Dynamics of the leading process, nucleus, and Golgi apparatus of migrating cortical interneurons in living mouse embryos

Abstract

Precisely arranged cytoarchitectures such as layers and nuclei depend on neuronal migration, of which many in vitro studies have revealed the mode and underlying mechanisms. However, how neuronal migration is achieved in vivo remains unknown. Here we established an imaging system that allows direct visualization of cortical interneuron migration in living mouse embryos. We found that during nucleokinesis, translocation of the Golgi apparatus either precedes or occurs in parallel to that of the nucleus, suggesting the existence of both a Golgi/centrosome-dependent and -independent mechanism of nucleokinesis. Changes in migratory direction occur when the nucleus enters one of the leading process branches, which is accompanied by the retraction of other branches. The nucleus occasionally swings between two branches before translocating into one of them, the occurrence of which is most often preceded by Golgi apparatus translocation into that branch. These in vivo observations provide important insight into the mechanisms of neuronal migration and demonstrate the usefulness of our system for studying dynamic events in living animals.cortex | GABAergic interneuron | in utero electroporation | in vivo imaging N euronal migration is a critical step in the construction of the nervous system. During development, neurons migrate from the sites of their birth to their final destinations, where they form neuronal architectures, such as laminated structures and nuclei, that are necessary for information processing in the nervous system.Because neuronal migration is a dynamic phenomenon, realtime imaging could provide important insight into its mechanisms. Studies using real-time imaging of neurons in dissociated culture have demonstrated various aspects of neuronal migration, including the fact that radial fibers (1) and neighboring cells (2) act as the substrate for migration and the molecular mechanisms driving nucleokinesis (3, 4). Real-time imaging has also helped reveal the dynamics and roles of cytoplasmic organelles and cytoskeletal components, such as the centrosome (5-9), microtubules (9), and even of some molecules like calcium (10) and actin (7,11,12) during migration. In addition, slice and explant preparations have demonstrated aspects of migratory behaviors, including locomotion and translocation (13), branch-induced changes in migratory direction (14), multidirectional migration (15), random-walk-like behavior (16), pausing behavior, and the transition from tangential to radial migration (17).However, as informative as these neural migration studies are, they are limited because they are all in vitro and do not necessarily show neuronal behaviors in vivo. Here, we observed migrating neurons in the brain of living mouse embryos and analyzed their behavior, including the dynamics of the nucleus and the Golgi apparatus. We took advantage of the fact that cortical interneurons tangentially migrate near the surface of the cortex. These neurons are generated in the ganglionic eminences (GEs) in the basal forebrain a...

CLASP2 Links Reelin to the Cytoskeleton during Neocortical Development

“…At P0, our results show that CLASP2 knockdown caused a reduction in the distance between the nucleus and centrosome when compared to controls (Figure 6A). Similar to previous reports, we found co-localization of Golgi and centrosome markers in both control and CLASP2 shRNA electroporated cells (Yanagida et al, 2012). The Golgi and centrosome complex was localized to the leading process in ~80% of GFP-positive control cells; however, following CLASP2 knockdown, there was a mislocalization of this complex with a higher percentage of cells with Golgi and centrosome staining adjacent to the nucleus (Figure 6B and 6C).…”

“…The positioning of the centrosome and Golgi apparatus is a key step in selecting migratory direction, and their translocation into the leading neurite precedes cell movement during neuronal migration (Yanagida et al, 2012; Sakakibara et al, 2013). Because CLASP2 is enriched at both the Golgi and centrosome, two organelles that control cell polarity (Miller et al, 2009; Beffert et al, 2012), we examined whether CLASP2 expression influenced the position and shape of the centrosome/Golgi complex during neuronal migration.…”

Fusion of Regionally Specified hPSC-Derived Organoids Models Human Brain Development and Interneuron Migration

“…Importantly, cytoplasm elongation was often observed preceding nucleokinesis (Figure 7G). These behaviors are remarkably similar to previous reports of interneuron migration in live mouse embryos, mouse MGE explant models, and postmortem human brain sections (Bellion et al, 2005; Ma et al, 2013; Paredes et al, 2016; Yanagida et al, 2012), demonstrating the suitability of using hfMCOs to study human interneuron migration.…”

“…We found that soma translocation of interneuron progenitors was dramatically decreased with 50 μM blebbistatin treatment (10.55 ± 2.25%, n=2 hfMCOs, 63 cells in total, mean ± SD) compared with control hfMCOs (41.23 ± 3.20%, n=4 hfMCOs, 79 cells in total, mean ± SD) within 10 hours, and 100 μM blebbistatin treatment completely abolished migration (n=2 hfMCOs, 44 cells in total, mean ± SD) (Figure 7H and 7I; Movie S5 and S6). The average migrating speed of interneuron progenitors was 0.19 ± 0.01 μm/min (n=35 cells from 4 hfMCOs, mean ± SD) (Figure 7J), which was close to the speed observed from living embryo but slower than that obtained from MGE explant culture (Bellion et al, 2005; Yanagida et al, 2012), indicating that the cellular environment of hfMCOs might closely approximate their in vivo counterparts. With myosin II inhibition, the migration speed largely decreased (Figure 7J).…”

Bcl11a (Ctip1) Controls Migration of Cortical Projection Neurons through Regulation of Sema3c

“…Co-electroporation of Cre-IRES-GFP, together with a vector containing Bcl11a-XL cDNA, was sufficient to completely normalize the ratio of multi-to bipolar morphologies ( Figures 3A-3C). To further characterize polarization in Bcl11a mutant cortical neurons, we labeled the Golgi apparatus with GFP-tagged galactosyltransferase (Jossin and Cooper, 2011;Yanagida et al, 2012) and quantified the proportion of Bcl11a mutant and control cells with the Golgi apparatus facing the CP ( Figure 3D). Loss of Bcl11a resulted in a significant decrease of cells with a radially oriented Golgi apparatus already at E16.5 ( Figures 3D and 3E), supporting a role of Bcl11a in cortical neuron polarization.…”