Chi-Bin Chien - US grants

As axons pathfind to their targets in the developing nervous system, they are guided by a complex environment of positive and negative cues, which are sensed and transduced by the machinery of the growth molecules involved in pathfinding, but many remain unknown. A large- scale genetic screen in zebrafish has isolated approximately -25 genes required for the projection of retinal axons to the tectum. Of these, the ashtray mutant shows the most severe and specific pathfinding phenotype. Retinal axons in ashtray seem to ignore many guidance cues: they cross the midline repeatedly, and project anteriorly to forebrain and posteriorly to hindbrain as well as to their normal target. There is no general derangement of brain patterning, and the early axon scaffold is undisturbed. Embryonic eye transplants show that ashtray acts eye- autonomously. Thus, the ashtray gene product is likely to be an axon pathfinding molecule required in retinal axons. We have genetically mapped ashtray at high resolution, and found it maps very close to a novel zebrafish roundabout homolog, zRobo1A. The experiments proposed here are designed to understand how ashtray acts during retinal axon pathfinding and to elucidate the molecular nature of the gene. First, axons will be labeled in fixed embryos and growing axons observed in live embryos to determine when and where ashtray axons misroute. Cell type-specific markers will also be used to check whether ashtray acts to change retinal ganglion cell fate. Second, single- cell ashtray is required at distinct choice points during pathfinding. Third, six specific sets of non-retinal axons that normally exhibit a wide variety of behaviors will be labeled to test whether they are affected in ashtray. Fourth, the ashtray gene will be cloned. We will first test whether it is a defect in zRobo1A. If not, we have begun a genomic walk and will clone the gene positionally. In summary, this project will advance our understanding of how wildtype retinal axons pathfind; illuminate where, when, and how the behavior of single zebrafish retinal axons requires the function of ashtray, be established for the future analysis of all the zebrafish retinotectal mutants, which promise to yield important insights into the genetic control of visual system development.

Chi-Bin Chien

Lay Abstract
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To form the correct circuitry in the developing nervous system,
billions of nerve cells must send their axons (individual nerve
fibers) over long distances to find their specific target cells. How
these axons find their targest is one of the fundamental questions of
developmental neuroscience. When many axons take similar pathways and
form a nerve bundle or axon tract, individual axons are often highly
organized within the larger tract. How such sorting within axon
tracts is achieved is a basic question of axon guidance that has been
studied very little.

In the visual system, retinal axons exit the eye and form the optic
nerve, where they are organized in a very precise array according to
their point of origin. Shortly after passing through the optic
chiasm, these retinal axons reorganize in a characteristic way, so
that they are sorted out according to a different order as they grow
through the optic tract on their way to visual centers in the brain.
This sorting of retinal axons in the optic tract allow the proper
formation of normal visual connections, and is therefore critical for
normal vision.

This project will study the mechanisms of axon sorting in the visual
system of the zebrafish, Danio rerio. The zebrafish visual system has
many fundamental similarities (both genetic and anatomical) to humans
and other vertebrates, and so the principles discovered here are
likely to have wide applicability. In a particular zebrafish mutant
strain, called boxer, a subset of the retinal axons fail to
reorganize normally within the optic tract. Thus, normal function of
the boxer gene is critical for normal development of the visual
system. The proposed experiments will analyze whether boxer function
is required in the eye or in the brain, and will clone boxer to
determine which mutated gene is responsible for the visual system
defect. These experiments will shed light on a critical aspect of how
the brain is properly wired during development.

[unreadable] DESCRIPTION (provided by applicant): For growing axons to find their targets in the developing brain, their growth cones must respond to both attractive and repulsive signals in the environment. It has recently been shown that growth cones can modulate their responses to particular signal, even switching from attraction to repulsion; however, it is not known if this occurs in vivo. The zebrafish visual system is uniquely suited for studying how growth cones integrate positive and negative signals in vivo, and testing how their responses to signals change as they pathfind. The proposed experiments study the roles of two classes of guidance cues, Slits and netrins, in retinal axon pathfinding. Slits are thought to signal repulsively through the Astray/Robo2 receptor, while netrins are thought to signal attractively through the DCC receptor. Slit/Robo2 and netrin/DCC are both known to guide retinal axons, but their roles in different parts of the pathway are unknown. A combination of forward-genetic, reverse-genetic, and transgenic approaches will be used to perturb Slit/Robo2 and netrin/DCC signaling in vivo, to test where in the retinal pathway these signals are important, and to test whether their roles change over the course of the pathway. The zebrafish retinotectal system not only allows visualization of retinal axons in vivo with exquisite resolution, but also allows precisely targeted perturbations of their in vivo environment. The results and techniques developed here will help lead to a comprehensive understanding of all the signals that guide retinal axon growth, and how these different signals interact. In summary, this project will illuminate where, when, and how the guidance of zebrafish retinal axons requires Slit/Robo2 and netrin/DCC signaling. The resulting knowledge of genetic and developmental mechanisms will be important for understanding human diseases, such as albinism, that affect optic axon guidance. This knowledge will also be critical for designing therapies to reverse optic nerve degeneration. More generally, this project will test broad principles of axon guidance which are important for understanding the wiring of the nervous system and the basis of inherited neurological disease.

DESCRIPTION (provided by applicant): Imaging of biological specimens has become an increasingly important tool in biomedical science, with the abilities to image live specimens and multiple fluorophores being especially important. We propose to purchase a Zeiss LSM510 confocal microscope system to be housed in the Cell Imaging Facility of the University of Utah School of Medicine. This confocal would replace an obsolete entry-level confocal (Olympus FVX) and improve the capabilities of the facility by adding a wide choice of excitation wavelengths, as well as fast switching between multiple emission filters. An environmentally controlled specimen chamber and motorized X-Y stage would particularly facilitate live-cell imaging experiments. No instrument with this set of features is currently available anywhere at the University of Utah. The core Cell Imaging Facility is centrally located in the School of Medicine, and is overseen by a fulltime Ph.D. director with postdoctoral experience. Under his direction the facility has been highly successful, with heavy use of the existing confocal microscopes (>35 hours/week/microscope). The director provides training and support to users in addition to maintaining the microscopes and administering their use. Furthermore, every year he teaches a didactic microscopy course together with the Principal Investigator. There is a strong institutional commitment to providing core microscopy facilities. The University of Utah fully funds the facility director's salary, and guarantees the cost of service contracts. The instrument's use would be monitored by an oversight committee, which would liaise with a committee that oversees all of the School of Medicine's core facilities. This instrument would primarily serve 7 major users from 4 different departments in the School of Medicine. These researchers are funded by 14 current R01, P01 or R37 NIH grants and are studying systems including cultured cells, C. elegans, and zebrafish. All of these projects involve live-cell imaging, and would benefit significantly from the improved flexibility in excitation and emission wavelengths offered by the proposed instrument.

DESCRIPTION (provided by applicant): Slit-Robo signaling is 1 of the main axon guidance signaling pathways that act to shape the circuitry of the nervous system. Recent work shows the critical role of Slit-Robo signaling in the development of the visual system and spinal cord in model organisms, and the corticospinal tract in humans. However, only a few effector genes have been implicated in this signaling pathway, and none of these effector genes have been studied genetically in vertebrates. In addition, it is poorly understood which structural domains of Robo receptors are critical for their in vivo function. This proposal will use a forward genetic screen searching for interactions (noncomplementation) with the zebrafish astray (robo2) mutant. By screening individual F1 larvae labeled with a GFP transgene, we will be able to screen 75,000 genomes in 3 years. A pilot screen of 5700 genomes has already yielded 12 putative mutants. This screen will (1) identify mutants in genes in the Slit-Robo pathway, then (2) genetically map these mutants and (3) conduct a preliminary phenotypic assessment analyzing multiple axon pathways. We expect to find 3 classes of mutants: (I) new mutant alleles of robo2, which we will identify and sequence; (II) temperature-sensitive robo2 alleles, which we will identify and characterize; and (III) mutations in genes that show strong genetic interactions with robo2, such as slit ligands or downstream effectors, which we will genetically map and analyze phenotypically. The zebrafish system offers unique advantages for studying vertebrate axon guidance, including the ability to trace the trajectories of single axons both in fixed tissue and using timelapse movies; the ability to generate temperature-sensitive alleles in a vertebrate; and the ability to carry out forward-genetic screens. The robo2 alleles isolated in this screen will be valuable for understanding the timing of Robo2 function (temperature-sensitive alleles) and the protein domains necessary for Robo2 signaling (missense or nonsense alleles). The interacting genes isolated will define a set of genes in the Robo2 signaling pathway, and allow us to study their function in vivo.

The patterning of the retina and its axonal output are critical for visual function, and the retinotectal proj- ection is a classic model of the development of two-dimensional neural maps. Retinal ganglion cells acquire positional coordinates along the dorsal-ventral (D-V) and anterior-posterior (A-P) axes, then translate these into graded expression of axon guidance molecules, which control topographic sorting in the optic tract and topographic targeting on the tectum. In the long term, we seek to understand how gradients of axonal beha- vior are generated. As a first step, we propose here a comprehensive analysis of the genes involved in the patterning of dorsal retina, which to date is poorly understood. Misexpression and dominant-negative experiments in chick and Xenopus have implicated BMP4, Tbx5, and ephrin-B2 in dorsal specification and D-V topography. However, the roles of Tbx5 and ephrin-B2 have not been tested by loss-of-function analysis, and our data show that more genes must be involved. We will analyze D-V retinal patterning using the zebrafish visual system, which is ideal for rapid and precise loss- of-function experiments to study retinal patterning and retinotectal topography. The main goals are: (1) to test the required roles of the known candidate genes bmp4, tbx5, and ephrin-B2 in specifying dorsal retinal fate and topographic projections;(2) to test the roles of new candidate genes we have identified, specifi- cally tcf7, other tbx genes, and ephrin-B1\ and (3) to identify new genes with a forward-genetic screen for mutants that disrupt dorsal retinal specification. Our preliminary data implicate Wnt signaling for the first time in D-V retinal patterning, and furthermore suggest crosstalk between the Wnt and BMP pathways. Building on our previous expertise in the zebrafish visual system, we will combine genetic experiments with the rapid and sophisticated embryological and phenotypic analysis possible in zebrafish. We will use ex- isting mutants and antisense morpholino oligonucleotides, as well as new mutants isolated by forward gene- tic and reverse genetic (TILLing) genetic screens. We will critically test the existing model of D-V retinal patterning, and extend it significantly by testing new hypotheses arising from our preliminary data, and by conducting an unbiased screen for required genes. These studies will address two fundamental questions in developmental neurobiology: How do regions of the nervous system acquire positional coordinates? and, How do they then make topographic connections with each other? Identifying the key genes in dorsal retina will lay the groundwork for future work on how these genes interact to generate topographic gradients. Relevance to public health. The development and structure of the visual system are remarkably conserv- ed across evolution from fish to humans. Therefore, these studies will shed light on how the human eye is formed and becomes patterned. This is of particular interest because defects in dorsoventral eye patterning are associated with human developmental disorders such as coloboma.

DESCRIPTION (provided by applicant): Animal behavior depends on the function of a large collection of overlapping neural circuits. To fully under- stand the circuit underlying a particular behavior, one must identify the neurons involved, determine what synaptic connections they make with each other, and measure their electrical responses during activation of the circuit. The zebrafish larva is an excellent system to study circuits: it has well-established behaviors, can be manipulated genetically, and most importantly, is transparent. By genetically expressing fluorescent reporters or light-activated channels, one can optically image neurons'morphology, connectivity, and activity, and even optically control their electrical activity, in an intact, living animal (Scott, 2009). What has been largely missing, until recently, are methods to express genes in particular neurons of interest. A powerful solution to this problem is provided by Gal4 "enhancer trap" screens in zebrafish (Scott et al., 2007;Asakawa et al., 2008). The Gal4 gene, which acts as a genetic trigger, is integrated randomly into the zebrafish genome;depending on where it lands, it will be turned on in a different set of cells (often including specific neuronal types), controlled by the regulatory elements of nearby genes. By screening through many Gal4 mutant lines, one can find lines that express in one's favorite neurons, then cross these to UAS "responder lines", so that fluorescent reporters or other genes are turned on in those neurons. This proposal will carry out a second-generation Gal4 enhancer trap screen with several improvements. (1) A new DNA trapping construct that not only expresses Gal4, but can be converted to instead express a different genetic switch, Cre recombinase. This will allow expression of genes in even more specific sets of cells by "intersecting" a Gal4 pattern with a Cre pattern. (2) An online database of Gal4 expression patterns, including three-dimensional views. This will allow collaborators, and eventually the zebrafish community at large, to quickly determine which lines may express in the tissues or neurons that they study. (3) A new public- domain 3D visualization package, FluoRender, that has been optimized for confocal microscopy data. This will improve and speed up documentation of expression patterns. (4) A "toolkit" of UAS responder lines, validated for uniform expression levels, to visualize neuronal shape and connectivity. Diencephalic dopaminergic neurons, for which no specific enhancer is yet known, will be analyzed as a test case. In summary, then, this project will generate a large number of well-characterized Gal4 enhancer trap lines and UAS responder lines, which will allow zebrafish neurobiologists as well as other zebrafish researchers to express genes of interest specifically in many different neuron classes and nonneural tissues. Techniques developed for intersectional gene expression, generation of UAS responders, and 3D visualization will also be widely applicable in the field. The project will significantly increase the utility of the Gal4-UAS method in zebrafish, aiding analysis of the development and function of many organs, in addition to neuronal circuits. PUBLIC HEALTH RELEVANCE: Neurological diseases ranging from autism, cerebral palsy, and Tourette's syndrome to Parkinson's disease are due to malfunction of neural circuits in the brain, yet in many cases the understanding of these circuits is only rudimentary. This proposal would develop genetic tools to study the zebrafish brain, which shares many organizational and even detailed features of the human brain, which will enable the analysis of the shape, synaptic connections, and electrical activity of nerve cells in many different circuits, with the long-term goal of understanding how these circuits may go wrong in human disease.