Activity of the RhoU/Wrch1 GTPase is critical for cranial neural crest cell migration

The neural crest (NC) is a stem cell-like population that arises at the border of neural and non-neural ectoderm. During development, NC undergoes an epithelio-mesenchymal transition (EMT), i.e. loss of epithelial junctions and acquisition of pro-migratory properties, invades the entire embryo and differentiates into a wide diversity of terminal tissues. We have studied the implication of Rho pathways in NC development and previously showed that RhoV is required for cranial neural crest (CNC) cell specification. We show here that the non-canonical Wnt response rhoU/wrch1 gene, closely related to rhoV, is also expressed in CNC cells but at later stages. Using both gain- and loss-of-function experiments, we demonstrate that the level of RhoU expression is critical for CNC cell migration and subsequent differentiation into craniofacial cartilages. In in vitro cultures, RhoU activates pathways that cooperate with PAK1 and Rac1 in epithelial adhesion, cell spreading and directional cell migration. These data support the conclusion that RhoU is an essential regulator of CNC cell migration.     

Rho GTPase Expression in Human Myeloid Cells

Myeloid cells are critical for innate immunity and the initiation of adaptive immunity. Strict regulation of the adhesive and migratory behavior is essential for proper functioning of these cells. Rho GTPases are important regulators of adhesion and migration; however, it is unknown which Rho GTPases are expressed in different myeloid cells. Here, we use a qPCR-based approach to investigate Rho GTPase expression in myeloid cells.We found that the mRNAs encoding Cdc42, RhoQ, Rac1, Rac2, RhoA and RhoC are the most abundant. In addition, RhoG, RhoB, RhoF and RhoV are expressed at low levels or only in specific cell types. More differentiated cells along the monocyte-lineage display lower levels of Cdc42 and RhoV, while RhoC mRNA is more abundant. In addition, the Rho GTPase expression profile changes during dendritic cell maturation with Rac1 being upregulated and Rac2 downregulated. Finally, GM-CSF stimulation, during macrophage and osteoclast differentiation, leads to high expression of Rac2, while M-CSF induces high levels of RhoA, showing that these cytokines induce a distinct pattern. Our data uncover cell type specific modulation of the Rho GTPase expression profile in hematopoietic stem cells and in more differentiated cells of the myeloid lineage.     

Signals derived from the underlying mesoderm are dispensable for zebrafish neural crest induction

Signals from the non-neural ectoderm, the neural ectoderm, and the underlying mesoderm have all been implicated in the induction of neural crest. Bone morphogenetic protein (BMP) signaling in particular has an important role in this process; however, it is unclear whether this activity of BMP is due to its effects on patterning the underlying mesoderm, to its ability to establish a competent neural plate boundary zone, or to the direct specification of neural crest at intermediate levels of activity within a BMP gradient. We show neural crest induction occurs in zebrafish in the absence of involuted mesoderm, indicating that this tissue and signals derived from it are dispensable for the formation of neural crest. Dorsal-involuted mesoderm is a major source of secreted BMP antagonists, and the activity of BMP signaling is thought to depend on the presence of the opposing activity of these antagonists. We find that the three BMP antagonists known to be expressed during gastrulation in zebrafish, noggin1, follistatin, and chordin, are dispensable for neural crest induction. These results suggest that mechanisms for restricting the spatio-temporal pattern of BMP expression may compensate for the loss of secreted BMP antagonist activity in establishing dorso-ventral patterning, neural induction, and the neural crest.     

Neural crest development is regulated by the transcription factor Sox9

The neural crest is a transient migratory population of stem cells derived from the dorsal neural folds at the border between neural and non-neural ectoderm. Following induction, prospective neural crest cells are segregated within the neuroepithelium and then delaminate from the neural tube and migrate into the periphery, where they generate multiple differentiated cell types. The intrinsic determinants that direct this process are not well defined. Group E Sox genes (Sox8, Sox9 and Sox10) are expressed in the prospective neural crest and Sox9 expression precedes expression of premigratory neural crest markers. Here, we show that group E Sox genes act at two distinct steps in neural crest differentiation. Forced expression of Sox9 promotes neural-crest-like properties in neural tube progenitors at the expense of central nervous system neuronal differentiation. Subsequently, in migratory neural crest cells, SoxE gene expression biases cells towards glial cell and melanocyte fate, and away from neuronal lineages. Although SoxE genes are sufficient to initiate neural crest development they do not efficiently induce the delamination of ectopic neural crest cells from the neural tube consistent with the idea that this event is independently controlled. Together, these data identify a role for group E Sox genes in the initiation of neural crest development and later SoxE genes influence the differentiation pathway adopted by migrating neural crest cells.     

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.     

Reiterated Wnt signaling during zebrafish neural crest development

While Wnt/beta-catenin signaling is known to be involved in the development of neural crest cells in zebrafish, it is unclear which Wnts are involved, and when they are required. To address these issues we employed a zebrafish line that was transgenic for an inducible inhibitor of Wnt/beta-catenin signaling, and inhibited endogenous Wnt/beta-catenin signaling at discrete times in development. Using this approach, we defined a critical period for Wnt signaling in the initial induction of neural crest, which is distinct from the later period of development when pigment cells are specified from neural crest. Blocking Wnt signaling during this early period interfered with neural crest formation without blocking development of dorsal spinal neurons. Transplantation experiments suggest that neural crest precursors must directly transduce a Wnt signal. With regard to identifying which endogenous Wnt is responsible for this initial critical period, we established that wnt8 is expressed in the appropriate time and place to participate in this process. Supporting a role for Wnt8, blocking its function with antisense morpholino oligonucleotides eliminates initial expression of neural crest markers. Taken together, these results demonstrate that Wnt signals are critical for the initial induction of zebrafish neural crest and suggest that this signaling pathway plays reiterated roles in its development.     

Neural crest induction by the canonical Wnt pathway can be dissociated from anterior-posterior neural patterning in Xenopus

While Wnt signaling is known to be involved in early steps of neural crest development, the mechanism remains unclear. Because Wnt signaling is able to posteriorize anterior neural tissues, neural crest induction by Wnts has been proposed to be an indirect consequence of posteriorization of neural tissues rather than a direct effect of Wnt signaling. To address the relationship between posteriorization and neural crest induction by Wnt signaling, we have used gain of function and loss of function approaches in Xenopus to modulate the level of Wnt signaling at multiple points in the pathway. We find that modulating the level of Wnt signaling allows separation of neural crest induction from the effects of Wnts on anterior-posterior neural patterning. We also find that activation of Wnt signaling induces ectopic neural crest in the anterior region without posteriorizing anterior neural tissues. In addition, Wnt signaling induces neural crest when its posteriorizing activity is blocked by inhibition of FGF signaling in neuralized explants. Finally, depletion of beta-catenin confirms that the canonical Wnt pathway is required for initial neural crest induction. While these observations do not exclude a role for posteriorizing signals in neural crest induction, our data, together with previous observations, strongly suggest that canonical Wnt signaling plays an essential and direct role in neural crest induction.     

Early expression of Tubulin Beta-III in avian cranial neural crest cells

Neural crest cells are a transient stem-like cell population that forms in the dorsal neural tube of vertebrate embryos and then migrates to various locations to differentiate into diverse derivatives such as craniofacial bone, cartilage, and the enteric and peripheral nervous systems. The current dogma of neural crest cell development suggests that there is a specific hierarchical gene regulatory network (GRN) that controls the induction, specification, and differentiation of these cells at specific developmental times. Our lab has identified that a marker of differentiated neurons, Tubulin Beta-III (TUBB3), is expressed in premigratory neural crest cells. TUBB3 has previously been identified as a major constituent of microtubules and is required for the proper guidance and maintenance of axons during development. Using the model organism, Gallus gallus, we have characterized the spatiotemporal localization of TUBB3 in early stages of development. Here we show TUBB3 is expressed in the developing neural plate, is upregulated in the pre-migratory cranial neural crest prior to cell delamination and migration, and it is maintained or upregulated in neurons in later developmental stages. We believe that TUBB3 likely has a role in early neural crest formation and migration separate from its role in neurogenesis.     

Frizzled7 mediates canonical Wnt signaling in neural crest induction

The neural crest is a multipotent cell population that migrates from the dorsal edge of the neural tube to various parts of the embryo where it differentiates into a remarkable variety of different cell types. Initial induction of neural crest is mediated by a combination of BMP, Wnt, FGF, Retinoic acid and Notch/Delta signaling. The two-signal model for neural crest induction suggests that BMP signaling induces the competence to become neural crest. The second signal involves Wnt acting through the canonical pathway and leads to expression of neural crest markers such as slug. Wnt signals from the neural plate, non-neural ectoderm and paraxial mesoderm have all been suggested to play a role in neural crest induction. We show that Xenopus frizzled7 (Xfz7) is expressed in the dorsal ectoderm including early neural crest progenitors and is a key mediator of the Wnt inductive signal. We demonstrate that Xfz7 expression is induced in response to a BMP antagonist, noggin, and that Xfz7 can induce neural crest specific genes in noggin-treated ectodermal explants (animal caps). Morpholino-mediated or dominant negative inhibition of Xfz7 inhibits Wnt induced Xslug expression in the animal cap assay and in the whole embryo leading to a loss of neural crest derived pigment cells. Full-length Xfz7 rescues the morpholino-induced phenotype, as does activated beta-catenin, suggesting that Xfz7 is signaling through the canonical pathway. We therefore demonstrate that Xfz7 is regulated by BMP antagonism and is required for neural crest induction by Wnt in the developing vertebrate embryo.     

Mutations in the Effector Domain of RhoV GTPase Impair Its Binding to Pak1 Protein Kinase

Atypical RhoV GTPase (Chp/Wrch-2) is a member of the human Rho GTPase family, which belongs to the superfamily of Ras-related small GTPases. The biological functions of RhoV, regulation of its activity, and mechanisms of its action remain largely unexplored. Rho GTPases regulate a wide range of cellular processes by interacting with protein targets called effectors. Several putative RhoV effectors have been identified, including protein kinases of the Pak (p21-activated kinase) family: Pak1, Pak2, Pak4, and Pak6. RhoV GTPase activates Pak1 protein kinase and simultaneously induces its ubiquitin-dependent degradation. Pak1 regulates E-cadherin localization at adherens junctions downstream of RhoV during gastrulation in fish. The effector domain of RhoV mediates its binding to the CRIB (Cdc42/Rac1 interactive binding) motif in the N-terminal p21-binding domain (PBD) of Pak6 protein kinase. The role of the RhoV effector domain in mediating interaction with Pak1 has not been studied. This study has identified mutations in the effector domain of RhoV GTPase (Y60K, T63A, L65A, and D66A) that impair its interaction with Pak1 in the GST-PAK-PBD pull-down assay and coimmunoprecipitation. Our results suggest that the effector domain of RhoV mediates its binding to Pak1, complementing the current view of the molecular basics of RhoV binding to effectors of the Pak family. These data lay the basis for further studies on the role of Pak1 in RhoV-activated signaling pathways and cellular processes.