Elsevier

Neuroscience Research

Volume 57, Issue 1, January 2007, Pages 40-49
Neuroscience Research

Migration and nucleogenesis of mouse precerebellar neurons visualized by in utero electroporation of a green fluorescent protein gene

https://doi.org/10.1016/j.neures.2006.09.010Get rights and content

Abstract

Neural migration is a critical step for accurate CNS development, but the molecular mechanisms that regulate migration, settlement and nucleogenesis remain largely unknown. The precerebellar neurons (PCNs), generated in the lower rhombic lip (LRL), migrate towards their destinations: some neurons form the pontine gray nucleus (PGN) and reticulotegmental nucleus (RTN) in the ipsilateral pons, while others form the lateral reticular and external cuneate nuclei in the contralateral medulla after crossing the midline. Here, by introducing an EGFP gene into a unilateral LRL of mouse embryos by in utero electroporation, we specifically labeled and tracked the PCNs in vivo. We found that a substantial number of the labeled neurons crossed the midline and formed PGN/RTN on the contralateral side. In addition, we found that a subpopulation of the interpolar subnucleus of the spinal trigeminal nucleus, which projects the axons to the cerebellum, was one of the PCNs derived from the LRL. Furthermore, because the electroporated mice were born and grew up healthy, we could visualize the PCNs and their mossy fibers in the adult brain. Therefore, the EGFP labeling of PCNs can be applied to studying the physiology of the mossy fiber system as well as PCN development in embryos.

Introduction

Neuronal migration plays an important role in proper patterning of the neural circuit during development. In the developing nervous system, many immature neurons migrate from the sites of origin to their final destinations (Hatten, 1999, Marin and Rubenstein, 2003). In the cerebral cortex, the pyramidal neurons are born in the ventricular zone and migrate radially to establish the layered structure, while the cortical interneurons, generated in the ganglionic eminences, take a tangential path into the cortex (Marin and Rubenstein, 2003, Kriegstein and Noctor, 2004). In the cerebellum, the granule cells, generated in the upper rhombic lip, migrate over the surface of the cerebellar anlage to form the external granular layer, and then migrate radially toward the internal granular layer (Hatten, 1999, Komuro et al., 2001). Previous studies have identified multiple signaling molecules and their receptor systems that guide migrating neurons towards their specific destinations (Hatten, 1999, Marin and Rubenstein, 2003), but the molecular mechanisms that regulate the mode of movement, the transition from tangential-to-radial migration, settlement in definite sites, and formation of nuclei remain largely unknown.

The precerebellar neurons (PCNs), which are collectively defined as the neurons sending afferents to the cerebellum, provide a good model for studying migration and nucleogenesis during development. PCNs form several different nuclei in the brainstem, including the pontine gray nucleus (PGN) and reticulotegmental nucleus (RTN) in the pons, and the external cuneate nucleus (ECN), lateral reticular nucleus (LRN), and inferior olivary nucleus (ION) in the medulla oblongata (Altman and Bayer, 1996). ION neurons project climbing fibers, while the other neurons project mossy fibers to the cerebellum. The early studies using sequential 3H-thymidine radiograms in rats revealed that the PCNs originate from the dorsal rhombencephalic neuroepithelium, which is called the lower rhombic lip (LRL), and migrate ventrally toward their destinations (Altman and Bayer, 1996). These studies demonstrated that the migrating PCNs take three different paths: the PGN and RTN neurons take the anterior extramural migratory stream (AES) toward the pontine region, while the ECN and LRN neurons take the posterior extramural migratory stream (PES) toward the medulla region, and only the ION neurons take the intramural circumferential pathway in the medulla (Altman and Bayer, 1987a, Altman and Bayer, 1987b, Altman and Bayer, 1996). Based on these sequential autoradiographic data, it was concluded that the LRN/ECN neurons cross the midline to form the nuclei on the side contralateral to the side of their origin, while the PGN/RTN neurons form nuclei on the ipsilateral side (Altman and Bayer, 1987a, Altman and Bayer, 1987b, Altman and Bayer, 1996). More direct evidence for the contralateral origin of the LRN/ECN neurons was later obtained by in vitro culture studies (Kyriakopoulou et al., 2002, Taniguchi et al., 2002), and by an in vivo study using electroporation of the enhanced green fluorescent protein (EGFP) gene (Kawauchi et al., 2006).

Recently, a molecular fate mapping method using recombinase-based marking of certain cell lineages was developed. Transgenic expression of a recombinase in mice using the promotors of Math1, Wnt1 or Pax6, in combination with a reporter gene, β-galactosidase, was successfully used to mark and trace the rhombic lip-derived neurons (Rodriguez and Dymecki, 2000, Landsberg et al., 2005, Machold and Fishell, 2005, Wang et al., 2005). These studies showed that the cerebellar and cochlear granular neurons, the mossy fiber-projecting PCNs, and the climbing fiber-projecting ION neurons originate from the rhombic lip. Together with the previous thymidine radiograms, they revealed the origin, migratory pathways, and timing of nucleogenesis. However, these previous studies did not precisely determine whether all the PGN/RTN neurons remain on the ipsilateral side without crossing the midline, because the PCNs originating from both sides, which are labeled similarly, are intermingled and cannot be distinguished after crossing the midline. This issue was addressed by Kawauchi et al. (2006), who, marking the LRL by electroporation, clearly demonstrated that the PGN/RTN neurons were derived from the bilateral LRL.

In this study, we applied in utero electroporation to directly visualize the migration and nucleogenesis of PCNs in the mouse by introducing EGFP into a unilateral LRL. In addition to confirming that a substantial number of the PGN/RTN neurons are originated from the contralateral side, we show that a subpopulation of the interpolar subnucleus of the spinal trigeminal nucleus, which project axons to the cerebellum, are among the PCNs derived from the LRL. Furthermore, strong labeling of PCNs and their mossy fibers is observed in the adult brain. Our findings demonstrate that neuronal labeling by in utero as well as ex utero electroporation is useful for studying PCN development and the physiology of the mossy fiber system.

Section snippets

Animals

Timed pregnant ICR mice were purchased from CLEA Japan (Tokyo, Japan). Noon of the day when a vaginal plug was observed was taken as embryonic day 0.5 (E0.5). For in utero electroporation, pregnant mice were anesthetized by intraperitoneal injection of sodium pentobarbital (70 mg/kg body weight). For transcardial perfusion, the mice were deeply anesthetized by intraperitoneal injection of an excess of sodium pentobarbital. All the animal experiments were approved by the animal care and use

Unilateral EGFP labeling of PCNs by in utero electroporation

PCNs are generated in the LRL, the most alar part of the hindbrain, and migrate circumferentially to form distinct nuclei in the brainstem. In order to directly track the migration of PCNs in vivo, we applied an in utero electroporation method to introduce the EGFP gene into the LRL region (Fig. 1A). We electroporated mouse embryos at embryonic day 12.5 (E12.5) to efficiently label the mossy fiber-projecting PCNs, because birthdating (Taber-Pierce, 1973) and fate mapping (Rodriguez and Dymecki,

Discussion

In this study, we visualized PCNs in the mouse using in utero electroporation of an EGFP gene into the LRL region of the E12.5 embryos. Similar methods of labeling the LRL-derived PCNs using in ovo electroporation in chick embryos or ex utero electroporation in mouse embryos were reported recently (Ono et al., 2004, Kawauchi et al., 2006). EGFP labeling of PCNs has several advantages over birthdating (Altman and Bayer, 1987a, Altman and Bayer, 1987b) and molecular fate mapping (Rodriguez and

Acknowledgements

We thank M. Okabe (Osaka University) for an EGFP-expression plasmid, pCX-EGFP, T. Saito for guidance regarding electroporation, and M. Yamamoto for critical reading of the manuscript. This work was supported in part by JSPS Research Fellowships for Young Scientists, and Grants-in-Aid for Scientific Research on Priority Areas and the 21st Century COE program from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

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