Biology subjects

Mastick, G. S.

Publications and source records attributed to Mastick, G. S..

4 recordsLinked to original sources

bioRxiv2019

Slit/Robo signals prevent spinal motor neuron emigration by organizing the spinal cord basement membrane

The developing spinal cord builds a boundary between the CNS and the periphery, in the form of a basement membrane. The spinal cord basement membrane is a barrier that retains CNS neuron cell bodies, while being selectively permeable to specific axon types. Spinal motor neuron cell bodies are located in the ventral neural tube next to the floor plate and project their axons out through the basement membrane to peripheral targets. However, little is known about how spinal motor neuron cell bodies are retained inside the ventral neural tube, while their axons can exit. In previous work, we found that disruption of Slit/Robo signals caused motor neuron emigration outside the spinal cord. In the current study, we investigate how Slit/Robo signals are necessary to keep spinal motor neurons within the neural tube. Our findings show that when Slit/Robo signals were removed from motor neurons, they migrated outside the spinal cord. Furthermore, this emigration was associated with abnormal basement membrane protein expression in the ventral spinal cord. Using Robo2 and Slit2 conditional mutants, we found that motor neuron-derived Slit/Robo signals were required to set up a normal basement membrane in the spinal cord. Together, our results suggest that motor neurons produce Slit signals that are required for the basement membrane assembly to retain motor neuron cell bodies within the spinal cord.

developmental biology↗

bioRxiv2019

Precise removal of Calm1 long 3′ UTR isoform by CRISPR-Cas9 genome editing impairs dorsal root ganglion development in mice.

Most mammalian genes are subject to Alternative cleavage and PolyAdenylation (APA), often resulting in alternative length 3' UTR isoforms. Thousands of extended or long 3' UTR variants are preferentially expressed in neuron-enriched tissues of metazoans. However, the in vivo functions of these long 3' UTR isoforms are largely unknown. Calmodulin 1 (Calm1) is a key integrator of calcium signaling that is required for correct neural development. Calm1 generates short (Calm1-S) and long 3' UTR (Calm1-L) mRNA isoforms via APA. We found Calm1-S to be broadly expressed across mouse tissues, whereas Calm1-L expression was largely restricted to neural tissues, including the dorsal root ganglion (DRG). Using CRISPR-Cas9 genome editing, a series of mouse deletion lines were generated that successfully eliminated expression of Calm1-L while maintaining expression of Calm1-S. One of these lines, Calm1{Delta}3' UTR, carried a 163 bp deletion surrounding the distal polyA site. Examination of Calm1{Delta}3' UTR embryos revealed disrupted development of the DRG. In Calm1{Delta}3' UTR DRG explant cultures undergoing axon outgrowth, we observed a dramatic increase in axon fasciculation. These results demonstrate a physiological role for Calm1-L in DRG development, and more generally, establish a genome-editing strategy to study in vivo functions of long 3' UTR isoforms. Author SummaryMore than half of all human genes generate alternative mRNA isoforms which differ in the length of their 3 Untranslated regions (3 UTRs). Through a process called Alternative Cleavage and Polyadenylation thousands of broadly expressed genes preferentially express long 3 UTR variants in brain tissues whereas their short 3 UTR counterparts are more broadly expressed. A challenge to study the functions of these transcripts has been to generate loss of function mutant animals that lack a long 3 UTR isoform but maintain expression of the corresponding short 3 UTR isoform. Here, we used the precise, rapid, and efficient approach of CRISPR genome-editing to generate long 3 UTR mutant mice. These mice, which do not express the long 3 UTR of the Calmodulin 1 (Calm1) gene, exhibit impairment in the development of sensory neurons, including increased fasciculation of axons and aberrant cell body migration. This finding is important because it provides conclusive genetic evidence for a neural function of a long 3 UTR isoform in an animal. The CRISPR genome-editing approach used here can be applied to the study of neuron-enriched long 3 UTR isoforms, which number in the thousands and have largely unexplored functions.

genetics↗

bioRxiv2018

Oculomotor nerve requires an early interaction with muscle precursors for nerve guidance and branch patterning

Muscle function is dependent on innervation by the correct motor nerves. Motor nerves are composed of motor axons that extend through peripheral tissues as a compact bundle, but then diverge to create nerve branches to specific muscle targets. A transition point typically occurs as motor nerves grow near their targets, where the fasciculated nerve halts further growth, then later initiates branching to muscles. The motor nerve transition point is potentially an intermediate target acting as a guidepost to present specific cellular and molecular signals for navigation. Here we describe the navigation of the oculomotor nerve with respect to eye muscle precursor cells in mouse embryos. We found that the oculomotor nerve initially grew to the eye three days prior to the appearance of any eye muscles. The oculomotor axons spread to form a plexus within a mass of eye muscle precursors, then the nerve growth paused for more than two days. This plexus persisted during primary extraocular myogenesis, with a subsequent phase in which the nerve branched out to specific muscles. To test the functional significance of the nerve-precursor contact in the plexus, we genetically ablated muscle precursors early in nerve development, prior to nerve contact. Ablation of muscle precursors resulted in oculomotor nerve fibers failing to stop to form the plexus, but instead growing past the eye. In contrast, ablating the precursor pool at later stages, after the nerve has contacted the precursor cells, results in ectopic branching restricted near the eye. These results demonstrate that muscle precursors act as an intermediate target for nerve guidance, and are required for the oculomotor nerve to transition between nerve growth and distinct stages of terminal axon branching.

neuroscience↗

bioRxiv2018

Robo1 and 2 repellent receptors cooperate to guide facial neuron cell migration and axon projections in the embryonic mouse hindbrain

BackgroundThe facial nerve is necessary for our ability to eat, speak, and make facial expressions. Both the axons and cell bodies of the facial nerve undergo a complex embryonic migration pattern involving migration of the cell bodies caudally and tangentially through rhombomeres, and simultaneously the axons projecting to exit the hindbrain to form the facial nerve.\n\nResultsOur goal in this study was to test the functions of the chemorepulsive receptors Robo1 and Robo2 in facial neuron and axon migration by analyzing genetically marked motor neurons in double mutant mouse embryos through the migration time course, E10.0-E13.5. In Robo1/2 double mutants, axon and cell body migration errors were more severe than in single mutants. Most axons did not make it to their motor exit point, and instead projected into and longitudinally within the floor plate. Surprisingly, some facial neurons had bifurcated axons that either exited or projected into the floor plate. At the same time, a subset of mutant facial cell bodies failed to migrate caudally, and instead shifted into the floor plate.\n\nConclusionsRobo1 and Robo2 have redundant functions to guide multiple aspects of the complex cell migration of the facial nucleus, as well as regulating axon trajectories and suppressing formation of ectopic axons.\n\nBullet pointsO_LIRobo1 and 2 repellent receptors were tested for functions in facial neuron cell body and axon migration in the developing E10.0-E13.5 hindbrain of double mutant mouse embryos.\nC_LIO_LIIn Robo1;Robo2 double mutant embryos, facial motor neuron cell bodies and axons abnormally grew into the hindbrain floor plate, with stronger effects than in single Robo mutants.\nC_LIO_LIMany facial nerve axons abnormally bifurcated, failed to project to their proper exit point, and instead projected longitudinally within the floor plate.\nC_LIO_LIFacial neuron cell body tangential migration was disrupted.\nC_LIO_LIRobo receptor-mediated repulsion is required for proper migration of both facial axon projections and cell body migrations.\nC_LI\n\nFunding was provided to G.S.M. by NIH R01 NS054740, R21 NS077169, and R01 EY025205. Core facilities at the University of Nevada Reno campus are supported by NIH COBREs GM103650 and GM103554, and Nevada INBRE GM103440.

neuroscience↗