In this study, we have shown that during gastrulation stages, endodermal cells undergo developmentally regulated changes in migration behavior, which are driven by corresponding changes in actin cytoskeletal dynamics. We have also shown that the increased actin dynamics and random motility of cells during early gastrulation stages depend on Nodal signaling and Rac1 activity. Furthermore, we showed that Nodal signaling induces the expression of the Rac-specific GEF prex1 and that Prex1 functions downstream of Nodal signaling to promote random migration at early gastrulation stages. Together, these observations indicate that the early random migration of endodermal cells is driven by Nodal-induced Rac1 activation.
Interestingly, our data also suggest that the transition to directed migration during late gastrulation may not be simply a result of down-regulation of Nodal and/or Rac1 signaling. First, we observed that Rac1 activity increases rather than decreases during late gastrulation (Fig. S4 I). This increase in Rac1 activity may correlate with the onset of Cxcl12–Cxcr4 chemokine signaling (Mizoguchi et al., 2008
), which has been reported to signal through Rac1 (Xu et al., 2012
). Second, when we examined endodermal cell migration during late gastrulation in Nodal- or Rac1-inhibited embryos, we found that although cell migration was not severely affected, directional persistence was slightly increased (Fig. S4, C and G). This result suggests that Nodal-dependent signals may still be operating to promote random motility, but, at late stages, they are now superseded by directional cues provided by putative chemoattractants such as Cxcl12. Therefore, we propose a model in which Nodal, via Prex1, induces global Rac1 activation, which results in directionally random cell migration during early gastrulation stages. Then, as endodermal cells become responsive to directional cues during late gastrulation, these cues may lead to strongly polarized Rac1 activation that overwhelms the Nodal-dependent global Rac1 activation, leading to highly persistent, dorsal-directed migration. Thus, we speculate that by promoting global Rac1 activation, the function of Nodal/Prex1 during early gastrulation stages is to generate noise in the subcellular distribution of activated Rac1, ensuring that endodermal cells do not inappropriately respond to weak directional cues that may be present at these stages (perhaps guiding mesodermal cell migration). Our observations that loss of Nodal or Rac1 signaling during early gastrulation stages leads to increased directional persistence could be a result of the unmasking of these weak polarization signals that would normally be overwhelmed by the global Rac1 activity induced by high Nodal signaling at these early stages. This model is also consistent with our cell transplantation results in which precociously inducing persistent migration by DN Rac1 expression results in the mistargeting of endodermal cells to mesodermal tissues. Notably, our observations differ from cell culture studies in which decreasing Rac1 activity was sufficient to switch cells from random to persistent migration (Pankov et al., 2005
). Although such a simple signaling mechanism may indeed be sufficient to regulate migratory behaviors under basic cell culture conditions, our results illustrate the complexity of regulating cell migration in the dynamic environment of the developing embryo.
The best-characterized role for Nodal signaling during endoderm development has been the induction of endoderm-specific transcription factor genes. Although it has been previously suggested that Nodal may regulate cell movement (Yokota et al., 2003
; Pézeron et al., 2008
), the mechanisms by which Nodal could affect cell motility were unknown. Here, we have shown that inhibition of Nodal signaling not only slowed cell migration velocity and increased migration persistence but also suppressed actin dynamics and Rac1 activity. We have further identified the Rac-GEF Prex1 as a downstream target of Nodal signaling. Rac1 is a well-known regulator of actin polymerization and cell migration both in vitro (Gardiner et al., 2002
; Srinivasan et al., 2003
; Pankov et al., 2005
; Woo and Gomez, 2006
) and in vivo (Li et al., 2002
; Kardash et al., 2010
; Yoo et al., 2010
), and it has also recently been shown to be crucial for the cell movements underlying gastrulation in mouse (Migeotte et al., 2011
). Although our results suggest that the Nodal-dependent Rac1 activity we observed is a result of increased expression of Prex1, Rac1 may be activated via a transcription-independent pathway as well. We observed that acute SB-505124 treatment lasting as little as 15 min was sufficient to alter cell migration behavior (Fig. S5
). Indeed, other TGF-β ligands have been to shown to induce both rapid Rho GTPase activation that is Smad independent as well as sustained increases in Rho activity that involve gene transcription (Kardassis et al., 2009
). It is also very likely that other cytoskeletal regulatory proteins besides Rac1 are involved in endoderm morphogenesis. Indeed, in our microarray analysis, we identified several genes associated with cell migration and cytoskeletal dynamics as potential targets of Nodal signaling (Fig. S3 A). In addition, a study using a proteomics-based approach identified at least four cytoskeleton-associated proteins that are differentially regulated between mesendodermal and ectodermal cells (Link et al., 2006
); one of these proteins, Ezrin, was demonstrated to function during the migration of prechordal plate progenitor cells by regulating membrane protrusion (Diz-Muñoz et al., 2010
). Future studies will no doubt identify additional cytoskeletal regulators important for tissue morphogenesis and organ development.
In this study, we provide evidence that prex1
is transcriptionally regulated by Nodal signaling. However, GEFs are also subject to posttranscriptional regulation. Although most GEFs are regulated by phosphorylation (Rossman et al., 2005
), Prex1 is synergistically activated by PIP3
and Gβγ (Welch et al., 2002
; Barber et al., 2007
; Zhao et al., 2007
). In neutrophils, Prex1 is thought to act as a coincidence detector that allows for high levels of Rac activation when both second messengers are generated (Weiner, 2002
), as occurs when G-protein–coupled chemokine receptors are activated (Stephens et al., 1997
). Zebrafish endodermal cells also express chemokine receptors, primarily Cxcr4a (Mizoguchi et al., 2008
; Nair and Schilling, 2008
). SDF-1–Cxcr4 signaling in primordial germ cells was recently shown to activate Rac1 in a Gβγ-dependent manner (Xu et al., 2012
), making it very likely that Prex1 lies directly in this signaling pathway. However, in terms of endoderm development, several questions remain about the role of Prex1. First, to what extent are both PIP3
and Gβγ necessary for Prex1 function in vivo? Mizoguchi et al. (2008)
suggested that phosphoinositide signaling is not highly active in migrating endodermal cells, and it may be possible to activate Prex1 with Gβγ alone, especially under conditions of low PIP3
concentrations (Welch et al., 2002
). If PIP3
and/or Gβγ are required for full Prex1 activity, are they generated downstream of receptors such as Cxcr4, and, if so, how do those signaling pathways interact with Nodal signaling? Given that most studies of Prex1 to date have used neutrophils in culture, the developing zebrafish endoderm may represent a useful system to probe important questions about Prex1 function in vivo.
In the double transplantation experiments, we observed that some cells in which random migration was suppressed by DN Rac1 expression seemed unable to maintain endodermal identity and instead contributed to mesodermal tissues. Although we interpret these results as being a result of the suppression of random migration during early gastrulation, it is also possible that DN Rac1 impairs cell movements before gastrulation, such as epiboly and ingression, which could aberrantly place cells in the mesodermal layer. However, although we did observe some endodermal cells that apparently failed to ingress in Prex1 MO–injected embryos, we did not see a similar effect with the low-level DN Rac expression used throughout this study, suggesting that pregastrulation movements are relatively unaffected. Thus, based on our time-lapse analyses, we propose that DN Rac1 expression precociously induces persistent migration, causing cells to more efficiently reach the dorsal side of the embryo. Once there, they may inappropriately interact with mesodermal cells or mesoderm differentiation signals. It is also possible that Rac1 is required for later aspects of endoderm morphogenesis, such as cell–cell adhesion during endodermal sheet formation, which may also affect the ability of Rac-deficient cells to remain within the endoderm.
The ability of cells to switch their migratory behavior has been observed in many different cell types and model systems (Bak and Fraser, 2003
; Wolf et al., 2003
; Pankov et al., 2005
; Pézeron et al., 2008
; Sanz-Moreno et al., 2008
). In general, it is thought that random migration plays either a dispersive or exploratory role, whereas persistent migration promotes rapid and efficient translocation. The need for multiple modes of migration may be crucial not only during development but in the adult as well. Most notably, processes such as wound healing and axon regeneration require cells to switch from a stationary state to a migratory one. Additionally, different types of invasive tumor cells are characterized by different migratory behaviors (Madsen and Sahai, 2010
); some cells are even able to switch between multiple migration modes (Sanz-Moreno et al., 2008
), which can impact the efficacy of drugs meant to block metastasis (Wolf et al., 2003
; Micuda et al., 2010
). Therefore, the findings presented in this study have clear implications beyond developmental processes.