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

Abstract

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.

The effects of modifying RhoA and Rac1 activities on heterotypic contact inhibition of locomotion

“…However, neither dominant active nor dominant negative Cdc42 influenced the heterotypic CIL of NIH3T3 cells in our system. Similarly, Cdc42 does not appear to be involved in the polarization and protrusion formation of NC cells [38].…”

Directional cell migration in vivo

“…[8][9][10] The neural crest is an embryonic population of cells that are specified at the border between the neural plate and the epidermis. 11 Upon induction neural crest cells undergo an epithelial to mesenchymal transition, 12 detach from the neural tube and migrate following defined pathways that eventually allow them to colonize almost the entire embryo.…”

“…More importantly, we have been able to show that the same molecular machinery controls directional migration in vivo. 9,10 One of the key factors that controls directional migration of NC cells is the Planar Cell Polarity (PCP) or non-canonical Wnt signaling pathway. 9,10,16 PCP signaling was first described in Drosophila, where a number of mutations were identified that disrupt the formation of bristles and hairs on the adult cuticle.…”

“…9 Recently this role has also been extended to zebrafish where directional migration of neural crest is severely disrupted in the PCP mutant trilobite (strabismus) and in embryos injected with a dominant negative form of Dsh or a morpholino against wnt5a, 10 with no effect in neural crest cell motility. 9,10 Two factors, pescadillo and syndecan-4 that have recently been proposed as modulators of the PCP signaling, 20,21 are also required for NC migration. 10,21 Taken together, these data point to an essential role for PCP signaling in neural crest migration.…”

“…9,10 Two factors, pescadillo and syndecan-4 that have recently been proposed as modulators of the PCP signaling, 20,21 are also required for NC migration. 10,21 Taken together, these data point to an essential role for PCP signaling in neural crest migration.…”