Overexpression of a slit homologue impairs convergent extension of the mesoderm and causes cyclopia in embryonic zebrafish.

Slit is expressed in the midline of the central nervous system both in vertebrates and invertebrates. In Drosophila, it is the midline repellent acting as a ligand for the Roundabout (Robo) protein, the repulsive receptor which is expressed on the growth cones of the commissural neurons. We have isolated cDNA fragments of the zebrafish slit2 and slit3 homologues and found that both genes start to be expressed by the midgastrula stage well before the axonogenesis begins in the nervous system, both in the axial mesoderm, and slit2 in the anterior margin of the neural plate and slit3 in the polster at the anterior end of the prechordal mesoderm. Later, expression of slit2 mRNA is detected mainly in midline structures such as the floor plate cells and the hypochord, and in the anterior margins of the neural plates in the zebrafish embryo, while slit3 expression is observed in the anterior margin of the prechordal plate, the floorplate cells in the hindbrain, and the motor neurons both in the hindbrain and the spinal cord. To study the role of Slit in early embryos, we overexpressed Slit2 in the whole embryos either by injection of its mRNA into one-cell stage embryos or by heat-shock treatment of the transgenic embryos which carries the slit2 gene under control of the heat-shock promoter. Overexpression of Slit2 in such ways impaired the convergent extension movement of the mesoderm and the rostral migration of the cells in the dorsal diencephalon and resulted in cyclopia. Our results shed light on a novel aspect of Slit function as a regulatory factor of mesodermal cell movement during gastrulation.

Laser-induced gene expression in specific cells of transgenic zebrafish.

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

Molecular cloning, expression, and activity of zebrafish semaphorin Z1a.

Semaphorins/collapsins are a large family of secreted and cell surface molecules that are thought to guide growth cones to their targets. Although some members are clearly repulsive to specific growth cones in vitro, the in vivo role of many of these molecules in vertebrate embryos is still unclear. As a first step towards clarifying the in vivo role of semaphorins/collapsins, we analyzed semaZ1a in the simple and well-characterized zebrafish embryo. SemaZ1a is a secreted molecule that is highly homologous to Sema III/D/collapsin-1, and it can collapse chick dorsal root ganglion growth cones in vitro. It is expressed in highly specific patterns within the developing embryo, which suggests that it influences outgrowth by a variety of growth cones including those of the posterior lateral line ganglion. Consistent with this hypothesis, the peripherally extending growth cones of posterior lateral line neurons retract and partially collapse during normal outgrowth.

Analysis of a Zebrafish semaphorin reveals potential functions in vivo.

The semaphorin/collapsin gene family is a large and diverse family encoding both secreted and transmembrane proteins, some of which are thought to act as repulsive axon guidance molecules. However, the function of most semaphorins is still unknown. We have cloned and characterized several semaphorins in the zebrafish in order to assess their in vivo function. Zebrafish semaZ2 is expressed in a dynamic and restricted pattern during the period of axon outgrowth that indicates potential roles in the guidance of several axon pathways. Analysis of mutant zebrafish with reduced semaZ2 expression reveals axon pathfinding errors that implicate SemaZ2 in normal guidance.

Molecular cloning and expression of two novel zebrafish semaphorins.

The large, conserved semaphorin/collapsin gene family encodes putative axon guidance molecules. We describe the cloning and expression of two n ovel zebrafish semaphorins that represent an increase in the size and diversity of the family. These semaphorins are expressed in unique and dynamic patterns during development.

Selective neurite outgrowth of cultured cortical neurons on specific regions of brain cryostat sections.

During development, axons of the mammalian cerebral cortex show a high degree of selectivity in their growth into specific regions of the central nervous system (CNS). A number of studies have shown that growing axons are guided by permissive or inhibitory membrane-bound molecules. Cryostat sections of the developing brain provide a useful assay to investigate possible membrane-bound guidance cues because such cues are retained in their normal in situ locations in specific regions of the CNS. Moreover, cryostat sections can also be subjected to various treatments that affect membrane-bound molecules. Therefore, to determine the ability of such cues to regulate the growth and guidance of cortical neurites into specific brain regions at different stages of development, we used an in vitro assay system in which explants from newborn hamster cortex were plated onto various regions of cryostat sections from developing and adult hamster brain. Neurite outgrowth from cortical explants onto the cryostat sections was visualized with a fluorescent vital dye. Results showed first that cortical neurites grew robustly on neonatal cryostat sections but only sparsely on sections from adult hamster. Second, cortical neurites grew preferentially on regions of the neonatal sections such as the cortex, basal ganglia, brainstem, thalamus, and colliculus, which are either pathways or targets for cortical axons in vivo. In contrast, cortical neurites avoided growing on the cerebellum and olfactory bulb, which are neither targets nor pathways for cortical neurites in vivo. Results also showed that cortical neurites extending onto cortical regions of neonatal sections preferred to grow along the radial axis of the cortex. Finally, heat treatment of the neonatal sections drastically reduced cortical neurite outgrowth. Taken together, these results suggest that the growth and guidance of cortical neurites is influenced by substrate-bound, developmentally regulated, heat-sensitive guidance cues preserved in the cryostat sections.

Dynamic behaviors of growth cones extending in the corpus callosum of living cortical brain slices observed with video microscopy.

During development, axons of the mammalian corpus callosum must navigate across the midline to establish connections with corresponding targets in the contralateral cerebral cortex. To gain insight into how growth cones of callosal axons respond to putative guidance cues along this CNS pathway, we have used time-lapse video microscopy to observe dynamic behaviors of individual callosal growth cones extending in living brain slices from neonatal hamster sensorimotor cortex. Crystals of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) were inserted into the cortex in vivo to label small populations of callosal axons and their growth cones. Subsequently, 400 microns brain slices that included the injection site, the corpus callosum, and the target cortex were placed in culture and viewed under low-light-level conditions with a silicon-intensified target (SIT) camera. Time-lapse video observations revealed striking differences in growth cone behaviors in different regions of the callosal pathway. In the tract, which is defined as the region of the callosal pathway from the injection site to the corresponding target cortex, growth cones advanced rapidly, displaying continual lamellipodial shape changes and filopodial exploration. Forward advance was sometimes interrupted by brief pauses or retraction. Growth cones in the target cortex had almost uniform compact shapes that were consistently smaller than those in the tract. In cortex, axons adhered to straight radial trajectories and their growth cones extended at only half the speed of those in the tract. Growth cones in subtarget regions of the callosum beneath cortical targets displayed complex behaviors characterized by long pauses, extension of transitory branches, and repeated cycles of collapse, withdrawal, and resurgence. Video observations suggested that extension of axons into cortical targets could occur by interstitial branching from callosal axons rather than by turning behaviors of the primary growth cones. These results suggest the existence of guidance cues distinct for each of these callosal regions that elicit characteristic growth cone behaviors.

Meeting the challenge of multiple academic roles through a nursing center practice model.

Filling the multiple requirements of academic life--to teach, to conduct research, and to give service to the community--can be difficult for any academician. When the educator is also a nurse, meshing academic life with that of a practicing professional adds to the difficulties. Maintaining control and managing resources for practice can be time consuming and wearisome. The authors use quotes from the sociologist Paul Starr to describe and examine this professional dilemma. The process they followed in implementing a practice model in a community nursing center is described. Using this approach teaching, research, and service requirements were blended with practice for greater professional control and more productive scholarly endeavors.