RESUMEN
Axon growth and branching, and development of neuronal polarity are critically dependent on proper organization and dynamics of the microtubule (MT) cytoskeleton. MTs must organize with correct polarity for delivery of diverse cargos to appropriate subcellular locations, yet the molecular mechanisms regulating MT polarity remain poorly understood. Moreover, how an actively branching axon reorganizes MTs to direct their plus ends distally at branch points is unknown. We used high-speed, in vivo imaging of polymerizing MT plus ends to characterize MT dynamics in developing sensory axon arbors in zebrafish embryos. We find that axonal MTs are highly dynamic throughout development, and that the peripheral and central axons of sensory neurons show differences in MT behaviors. Furthermore, we show that Calsyntenin-1 (Clstn-1), a kinesin adaptor required for sensory axon branching, also regulates MT polarity in developing axon arbors. In wild type neurons the vast majority of MTs are directed in the correct plus-end-distal orientation from early stages of development. Loss of Clstn-1 causes an increase in MTs polymerizing in the retrograde direction. These misoriented MTs most often are found near growth cones and branch points, suggesting Clstn-1 is particularly important for organizing MT polarity at these locations. Together, our results suggest that Clstn-1, in addition to regulating kinesin-mediated cargo transport, also organizes the underlying MT highway during axon arbor development.
RESUMEN
The midbrain dopaminergic neuronal groups A8, A9, A10, and A10dc occupy, respectively, the retrorubral field (RRF), substantia nigra compacta (SNc), ventral tegmental area (VTA), and ventrolateral periaqueductal gray (PAGvl). Collectively, these structures give rise to a mixed dopaminergic and nondopaminergic projection system that essentially permits adaptive behavior. However, knowledge is incomplete regarding how the afferents of these structures are organized. Although the VTA is known to receive numerous afferents from cortex, basal forebrain, and brainstem and the SNc is widely perceived as receiving inputs mainly from the striatum, the afferents of the RRF and PAGvl have yet to be assessed comprehensively. This study was performed to provide an account of those connections and to seek a better understanding of how afferents might contribute to the functional interrelatedness of the VTA, SNc, RRF, and PAGvl. Ventral midbrain structures received injections of retrograde tracer, and the resulting retrogradely labeled structures were targeted with injections of anterogradely transported Phaseolus vulgaris leucoagglutinin. Whereas all injections of retrograde tracer into the VTA, SNc, RRF, or PAGvl produced labeling in many structures extending from the cortex to caudal brainstem, pronounced labeling of structures making up the central division of the extended amygdala occurred following injections that involved the RRF and PAGvl. The anterograde tracing supported this finding, and the combination of retrograde and anterograde labeling data also confirmed reports from other groups indicating that the SNc receives robust input from many of the same structures that innervate the VTA, RRF, and PAGvl.
