the movements displayed by the extracellular matrix, stained with a fluorescently labeled anti-fibronectin antibody, from the cell displacements tracked by H2B-GFP. The remaining cell movements showed random instead of directional motility. Moreover, by electroporating an inducible (in order to avoid early developmental events) dominant negative FGF receptor in the PSM and tracking cell movements by time-lapse imaging, Benazeraf and coworkers found that cell motility and the concomitant elongation depend on FGF signaling. In addition, when they overexpressed FGF8 in the cells of the PSM, cell motility at the anterior PSM increases, disrupting the motility gradient and reducing tissue elongation. Analyzing a long series of 13 time-lapse movies, Benazeraf and colleagues (2010) proposed a model of PSM cell motility where they concluded that the FGF-dependent gradient of random cell motility along the PSM causes the opposed cell density gradient that directs the elongation toward the posterior end of the embryo. Previous studies from the same group demonstrated years before that the posterior-to-anterior motility gradient in the PSM was regulated by the Mapk/Erk pathway and that this pathway was in turn controlled by FGF signaling (Delfini et al. 2005). For this they performed live imaging of mosaic chicken PSM cells electroporated with the pCIG vector (that contains GFP as a reporter) fused to the MKKlca or MKKlr/n constructs, that contained a constitutively active or a dominant negative form of MKK1 (the МАРК kinase), respectively. They compared velocity, cell directionality, and cell dispersion using time-lapse imaging and cell tracking, finding that activated MAPK/ERK signaling increases cell velocity and dispersion. On the other hand, MKKl-inhibited cells were slower and formed patches that distinguished them from the WT/non- electroporated cells (Delfini et al. 2005). Taken together, these findings suggest that cell migration during posterior growth in vertebrates is not directed by mechanisms such as cell polarization toward or against chemical stimuli but by an FGF gradient that, via Mapk/Erk, maintains single cell movement without specific directionality but depends on the levels of FGF/Erk activity. This generates more cell displacement at the posterior that triggers a reduction in cell density, which directs collective cell migration from high to low density by “simple” diffusion (Delfini et al. 2005; Benazeraf et al. 2010; Benazeraf and Pourquie 2013).

Time-lapse imaging of zebrafish PSM cells has been used mainly to visualize the oscillatory expression of the cyclic gene herl (Delaune et al. 2012; Soroldoni et al. 2014; Shih et al. 2015). Some of the cellular processes involved in axial elongation and segmentation were covered using live imaging approaches by at least two studies. Cell division (Horikawa et al. 2006) and cell motility (Lawton et al. 2013) were analyzed in the context of their effect on the synchronization of oscillations (commented before) or cell motility through different regions of the tailbud, respectively. The time-lapses and cell tracking performed by Lawton and colleagues in zebrafish embryos injected with nlsRFP mRNA showed a transition in the coherence of cell flow from the dorsal medial zone (DMZ) to the progenitor zone (PZ) and then to the PSM. The velocity during the collective cell migration decreases, revealing changes in tissue fluidity, in addition to the loss of coherence between cells, which means that neighbor cells within the PZ and PSM migrate in different directions compared to the DMZ (Lawton et al. 2013). Moderate reduction of the Wnt and FGF signaling pathways by injecting notumla (Wnt inhibitor) mRNA or incubating embryos with the FGF inhibitor SU5402 allowed authors to visualize the effect of these signaling pathways on cell fluidity within the tailbud using time-lapse microscopy. The reduction in Wnt or FGF signaling mainly showed a loss of cell migration coherence within the DMZ, without affecting this parameter in the PZ or PSM regions.

The question that arises after understanding some of the cell behaviors that take place during vertebrate posterior growth and axial elongation is how they are coordinated with the establishment of the segmental patterning, especially because the same signaling pathways appear to be implied in both processes and cellular dynamics are possibly influencing (and influenced by) the molecular behavior of the cells. Given that the use of live imaging is still in the early stages, the expectations are as high as the growing number of research groups interested in the molecular and cellular mechanisms underlying axial elongation and segmentation in vertebrates (and arthropods, later below).

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