Howard Sirotkin - US grants

The nature of the inductive signaling networks that pattern the vertebrate neuroectoderm is a central problem in developmental biology. The induction of the floor plate differentiation by the underlying notochord is a paradigm of the signaling events that establish the dorsoventral axis of the neural tube. Floor plate induction is mediated by Sonic hedgehog, but much remains unknown about the mechanisms by which the floor plate cell fate is specified. I propose to learn about the basis of floor plate specification with a cellular and molecular analysis of the zebrafish (Danio rerio) mutant cyclops (cyc), which deletes the floor plate and other ventral neuroectodermal cell types. This proposal will test the hypothesis that cyc has an essential function in floor plate specification, perhaps mediating the inductive action of Sonic hedgehog.. The aims of this proposal are (1) to characterize floor plate precursors in wild-type and cyc mutant embryos using fate mapping techniques, and (2) to molecularly identify the cyc gene by positional cloning. These experiments will provide critical information about the specification of midline cell types in wild-type embryos, and the role that cyc plays in the process.

[unreadable] DESCRIPTION (provided by applicant): The diversity of cell types within the vertebrate nervous system depends on patterning events that occur at early stages of development. The specification and patterning of neural tissue is closely coupled to the development of the other germ layers. The mesoderm and endoderm are important sources of signals that induce neural tissue and establish asymmetries within the neural plate. In this proposal, we seek to utilize the potent genetic and cellular methodologies available in the zebrafish to study patterning of the neural ectoderm. The zebrafish is well suited to this analysis. Zebrafish embryos are transparent and embryonic development occurs rapidly. These attributes foster detailed observation of normal and aberrant embryonic development. Zebrafish produce large numbers of offspring, which in addition to facilitating phenotypic characterization, enhances genetic analysis. The proposed experiments utilize several well characterized zebrafish mutations to investigate the molecular mechanisms that induce and pattern neural tissue. The general approach is to account for all the signals that generate anterior and posterior neural tissue. Models for both neural induction and patterning will be tested. A genetic screen is proposed to identify novel loci that disrupt anterior neural specification. The screen takes advantage of the ability to generate haploid zebrafish embryos in order to increases the throughput of the screen. There are two components to the screen: a morphology based approach to identify enhancers of a mutation (bozozok) which disrupts anterior neural patterning and an in situ based effort to detect alterations of the expression domains of the phox2a transcription factor. One promising mutation identified in a pilot screen alters anterior neural patterning and will be studied in detail. Because all vertebrates share fundamental similarities in the organization of their nervous systems, understanding the genetic networks that govern neural patterning in zebrafish will provide important insights into development of other species, including humans. Several zebrafish mutations have that disrupt embryonic development have anterior neural defects similar to a common human congenital abnormality, holoprosencephaly, and share similar etiologies. Deciphering the mechanisms of vertebrate axis formation may also provide insight into the causes other human developmental disorders. [unreadable] [unreadable]

DESCRIPTION (Provided by Applicant): One fundamental method to investigate biological processes is by loss-of-function studies. The goal of these efforts is to determine the activity of a gene by disrupting that gene and monitoring the effects on a biological system. Recently, gene targeting technology has been developed using zinc finger nucleases (ZFNs) as a method to mutate specific genes. The approach involves engineering zinc finger DNA binding domains to recognize specific target sequences. These domains can then be tethered to the FOK1 exonuclease domains to generate a fusion protein that can induce double stranded DNA breaks near the zinc finger binding sites. ZFNs have been successfully used to disrupted genes in a variety of systems ranging from plants to mammals. Thus far, only a relatively small number of laboratories have reported successful efforts to employ ZFNs. The use of ZFNs has been curtailed by the difficulty in generating zinc finger domains that bind to specific target sequences, and by the high cost of commercially available ZFNs. In preliminary studies, computer aided modular design has been an effective means to engineer functional ZFNs. The experiments in this application evaluate the effectiveness of a strategy to use modular ZFN design to generate germ-line mutations using zebrafish as a model system. Zebrafish is a powerful system for genetic, cellular, and molecular studies, and the ability to routinely perform gene targeting studies would be a major advance. The methods in this application are broadly applicable to biomedical research in both cell culture systems and model organisms. RELEVANCE: The human genome project has identified thousands of novel genes, but the activity of many of these genes remains unknown. The ability to disrupt specific genes is a powerful means to determine the function of these genes. The experiments in this application evaluate the effectiveness of one means to accomplish this goal using model organisms and cell culture systems.

It is imperative that we develop rational and systematic approaches to drug discovery to treat Parkinson's and other neurological disorders. Rodent models are essential to this process, but they may not be the optimal starting place. The LRRK2 G2019S allele is the most common Parkinson's disease mutation and corresponding animal models have been developed in a variety of organisms. While the existing models are valuable, we engineered a zebrafish LRRK2 G2019S line that is a uniquely powerful starting place to accelerate PD therapeutics. The experimental flexibility of zebrafish including rapid gene targeting and unparalleled high-resolution functional brain imaging empowers studies of molecular, cellular and physiological mechanism of disease. However, the true power of zebrafish to promote PD research lies in the ability to conduct transformative experiments that cannot readily be done with other models. The ability to culture larvae in 96-well dishes and introduce test compounds via the water enables high throughput screening of small molecules using behavioral readouts. Tens of thousands of compounds can be assayed to identify potential therapeutic agents that can then be further refined to streamline the drug discovery pipeline. Our long-term goal is to perform such a screen to search for compounds that modulate Parkinsonian related behaviors using a zebrafish PD model. In order for this approach to be effective, behaviors that reflect those in patients must first be demonstrated in our LRRK2 G2019S mutant. The objectives of this study are to assay PD-related behaviors and dopaminergic populations in the zebrafish LRRK2 G2019S mutants to determine whether this model is a suitable substrate for high throughput chemical screens. If successful, this approach would go beyond identification of direct LRRK2 inhibitors and enable isolation of compounds that influence other processes to compensate for LRRK2 dysfunction.        

PROJECT SUMMARY/ABSTRACT NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the brain. Many higher order neural processes including synaptogenesis and the synaptic plasticity underlining learning and memory depend on NMDAR-mediated transmission. The NMDAR GluN2B subunit is critically involved in early brain development. Accordingly, missense and nonsense mutations in GRIN2B, the gene encoding GluN2B, are associated with autism spectrum disorder, intellectual disability, and schizophrenia among other neurodevelopmental disorders. The specific role of GluN2B in brain development and the causal link between GluN2B dysfunction and these diverse disorders is unknown. Our goal, taking advantage of the power of zebrafish, is to identify the role of GluN2B in brain development and neurodevelopmental disorders and to pioneer new therapies to treat such disorders. We must first establish the use of zebrafish as a model system to study GluN2B in neurodevelopmental disorders. To do so, we will test whether zebrafish GluN2B (zGluN2B) functions similarly to human GluN2B and if it displays a comparable pharmacology (Aim#1). In addition, we need to identify GluN2B-dependent zebrafish behaviors (Aim#2), which provide a pathway to study circuit development and a substrate for behaviorally-based drug screens. In Aim 1, we will use heterologous expression zebrafish and human NMDAR subunits in HEK293 cells and patch clamp electrophysiology to characterize functional and pharmacological properties. In Aim 2, we will examine how knockout of zebrafish GluN2B affects behaviors associated with human neurodevelopmental disorders. In the long-term, zebrafish have the potential to provide a platform to study the role of GluN2B in brain development and the effects of human GRIN2B missense mutations on neural circuit development and carry out behaviorally based high throughput small molecule drug screens to counter the effects of GluN2B dysfunction.