A major focus of the lab is investigating the biological basis of motoneuron diseases. In particular, we focus on two human motoneuron diseases, spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). We are also interested in the genetic and molecular cues that guide motor axons to their target muscles and facilitate motoneuron development. For all of our studies, we use zebrafish as a vertebrate model organism due to its well characterized nervous system and its relatively simple neuromuscular organization. For example, during the first day of development only three primary motoneurons innervate the developing myotome. During the second and subsequent days of development secondary motor axons fasciculate with these primary motor axons to form a dorsal, ventral, and intermediate nerve. This organization is maintained as the fish develops allowing analysis of nerves over time and at later stages of development. Because we can study motoneurons from their earliest stages of development through adulthood, we can ask what happens to motoneurons during the disease process. Moreover, by modeling these diseases in zebrafish, we can develop new approaches to identifying ways to alleviate the disease process. For example, we can perform genetic and drug screens to identify novel drug targets.
The main projects in the lab are:
What is the biological basis of the motoneuron disease Spinal Muscular Atrophy (SMA)?
Our understanding of the genetics and development of motoneurons puts us in an excellent position to address the biological basis of motoneuron diseases. We have had a long-standing interest in the motoneuron disease Spinal Muscular Atrophy (SMA); a motoneuron degenerative disease caused by mutations in the survival motoneuron gene (smn). SMA is caused by low levels of the SMN protein. Although the ubiquitously expressed Smn protein has been implicated in snRNP (RNA and protein) complex essential for mRNA splicing, it remains unclear why low Smn levels compromise motoneurons. Using protein knockdown technology (anti-sense morpholinos), we decreased the amount of Smn present during zebrafish development and found dramatic defects in motor axon outgrowth and guidance. In particular, motor axons were truncated and excessively branched. We have gone on to generate maternal:zygotic (mz) smn mutants and show the defects in motor axon outgrowth are due to poor motoneuron development. Using a conditional transgene on the mutant background, we show that Smn protein is needed very early in motoneuron development. We are now focused on understanding how Smn functions in motoneuron development and how this affects motor circuit formation.
Modeling ALS in zebrafish
Amyotrophic lateral sclerosis is an adult onset, fatal, motoneuron degenerative disease that has no cure and limited therapies. While the majority of ALS cases have no known genetic component, 1-2% are caused by mutations in the SOD1 gene. Changes in 74 of the 154 amino acid protein causes a form of ALS indistinguishable from sporadic ALS thus serving as a way to model ALS in animals. To date, only a mouse model of SOD1 ALS has been generated. Major questions regarding the toxicity of the mutant SOD1 forms, how they cause motoneuron death, where they are functioning, and what genetic pathways they act in remain unanswered. Using the zebrafish Sod1 gene, we have generated Sod G93R transgenic zebrafish that recapitulate the major hallmarks of ALS. Our goal is to understand the early changes that happen in the spinal cord of these animals as a way to better understanding the mechanism of motoneuron dysfunction in ALS.
What genes define motor axon outgrowth?
The process of axon outgrowth occurs due to the integration of signals from the environment received by the growth cone. Using forward genetics, we have identified two mutations that cause motor axons to stall and fail to extend into distal target regions. Although these mutations have a similar phenotype, our analysis reveals that they are disrupting distinctly different aspects of motor axon guidance. One of these mutations, stumpy, causes all motor axon growth cones to stall at intermediate targets; regions along axon pathways where growth cones pause, branch, or turn suggesting that information is being imparted. Cloning has revealed that stumpy encodes the collagen 19A gene and it is expressed at intermediate targets in the myotome. The other mutation, topped, has a very specific phenotype where the CaP motoneuron is severely delayed in growing into the ventral myotome. Genetic mosaic analysis revealed that Topped is functioning in the ventromedial fast muscle. These data suggest that topped is the ventral cue that enables motor axons to extend into the ventral myotome. This finding is significant in that it strongly supports the idea that growth cones recognize unique myotome regions based on the presence of particular molecules. We are now in the process of cloning the topped gene and identifying other molecules involved in motor axon outgrowth. Our goal is to better understand how these genes are controlling motor axon outgrowth during development.
Using zebrafish to study glioblastoma
Glioblastoma (GBM) is a deadly brain cancer with few effective drug treatments available stressing the need to better understand the biology of these highly aggressive tumors. To facilitate analysis of glioblastoma tumor cell behavior in-vivo in real time and to develop new approaches for GBM drug screens, we have generated and standardized a xenotransplant model of GBM in zebrafish. Two patient-derived GBM cell lines, serum grown adherent cells (X12) and neurospheres (GBM9), were transplanted into the midbrain region of embryonic zebrafish. Analysis of larvae over time showed progressive brain tumor growth and premature death with both cell lines, however, fewer GBM9 cells were needed to cause tumor growth and lethality. Approximately half of the cells in both xenotransplants were dividing whereas control mouse neural stem cells failed to engraft and were cleared from the brain. Few GBM9 cells expressed GFAP or vimentin, markers of more differentiated cells, early, but this number increased significantly during tumor growth indicating that GBM9 cells undergo differentiation in vivo. In contrast the vast majority of serum grown X12 cells expressed GFAP and vimentin at the earliest times examined post transplant and this population remained high. Both cell types also contained Sox2-expressing cells indicative of stem cells. To determine whether in-vivo GBM9 tumors were responsive to currently used therapeutics, we treated transplanted larvae with either temozolomide or bortezomib and found a reduction in tumor volume in-vivo and an increase in survival. Thus, we have successfully generated and standardized an orthotopic model to study glioma cell biology in-vivo as well as to perform drug screens. We are using this model to investigate the role of cell migration in GBM, the effect of treatment on tumor cell heterogeneity, and as a model for GBM9 drug screens.
Tools and Techniques
Genetics and molecular biology: working with mutant zebrafish lines, generating transgenic lines, generating targeted knockouts using CRISPR/Cas9
Cell biology and microscopy: Confocal and compound microscopy, analysis of axonal outgrowth and axon morphology, characterization of synapses, analysis of the motor circuit
Biochemistry: western blots, co-IPs, mass spectrophotometry
RNA biology: RNAseq, RNA in situ hybridization and cellular localization
For a list of current and former publications, please follow the link to PubMed: http://www.ncbi.nlm.nih.gov/pubmed/?term=beattie+ce