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The homeobox gene mbx is first activated at the end of gastrulation in zebrafish in the presumptive forebrain and midbrain region. During somitogenesis stages, the anterior expression of mbx, which partly overlaps the future eye field, gradually decreases, while midbrain expression intensifies and becomes restricted to the presumptive tectum. Knockdown of mbx expression by morpholino antisense oligonucleotides (mbx-MO) leads to a reduction in the size of the eyes and tectum. Expression domains of rx1 and pax6 in the eye field and of mab21l2 in the eye field and tectum anlage were reduced in size in mbx-MO-injected embryos by somitogenesis stages. Further, induction of islet1 and lim3 expression in the eye at 2 days postfertilization (dpf) was suppressed in mbx-MO-injected embryos. In mbx-MO-injected embryos at 2-5 dpf, the lamination of the eye was disorganized and the number of retinal axons was substantially reduced, but the few remaining axons navigated appropriately to the contralateral rectum. A chimeric protein composed of the Mbx DNA-binding domain and the VP16 activation domain affected eye and rectum development similarly to mbx-MO knockdown, suggesting that Mbx acts as a transcriptional repressor in the zebrafish embryo. Based on these data, we propose that the mbx homeobox gene is required for the development of the eyes and rectum. Key Words: mbx; homeobox gene; eye; rectum; midbrain-hindbrain boundary; morpholino.

Abstract Background Growth cone navigation across the vertebrate midline is critical in the establishment of nervous system connectivity. While midline crossing is achieved through coordinated signaling of attractive and repulsive cues, this has never been demonstrated at the single cell level. Further, though growth cone responsiveness to guidance cues changes after crossing the midline, it is unclear whether midline crossing itself is required for subsequent guidance decisions in vivo. In the zebrafish, spinal commissures are initially formed by a pioneer neuron called CoPA (Commissural Primary Ascending). Unlike in other vertebrate models, CoPA navigates the midline alone, allowing for single-cell analysis of axon guidance mechanisms. Results We provide evidence that CoPA expresses the known axon guidance receptors dcc, robo3 and robo2. Using loss of function mutants and gene knockdown, we show that the functions of these genes are evolutionarily conserved in teleosts and that they are used consecutively by CoPA neurons. We also reveal novel roles for robo2 and robo3 in maintaining commissure structure. When midline crossing is prevented in robo3 mutants and dcc gene knockdown, ipsilaterally projecting neurons respond to postcrossing guidance cues. Furthermore, DCC inhibits Robo2 function before midline crossing to allow a midline approach and crossing. Conclusions Our results demonstrate that midline crossing is not required for subsequent guidance decisions by pioneer axons and that this is due, in part, to DCC inhibition of Robo2 function prior to midline crossing.

Abstract Background Many neurons in the central nervous system, including retinal ganglion cells (RGCs), possess asymmetric dendritic arbors oriented toward their presynaptic partners. How such dendritic arbors become biased during development in vivo is not well understood. Dendritic arbors may become oriented by directed outgrowth or by reorganization of an initially unbiased arbor. To distinguish between these possibilities, we imaged the dynamic behavior of zebrafish RGC dendrites during development in vivo. We then addressed how cell positioning within the retina, altered in heart-and-soul (has) mutants, affects RGC dendritic orientation. Results In vivo multiphoton time-lapse analysis revealed that RGC dendrites initially exhibit exploratory behavior in multiple directions but progressively become apically oriented. The lifetimes of basal and apical dendrites were generally comparable before and during the period when arbors became biased. However, with maturation, the addition and extension rates of basal dendrites were slower than those of the apical dendrites. Oriented dendritic arbors were also found in misplaced RGCs of the has retina but there was no preferred orientation amongst the population. However, has RGCs always projected dendrites toward nearby neuropil where amacrine and bipolar cell neurites also terminated. Chimera analysis showed that the abnormal dendritic organization of RGCs in the mutant was non-cell autonomous. Conclusions Our observations show that RGC dendritic arbors acquire an apical orientation by selective and gradual restriction of dendrite addition to the apical side of the cell body, rather than by preferential dendrite stabilization or elimination. A biased arbor emerges at a stage when many of the dendritic processes still appear exploratory. The generation of an oriented RGC dendritic arbor is likely to be determined by cell-extrinsic cues. Such cues are unlikely to be localized to the basal lamina of the inner retina, but rather may be provided by cells presynaptic to the RGCs.

Abstract In the absence of external cues, neurons in vitro polarize by using intrinsic mechanisms. For example, cultured hippocampal neurons extend arbitrarily oriented neurites and then one of these, usually the one nearest the centrosome, begins to grow more quickly than the others. This neurite becomes the axon as it accumulates molecular components of the apical junctional complex. All the other neurites become dendrites. It is unclear, however, whether neurons in vivo, which differentiate within a polarized epithelium, break symmetry by using similar intrinsic mechanisms. To investigate this, we use four-dimensional microscopy of developing retinal ganglion cells (RGCs) in live zebrafish embryos. We find that the situation is indeed very different in vivo, where axons emerge directly from uniformly polarized cells in the absence of other neurites. In vivo, moreover, components of the apical complex do not localize to the emerging axon, nor does the centrosome predict the site of axon emergence. Mosaic analysis in four dimensions, using mutants in which neuroepithelial polarity is disrupted, indicates that extrinsic factors such as access to the basal lamina are critical for normal axon emergence from RGCs in vivo.