Current Progress in Neural Crest Cell Motility and Migration and Future Prospects for the Zebrafish Model System

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Nov, 2003  |  Pubmed ID: 14579388

The neural crest is a unique population of cells that contributes to the formation of diverse cell types, including craniofacial cartilage, peripheral neurons, the cardiac outflow tract, and pigment cells. Neural crest cells (NCCs) are specified within the neuroepithelium, undergo an epithelial-to-mesenchymal transition, and migrate to target destinations throughout the embryo. Here, we review current understanding of two steps in NCC development, both of which involve NCC motility. The first is NCC delamination from the neuroepithelium and the changes in cell adhesion and the cytoskeleton necessary for the initiation of migration. The second is NCC migration and the signals that guide NCCs along specific migratory pathways. We illustrate the strength of the zebrafish, Danio rerio, as a model organism to study NCC motility. The zebrafish is particularly well suited for the study of neural crest motility because of the ability to combine genetic manipulation with live imaging of migrating NCCs.

Semaphorin3D Guides Retinal Axons Along the Dorsoventral Axis of the Tectum

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2004  |  Pubmed ID: 14724229

We examined the role of Sema3D, a semaphorin of previously unknown function, in guiding retinal ganglion cell (RGC) axons to the optic tectum in the developing zebrafish. Sema3D is expressed more strongly in the ventral versus dorsal tectum, suggesting that it may participate in guiding RGC axons along the dorsoventral axis of the tectum. Ubiquitous misexpression of Sema3D in transgenic zebrafish inhibits ventral but not dorsal RGC axon growth. In addition, ventral RGC axons avoid or stop at individual cells misexpressing Sema3D along their pathway. Sema3D ubiquitous misexpression at later stages also causes ventral RGC axon arbors to spread more widely along the dorsoventral axis of the tectum. Knock-down of Sema3D with morpholino antisense causes ventral RGC axons to extend aberrantly into the ventral tectum. These results suggest that Sema3D in the ventral tectum normally acts to inhibit ventral RGCs from extending into ventral tectum, ensuring their correct innervation of dorsal tectum.

Repulsion and Attraction of Axons by Semaphorin3D Are Mediated by Different Neuropilins in Vivo

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2004  |  Pubmed ID: 15456815

Class 3 semaphorins are known to repel and/or sometimes attract axons; however, their role in guiding developing axons in the CNS in vivo is still essentially unknown. We investigated the role of Semaphorin3D (Sema3D) in the formation of the early axon pathways in the zebrafish CNS. Morpholino knock-down shows that Sema3D is essential for the correct formation of two early axon pathways. Sema3D appears to guide axons of the nucleus of the medial longitudinal fasciculus (nucMLF) by repulsion and modulation of fasciculation. In contrast, Sema3D appears to be attractive to telencephalic neurons that form the anterior commissure (AC). Knock-down of Neuropilin-1A (Npn-1A) phenocopied the effects of Sema3D knock-down on the nucMLF axons, and knock-down of either Npn-1A or Npn-2B phenocopied the defects of the AC. Furthermore, simultaneous partial knock-down experiments demonstrated genetic interactions among Sema3D, Npn-1A, and Npn-2B. Together, these data support the hypothesis that Sema3D may act as a repellent through receptors containing Npn-1A and as an attractant via receptors containing Npn-1A and Npn-2B.

Developmental Expression of Sema3G, a Novel Zebrafish Semaphorin

Gene Expression Patterns : GEP. Jun, 2005  |  Pubmed ID: 15939377

The semaphorins are a large, evolutionarily conserved family of signaling molecules with broad functions during development. The class 3 semaphorins are a subclass of secreted semaphorins found in vertebrates. There have been six class 3 semaphorins identified to date (sema3A to sema3F) and some have been shown to function in axon guidance and cardiovascular development. However, the functions of many class 3 semaphorins and their potential interactions in vivo are still not well understood. As a step toward understanding the actions of all class 3 semaphorins in vivo, we have cloned and analyzed the developmental expression pattern of a novel zebrafish class 3 semaphorin, sema3H [corrected] sema3H [corrected] is expressed in a dynamic pattern throughout the first 3 days of development. It is expressed in the adaxial cells of the somite during somitogenesis. In the brain, sema3H [corrected] is expressed in cell clusters in the midbrain and diencephalon, and is expressed in the telencephalon in close proximity to the olfactory epithelium. sema3H [corrected] also is expressed in the pharyngeal arches, the pectoral fin bud, and the developing pronephros. These results provide a basis for studying how expression of multiple semaphorins could be essential for aspects of early development.

Central and Peripheral Axon Branches from One Neuron Are Guided Differentially by Semaphorin3D and Transient Axonal Glycoprotein-1

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2005  |  Pubmed ID: 16280593

For multiple axons from one neuron to extend in different directions to unique targets, the growth cones of each axon must have distinct responses to guidance cues. However, the mechanisms by which separate axon branches are guided along different pathways are mainly unknown. Zebrafish Rohon-Beard (R-B) sensory neurons extend central axon branches in the spinal cord and peripheral axons to the epidermis. To investigate the differential guidance mechanisms of the central versus peripheral R-B axon branches, we used live-growth cone imaging in vivo combined with manipulation of individual guidance molecules. We show that a semaphorin expressed at the dorsal spinal cord midline, Semaphorin3D (Sema3D), may act to repel the peripheral axons out of the spinal cord. Sema3D knock-down reduces the number of peripheral axons. Remarkably, Sema3D ectopic expression repels and induces branching of peripheral axons in vivo but has no effect on central axons from the same neurons. Conversely, central axons require a growth-promoting molecule, transient axonal glycoprotein-1 (TAG-1), to advance, whereas peripheral axons do not. After TAG-1 knock-down, central growth cones display extensive protrusive activity but make little forward advance. TAG-1 knock-down has no effect on the motility or advance of peripheral growth cones. These experiments show how Sema3D and TAG-1 regulate the motility and behavior of growth cones extending in their natural in vivo environment and demonstrate that two different axon branches from one neuron respond differently to guidance cues in vivo.

Semaphorin 3d Guides Laterality of Retinal Ganglion Cell Projections in Zebrafish

Development (Cambridge, England). Mar, 2006  |  Pubmed ID: 16467361

The optic chiasm is an important choice point at which retinal ganglion cell (RGC) axons either cross the midline to innervate the contralateral brain or turn back to innervate the ipsilateral brain. Guidance cues that regulate this decision, particularly those directing the midline crossing of contralateral axons, are still not well understood. Here we show that Sema3d, a secreted semaphorin expressed at the midline, guides the crossing of RGC axons in zebrafish. Both Sema3d knockdown and ubiquitous overexpression induced aberrant ipsilateral projections, suggesting that Sema3d normally guides axons into the contralateral optic tract. Live imaging in vivo showed that RGC growth cones responded to ubiquitous Sema3d overexpression by pausing for extended periods and increasing their exploratory behavior at the midline, suggesting that Sema3d overexpression causes the midline environment to become less favorable for RGC axon extension. Interestingly, Sema3d overexpression did not affect growth cone behaviors before the midline, suggesting that RGC axons normally respond to Sema3d only upon reaching the midline. After Sema3d knockdown, growth cones grew across the midline but then paused or repeatedly retracted, impairing their ability to leave the midline region. Our results indicate that a proper balance of Sema3d is needed at the midline for the progression of RGC axons from the chiasm midline into the contralateral optic tract.

American College of Clinical Pharmacy's Vision of the Future: Postgraduate Pharmacy Residency Training As a Prerequisite for Direct Patient Care Practice

Pharmacotherapy. May, 2006  |  Pubmed ID: 16637798

Leflunomide and Warfarin Interaction: Case Report and Review of the Literature

Pharmacotherapy. Jun, 2006  |  Pubmed ID: 16716139

A 61-year-old Caucasian woman receiving long-term anticoagulation with warfarin for recurrent thromboembolism and atrial fibrillation was found to have an elevated international normalized ratio (INR) after she started leflunomide therapy for rheumatoid arthritis. Her INR had been stable for 4 months before this event. The patient required an overall decrease of 22% in her weekly warfarin dose to maintain a therapeutic INR within the goal range of 2.0-3.0 after adding leflunomide therapy. A comprehensive PubMed/MEDLINE search was conducted to identify literature addressing the potential interaction between warfarin and leflunomide. Evidence describing the interaction and its potential mechanism was limited to one published case report and to in vitro data, respectively. Our case report provides additional support that such an interaction exists and that it was at least partly responsible for the subsequent increase in the patient's INR. Therefore, continued evaluation and documentation of this potential drug interaction is imperative. To reduce the risk of adverse effects related to excessive anticoagulation with the start of leflunomide in patients taking warfarin, clinicians should increase their frequency of INR monitoring and adjust the warfarin dosage accordingly to maintain therapeutic anticoagulation.

Repulsion or Adhesion: Receptors Make the Call

Current Opinion in Cell Biology. Oct, 2006  |  Pubmed ID: 16930978

Repulsive signaling plays a prominent role in regulating cell-cell interactions and is fundamental to multiple developmental processes. A proper balance between repulsion from and adhesion to other cells or the extracellular matrix is also important. Semaphorin-Plexin and ephrin-Eph ligand-receptor pairs compose two major repulsive signaling systems. Recent advances have elucidated mechanisms by which Semaphorin-Plexin and ephrin-Eph signaling control repulsion versus adhesion. Semaphorins act through a complex signaling pathway to inhibit integrin-mediated adhesion, allowing cell repulsion. Ephrin-Eph interactions can directly mediate cell adhesion and several mechanisms control whether ephrin-Eph binding and signaling induces repulsion or adhesion.

Semaphorin 3d Promotes Cell Proliferation and Neural Crest Cell Development Downstream of TCF in the Zebrafish Hindbrain

Development (Cambridge, England). Oct, 2006  |  Pubmed ID: 16971468

Neural crest cells (NCCs) are pluripotent migratory cells that are crucial to the development of the peripheral nervous system, pigment cells and craniofacial cartilage and bone. NCCs are specified within the dorsal ectoderm and undergo an epithelial to mesenchymal transition (EMT) in order to migrate to target destinations where they differentiate. Here we report a role for a member of the semaphorin family of cell guidance molecules in NCC development. Morpholino-mediated knockdown of Sema3d inhibits the proliferation of hindbrain neuroepithelial cells. In addition, Sema3d knockdown reduces markers of migratory NCCs and disrupts NCC-derived tissues. Similarly, expression of a dominant-repressor form of TCF (DeltaTCF) reduces hindbrain cell proliferation and leads to a disruption of migratory NCC markers. Moreover, expression of DeltaTCF downregulates sema3d RNA expression. Finally, Sema3d overexpression rescues reduced proliferation caused by DeltaTCF expression, suggesting that Sema3d lies downstream of Wnt/TCF signaling in the molecular pathway thought to control cell cycle in NCC precursors.

Zebrafish Bashful/laminin-alpha 1 Mutants Exhibit Multiple Axon Guidance Defects

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jan, 2006  |  Pubmed ID: 16261616

Laminin is known to provide a highly permissive substratum and in some cases directional information for axon outgrowth in vitro. However, there is still little known about laminin function in guiding axons in vivo. We investigated the axon guidance role of laminin-alpha1 in the developing zebrafish nervous system. Analysis of zebrafish bashful (bal)/laminin-a1 mutants revealed multiple functions for laminin-alpha1 in the outgrowth and guidance of central nervous system (CNS) axons. Most CNS axon pathways are defective in bal embryos. Some axon types, including retinal ganglion cell axons, early forebrain axons, and hindbrain reticulospinal axons, make specific pathfinding errors, suggesting laminin-alpha1 is required for directional decisions. Other axon tracts are defasciculated or not fully extended in bal embryos, suggesting a function for laminin-alpha1 in regulating adhesion or providing a permissive substratum for growth. In addition, some neurons have excessively branched axons in bal, indicating a potential role for laminin-alpha1 in branching. In contrast to CNS axons, most peripheral axons appear normal in bal mutants. Our results, thus, reveal important and diverse functions for laminin-alpha1 in guiding developing axons in vivo.

Semaphorin3D Regulates Axon Axon Interactions by Modulating Levels of L1 Cell Adhesion Molecule

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2007  |  Pubmed ID: 17804626

The decision of a growing axon to selectively fasciculate with and defasciculate from other axons is critical for axon pathfinding and target innervation. Fasciculation can be regulated by cell adhesion molecules that modulate interaxonal adhesion and repulsive molecules, expressed by surrounding tissues that channel axons together. Here we describe crosstalk between molecules that mediate these mechanisms. We show that Semaphorin3D (Sema3D), a classic repulsive molecule, promotes fasciculation by regulating L1 CAM levels and axon-axon interactions rather than by creating a repulsive surround. Knockdown experiments show that Sema3D and L1 genetically interact to promote fasciculation. Sema3D overexpression increases and Sema3D knockdown decreases levels of axonal L1 protein. Moreover, excess L1 rescues defasciculation caused by the loss of Sema3D. In vivo time-lapse imaging reveals that Sema3D or L1 knockdown cause identical defects in growth cone behaviors during axon-axon interactions, consistent with a loss of adhesion. These results reveal a novel mechanism by which a semaphorin promotes fasciculation and modulates axon-axon interactions by regulating an adhesion molecule.

Developing a Business-practice Model for Pharmacy Services in Ambulatory Settings

Pharmacotherapy. Feb, 2008  |  Pubmed ID: 18225974

A business-practice model is a guide, or toolkit, to assist managers and clinical pharmacy practitioners in the exploration, proposal, development and implementation of new clinical pharmacy services and/or the enhancement of existing services. This document was developed by the American College of Clinical Pharmacy Task Force on Ambulatory Practice to assist clinical pharmacy practitioners and administrators in the development of business-practice models for new and existing clinical pharmacy services in ambulatory settings. This document provides detailed instructions, examples, and resources on conducting a market assessment and a needs assessment, types of clinical services, operations, legal and regulatory issues, marketing and promotion, service development and exit plan, evaluation of service outcomes, and financial considerations in the development of a clinical pharmacy service in the ambulatory environment. Available literature is summarized, and an appendix provides valuable citations and resources. As ambulatory care practices continue to evolve, there will be increased knowledge of how to initiate and expand the services. This document is intended to serve as an essential resource to assist in the growth and development of clinical pharmacy services in the ambulatory environment.

Transient Axonal Glycoprotein-1 (TAG-1) and Laminin-alpha1 Regulate Dynamic Growth Cone Behaviors and Initial Axon Direction in Vivo

Neural Development. 2008  |  Pubmed ID: 18289389

How axon guidance signals regulate growth cone behavior and guidance decisions in the complex in vivo environment of the central nervous system is not well understood. We have taken advantage of the unique features of the zebrafish embryo to visualize dynamic growth cone behaviors and analyze guidance mechanisms of axons emerging from a central brain nucleus in vivo.

The Eye Organizes Neural Crest Cell Migration

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jun, 2008  |  Pubmed ID: 18498099

In the anterior vertebrate head, a population of neural crest cells (NCCs) migrates to the periocular mesenchyme and makes critical contributions to the developing eye and orbit. Improper migration and differentiation of these NCCs have been implicated in human diseases such as congenital glaucoma and anterior segment dysgenesis syndromes. The mechanisms by which these cells migrate to their target tissues within and around the eye are not well understood. We present a fate map of zebrafish diencephalic and mesencephalic NCC contributions to the eye and orbit. The fate map closely resembles that in chick and mice, demonstrating evolutionary conservation. To gain insight into the mechanisms of anterior NCC guidance, we used the eyeless mutant chokh/rx3. We show that, in chokh mutants, dorsal anterior NCC migration is severely disorganized. Time-lapse analysis shows that NCCs have significantly reduced migration rates and directionality in chokh mutants.

Rho-kinase and Myosin II Affect Dynamic Neural Crest Cell Behaviors During Epithelial to Mesenchymal Transition in Vivo

Developmental Biology. Dec, 2008  |  Pubmed ID: 18926812

The induction and migration of neural crest cells (NCCs) are essential to the development of craniofacial structures and the peripheral nervous system. A critical step in the development of NCCs is the epithelial to mesenchymal transition (EMT) that they undergo in order to initiate migration. Several transcription factors are important for the NCC EMT. However, less is known about the effectors regulating changes in cell adhesion, the cytoskeleton, and cell motility associated with the EMT or about specific changes in the behavior of cells undergoing EMT in vivo. We used time-lapse imaging of NCCs in the zebrafish hindbrain to show that NCCs undergo a stereotypical series of behaviors during EMT. We find that loss of cell adhesion and membrane blebbing precede filopodial extension and the onset of migration. Live imaging of actin dynamics shows that actin localizes differently in blebs and filopodia. Moreover, we find that disruption of myosin II or Rho-kinase (ROCK) activity inhibits NCC blebbing and causes reduced NCC EMT. These data reveal roles for myosin II and ROCK in NCC EMT in vivo, and provide a detailed characterization of NCC behavior during EMT that will form a basis for further mechanistic studies.

The Cell Adhesion Molecule Tag1, Transmembrane Protein Stbm/Vangl2, and Lamininalpha1 Exhibit Genetic Interactions During Migration of Facial Branchiomotor Neurons in Zebrafish

Developmental Biology. Jan, 2009  |  Pubmed ID: 19013446

Interactions between a neuron and its environment play a major role in neuronal migration. We show here that the cell adhesion molecule Transient Axonal Glycoprotein (Tag1) is necessary for the migration of the facial branchiomotor neurons (FBMNs) in the zebrafish hindbrain. In tag1 morphant embryos, FBMN migration is specifically blocked, with no effect on organization or patterning of other hindbrain neurons. Furthermore, using suboptimal morpholino doses and genetic mutants, we found that tag1, lamininalpha1 (lama1) and stbm, which encodes a transmembrane protein Vangl2, exhibit pairwise genetic interactions for FBMN migration. Using time-lapse analyses, we found that FBMNs are affected similarly in all three single morphant embryos, with an inability to extend protrusions in a specific direction, and resulting in the failure of caudal migration. These data suggest that tag1, lama1 and vangl2 participate in a common mechanism that integrates signaling between the FBMN and its environment to regulate migration.

Muscle Contractions Guide Rohon-beard Peripheral Sensory Axons

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Oct, 2009  |  Pubmed ID: 19846707

Multiple molecular cues guide neuronal axons to their targets during development. Previous studies in vitro have shown that mechanical stimulation also can affect axon growth; however, whether mechanical force contributes to axon guidance in vivo is unknown. We investigated the role of muscle contractions in the guidance of zebrafish peripheral Rohon-Beard (RB) sensory axons in vivo. We analyzed several mutants that affect muscle contraction through different molecular pathways, including a new mutant allele of the titin a (pik) gene, mutants that affect the hedgehog signaling pathway, and a nicotinic acetylcholine receptor mutant. We found RB axon defects in these mutants, the severity of which appeared to correlate with the extent of muscle contraction loss. These axons extend between the muscle and skin and normally have ventral trajectories and repel each other on contact. RB peripheral axons in muscle mutants extend longitudinally instead of ventrally, and the axons fail to repel one another on contact. In addition, we showed that limiting muscle movements by embedding embryos in agarose caused similar defects in peripheral RB axon guidance. This work suggests that the mechanical forces generated by muscle contractions are necessary for proper sensory axon pathfinding in vivo.

Control of Neural Crest Cell Behavior and Migration: Insights from Live Imaging

Cell Adhesion & Migration. Oct-Dec, 2010  |  Pubmed ID: 20671421

Neural crest cells (NCCs) are a remarkable, dynamic group of cells that travel long distances in the embryo to reach their target sites. They are responsible for the formation of craniofacial bones and cartilage, neurons and glia in the peripheral nervous system, and pigment cells. Live imaging of NCCs as they traverse the embryo has been critical to increasing our knowledge of their biology. NCCs exhibit multiple behaviors and communicate with each other and their environment along each step of their journey. Imaging combined with molecular manipulations has led to insights into the mechanisms controlling these behaviors. In this review, we highlight studies that have used live imaging to provide novel insight into NCC migration and discuss how continued use of such techniques can advance our understanding of NCC biology.

Regulation of Cell Adhesions and Motility During Initiation of Neural Crest Migration

Current Opinion in Neurobiology. Feb, 2011  |  Pubmed ID: 20970990

Accurate neural crest cell (NCC) migration requires tight control of cell adhesions, cytoskeletal dynamics and cell motility. Cadherins and RhoGTPases are critical molecular players that regulate adhesions and motility during initial delamination of NCCs from the neuroepithelium. Recent studies have revealed multiple functions for these molecules and suggest that a precise balance of their activity is crucial. RhoGTPase appears to regulate both cell adhesions and protrusive forces during NCC delamination. Increasing evidence shows that cadherins are multi-functional proteins with novel, adhesion-independent signaling functions that control NCC motility during both delamination and migration. These functions are often regulated by specific proteolytic cleavage of cadherins. After NCC delamination, planar cell polarity signaling acts via RhoGTPases to control NCC protrusions and migration direction.

In Vivo Imaging of Cell Behaviors and F-actin Reveals LIM-HD Transcription Factor Regulation of Peripheral Versus Central Sensory Axon Development

Neural Development. 2011  |  Pubmed ID: 21619654

Development of specific neuronal morphology requires precise control over cell motility processes, including axon formation, outgrowth and branching. Dynamic remodeling of the filamentous actin (F-actin) cytoskeleton is critical for these processes; however, little is known about the mechanisms controlling motile axon behaviors and F-actin dynamics in vivo. Neuronal structure is specified in part by intrinsic transcription factor activity, yet the molecular and cellular steps between transcription and axon behavior are not well understood. Zebrafish Rohon-Beard (RB) sensory neurons have a unique morphology, with central axons that extend in the spinal cord and a peripheral axon that innervates the skin. LIM homeodomain (LIM-HD) transcription factor activity is required for formation of peripheral RB axons. To understand how neuronal morphogenesis is controlled in vivo and how LIM-HD transcription factor activity differentially regulates peripheral versus central axons, we used live imaging of axon behavior and F-actin distribution in vivo.