One of the pivotal events in neural development is compartmentalization, wherein the neural tissue divides into domains and undergoes functional differentiation. For example, midbrain-hindbrain boundary (MHB) formation and subsequent isthmus development are key steps in cerebellar development. Although several regulatory mechanisms are known to underlie this event, little is known about cellular behaviors. In this study, to examine the cellular dynamics around the MHB region, we performed confocal time-lapse imaging in zebrafish embryos to track cell populations in the neural tube via 4D analysis. We used a transgenic line wherein enhanced green fluorescent protein (EGFP) expression is driven by the gastrulation brain homeobox 2 (gbx2) enhancer, which is involved in MHB maintenance. 4D time-lapse imaging of 5-20 h revealed a novel pattern in cell migration: a dynamic ventrocaudally directed migration from the MHB region toward the hindbrain. Furthermore, in the hindbrain region, these EGFP-positive cells altered their shapes and extended the axons. Immunohistochemical analysis and retrograde labeling showed that these cells in the hindbrain were in the process of neuronal differentiation, including reticulospinal neurons. These results revealed the dynamic and two-step behavior and possible fate of the cell population, which are linked to brain compartmentalization, leading to a deeper understanding of brain development and formation of neuronal circuits.
Optical measurement of membrane potentials enables fast, direct and simultaneous detection of membrane potentials from a population of neurons, providing a desirable approach for functional analysis of neuronal circuits. Here, we applied recently developed genetically encoded voltage indicators, ASAP1 (Accelerated Sensor of Action Potentials 1) and QuasAr2 (Quality superior to Arch 2), to zebrafish, an ideal model system for studying neurogenesis. To achieve this, we established transgenic lines which express the voltage sensors, and showed that ASAP1 is expressed in zebrafish neurons. To examine whether neuronal activity could be detected by ASAP1, we performed whole-cerebellum imaging, showing that depolarization was detected widely in the cerebellum and optic tectum upon electrical stimulation. Spontaneous activity in the spinal cord was also detected by ASAP1 imaging at single-cell resolution as well as at the neuronal population level. These responses mostly disappeared following treatment with tetrodotoxin, indicating that ASAP1 enabled optical measurement of neuronal activity in the zebrafish brain. Combining this method with other approaches, such as optogenetics and behavioural analysis may facilitate a deeper understanding of the functional organization of brain circuitry and its development.