Mmp17b Is Essential for Proper Neural Crest Cell Migration In Vivo

The extracellular matrix plays a critical role in neural crest (NC) cell migration. In this study, we characterize the contribution of the novel GPI-linked matrix metalloproteinase (MMP) zebrafish mmp17b. Mmp17b is expressed post-gastrulation in the developing NC. Morpholino inactivation of mmp17b function, or chemical inhibition of MMP activity results in aberrant NC cell migration with minimal change in NC proliferation or apoptosis. Intriguingly, a GPI anchored protein with metalloproteinase inhibitor properties, Reversion-inducing-Cysteine-rich protein with Kazal motifs (RECK), which has previously been implicated in NC development, is expressed in close apposition to NC cells expressing mmp17b, raising the possibility that these two gene products interact. Consistent with this possibility, embryos silenced for mmp17b show defective development of the dorsal root ganglia (DRG), a crest-derived structure affected in RECK mutant fish sensory deprived (sdp). Taken together, this study has identified the first pair of MMP, and their putative MMP inhibitor RECK that functions together in NC cell migration.     

DNA Methyltransferase 3b Is Dispensable for Mouse Neural Crest Development

The neural crest is a population of multipotent cells that migrates extensively throughout vertebrate embryos to form diverse structures. Mice mutant for the de novo DNA methyltransferase DNMT3b exhibit defects in two neural crest derivatives, the craniofacial skeleton and cardiac ventricular septum, suggesting that DNMT3b activity is necessary for neural crest development. Nevertheless, the requirement for DNMT3b specifically in neural crest cells, as opposed to interacting cell types, has not been determined. Using a conditional DNMT3b allele crossed to the neural crest cre drivers Wnt1-cre and Sox10-cre, neural crest DNMT3b mutants were generated. In both neural crest-specific and fully DNMT3b-mutant embryos, cranial neural crest cells exhibited only subtle migration defects, with increased numbers of dispersed cells trailing organized streams in the head. In spite of this, the resulting cranial ganglia, craniofacial skeleton, and heart developed normally when neural crest cells lacked DNMT3b. This indicates that DNTM3b is not necessary in cranial neural crest cells for their development. We conclude that defects in neural crest derivatives in DNMT3b mutant mice reflect a requirement for DNMT3b in lineages such as the branchial arch mesendoderm or the cardiac mesoderm that interact with neural crest cells during formation of these structures.     

Beta-Actin Is Required for Proper Mouse Neural Crest Ontogeny

The mouse genome consists of six functional actin genes of which the expression patterns are temporally and spatially regulated during development and in the adult organism. Deletion of beta-actin in mouse is lethal during embryonic development, although there is compensatory expression of other actin isoforms. This suggests different isoform specific functions and, more in particular, an important function for beta-actin during early mammalian development. We here report a role for beta-actin during neural crest ontogeny. Although beta-actin null neural crest cells show expression of neural crest markers, less cells delaminate and their migration arrests shortly after. These phenotypes were associated with elevated apoptosis levels in neural crest cells, whereas proliferation levels were unchanged. Specifically the pre-migratory neural crest cells displayed higher levels of apoptosis, suggesting increased apoptosis in the neural tube accounts for the decreased amount of migrating neural crest cells seen in the beta-actin null embryos. These cells additionally displayed a lack of membrane bound N-cadherin and dramatic decrease in cadherin-11 expression which was more pronounced in the pre-migratory neural crest population, potentially indicating linkage between the cadherin-11 expression and apoptosis. By inhibiting ROCK ex vivo, the knockout neural crest cells regained migratory capacity and cadherin-11 expression was upregulated. We conclude that the presence of beta-actin is vital for survival, specifically of pre-migratory neural crest cells, their proper emigration from the neural tube and their subsequent migration. Furthermore, the absence of beta-actin affects cadherin-11 and N-cadherin function, which could partly be alleviated by ROCK inhibition, situating the Rho-ROCK signaling in a feedback loop with cadherin-11.     

The Wnt Co-Receptor Lrp5 Is Required for Cranial Neural Crest Cell Migration in Zebrafish

During vertebrate neurulation, cranial neural crest cells (CNCCs) undergo epithelial to mesenchymal transition (EMT), delaminate from the neural plate border, and migrate as separate streams into different cranial regions. There, they differentiate into distinct parts of the craniofacial skeleton. Canonical Wnt signaling has been shown to be essential for this process at different levels but the involved receptors remained unclear. Here we show that the frizzled co-receptor low-density-lipoprotein (LDL) receptor-related protein 5 (Lrp5) plays a crucial role in CNCC migration and morphogenesis of the cranial skeleton. Early during induction and migration of CNCCs, lrp5 is expressed ubiquitously but later gets restricted to CNCC derivatives in the ventral head region besides different regions in the CNS. A knock-down of lrp5 does not interfere with induction of CNCCs but leads to reduced proliferation of premigratory CNCCs. In addition, cell migration is disrupted as CNCCs are found in clusters at ectopic positions in the dorsomedial neuroepithelium after lrp5 knock-down and transient CRISPR/Cas9 gene editing. These migratory defects consequently result in malformations of the craniofacial skeleton. To date, Lrp5 has mainly been associated with bone homeostasis in mammals. Here we show that in zebrafish, lrp5 also controls cell migration during early morphogenetic processes and contributes to shaping the craniofacial skeleton.     

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.Download video file.^{(94M, mp4)}     

Neural Crest Cell Fate

The neural crest, the intriguing cell population that gives rise to a panoply of derivatives in the vertebrate embryo, including the mesenchymal structures in the head, melanocytes and most of the peripheral nervous system, still proves to be an important yet enigmatic developmental cell population to study with applications in stem cell biology, cancer biology and clinical medicine. Albeit our knowledge base is rich due to a strong history of experimentation, the fact that we have yet to decipher so many key aspects of neural crest cell (NCC) behavior speaks to the challenging complexity of this transient yet vital cell population. With the advent of new fluorescent tracing techniques, we have reexamined the migratory behaviors and ultimate fate of ventrally migrating avian NCCs within a late wave of emigration and identified a subpopulation of lineally restricted NCCs who migrate to the contralateral dorsal root ganglia (DRG) and therein give rise to mitotically active progenitor cells that ultimately produce the majority of the nociceptive sensory neurons in the DRG. These data provide evidence for the fate prespecification of subsets of NCCs while still resident in the neural tube.     

Cadherin-11 Mediates Contact Inhibition of Locomotion during Xenopus Neural Crest Cell Migration

Collective cell migration is an essential feature both in embryonic development and cancer progression. The molecular mechanisms of these coordinated directional cell movements still need to be elucidated. The migration of cranial neural crest (CNC) cells during embryogenesis is an excellent model for collective cell migration in vivo. These highly motile and multipotent cells migrate directionally on defined routes throughout the embryo. Interestingly, local cell-cell interactions seem to be the key force for directionality. CNC cells can change their migration direction by a repulsive cell response called contact inhibition of locomotion (CIL). Cell protrusions collapse upon homotypic cell-cell contact and internal repolarization leads to formation of new protrusions toward cell-free regions. Wnt/PCP signaling was shown to mediate activation of small RhoGTPase RhoA and inhibition of cell protrusions at the contact side. However, the mechanism how a cell recognizes the contact is poorly understood. Here, we demonstrate that Xenopus cadherin-11 (Xcad-11) mediated cell-cell adhesion is necessary in CIL for directional and collective migration of CNC cells. Reduction of Xcad-11 adhesive function resulted in higher invasiveness of CNC due to loss of CIL. Additionally, transplantation analyses revealed that CNC migratory behaviour in vivo is non-directional and incomplete when Xcad-11 adhesive function is impaired. Blocking Wnt/PCP signaling led to similar results underlining the importance of Xcad-11 in the mechanism of CIL and directional migration of CNC.     

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition ^1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells ^1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components ³. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration ⁴. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands ^5-8. However, not until recently have any chemoattractants of trunk NCCs been identified ⁹. Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere¹⁰). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.     

Calponin 2 Acts As an Effector of Noncanonical Wnt-Mediated Cell Polarization during Neural Crest Cell Migration

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Highlights? Cnn2 is expressed in NCCs and required for their migration in frogs and chicks ? Cnn2 is inactivated by noncanonical Wnt signaling ? Loss of Cnn2 causes a switch from cortical actin to central stress fibers ? Cnn2 polarizes the actin cytoskeleton downstream of PCP     

A Novel Role for Lh3 Dependent ECM Modifications during Neural Crest Cell Migration in Zebrafish

During vertebrate development, trunk neural crest cells delaminate along the entire length of the dorsal neural tube and initially migrate as a non-segmented sheet. As they enter the somites, neural crest cells rearrange into spatially restricted segmental streams. Extracellular matrix components are likely to play critical roles in this transition from a sheet-like to a stream-like mode of migration, yet the extracellular matrix components and their modifying enzymes critical for this transition are largely unknown. Here, we identified the glycosyltransferase Lh3, known to modify extracellular matrix components, and its presumptive substrate Collagen18A1, to provide extrinsic signals critical for neural crest cells to transition from a sheet-like migration behavior to migrating as a segmental stream. Using live cell imaging we show that in lh3 null mutants, neural crest cells fail to transition from a sheet to a stream, and that they consequently enter the somites as multiple streams, or stall shortly after entering the somites. Moreover, we demonstrate that transgenic expression of lh3 in a small subset of somitic cells adjacent to where neural crest cells switch from sheet to stream migration restores segmental neural crest cell migration. Finally, we show that knockdown of the presumptive Lh3 substrate Collagen18A1 recapitulates the neural crest cell migration defects observed in lh3 mutants, consistent with the notion that Lh3 exerts its effect on neural crest cell migration by regulating post-translational modifications of Collagen18A1. Together these data suggest that Lh3–Collagen18A1 dependent ECM modifications regulate the transition of trunk neural crest cells from a non-segmental sheet like migration mode to a segmental stream migration mode.     

Fibronectin Combined with Stem Cell Factor Plays an Important Role in Melanocyte Proliferation,Differentiation and Migration in Cultured Mouse Neural Crest Cells

Stem cell factor (SCF) is essential to the migration and differentiation of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Using a neural crest cell (NCC) primary culture system from wild‐type mice, we previously demonstrated that KIT‐positive and/or L ‐3, 4‐dihydroxyphenylalanine (DOPA)‐positive melanocyte precursors proliferate following the addition of SCF to the culture medium. Extracellular matrix (ECM) proteins are considered to play a role in the migration and differentiation of various cells including melanocytes. We cultured mouse NCCs in the presence of SCF in individual wells coated with ECM; fibronectin (FN), collagen I (CLI), chondroitin sulphate, or dermatan sulphate. More KIT‐positive cells and DOPA‐positive cells were detected in the presence of SCF on ECM‐coated wells than on non‐coated wells. A statistically significant increase in DOPA‐positive cells was evident in FN and CLI wells. In contrast, in the absence of SCF, few DOPA‐positive cells and KIT‐positive cells were detected on either the ECM‐coated or non‐coated wells. We concluded that ECM affect melanocyte proliferation and development in the presence of SCF. To determine the key site of FN function, RGDS peptides in the FN sequence, which supports spreading of NCCs, were added to the NCC culture. The number of DOPA‐positive cells decreased with RGDS concentration in a dose‐dependent fashion. Immunohistochemical staining revealed the presence of integrin a5, a receptor of RGDS, in NCCs. These results suggest the RGDS domain of FN plays a contributory role as an active site in the induction of FN function in NCCs. In addition, we examined the effect of FN with SCF on the NCC migration by measuring cluster size, and found an increase in size following treatment with FN.     

miR-204 Targeting of Ankrd13A Controls Both Mesenchymal Neural Crest and Lens Cell Migration

Loss of cell adhesion and enhancement of cell motility contribute to epithelial-to-mesenchymal transition during development. These processes are related to a) rearrangement of cell-cell and cell-substrate adhesion molecules; b) cross talk between extra-cellular matrix and internal cytoskeleton through focal adhesion molecules. Focal adhesions are stringently regulated transient structures implicated in cell adhesion, spreading and motility during tissue development. Importantly, despite the extensive elucidation of the molecular composition of focal adhesions, the complex regulation of their dynamics is largely unclear. Here, we demonstrate, using live-imaging in medaka, that the microRNA miR-204 promotes both mesenchymal neural crest and lens cell migration and elongation. Overexpression of miR-204 results in upregulated cell motility, while morpholino-mediated ablation of miR-204 activity causes abnormal lens morphogenesis and neural crest cell mislocalization. Using a variety of in vivo and in vitro approaches, we demonstrate that these actions are mediated by the direct targeting of the Ankrd13A gene, which in turn controls focal cell adhesion formation and distribution. Significantly, in vivo restoration of abnormally elevated levels of Ankrd13A resulting from miR-204 inactivation rescued the aberrant lens phenotype in medaka fish. These data uncover, for the first time in vivo, the role of a microRNA in developmental control of mesenchymal cell migration and highlight miR-204 as a “master regulator” of the molecular networks that regulate lens morphogenesis in vertebrates.     

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)^1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube⁴. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage^5-13, melanocytes^11,14-20, neurons and glia^21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo^11,33-37, 2) signals from neighboring cells as they migrate^38-44, and 3) the microenvironment of their ultimate destination within the embryo^45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis^2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample^2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed⁴⁷.Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody^48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis⁴⁵. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation⁴⁰, injection of inhibitory molecules^57,58, or genetic manipulation via electroporation of expression plasmids^59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo''s developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.⁶²     

Spatial Analysis of Multi-species Exclusion Processes: Application to Neural Crest Cell Migration in the Embryonic Gut

Hindbrain (vagal) neural crest cells become relatively uniformly distributed along the embryonic intestine during the rostral to caudal colonization wave which forms the enteric nervous system (ENS). When vagal neural crest cells are labeled before migration in avian embryos by in ovo electroporation, the distribution of labeled neural crest cells in the ENS varies vastly. In some cases, the labeled neural crest cells appear evenly distributed and interspersed with unlabeled neural crest cells along the entire intestine. However, in most specimens, labeled cells occur in relatively discrete patches of varying position, area, and cell number. To determine reasons for these differences, we use a discrete cellular automata (CA) model incorporating the underlying cellular processes of neural crest cell movement and proliferation on a growing domain, representing the elongation of the intestine during development. We use multi-species CA agents corresponding to labeled and unlabeled neural crest cells. The spatial distributions of the CA agents are quantified in terms of an index. This investigation suggests that (i) the percentage of the initial neural crest cell population that is labeled and (ii) the ratio of cell proliferation to motility are the two key parameters producing the extreme differences in spatial distributions observed in avian embryos.