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Gene Expression Patterns 8 (2008) 307–310

Expression of MEGAP mRNA during embryonic development

Robert Waltereit, Sandra Kautt, Dusan Bartsch *

Central Institute of Mental Health, Department of Molecular Biology, University of Heidelberg, J5, 68159 Mannheim, Germany

Received 28 December 2007; accepted 28 January 2008Available online 2 February 2008

Abstract

Mental retardation (MR) affects up to two percent of the population and is usually accompanied by impaired learning and memory.MR is frequently associated with mutations of genes involved in Rho signal transduction cascades. Rho signal transduction pathwaysregulate axonal and dendritic growth during embryonic development and morphological adaptations of dendritic spines during synapticplasticity in adult neurons. The MEGAP/SRGAP3 protein is part of the Rho signal transduction cascades. Mutation of MEGAP (Men-tal disorder associated GTPase-activating protein) gene is present in some patients who have MR, ataxia and monosomy for the distalportion of the short arm of chromosome 3. Here, we investigated the pattern of mouse MEGAP mRNA expression during embryonicdevelopment. Mouse embryos and brain sections from stage E10 to adults were analysed by in-situ hybridisation for MEGAP transcriptin whole-mount and in brain sections. We demonstrate here robust expression of MEGAP mRNA in the whole central nervous system ofE10 embryos. At E12, we also detected MEGAP in dorsal root ganglia. This pattern narrows in adult animals into an expression mainlyin hippocampus and cortex. Our findings support the presumed role of MEGAP in Rho-associated MR.� 2008 Elsevier B.V. All rights reserved.

Keywords: Nervous system; Cortex; Hippocampus; Mental retardation; Ataxia; MEGAP; SRGAP3; Rho proteins; RhoA; Rac1; Cdc42; Actin cytosk-eleton; Neuronal migration; Synaptic plasticity

1. Results and discussion

The 3p� syndrome is characterized by severe mentalretardation and ataxia. Deficiency of the 3p25.3 band iscritical to produce the main clinical manifestations of the3p� syndrome (Narahara et al., 1990). MEGAP, also calledSRGAP3 (Slit-Robo GTPase-activating protein 3), wasfound to be heterozygously deleted in at least some 3p�

patients (Endris et al., 2002). RhoA, Rac1 and Cdc42 arethe most prominent members of the family of Rho pro-teins, which regulate the dynamics of the actin cytoskeleton(Schwartz, 2004). In neurons, Rho proteins modulate neu-rite outgrowth and neuronal migration during develop-ment and synaptic plasticity in the adult brain (Goveket al., 2005). MEGAP is a GTPase activating protein(GAP) of Rac1 and Cdc42 (Endris et al., 2002; Yang

1567-133X/$ - see front matter � 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.gep.2008.01.006

* Corresponding author. Tel.: +49 621 1703 6202; fax: +49 621 17036205.

E-mail address: dusan.bartsch@zi-mannheim.de (D. Bartsch).

et al., 2006), which means that it modulates the activityof Rho proteins. Thus, haploinsufficiency of MEGAPcould be the cause of MR and ataxia in 3p� patients.

In humans, Northern blot studies have shown thatMEGAP mRNA is strongly expressed in adult brain, espe-cially in cortex, hippocampus, amygdala, substantia nigra,thalamus and cerebellum, but not in medulla oblongata,spinal chord, putamen, caudate nucleus, corpus callosumand subthalamic nucleus. There is also weak expressionin kidney, but not in heart, muscle, colon, thymus, spleen,liver, intestine, placenta, lung or leucocytes. In human fetaltissue, it is strongly expressed in brain, very weakly in kid-ney, but not in lung or liver (Endris et al., 2002).

These findings suggest that, at least in human adults,expression of MEGAP mRNA is mostly restricted to thebrain. However, it is only partially known what the expres-sion pattern of MEGAP mRNA is during embryonic devel-opment. Moreover, it is unclear at this point whetherMEGAP is always exclusively expressed in the nervous sys-tem, or if it is expressed during embryonic development in

Fig. 1. Expression of MEGAP mRNA in E10 and E12 embryos. (A–D)E10, (E–H) E12. (A) Whole-mount in-situ hybridisation with MEGAPsense probe. (B and E–F) Whole-mount in-situ hybridisation withMEGAP anti-sense probe. (C–D and G–H) Microtome section ofparaffin-embedded embryos after whole-mount in-situ hybridisation withMEGAP anti-sense probe. DRG: dorsal root ganglia. FV, fourthventricle; ME, mesencephalon; MV, mesencephalic ventricle; NL, neurallumen; RE, rhombencephalon; SC, spinal chord; TE, telencephalon; TV,telencephalic ventricle.

308 R. Waltereit et al. / Gene Expression Patterns 8 (2008) 307–310

other tissues as well. This is of interest to better understandthe development of mental retardation and ataxia in 3p�

patients. Mouse MEGAP shares 92% nucleotide and 97%protein homology with the human gene (Endris et al.,2002). We chose mouse embryos to analyse the expressionpattern of MEGAP mRNA during embryonic development.

1.1. Expression of MEGAP mRNA during embryonic

development and adult age

Mouse embryos at the stages E10, E12 and E15 werestained by non-radioactive whole-mount in-situ hybridisa-tion with MEGAP sense (E10) and anti-sense probe.Embryos at the stages E10 and E12 were embedded in par-affin and slice sectioned. At the postnatal stages P5 andP100, mouse brains were dissected with a vibratome andstained as free-floating sections by hybridisation withMEGAP sense and anti-sense probe. The staining resultedfor all specimens in a robust and specific signal.

1.1.1. Embryonic stage E10

There is robust and specific staining of the neuroepithe-lium of the whole brain and spinal chord (Fig. 1A–D).Other parts of the embryo do not show any specific signal.There is some crystallised staining in the neural lumen ofthe specimen presented here (Fig. 1D) which seems to bean artefact.

1.1.2. Embryonic stage E12

The whole brain shows strong MEGAP signal (Fig. 1E–H). There is also strong and specific staining of the spinalchord, however only close to midline. In the transversalsections, the dorsal root ganglia express MEGAP signal.Some residual crystallised staining in the telencephalicand fourth ventricle of the specimen presented here appearsas an artefact (Fig. 1G–H).

1.1.3. Embryonic stage E15

The embryo expresses strong signal in cortex (Fig. 2).There is also specific, but weaker staining in mesencepha-lon. The eye shows some specific signal, which was repro-duced by staining of transversal sections of E15 brains(data not shown). The lumbar spinal chord expresses somespecific and robust staining. Finally, there is strong signalin the area of the whiskers.

1.1.4. Postnatal stage P5The coronary section shows strong staining in neocortex

(Fig. 3B) and in the pyramidal neurons of the hippocampus(Fig. 3D). However, there is only weak staining in the den-tate gyrus. The basal ganglia robustly express MEGAPmRNA, albeit to a lesser extent than neocortex and hippo-campus (Fig. 3B).

1.1.5. Adult stage P100The coronary sections show strong signal in neocortex

(Fig. 3F). The pyramidal neurons and the dentate gyrus

Fig. 2. Expression of MEGAP mRNA in E15 embryos. (A–B) Whole-mount in-situ hybridisation of embryos with MEGAP anti-sense probe. C,cortex; E, eye; M, mesencephalon; SC, spinal chord; W, whiskers.

Fig. 3. Expression of MEGAP mRNA in postnatal and adult brain. (A–D) P5, (E–H) P100. (A, C, E and G) Whole-mount in-situ hybridisation of100 lm vibratome sections with MEGAP sense probe. (B, D, F and H)Whole-mount in-situ hybridisation of vibratome sections with MEGAPanti-sense probe. (C, D, G and H) Magnification of the hippocampal area.BG, basal ganglia; DG, dentate gyrus of the hippocampus; NC, neocortex;PY, pyramidal cell layer of the hippocampus.

R. Waltereit et al. / Gene Expression Patterns 8 (2008) 307–310 309

of hippocampus robustly express MEGAP mRNA(Fig. 3H). There is no or only weak signal in the basal gan-glia (Fig. 3F).

1.2. Discussion

The results demonstrate robust expression of MEGAPmRNA in the whole central nervous system, and duringembryonic development also in dorsal root ganglia. Therewas no expression of MEGAP mRNA detectable outsidethe nervous system. With progression into adult age, thepattern of MEGAP transcription narrowed more and moreinto circumscribed regions within the brain.

From E10 to P100, there was strong expression ofMEGAP mRNA in neocortex. At stages P5 and P100,we observed also strong expression in hippocampus.Whereas the pyramidal neurons already showed robuststaining at stage P5, the dentate gyrus seemed to be theonly region where MEGAP expression became strongerwith maturation of the brain: At P5 the signal in dentategyrus was weak, at P100 the signal was strong. In all otherregions of the central nervous system MEGAP signalseemed to attenuate or even disappear with time: At E10there was strong expression of MEGAP in the primordiumof the spinal chord. At E12 there was some specific signal inthe spinal chord and still strong signal in rhombencephalonand mesencephalon, which attenuated at E15. At P5expression was robust in basal ganglia. At P100 this expres-sion had almost disappeared.

For adult age and for the regions which were analysed infetal tissue (Endris et al., 2002), the previously describedexpression pattern in human tissue corresponds with theexpression pattern found here for embryonic and adultmouse tissue. Our data describe the expression of MEGAPmRNA from E10 to P100. We did not analyse the expres-sion during early embryonic development.

MEGAP acts as a GAP of Rac1 and Cdc42 (Endriset al., 2002; Yang et al., 2006). In mouse embryos, Rac1and Cdc42 transcripts are expressed before preimplanta-tion (Natale and Watson, 2002). Rac1 and Cdc42 mRNAare robustly expressed in rat neocortex at stages E16,E18, P0 and P7 (Threadgill et al., 1997). In chick embryosat stage E6.5, Rac1 mRNA is strongly expressed in thewhole central nervous system and also in dorsal root gan-glia (Malosio et al., 1997). To our knowledge, the develop-mental expression pattern of Cdc42 mRNA has not beenanalysed so far in vertebrate embryos. However, by com-parison of the expression patterns of Rac1 and Cdc42 asreported in the literature and MEGAP as described here,MEGAP mRNA seems to have overlapping expressionwith Rac1 and Cdc42 transcripts.

MEGAP mRNA is expressed during embryonic devel-opment in the whole central nervous system and addition-ally in dorsal root ganglia, which are part of the peripheralnervous system. During adult age, MEGAP is expressed inthose regions of the brain where most synaptic plasticityoccurs: in neocortex and in hippocampus. Because of thestructure of the protein domains and its function as aGAP of Rac1 and Cdc42 (Endris et al., 2002; Yanget al., 2006), MEGAP has a proposed role in actin cytoskel-eton dynamics. We propose that MEGAP could be

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involved in neuronal migration during embryonic develop-ment and in synaptic plasticity during adult age. Theexpression pattern of MEGAP mRNA as described heresupports this role. It may be speculated that ataxia in3p� patients could be due to a migration deficit in thespinal chord. In addition, the expression pattern ofMEGAP supports the idea that mental retardation in3p� patients could first be caused by migration deficits inthe developing brain and later by deficits in synapticplasticity.

2. Experimental procedures

2.1. Non-radioactive MEGAP riboprobe

Sense and anti-sense riboprobes for MEGAP were prepared by in vitrotranscription of mouse cDNA encoding for exons 1–19 labelled with DIG-11-UTP as described (Endris et al., 2002).

2.2. Whole-mount in-situ hybridization

Embryos (E10, E12 and E15) were prepared from timed pregnantNMRI mice (Charles River, Sulzfeld, Germany) and fixed in 4% parafor-maldehyde. The brains of postnatal mice (P5 and P100) were fixed in 4%paraformaldehyde and sectioned into 100-lm slices using a vibratome(Leica VT 1000 S). After proteinase K treatment, embryos and floatingsections were hybridized overnight at 68 �C with DIG-labelled riboprobefollowed by hybridization with alkaline phosphatase conjugated anti-DIG antibody (Roche, Mannheim, Germany). The signal was detectedwith NBT/BCIP-substrate (Dako, Hamburg, Germany).

2.3. Paraffin sections

After whole-mount in-situ hybridisation, over-stained embryos (4 daysin NBT/BCIP-substrate) were embedded in paraffin using standard meth-ods. Microtome sections had a depth of 10 lm.

2.4. Interpretation of signals

Of all findings presented here, there were results from at least three dif-ferent embryos or brains showing similar patterns of MEGAP expression.The anatomical distribution of the signals was analysed using standardreferences (Kaufman, 1995; Paxinos and Franklin, 2001).

Acknowledgements

We thank Dr. Gudrun Rappold for providing a mouseMEGAP cDNA clone and Karina Borowski for technicalsupport. This research was supported by a EU IP Inte-grated technologies for in-vivo molecular imaging(LSHG-CT-2003-503259) research grant to D.B.

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