Lara D. Hutson

Pathfinding and Error Correction by Retinal Axons: The Role of astray/robo2

To address how the highly stereotyped retinotectal pathway develops in zebrafish, we used fixed-tissue and time-lapse imaging to analyze morphology and behavior of wild-type and mutant retinal growth cones. Wild-type growth cones increase in complexity and pause at the midline. Intriguingly, they make occasional ipsilateral projections and other pathfinding errors, which are always eventually corrected. In the astray/robo2 mutant, growth cones are larger and more complex than wild-type. astray axons make midline errors not seen in wild-type, as well as errors both before and after the midline. astray errors are rarely corrected. The presumed Robo ligands Slit2 and Slit3 are expressed near the pathway in patterns consistent with their mediating pathfinding through Robo2. Thus, Robo2 does not control midline crossing of retinal axons, but rather shapes their pathway, by both preventing and correcting pathfinding errors.

Robo2 is required for establishment of a precise glomerular map in the zebrafish olfactory system

Olfactory sensory neurons (OSNs) expressing a given odorant receptor project their axons to specific glomeruli, creating a topographic odor map in the olfactory bulb (OB). The mechanisms underlying axonal pathfinding of OSNs to their precise targets are not fully understood. Here, we demonstrate that Robo2/Slit signaling functions to guide nascent olfactory axons to the OB primordium in zebrafish. robo2 is transiently expressed in the olfactory placode during the initial phase of olfactory axon pathfinding. In the robo2 mutant, astray (ast), early growing olfactory axons misroute ventromedially or posteriorly, and often penetrate into the diencephalon without reaching the OB primordium. Four zebrafish Slit homologs are expressed in regions adjacent to the olfactory axon trajectory, consistent with their role as repulsive ligands for Robo2. Masking of endogenous Slit gradients by ubiquitous misexpression of Slit2 in transgenic fish causes posterior pathfinding errors that resemble the ast phenotype. We also found that the spatial arrangement of glomeruli in OB is perturbed in ast adults, suggesting an essential role for the initial olfactory axon scaffold in determining a topographic glomerular map. These data provide functional evidence for Robo2/Slit signaling in the establishment of olfactory neural circuitry in zebrafish.

Two divergent slit1 genes in zebrafish

Members of the Slit family regulate axon guidance and cell migration. To date, three vertebrate slit1 genes have been identified in mammals and orthologs of two, slit2 and slit3, have been identified in zebrafish. Here, we describe the cloning of full‐length cDNAs for two zebrafish slit orthologs, slit1a and slit1b. Both predicted proteins contain the conserved motifs that characterize other vertebrate Slits. slit1a and slit1b are both expressed in the midline, hypochord, telencephalon, and hindbrain. Apart from these shared expression domains, however, their expression patterns largely differ. Whereas slit1a is expressed broadly in the central nervous system (CNS) and in the somites, pectoral fin buds, tail bud, and caudal fin folds, slit1b is expressed in the olfactory system throughout embryonic and larval development, and in the retina during larval stages. Their expression patterns, particularly that of slit1a, suggest that Slit proteins may have roles in tissue morphogenesis in addition to their established roles in axon guidance and cell migration. Developmental Dynamics, 2003.

Wiring the zebrafish: Axon guidance and synaptogenesis

Many zebrafish mutants have specific defects in axon guidance or synaptogenesis, particularly in the retinotectal and motor systems. Several mutants have now been characterized in detail and/or cloned. A combination of genetic studies, in vivo imaging and new techniques for misexpressing genes or blocking their function promises to reveal the molecules and principles that govern wiring of the vertebrate nervous system.

Developmental expression patterns of the zebrafish small heat shock proteins

Small heat shock proteins (sHSPs), or α‐crystallins, are low‐molecular weight proteins found in every kingdom and nearly every species examined to date. Many, if not all, sHSPs act as molecular chaperones. Several also have functions independent of their chaperone activity, and at least a few are expressed in specific spatiotemporal patterns during embryonic and/or juvenile stages, suggesting specific roles during development. To date, however, no one has systematically characterized the expression patterns of all of the sHSPs during development in any organism. We have characterized the normal heat shock‐induced expression patterns of all 13 zebrafish sHSPs during development. Seven of the sHSPs are expressed in a tissue‐specific manner during development, and five are upregulated by heat shock. The results of these studies provide a foundation for analysis of sHSP function during normal development and their roles in protecting cells from the effects environmental stressors. Developmental Dynamics 237:454–463, 2008.

Modular laboratory exercises to analyze the development of zebrafish motor behavior.

The embryonic zebrafish is an excellent research model to examine the neural networks that coordinate locomotive behavior. It demonstrates robust locomotive behavior early in development, its nervous system is relatively simple and accessible compared to mammalian systems, and there are mutants available with specific molecular and motor deficits. We have developed a series of four exercises that provide students with a basic understanding of locomotive behavior development, nervous system organization, development of neurotransmitter responsiveness, and genetics. The first two exercises can be performed in one 3-h laboratory period, and the third and fourth exercises, which build on the first two, can be completed in one or two subsequent periods. In the first exercise, students observe and quantify two distinct behaviors that characterize different developmental stages, spontaneous movement, and touch-evoked tail coiling. In the second, the students use a pharmacological approach to determine if the neurotransmitter glycine is required for the embryo to perform each behavior. In the third, they use simple lesions to assess whether the brain is required for each type of behavior. In the fourth, the students examine bandoneon, a zebrafish motility mutant that has a glycine receptor defect, by observing its behavior during spontaneous movement and touch-evoked tail coiling, performing lesions, and applying pharmacological drugs. These exercises are readily adaptable, such that portions can be omitted or expanded to examine other neurotransmitter systems or later stages of locomotive behavior development.

Analyzing Axon Guidance in the Zebrafish Retinotectal System

Publisher Summary This chapter analyzes axon guidance in the Zebrafish retinotectal system. The chapter describes the strategies that have been used to observe and to perturb retinotectal development in the zebrafish. It begins with a brief overview of the genetic control of retinal axon guidance. This chapter then describes the methods for labeling and observing retinal axons. Moreover, this chapter explains the methods for perturbing retinotectal development. Furthermore, multiphoton excitation has two main advantages: (1) reduced photobleaching and phototoxicity, since multiphoton absorption is restricted to the plane of focus; and (2) improved brightness and resolution in thick or scattering tissues, since infrared light scatters less than visible light and nondescanned detectors can make use of the scattered emitted light. Thus, it is ideally suited to imaging cell behavior in live thick samples, such as the intact zebrafish larva and in particular, in the retinotectal system. Finally, this chapter concludes with a brief discussion of the methods likely to be important in the future. The methods described here for the retinotectal system are also useful, either directly or with some modifications, for studying the development of other axon tracts.

Small heat shock proteins are necessary for heart migration and laterality determination in zebrafish.

Small heat shock proteins (sHsps) regulate cellular functions not only under stress, but also during normal development, when they are expressed in organ-specific patterns. Here we demonstrate that two small heat shock proteins expressed in embryonic zebrafish heart, hspb7 and hspb12, have roles in the development of left-right asymmetry. In zebrafish, laterality is determined by the motility of cilia in Kupffers vesicle (KV), where hspb7 is expressed; knockdown of hspb7 causes laterality defects by disrupting the motility of these cilia. In embryos with reduced hspb7, the axonemes of KV cilia have a 9+0 structure, while control embyros have a predominately 9+2 structure. Reduction of either hspb7 or hspb12 alters the expression pattern of genes that propagate the signals that establish left-right asymmetry: the nodal-related gene southpaw (spaw) in the lateral plate mesoderm, and its downstream targets pitx2, lefty1 and lefty2. Partial depletion of hspb7 causes concordant heart, brain and visceral laterality defects, indicating that loss of KV cilia motility leads to coordinated but randomized laterality. Reducing hspb12 leads to similar alterations in the expression of downstream laterality genes, but at a lower penetrance. Simultaneous reduction of hspb7 and hspb12 randomizes heart, brain and visceral laterality, suggesting that these two genes have partially redundant functions in the establishment of left-right asymmetry. In addition, both hspb7 and hspb12 are expressed in the precardiac mesoderm and in the yolk syncytial layer, which supports the migration and fusion of mesodermal cardiac precursors. In embryos in which the reduction of hspb7 or hspb12 was limited to the yolk, migration defects predominated, suggesting that the yolk expression of these genes rather than heart expression is responsible for the migration defects.

Vincristine and bortezomib cause axon outgrowth and behavioral defects in larval zebrafish.

Peripheral neuropathy is a common side effect of a number of pharmaceutical compounds, including several chemotherapy drugs. Among these are vincristine sulfate, a mitotic inhibitor used to treat a variety of leukemias, lymphomas, and other cancers, and bortezomib, a 26S proteasome inhibitor used primarily to treat relapsed multiple myeloma and mantle cell lymphoma. To gain insight into the mechanisms by which these compounds act, we tested their effects in zebrafish. Vincristine or bortezomib given during late embryonic development caused significant defects at both behavioral and cellular levels. Intriguingly, the effects of the two drugs appear to be distinct. Vincristine causes uncoordinated swimming behavior, which is coupled with a reduction in the density of sensory innervation and overall size of motor axon arbors. Bortezomib, in contrast, increases the duration and amplitude of muscle contractions associated with escape swimming, which is coupled with a preferential reduction in fine processes and branches of sensory and motor axons. These results demonstrate that zebrafish is a convenient in vivo assay system for screening potential pharmaceutical compounds for neurotoxic side effects, and they provide an important step toward understanding how vincristine and bortezomib cause peripheral neuropathy.

Making an impact: Zebrafish in education

Science and technology have fundamentally changed the world within our lifetimes. Ideas that once seemed to belong only to the realm of science fiction now are within reach. In the 2000 Arnold Schwarzenegger movie The 6th Day elderly and beloved pets are routinely cloned. Less than a decade after the first successful cloning of Dolly the sheep, a myriad of mammalian species have been cloned. Techniques such as stem cell therapy, genetic engineering, and RNAimediated gene regulation offer potential cures that even the doctors on Star Trek might be proud of. Along with incredible advancements, there has been an increasing concern over the state of science and mathematics education. An adequately educated populace is going to be required for economic success, for national security, and for citizens to make informed choices about their personal health and the use of new technologies. The zebrafish model system offers exciting possibilities for improving science education in the classroom and through outreach by scientists to the surrounding community. Together, these learning experiences offer the opportunity to recruit and encourage the next generation of scientists and increase communication between scientists and citizens. The zebrafish model system was relatively recently introduced to the scene (http:==www.neuro.uoregon.edu=k12= george_streisinger.html). Thus, while there is not a long history of using zebrafish as an educational tool, teacherscientists around the world are developing many exciting methods using zebrafish in laboratory courses and in outreach (for instance, see Zebrafish for K-12 at http:==www .neuro.uoregon.edu=k12=zfk12.html; Zebrafish in the Classroom at http:==www.zfic.org; and Bioeyes at http:==www .jefferson.edu=bioeyes=). Discussions at the ‘‘Zebrafish in the Classroom’’ workshop at the 2008 meeting of Zebrafish Development and Genetics (Madison, WI) led to a consensus that for zebrafish to make a strong impact on education, we needed a broader venue for communicating educational methods and ideas. This special issue of Zebrafish is one of the first efforts to answer that call. The goal was to create a resource that (1) is widely available and (2) contains articles that are peer reviewed, thus providing due credit to those developing the educational resources. Through generous support from Zebrafish editor Dr. Steve Ekker, Mary Ann Liebert, Inc., and many donors, we have achieved both of these goals. Experiential learning, which involves not just active learning, but also reflection and analysis of the experience, and then applying what was learned, may be the ideal approach to teaching students how to approach science. Zebrafish is an excellent system for using active and=or experiential approaches to teach developmental biology, genetics, neurobiology, and even behavior at many levels, from grade school to beginning undergraduate laboratories to courses teaching advanced concepts. The list of characteristics that make zebrafish an excellent classroom system is a long one. Just a few of the advantages are that zebrafish are cost effective, easy to maintain, and very reliable at producing large numbers of embryos on demand. In addition, a wide range of wild-type, mutant, and transgenic strains are now widely available. The large numbers of embryos that can be obtained from a modest number of adult pairs, the speed and predictability of their development, and the translucency and relatively large size of the embryos and larvae make it possible for students in the classroom to carry out their own hypothesis-driven experiments. While our potential to impact learning in the classroom is at an all-time high, schools are increasingly reducing or eliminating laboratories in response to funding cuts. Outreach from members of the zebrafish community have begun to fill this void. In addition to giving students valuable learning experiences, outreach activities can serve to increase public understanding on controversial issues, such as genetically modified foods and embryonic stem cells. Outreach programs can build bridges between universities and the surrounding communities, break down stereotypes about scientists, and offer valuable opportunities for undergraduate and graduate students to mentor others. These experiences can be extremely rewarding for the scientists as well as the teachers and the students with whom they interact. We particularly want to emphasize the many resources that are available for those who are interested in using zebrafish for the first time. One of the foremost challenges in using zebrafish in the classroom laboratory is establishing a zebrafish colony that is sufficiently robust to provide fertilized eggs on a regular basis. A colony can be as simple as a single tank or aquarium, using a mesh-covered pyrex tray as an eggcollecting vehicle (described in Emran et al.) or as complex as a benchtop, single-rack, or multi-rack recirculating system. Recirculating systems are available from several vendors, the support of whom has made it possible for us to provide immediate open access to this issue. Zebrafish can be obtained from many pet stores and biological supply companies, but for fish that are more likely to be pathogen-free and for specific strains, mutants, or transgenic lines, it is usually worth the extra cost to get zebrafish from the Zebrafish ZEBRAFISH Volume 6, Number 2, 2009 a Mary Ann Liebert, Inc. DOI: 10.1089=zeb.2009.9996