Further Development 11.4: The Forces of Convergent Extension

Amphibians and Fish

The first force is a polarized cell cohesion, wherein the involuted mesodermal cells send out bipolar protrusions to contact one another. These acts of “reaching out” are not random, but occur oriented toward the midline of the embryo and require an extracellular fibronectin matrix (Goto et al. 2005; Davidson et al. 2008). These intercalations, both mediolaterally and radially, are stabilized by the planar cell polarity (PCP) pathway that is initiated by Wnts independent of β-catenin signaling (see Chapter 4; Jessen et al. 2002; Shindo and Wallingford 2014; Ossipova et al. 2015). Manipulation of conserved PCP pathway components results in the loss of bipolar protrusions and inhibition of convergent extension (Figure 1; Darken et al. 2002; Goto et al. 2005).

The second force is differential cell cohesion. During gastrulation, the genes encoding the adhesion proteins paraxial protocadherin and axial protocadherin become expressed specifically in the paraxial (somite-forming; see Chapter 17) mesoderm and the notochord, respectively. An experimental dominant-negative form of axial protocadherin prevents the presumptive notochord cells from sorting out from the paraxial mesoderm and blocks normal axis formation. A dominant-negative paraxial protocadherin (which is secreted instead of being bound to the cell membrane) prevents convergent extension (Kim et al. 1998; Kuroda et al. 2002).¹ Moreover, the expression domain of paraxial protocadherin characterizes the trunk mesodermal cells, which undergo convergent extension, distinguishing them from the head mesodermal cells, which do not undergo convergent extension.

A third factor regulating convergent extension is calcium flux. Wallingford and colleagues (2001) found that dramatic waves of calcium ions (Ca²⁺) surge across the dorsal tissues undergoing convergent extension, causing waves of contraction within the tissue. Ca²⁺ is released from intracellular stores and is required for convergent extension. If Ca²⁺ release is blocked, normal cell specification still occurs, but the dorsal mesoderm neither converges nor extends. These findings support a model for convergent extension wherein regulatory proteins cause changes in the outer surface of the tissue and generate mechanical traction forces that either prevent or encourage cell migration (Beloussov et al. 2006; Davidson et al. 2008; Kornikova et al. 2009).

¹ Dominant-negative proteins are mutated forms of the wild-type protein that interfere with the normal functioning of the wild-type protein. Thus, a dominant-negative protein will have an effect similar to a loss-of-function mutation in the gene that encodes the protein.

**Figure 1** PCP-mediated bipolar protrusions drive mediolateral intercalation. Open-faced dorsal mesoderm “Keller” explants were created so that fluorescently labeled cells could be imaged live on a laser scanning confocal microscope. (A) Gap43-GFP (a fusion protein with green fluorescent protein that results in the fluorescent labeling of cell membranes) was used to visualize the shapes of cells, and contrasts with the red fluorescent labeling of the cell’s contacts with the fibronectin-coated microscope slide. Note the bipolar lamellipodia in the control (arrows, left), which are absent when PCP signaling is inhibited by overexpressing the pathway antagonist *strabismus*/*van gogh* (arrows, right). (B) Quantitative analysis displayed on rose diagrams shows the direction of bipolar protrusive activity from the cells’ centers and the frequency (indicated by percentages) with which this occurred over the 30-minute period of observation. Note the lack of unipolar lamellipodia activity revealed by the analysis. ant, anterior; pos, posterior; ml, mediolateral. (B after T. Goto et al. 2005. *Curr Biol* 15: 787–793.)

Literature Cited

Beloussov, L. V., N. N. Luchinskaya, A. S. Ermakov and N. S. Glagoleva. 2006. Gastrulation in amphibian embryos, regarded as a succession of biomechanical feedback events. Int. J. Dev. Biol. 50: 113–122.
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Darken, R. S., A. M. Scola, A. S. Rakeman, G. Das, M. Mlodzik and P. A. Wilson. 2002. The planar polarity gene strabismus regulates convergent extension movements in Xenopus. EMBO J. 21: 976–85.

Davidson, L. A., B. D. Dzamba, R. Keller and D. W. DeSimone. 2008. Live imaging of cell protrusive activity, and extracellular matrix assembly and remodeling during morphogenesis in the frog, Xenopus laevis. Dev. Dyn. 237: 2684–2692.
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Goto, T., L. Davidson, M. Asashima and R. Keller. 2005. Planar cell polarity genes regulate polarized extracellular matrix deposition during frog gastrulation. Curr. Biol. 15: 787–793.
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Jessen, J. R., J. Topczewski, S. Bingham, D. S. Sepich, F. Marlow, A. Chandrasekhar and L. Solnica-Krezel. 2002. Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements. Nat. Cell Biol. 4: 610–615.

Kim, S.-H., A. Yamamoto, T. Bouwmeester, E. Agius and E. M. De Robertis. 1998. The role of paraxial protocadherin in selective adhesion and cell movements of the mesoderm during Xenopus gastrulation. Development 125: 4681–4691.
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Kornikova, E. S, E. G. Korvin-Pavlovskaya and L. V. Beloussov. 2009. Relocations of cell convergence sites and formation of pharyngula-like shapes in mechanically relaxed Xenopus embryos. Dev. Genes Evol. 219: 1–10.
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Kuroda, H., M. Inui, K. Sugimoto, T. Hayata and M. Asashima. 2002. Axial protocadherin is a mediator of prenotochord cell sorting in Xenopus. Dev. Biol. 244: 267–277.
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Ossipova, O., C. W. Chu, J. Fillatre, B. K. Brott, K. Itoh, and S. Y. Sokol. 2015. The involvement of PCP proteins in radial cell intercalations during Xenopus embryonic development. Dev. Biol. 408: 316–327.
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Shindo, A. and J. B. Wallingford. 2014. PCP and septins compartmentalize cortical actomyosin to direct collective cell movement. Science 343: 649–652.
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Wallingford, J. B., A. J. Ewald, R. M. Harland and S. E. Fraser. 2001. Calcium signaling during convergent extension in Xenopus. Curr. Biol. 11: 652–661.
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