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.

References (40)

  • M. Santacana et al.

    Development of the main efferent cells of the olfactory bulb and of the bulbar component of the anterior commissure

    Brain Res. Dev. Brain Res.

    (1992)
  • M.T. Shipley et al.

    The connections of the mouse olfactory bulb: a study using orthograde and retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase

    Brain Res. Bull.

    (1984)
  • P. Wei et al.

    Defective vascular morphogenesis and mid-gestation embryonic death in mice lacking RA-GEF-1

    Biochem. Biophys. Res. Commun.

    (2007)
  • S.E. Bilasy et al.

    Dorsal telencephalon-specific RA-GEF-1 knockout mice develop heterotopic cortical mass and commissural fiber defect

    Eur. J. Neurosci.

    (2009)
  • N. Buonviso et al.

    The projections of mitral cells from small local regions of the olfactory bulb: an anterograde tracing study using PHA-L (Phaseolus vulgaris Leucoagglutinin)

    Eur. J. Neurosci.

    (1991)
  • J. de Olmos et al.

    The afferent connections of the main and the accessory olfactory bulb formations in the rat: an experimental HRP-study

    J. Comp. Neurol.

    (1978)
  • J. Falk et al.

    Dual functional activity of semaphorin 3B is required for positioning the anterior commissure

    Neuron

    (2005)
  • A.N. Francis et al.

    Abnormalities of the corpus callosum in non-psychotic high-risk offspring of schizophrenia patients

    Psychiatry Res.

    (2010)
  • S. Hisata et al.

    Rap1-PDZ-GEF1 interacts with a neurotrophin receptor at late endosomes, leading to sustained activation of Rap1 and ERK and neurite outgrowth

    J. Cell Biol.

    (2007)
  • S.M. Islam et al.

    Draxin, a repulsive guidance protein for spinal cord and forebrain commissures

    Science

    (2009)
  • Cited by (18)

    • Expression of the guanine nucleotide exchange factor, RAPGEF5, during mouse and human embryogenesis

      2019, Gene Expression Patterns
      Citation Excerpt :

      RAPGEF5 expression was also observed in the spinal cord, notochord and vertebral body (Fig. 6C5). Rap GTPases and their regulators, including several RAPGEFs, are strongly associated with neural development and function (Bilasy et al., 2011; Franco et al., 2011; Fu et al., 2007; Jossin, 2011; Jossin and Cooper, 2011; Schwamborn and Puschel, 2004; Shah et al., 2017; Srivastava et al., 2012; Woolfrey et al., 2009; Ye et al., 2014; Zhu et al., 2002). Interestingly, Rapgef5 expression was previously reported in the developing brain, and alterations in Rapgef5 expression have been associated with impaired development of GABAergic neurons in the telencephalon and bipolar disorder (Bithell et al., 2003; Rebhun et al., 2000).

    • Functional Implications of miR-19 in the Migration of Newborn Neurons in the Adult Brain

      2016, Neuron
      Citation Excerpt :

      In addition, we confirmed that miR-19 was sufficient to suppress Rapgef2 without any additive function of other miRNAs from the same cluster (Figures S4F–S4I). Rapgef2 has been shown to be involved in cell adhesion by specifically activating Rap1 and Rap2 (Boettner and Van Aelst, 2009; Rebhun et al., 2000), and effects of Rapgef2 on neuronal cell migration in the developing brain have been reported (Bilasy et al., 2009, 2011; Ye et al., 2014). To determine whether Rapgef2 influenced migration and/or maturation of newborn neurons in the adult hippocampus as a downstream gene of miR-19, three different shRNAs were designed to target endogenous Rapgef2 (shRapgef2 #1–3) (Figure S5A).

    • Rap1gap2 regulates axon outgrowth in olfactory sensory neurons

      2012, Molecular and Cellular Neuroscience
      Citation Excerpt :

      By P42, mRap1gap2 expression was not detectable in the OB (Fig. 5G). Rap1 has been shown to promote neurite outgrowth and branching in cortical neurons in vitro (Chen et al., 2005), and a conditional knockout of the Rap1 activator Rapgef2 resulted in a disruption of commissural axon extension across the cortical midline (Bilasy et al., 2011a). These findings imply that Rap1 activity is vital to proper axon outgrowth.

    View all citing articles on Scopus
    View full text