Mary C. Halloran

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

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.

Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo

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.

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

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.

Semaphorin3D Guides Retinal Axons along the Dorsoventral Axis of the Tectum

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.

Current progress in neural crest cell motility and migration and future prospects for the zebrafish model system

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. Developmental Dynamics, 2003.

The eye organizes neural crest cell migration

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. Developmental Dynamics 237:1645–1652, 2008.

Semaphorin 3d guides laterality of retinal ganglion cell projections in zebrafish

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.

Zebrafish bashful/laminin-α1 mutants exhibit multiple axon guidance defects

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‐α1 in the developing zebrafish nervous system. Analysis of zebrafish bashful (bal)/laminin‐a1 mutants revealed multiple functions for laminin‐α1 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‐α1 is required for directional decisions. Other axon tracts are defasciculated or not fully extended in bal embryos, suggesting a function for laminin‐α1 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‐α1 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‐α1 in guiding developing axons in vivo. Developmental Dynamics 235:213–224, 2006.

The cell adhesion molecule Tag1, transmembrane protein Stbm/ Vangl2, and Lamininα1 exhibit genetic interactions during migration of facial branchiomotor neurons in zebrafish

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.

Semaphorin3D regulates axon axon interactions by modulating levels of L1 cell adhesion molecule.

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.