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. 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, CaP (caudal primary), MiP (middle primary) and RoP (rostral primary) motoneurons innervate the developing myotome. CaP extends a ventral axon, MiP a dorsal axon, and RoP innervates the region in-between. 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.
There are three main projects in my lab:
What is the biological basis of the motoneuron diseases 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. Currently, we are establishing zebrafish as a model of 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 specifically compromises 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. By decreasing Smn in single motoneurons in living embryos, we found that Smn functions cell-autonomously with respect to motoneurons in this process. This data suggests that Smn is needed for normal motoneuron development. We also showed that more severe motor axon defects caused decreased longevity and disruptions at the neuromuscular junction (NMJ).
A central question in SMA is what function of SMN, when disrupted, leads to motoneuron dysfunction and disease. SMN has a well-characterized role in splicing, but data has also suggested that it could function in other ways in cells perhaps to assembly mRNA and proteins for transport and localized protein translation. To get at this, we asked what forms of Smn could rescue these motor axon defects. Using human SMN RNA we tested a number of both patient and synthetic mutations and co-injected these with the smn MO. We found that wild-type hSMN could rescue the motor axon defects, but no patient mutations could rescue when added at the same dose. Testing a number of different SMN forms, we found that SMN forms that had their snRNP properties in tact, could still not rescue the motor axon defects caused by low Smn. These studies suggest that for normal motor axon development that perhaps some other function beside snRNP is necessary. To further investigate this possibility, we have generated a genetic model of SMA in zebrafish that will allow us to perform more detailed and long range studies.
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. As there are no reported examples of ALS models in invertebrates, only rodent models of this disease exist thus limiting the type of experiments that can be performed. For example, although it is possible to map modifier genes that affect FALS, it is not possible to do modifier screens in mice. Using the zebrafish Sod1 gene, we have generated Sod G93A and G85R transgenic zebrafish. Two independent lines of G93A express mutant protein in brain and spinal cord at levels ~3-fold higher than wild type Sod. We used confocal microscopy to show that the fish display neuromuscular junction defects suggestive of muscle denervation. This is confirmed by electron microscopy (EM) showing both muscle and motoneuron degeneration. Since we were seeing defects in the neuromuscular system, we wanted to test the strength of the muscle. We did this by testing fish in a current swim tunnel. Using this approach we found that the transgenic lines over expressing mutant Sod protein, were unable to swim in strong currents like wild-types indicating that they had muscle weakness. Our data suggest that we have generated a new vertebrate model of ALS that will be useful for studies of ALS biology and as a tool for drug and genetic screening.
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. Using genetic mosaics we show that Stumpy function is needed both in the neuron and in the environment. Our hypothesis is that Stumpy enables growth cones to proceed past intermediate targets, perhaps by a de-adhesive mechanism. To test this hypothesis, we are cloning stumpy to reveal its molecular identity. 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.
