Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements.

Embryonic morphogenesis is driven by a suite of cell behaviours, including coordinated shape changes, cellular rearrangements and individual cell migrations, whose molecular determinants are largely unknown. In the zebrafish, Dani rerio, trilobite mutant embryos have defects in gastrulation movements and posterior migration of hindbrain neurons. Here, we have used positional cloning to demonstrate that trilobite mutations disrupt the transmembrane protein Strabismus (Stbm)/Van Gogh (Vang), previously associated with planar cell polarity (PCP) in Drosophila melanogaster, and PCP and canonical Wnt/beta-catenin signalling in vertebrates. Our genetic and molecular analyses argue that during gastrulation, trilobite interacts with the PCP pathway without affecting canonical Wnt signalling. Furthermore, trilobite may regulate neuronal migration independently of PCP molecules. We show that trilobite mediates polarization of distinct movement behaviours. During gastrulation convergence and extension movements, trilobite regulates mediolateral cell polarity underlying effective intercalation and directed dorsal migration at increasing velocities. In the hindbrain, trilobite controls effective migration of branchiomotor neurons towards posterior rhombomeres. Mosaic analyses show trilobite functions cell-autonomously and non-autonomously in gastrulae and the hindbrain. We propose Trilobite/Stbm mediates cellular interactions that confer directionality on distinct movements during vertebrate embryogenesis.

Neuronal development and migration in zebrafish hindbrain explants.

The zebrafish embryo is an excellent system for studying dynamic processes such as cell migration during vertebrate development. Dynamic analysis of neuronal migration in the zebrafish hindbrain has been hampered by morphogenetic movements in vivo, and by the impermeability of embryos. We have applied a recently reported technique of embryo explant culture to the analysis of neuronal development and migration in the zebrafish hindbrain. We show that hindbrain explants prepared at the somitogenesis stage undergo normal morphogenesis for at least 14 h in culture. Importantly, several aspects of hindbrain development such as patterning, neurogenesis, axon guidance, and neuronal migration are largely unaffected, inspite of increased cell death in explanted tissue. These results suggest that hindbrain explant culture can be employed effectively in zebrafish to analyze neuronal migration and other dynamic processes using pharmacological and imaging techniques.

Effects of ethanol on cultured embryonic neurons from the cerebral cortex of the rat.

Fetal alcohol syndrome is a serious disorder that causes lifelong learning, memory, and behavioral problems. In the current study, we determined the ethanol concentrations that produced detrimental effects on the development of embryonic cortical neurons because mental capacity seems to be proportional to the level of dendritic arborization. Neurons from fetal rat cortices were grown in culture in close proximity to a glial plane. The cells were treated with concentrations of ethanol ranging from 450 nM to 45 mM, and neurite outgrowth was subsequently quantified. A significant decrease in dendritic branching was observed at ethanol concentrations as low as 45 microM after 6 days of ethanol exposure in vitro, whereas changes in primary neurite outgrowth were observed at an ethanol concentration of 4.5 microM. This finding is of particular interest as it seems to indicate that occasional ethanol exposure is detrimental to cortical development at very low concentrations of ethanol.

Multiple mechanisms mediate motor neuron migration in the zebrafish hindbrain.

The transmembrane protein Van gogh-like 2 (Vangl2) is a component of the noncanonical Wnt/Planar Cell Polarity (PCP) signaling pathway, and is required for tangential migration of facial branchiomotor neurons (FBMNs) from rhombomere 4 (r4) to r5-r7 in the vertebrate hindbrain. Since vangl2 is expressed throughout the zebrafish hindbrain, it might also regulate motor neuron migration in other rhombomeres. We tested this hypothesis by examining whether migration of motor neurons out of r2 following ectopic hoxb1b expression was affected in vangl2(-) (trilobite) mutants. Hoxb1b specifies r4 identity, and when ectopically expressed transforms r2 to an "r4-like" compartment. Using time-lapse imaging, we show that GFP-expressing motor neurons in the r2/r3 region of a hoxb1b-overexpressing wild-type embryo migrate along the anterior-posterior (AP) axis. Furthermore, these cells express prickle1b (pk1b), a Wnt/PCP gene that is specifically expressed in FBMNs and is essential for their migration. Importantly, GFP-expressing motor neurons in the r2/r3 region of hoxb1b-overexpressing trilobite mutants and pk1b morphants often migrate, even though FBMNs in r4 of the same embryos fail to migrate longitudinally (tangentially) into r6 and r7. These observations suggest that tangentially migrating motor neurons in the anterior hindbrain (r1-r3) can use mechanisms that are independent of vangl2 and pk1b functions. Interestingly, analysis of tri; val double mutants also suggests a role for vangl2-independent factors in neuronal migration, since the valentino mutation partially suppresses the trilobite mutant migration defect. Together, the hoxb1b and val experiments suggest that multiple mechanisms regulate motor neuron migration along the AP axis of the zebrafish hindbrain.

The Zebrafish trilobite gene is essential for tangential migration of branchiomotor neurons.

Newborn neurons migrate extensively in the radial and tangential directions to organize the developing vertebrate nervous system. We show here that mutations in zebrafish trilobite (tri) that affect gastrulation-associated cell movements also eliminate tangential migration of motor neurons in the hindbrain. In the wild-type hindbrain, facial (nVII) and glossopharyngeal (nIX) motor neurons are induced in rhombomeres 4 and 6, respectively, and migrate tangentially into r6 and r7 (nVII) and r7 (nIX). In all three tri alleles examined, although normal numbers of motor neurons are induced, nVII motor neurons are found exclusively in r4, and nIX-like motor neurons are found exclusively in r6. The migration of other neuronal and nonneuronal cell types is unaffected in tri mutants. Rhombomere formation and the development of other hindbrain neurons are also unaffected in tri mutants. Furthermore, tangential neuronal migration occurs normally in the gastrulation mutant knypek, indicating that the trilobite neuron phenotype does not arise nonspecifically from aberrant gastrulation-associated movements. We conclude that trilobite function is specifically required for two types of cell migration that occur at different stages of zebrafish development.

Neurogenic phenotype of mind bomb mutants leads to severe patterning defects in the zebrafish hindbrain.

Failure of Notch signaling in zebrafish mind bomb (mib) mutants results in a neurogenic phenotype where an overproduction of early differentiating neurons is accompanied by the loss of later-differentiating cell types. We have characterized in detail the hindbrain phenotype of mib mutants. Hindbrain branchiomotor neurons (BMNs) are reduced in number but not missing in mib mutants. In addition, BMN clusters are frequently fused across the midline in mutants. Mosaic analysis indicates that the BMN patterning and fusion defects in the mib hindbrain arise non-cell autonomously. Ventral midline signaling is defective in the mutant hindbrain, in part due to the differentiation of some midline cells into neural cells. Interestingly, while early hindbrain patterning appears normal in mib mutants, subsequent rhombomere-specific gene expression is completely lost. The defects in ventral midline signaling and rhombomere patterning are accompanied by an apparent loss of neuroepithelial cells in the mutant hindbrain. These observations suggest that, by regulating the differentiation of neuroepithelial cells into neurons, Notch signaling preserves a population of non-neuronal cells that are essential for maintaining patterning mechanisms in the developing neural tube.