Research report
Expression of the guanine nucleotide exchange factor, mr-gef, is regulated during the differentiation of specific subsets of telencephalic neurons

https://doi.org/10.1016/j.devbrainres.2003.09.017Get rights and content

Abstract

We have performed a screen combining subtractive hybridization with PCR to isolate genes that are regulated when neuroepithelial (NE) cells differentiate into neurons. From this screen, we have isolated a number of known genes that have not previously been associated with neurogenesis, together with several novel genes. Here we report that one of these genes, encoding a guanine nucleotide exchange factor (GEF), is regulated during the differentiation of distinct neuronal populations. We have cloned both rat and mouse GEF genes and shown that they are orthologs of the human gene, MR-GEF, which encodes a GEF that specifically activates the small GTPase, Rap1. We have therefore named the rat gene rat mr-gef (rmr-gef) and the mouse gene mouse mr-gef (mmr-gef). Here, we will collectively refer to these two rodent genes as mr-gef. Expression studies show that mr-gef is expressed by young neurons of the developing rodent CNS but not by progenitor cells in the ventricular zone (VZ). The expression pattern of mr-gef during early telencephalic neurogenesis is strikingly similar to that of GABA and the LIM homeobox gene Lhx6, a transcription factor expressed by GABAergic interneurons generated in the ventral telencephalon, some of which migrate into the cortex during development. These observations suggest that mr-gef encodes a protein that is part of a signaling pathway involved in telencephalic neurogenesis; particularly in the development of GABAergic interneurons.

Introduction

One of the major problems in developmental neurobiology is in understanding how the diverse range of cell-types present in the adult brain is generated during development. Recent studies have identified transcription factors that define specific domains within the telencephalic ventricular zone (VZ) and have shown that the VZ cells within each expression domain generate specific types of neurons [7], [17], [21], [33], [34], [39], [47], [49], [52], [66]. For instance, the transcription factors Dlx1, Dlx2, Mash1 and Nkx2.1 are almost exclusively expressed in the ventral telencephalic VZ (the lateral and medial ganglionic eminences, with Nkx2.1 expression restricted to the latter). These genes are required for the proper development of GABAergic neurons. In fact, most of the cortical GABAergic interneurons arise ventrally and migrate tangentially into the cortex during development, presumably because their development is dependent upon the expression of ventral-specific genes [1], [2], [3], [4], [12], [51], [60], [67]. Conversely, the glutamatergic neurons of the cortex are generated from the dorsal (cortical) telencephalic VZ [17], [59], which specifically expresses transcription factors including Pax6 and Emx2 [6], [7], [49], [56]. Thus, it appears that the type of neurons generated by telencephalic VZ cells in vivo depends upon the specific transcription factors expressed by those cells. Similar observations have been made in the developing spinal cord (reviewed in Ref. [54]). However, the cellular and molecular mechanisms that control the differentiation of progenitor cells into specific types of neurons are still poorly understood.

Culture studies on mutipotential progenitor cells isolated from the developing telencephalon have shown that these cells require external cues in order to differentiate into neurons or glial cells [9], [20], [22], [26], [32], [41]. We call these multipotential progenitor cells, neuroepithelial (NE) cells [61], [62] and have shown that platelet-derived growth factor (PDGF) induces these cells to differentiate into neurons [62]. To expand our knowledge of the molecular mechanisms involved in neuronal development, we have used this PDGF-induced differentiation of NE cells as the basis for a PCR-based subtractive hybridization screen to isolate genes whose expression is regulated during neuronal development. None of the genes that we have identified have previously been shown to be involved in neurogenesis and many are novel. From this screen we identified a gene that encodes a guanine nucleotide exchange factor (GEF). This rat gene is the ortholog of the human gene MR-GEF that specifically activates the small GTPase Rap1 [43]; we have therefore called the rat gene rmr-gef. The mouse ortholog of this gene, mmr-gef, is almost identical to the rat gene, and since experiments performed with rat and mouse tissues and probes yield the same results, we will refer to these rodent genes collectively as mr-gef. mr-gef is of particular interest as Rap1 has been shown to be involved in NGF-mediated neuronal differentiation of PC12 cells [65]. In addition, an EST (EST107040) identical to part of mr-gef had previously been isolated during a screen in PC12 cells for genes that are regulated when they differentiate into neurons in response to nerve growth factor (NGF) [31]. Studying mr-gef presents us with an opportunity to understand one of the signaling pathways utilized by NE cells during neuronal differentiation.

We present evidence that mr-gef is regulated during the differentiation of specific populations of neurons in the rodent brain. In the ventral telencephalon, the spatiotemporal expression of mr-gef coincides with that of Lhx6, a transcription factor involved in GABAergic neuronal development and is co-expressed with GABA in a large number of neurons of the ventral telencephalon. A few specific GEFs have been implicated in aspects of neuronal development including the drosophila Ras-GEF, Son of sevenless (SOS) [8] and the Rho-GEF, Kalirin-7, reported to affect neuronal morphogenesis and increase neurite extension in cultured cortical neurons [40]. Thus, MR-GEF (the protein product of the rmr-gef and mmr-gef genes) represents one of few GEFs so far shown to be involved in mammalian neurogenesis.

Section snippets

E14 NE cell cultures and PDGF subtractive hybridization screen

Cultures were prepared as described previously and grown in the presence or absence of PDGF [38], [62]. RNA was isolated by the guanidinium isothiocyanate/cesium pad method [13] and Poly A+RNA was purified. In all cultures exposed to PDGF, the ability of the factor to induce neuronal differentiation was verified by immunohistochemistry on sister cultures using the neuronal-specific markers TuJ1 (1:1000, Cambridge Bioscience) or MAP2 (1:200, Sigma). cDNAs were synthesized using commercially

Isolation of PDGF-regulated genes

NE cells from the E14 rat cerebral cortex differentiate into neurons when exposed to PDGF [62]. To identify genes that are involved in neurogenesis, a subtractive hybridization screen was performed using RNA isolated from NE cells grown in the presence or absence of PDGF (Fig. 1A). Briefly, cDNAs were synthesized from the +PDGF and −PDGF RNA pools and linkers were added. The +PDGF cDNA pool was then biotinylated in order to isolate down regulated sequences. Both pools were then denatured and

Discussion

Using a screen for genes regulated during neurogenesis, we have identified rmr-gef and mmr-gef, the rat and mouse orthologs of a human gene encoding the Rap1-GEF, MR-GEF [43]. The spatiotemporal expression profile of mr-gef in the rodent ventral telencephalon is consistent with this GEF playing a role in the development of specific populations of young neurons; particularly GABAergic neurons.

We have additionally isolated a number of known genes that have not previously been associated with

Acknowledgements

Our thanks go to Maria Grigoriou and Vassilis Pachnis for the Lhx6 cDNA probe. We would also like to thank Paris Ataliotis and Ian Everall for their helpful discussions and critical reading of the manuscript. This research was supported by the Wellcome Trust and the Medical Research Council (UK).

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