Elsevier

Neuroscience Research

Volume 71, Issue 3, November 2011, Pages 200-209
Neuroscience Research

RA-GEF-1 (Rapgef2) is essential for proper development of the midline commissures

https://doi.org/10.1016/j.neures.2011.08.004Get rights and content

Abstract

The cerebral hemispheres are directly connected by three major interhemispheric fibers: the corpus callosum, the anterior commissure, and the hippocampal commissure. RA-GEF-1 (also termed Rapgef2) is a guanine nucleotide exchange factor responsible for sustained activation of Rap1. We previously reported anatomical defects of the major forebrain commissures in the adult dorsal telencephalon-specific RA-GEF-1 conditional knockout (cKO) mice. In this study, we use neuroanatomical tracing and immunohistochemistry to study the formation of the commissural fibers during early postnatal development. DiI anterograde tracing reveals the inability of the callosal axons to cross the midline in cKO mice, thereby forming Probst bundles on the ipsilateral side, which is associated with the absence of the indusium griseum glia and the glial sling at the cortical midline. Wheat germ agglutinin-conjugated horseradish peroxidase retrograde tracing verifies the agenesis of the anterior commissure in cKO mice, and DiI anterograde tracing confirms the deviation of the fibers from their original tract. As for the hippocampal commissure, agenesis and hypoplasia are observed in its dorsal and ventral parts, respectively. These results indicate the essential role of RA-GEF-1 in the proper formation of the cerebral midline commissures.

Highlights

► Dorsal-telencephalon-specific RA-GEF-1 (cKO) mice develop commissural fiber defect. ► RA-GEF-1 is ubiquitously expressed in neurons but not in astrocytes. ► Probst bundles are formed at rostral levels of the cKO mice cerebral cortex. ► In cKO mice, indusium griseum glia and glial sling are absent at the cotical midline.

Introduction

The cerebral commissures integrate the neural activity of the left and right cerebral hemispheres. The neocortex is connected interhemispherically by three major axon tracts: the corpus callosum, the hippocampal commissure, and the anterior commissure. The corpus callosum is the largest fiber bundle in the brain, that is mainly composed of commissural axons derived from layers II/III and V neocortical neurons (Yorke and Caviness, 1975). The formation of the corpus callosum starts around embryonic day (E) 15.5 and requires the coordination of a series of events, including midline patterning, specification of commissural neurons, and axon guidance across the midline (Koester and O’Leary, 1993). In the embryonic telencephalon, midline glia guide the midline crossing of commissural axons. Midline glia are composed of three glial populations: indusium griseum glia (IGG), glial wedge (GW), and midline zipper glia (MZG) (Shu and Richards, 2001, Shu et al., 2003). The GW is born from radial glia cells by E13.5, followed by the IGG and MZG starting from E14.5 until E17.5. The correct morphogenesis of these glial populations along the midline is crucial for guiding the developing callosal commissure axons, although the underlying molecular mechanism is presently unclear. The glial sling (GS), a neuronal population that migrates from the subventricular zone and forms a U-shaped sling over which the pioneer axons extend, acts as a guidance substratum for the developing callosal axons (Silver and Ogawa, 1983).

The hippocampal commissure connects the hippocampi of the left and right hemispheres across the midline. It normally develops a day earlier than the corpus callosum, and callosal axons use the hippocampal axons as a guidance cue during midline crossing (Livy and Wahlsten, 1997). The anterior commissure is composed of posterior and anterior limbs which connect the piriform cortex and the olfactory bulbs, respectively (Falk et al., 2005).

RA-GEF-1 (PDZ-GEF1/Rapgef2/CNrasGEF/nRapGEP) is a guanine nucleotide exchange factor specific for small GTPases Rap1 and Rap2 (de Rooij et al., 1999, Liao et al., 1999, Ohtsuka et al., 1999, Pham et al., 2000). RA-GEF-1 acts not only as an upstream activator of Rap1 but also as an effector by direct association with Rap1-GTP through its RA domain, leading to the amplification of Rap1-mediated signaling (Liao et al., 1999, Liao et al., 2001). We recently reported the essential role of RA-GEF-1 in the proper development of the cerebral cortex through analysis of RA-GEF-1flox/flox;Emx1cre/+ conditional knockout (cKO) mice, in which RA-GEF-1 was specifically disrupted in the dorsal telencephalon (Bilasy et al., 2009). Anatomically, the adult cKO mice developed an ectopic cortical mass (ECM) extending throughout the rostro-caudal axis of the cerebral hemisphere. We also observed the enlargement of the lateral ventricles and the agenesis of interhemispheric connections, i.e., the corpus callosum, the dorsal hippocampal commissure, and the anterior commissure. Here, we use neuroanatomical tracing to study this commissural fiber defect during early postnatal development. This study reveals that the formation of the major forebrain fiber connections during mouse brain development depends on RA-GEF-1 function.

Section snippets

Animals and genotyping

RA-GEF-1 cKO (RA-GEF-1flox/flox;Emx1cre/+) mice and their control littermate (RA-GEF-1flox/flox and RA-GEF-1flox/+;Emx1cre/+) mice were generated as previously described (Bilasy et al., 2009). Mice were maintained on the C57BL/6 and ICR mixed background. Timed-pregnant females were obtained by placing male and female mice in the same cage overnight. The day of vaginal plug formation was designated as E0.5 and the date of birth was designated as postnatal day (P) 0. Mouse tail lysates were

RA-GEF-1 is essential for the formation of forebrain commissures at P0

First, we analyzed the defects of the forebrain commissures in RA-GEF-1 cKO mice at P0 in coronal (Fig. 1A–H), sagittal (Fig. 1M–P) and horizontal (Fig. 1Q–V) sections at various levels as indicated in Fig. 1W. HE staining showed the presence of the ECM underlying a thin homotopic cerebral cortex as previously observed in adult cKO mice (Bilasy et al., 2009; Fig. 1E–H). Moreover, the formation of the corpus callosum was impaired at rostral levels, longitudinal bundles of the callosal axons,

Discussion

Neuronal interhemispheric connections are created by axons traversing the midline at the corpus callosum, the hippocampal commissure, and the anterior commissure (Paul et al., 2007). We previously reported that dorsal telencephalon-specific RA-GEF-1 cKO mice exhibited agenesis of the corpus callosum, the dorsal hippocampal commissure, and the anterior commissure in adulthood, along with other cortical malformations (Bilasy et al., 2009).

In the present study, we investigated the formation of the

Acknowledgements

We thank Dr. T. Setsu and Mr. Y. Sakihama for their excellent technical assistance. This work was supported by Grants-in-Aid for Priority Areas 1701406 and 20016016, and for Scientific Research 20390080, and by Global COE Program A08 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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