Roberto Mayor

Contact inhibition of locomotion in vivo controls neural crest directional migration

Contact inhibition of locomotion was discovered by Abercrombie more than 50 years ago and describes the behaviour of fibroblast cells confronting each other in vitro, where they retract their protrusions and change direction on contact. Its failure was suggested to contribute to malignant invasion. However, the molecular basis of contact inhibition of locomotion and whether it also occurs in vivo are still unknown. Here we show that neural crest cells, a highly migratory and multipotent embryonic cell population, whose behaviour has been likened to malignant invasion, demonstrate contact inhibition of locomotion both in vivo and in vitro, and that this accounts for their directional migration. When two migrating neural crest cells meet, they stop, collapse their protrusions and change direction. In contrast, when a neural crest cell meets another cell type, it fails to display contact inhibition of locomotion; instead, it invades the other tissue, in the same manner as metastatic cancer cells. We show that inhibition of non-canonical Wnt signalling abolishes both contact inhibition of locomotion and the directionality of neural crest migration. Wnt-signalling members localize at the site of cell contact, leading to activation of RhoA in this region. These results provide the first example of contact inhibition of locomotion in vivo, provide an explanation for coherent directional migration of groups of cells and establish a previously unknown role for non-canonical Wnt signalling.

Collective Chemotaxis Requires Contact-Dependent Cell Polarity

Summary Directional collective migration is now a widely recognized mode of migration during embryogenesis and cancer. However, how a cluster of cells responds to chemoattractants is not fully understood. Neural crest cells are among the most motile cells in the embryo, and their behavior has been likened to malignant invasion. Here, we show that neural crest cells are collectively attracted toward the chemokine Sdf1. While not involved in initially polarizing cells, Sdf1 directionally stabilizes cell protrusions promoted by cell contact. At this cell contact, N-cadherin inhibits protrusion and Rac1 activity and in turn promotes protrusions and activation of Rac1 at the free edge. These results show a role for N-cadherin during contact inhibition of locomotion, and they reveal a mechanism of chemoattraction likely to function during both embryogenesis and cancer metastasis, whereby attractants such as Sdf1 amplify and stabilize contact-dependent cell polarity, resulting in directional collective migration.

THE INDUCTIVE PROPERTIES OF MESODERM SUGGEST THAT THE NEURAL CREST CELLS ARE SPECIFIED BY A BMP GRADIENT

We have analyzed the role of mesoderm in the induction of the neural crest in Xenopus using expression of neural plate (Xsox-2) and neural crest (Xslug and ADAM). Conjugation experiments using different kinds of mesoderm together with embryonic dissection experiments suggest that the dorsolateral mesoderm is capable of specifically inducing neural crest cells. Neural crest markers can be induced in competent ectoderm at varying distances from the inducing mesoderm, with dorsal tissue inducing neural crest at a distance while dorsolateral tissue only induces neural crest directly in adjacent ectoderm. The results suggest that dorsal mesoderm has a high level of inducer and dorsolateral mesoderm has a lower level, consistent with a inductive gradient. We explored the possible role of BMP and noggin in the generation of such a hypothetical gradient and found that: (1) progressively higher levels of BMP activity are sufficient for the specification of neural plate, neural crest, and nonneural cells, respectively; (2) progressively higher levels of noggin are able to induce neural crest at greater distances from the source of inducer; and (3) modification of the levels of BMP activity causes induction of the neural crest in absence of neural plate, suggesting independent induction of these two tissues. We propose a model in which a gradient of BMP activity is established in the ectoderm by interaction between BMP in the ectoderm and BMP inhibitors in the mesoderm. Neural crest is induced when a threshold level of BMP is attained in the ectoderm. The dorsolateral mesoderm produces either BMP inhibitors or a specific neural crest inducer, with low BMP activity inducing neural plate while high BMP activity induces epidermis.

Regulation of Msx genes by a Bmp gradient is essential for neural crest specification

There is evidence in Xenopus and zebrafish embryos that the neural crest/neural folds are specified at the border of the neural plate by a precise threshold concentration of a Bmp gradient. In order to understand the molecular mechanism by which a gradient of Bmp is able to specify the neural crest, we analyzed how the expression of Bmp targets, the Msx genes, is regulated and the role that Msx genes has in neural crest specification. As Msx genes are directly downstream of Bmp, we analyzed Msx gene expression after experimental modification in the level of Bmp activity by grafting a bead soaked with noggin into Xenopus embryos, by expressing in the ectoderm a dominant-negative Bmp4 or Bmp receptor in Xenopus and zebrafish embryos, and also through Bmp pathway component mutants in the zebrafish. All the results show that a reduction in the level of Bmp activity leads to an increase in the expression of Msx genes in the neural plate border. Interestingly, by reaching different levels of Bmp activity in animal cap ectoderm, we show that a specific concentration of Bmp induces msx1 expression to a level similar to that required to induce neural crest. Our results indicate that an intermediate level of Bmp activity specifies the expression of Msx genes in the neural fold region. In addition, we have analyzed the role that msx1 plays on neural crest specification. As msx1 has a role in dorsoventral pattering, we have carried out conditional gain- and loss-of-function experiments using different msx1 constructs fused to a glucocorticoid receptor element to avoid an early effect of this factor. We show that msx1 expression is able to induce all other early neural crest markers tested (snail, slug, foxd3) at the time of neural crest specification. Furthermore, the expression of a dominant negative of Msx genes leads to the inhibition of all the neural crest markers analyzed. It has been previously shown that snail is one of the earliest genes acting in the neural crest genetic cascade. In order to study the hierarchical relationship between msx1 and snail/slug we performed several rescue experiments using dominant negatives for these genes. The rescuing activity by snail and slug on neural crest development of the msx1 dominant negative, together with the inability of msx1 to rescue the dominant negatives of slug and snail strongly argue that msx1 is upstream of snail and slug in the genetic cascade that specifies the neural crest in the ectoderm. We propose a model where a gradient of Bmp activity specifies the expression of Msx genes in the neural folds, and that this expression is essential for the early specification of the neural crest.

Neural crest delamination and migration: From epithelium-to-mesenchyme transition to collective cell migration

After induction and specification in the ectoderm, at the border of the neural plate, the neural crest (NC) population leaves its original territory through a delamination process. Soon afterwards, the NC cells migrate throughout the embryo and colonize a myriad of tissues and organs where they settle and differentiate. The delamination involves a partial or complete epithelium-to-mesenchyme transition (EMT) regulated by a complex network of transcription factors including several proto-oncogenes. Studying the relationship between these genes at the time of emigration, and their individual or collective impact on cell behavior, provides valuable information about their role in EMT in other contexts such as cancer metastasis. During migration, NC cells are exposed to large number of positive and negative regulators that control where they go by generating permissive and restricted areas and by modulating their motility and directionality. In addition, as most NC cells migrate collectively, cell-cell interactions play a crucial role in polarizing the cells and interpreting external cues. Cell cooperation eventually generates an overall polarity to the population, leading to directional collective cell migration. This review will summarize our current knowledge on delamination, EMT and migration of NC cells using key examples from chicken, Xenopus, zebrafish and mouse embryos. Given the similarities between neural crest migration and cancer invasion, these cells may represent a useful model for understanding the mechanisms of metastasis.

Essential role of non-canonical Wnt signalling in neural crest migration

Migration of neural crest cells is an elaborate process that requires the delamination of cells from an epithelium and cell movement into an extracellular matrix. In this work, it is shown for the first time that the non-canonical Wnt signalling [planar cell polarity (PCP) or Wnt-Ca2+] pathway controls migration of neural crest cells. By using specific Dsh mutants, we show that the canonical Wnt signalling pathway is needed for neural crest induction, while the non-canonical Wnt pathway is required for neural crest migration. Grafts of neural crest tissue expressing non-canonical Dsh mutants, as well as neural crest cultured in vitro, indicate that the PCP pathway works in a cell-autonomous manner to control neural crest migration. Expression analysis of non-canonical Wnt ligands and their putative receptors show that Wnt11 is expressed in tissue adjacent to neural crest cells expressing the Wnt receptor Frizzled7 (Fz7). Furthermore, loss- and gain-of-function experiments reveal that Wnt11 plays an essential role in neural crest migration. Inhibition of neural crest migration by blocking Wnt11 activity can be rescued by intracellular activation of the non-canonical Wnt pathway. When Wnt11 is expressed opposite its normal site of expression, neural crest migration is blocked. Finally, time-lapse analysis of cell movement and cell protrusion in neural crest cultured in vitro shows that the PCP or Wnt-Ca2+ pathway directs the formation of lamellipodia and filopodia in the neural crest cells that are required for their delamination and/or migration.

Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA

Directed cell migration is crucial for development, but most of our current knowledge is derived from in vitro studies. We analyzed how neural crest (NC) cells migrate in the direction of their target during embryonic development. We show that the proteoglycan Syndecan-4 (Syn4) is expressed in the migrating neural crest of Xenopus and zebrafish embryos. Loss-of-function studies using an antisense morpholino against syn4 show that this molecule is required for NC migration, but not for NC induction. Inhibition of Syn4 does not affect the velocity of cell migration, but significantly reduces the directional migration of NC cells. Furthermore, we show that Syn4 and PCP signaling control the directional migration of NC cells by regulating the direction in which the cell protrusions are generated during migration. Finally, we perform FRET analysis of Cdc42, Rac and RhoA in vitro and in vivo after interfering with Syn4 and PCP signaling. This is the first time that FRET analysis of small GTPases has been performed in vivo. Our results show that Syn4 inhibits Rac activity, whereas PCP signaling promotes RhoA activity. In addition, we show that RhoA inhibits Rac in NC cells. We present a model in which Syn4 and PCP control directional NC migration by, at least in part, regulating membrane protrusions through the regulation of small GTPase activities.

Snail precedes slug in the genetic cascade required for the specification and migration of the Xenopus neural crest.

The complex sequence of inductive events responsible for the generation of the neural crest at the border between the neural plate and the epidermis, triggers a genetic cascade involving several families of transcription factors. Two members of the Snail family, Snail and Slug, have both been implicated in this cascade. In chick and Xenopus, loss- and gain-of-function experiments have provided evidence that Slug plays a key role in neural crest development. However, in contrast to the chick, Snail rather than Slug is expressed in the premigratory neural crest in the mouse and, in Xenopus, Snail precedes Slug expression in this population. Thus, in order to study the function of Snail in neural crest development in Xenopus, we have carried out conditional gain- and loss-of-function experiments using different Snail constructs fused to a glucocorticoid receptor element. We show that Snail is able to induce the expression of Slug and all other neural crest markers tested (Zic5, FoxD3, Twist and Ets1) at the time of specification. This activation is observed in whole embryos and in animal caps, in the absence of neural plate and mesodermal markers. We show that Snail is required for neural crest specification and migration and that it works as a transcriptional repressor. These functions have been previously attributed to Slug. However, Slug alone is unable to induce other neural crest markers in animal cap assays, and we show that Snail and Slug can be functionally equivalent when tested in overexpression studies. This suggests that, in Xenopus embryos, at least some of the functions previously attributed to Slug can be carried out by Snail. This is additionally supported by rescue experiments in embryos injected with dominant-negative constructs that indicate that Snail lies upstream of Slug in the genetic cascade leading to neural crest formation and that it plays a key role in crest development.

Sox10 is required for the early development of the prospective neural crest in Xenopus embryos

The Sox family of transcription factors has been implicated in the development of different tissues during embryogenesis. Several mutations in humans, mice, and zebrafish have shown that depletion of Sox10 activity produces defects in the development of neural crest derivatives, such as melanocytes, ganglia of the peripheral nervous system, and some specific cell types as glia. We have isolated the Xenopus homologue of the Sox10 gene. It is expressed in prospective neural crest and otic placode regions from the earliest stages of neural crest specification and in migrating cranial and trunk neural crest cells. Loss-of-function experiments using morpholino antisense oligos against Sox10 produce a loss of neural crest precursors and an enlargement of the surrounding neural plate and epidermis. This effect of Sox10 depletion is produced during some of the earliest steps of neural crest specification, as is shown by the inhibition in the expression of Slug and FoxD3, which are early markers of neural crest specification. In addition, we show that Sox10 depletion leads to an increase in apoptosis and a decrease in cell proliferation in the neural folds, suggesting that Sox10 could work as a survival as well as a specification factor in neural crest precursors during premigratory stages. Although some of the deficiencies found in the Waardenburg syndrome and in the Hirschprung disease could be associated with a failure of the development of crest derivatives during the late phase of its development, or even during adulthood, our results suggest that inhibition of Sox10 activity produces an earlier failure of neural crest precursors. In experiments where melanocytes and ganglia were induced in vivo and in vitro, we were able to block their development by inhibiting Sox10 activity. These results are compatible with an additional late role of Sox10 on development of neural crest derivatives, as it has been previously proposed. We show that Sox10 expression is dependent on FGF and Wnt activity, both in the neural crest and in the otic placode territories. Finally, in order to establish the position of Sox10 in the hierarchical cascade of gene activation required for neural crest specification, we used inducible forms of the wild type and dominant negatives for the Snail and Slug genes. Our results show that Snail is able to control Sox10 expression. However, the overexpression of Slug was not able to upregulate Sox10 expression. Taken together, these results indicate that Sox10 may lie between Snail and Slug in the genetic cascade that controls neural crest development.

Complement Fragment C3a Controls Mutual Cell Attraction during Collective Cell Migration

Summary Collective cell migration is a mode of movement crucial for morphogenesis and cancer metastasis. However, little is known about how migratory cells coordinate collectively. Here we show that mutual cell-cell attraction (named here coattraction) is required to maintain cohesive clusters of migrating mesenchymal cells. Coattraction can counterbalance the natural tendency of cells to disperse via mechanisms such as contact inhibition and epithelial-to-mesenchymal transition. Neural crest cells are coattracted via the complement fragment C3a and its receptor C3aR, revealing an unexpected role of complement proteins in early vertebrate development. Loss of coattraction disrupts collective and coordinated movements of these cells. We propose that coattraction and contact inhibition act in concert to allow cell collectives to self-organize and respond efficiently to external signals, such as chemoattractants and repellents.