MeCP2 functions largely cell-autonomously, but also non-cell-autonomously, in neuronal maturation and dendritic arborization of cortical pyramidal neurons

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Abstract

Rett syndrome is a human neurodevelopmental disorder presenting almost exclusively in female infants; it is the second most common cause of mental retardation in girls, after Down's syndrome. The identification in 1999 that mutation of the methyl-CpG-binding protein 2 (MECP2) gene on the X chromosome causes Rett syndrome has led to a rapid increase in understanding of the neurobiological basis of the disorder. However, much about the functional role of MeCP2, and the cellular phenotype of both patients with Rett syndrome and mutant Mecp2 mouse models, remains unclear.

Building on prior work in which we demonstrated that cortical layer 2/3 pyramidal neurons (primarily interhemispheric “callosal projection neurons” (CPN)) have reduced dendritic complexity and smaller somata in Mecp2-null mice, here we investigate whether Mecp2 loss-of-function affects neuronal maturation cell-autonomously and/or non-cell-autonomously by creating physical chimeras. We transplanted Mecp2-null or wild-type (wt) E17-18 cortical neuroblasts and immature neurons from mice constitutively expressing enhanced green fluorescent protein (eGFP) into wt P2-3 mouse cortices to generate chimeric cortices. Mecp2-null layer 2/3 pyramidal neurons in both Mecp2-null and wt neonatal cortices exhibit equivalent reduction in dendritic complexity, and are smaller than transplanted wt neurons, independent of recipient environment. These results indicate that the phenotype of Mecp2-null pyramidal neurons results largely from cell-autonomous mechanisms, with additional non-cell-autonomous effects. Dysregulation of MeCP2 target genes in individual neuronal populations such as CPN is likely centrally involved in Rett syndrome pathogenesis. Our results indicating MeCP2 function in the centrally affected projection neuron population of CPN themselves provide a foundation and motivation for identification of transcriptionally regulated MeCP2 target genes in developing CPN.

Introduction

Rett syndrome is a neurodevelopmental disorder presenting almost exclusively in girls, with a prevalence rate of one in 10,000 to 15,000 female births (Bienvenu and Chelly, 2006, Francke, 2006, Hagberg, 2002, Kishi and Macklis, 2005, Kriaucionis and Bird, 2003, Moretti and Zoghbi, 2006, Rett, 1966). After Down's syndrome, Rett syndrome is the second most common cause of mental retardation in girls (Ellaway and Christodoulou, 2001). Children with Rett syndrome exhibit mental retardation, autistic features, and a breathing disorder after 6–18 months of relatively normal development. In 1999, it was discovered that loss-of-function mutation of the methyl-CpG-binding protein 2 (MECP2) gene on the X chromosome causes Rett syndrome (Amir et al., 1999). MeCP2 is typically referred to as a transcriptional repressor that selectively binds methyl CpG dinucleotides in the mammalian genome, mediating transcriptional repression, though a recent report describes transcriptional activator activity (Chahrour et al., 2008). It is becoming widely accepted that many transcriptional regulators can function as both repressors and activators, depending on context and cofactors.

Mecp2-deficient mice display several phenotypic characteristics that mimic symptoms of Rett syndrome patients, offering a valuable investigative tool as a murine disease model (Chen et al., 2001, Guy et al., 2001, Lawson-Yuen et al., 2007, Pelka et al., 2006, Shahbazian et al., 2002). These phenotypes are known to be due to the loss of MeCP2 function in the central nervous system (CNS), because specific deletion of Mecp2 in the brain mimics the germline loss of Mecp2 (Chen et al., 2001, Guy et al., 2001). Importantly, recent evidence clearly indicates that gene silencing mechanisms and target genes of MeCP2 are quite different between neural and nonneural cells (Lunyak et al., 2002). MeCP2 expression increases with maturity in the developing cortex, and MeCP2 is broadly expressed in the adult mouse brain. While MeCP2 begins expression as neurons emerge from progenitors, and is strongly expressed in mature neurons (Kishi and Macklis, 2004), recent studies report that MeCP2 is also expressed in astroglia and oligodendroglia at low levels (Ballas et al., 2009, Maezawa et al., 2009). Despite the broad expression of MeCP2 at high levels in neurons, and at lower levels in glia of the CNS, recent results indicate that loss of Mecp2 function has distinct influences on different cell types (Adachi et al., 2009, Fyffe et al., 2008, Kishi and Macklis, 2004). Therefore, in order to understand the pathogenesis of Rett syndrome, it is critical to investigate MeCP2 function specifically in affected populations of CNS neurons. This approach has provided insight into MeCP2 function, including MeCP2 target genes in neurons (Bienvenu and Chelly, 2006), as well as activity-dependent Bdnf regulation by MeCP2 (Chen et al., 2003, Martinowich et al., 2003). However, due to lack of accurate in vitro models, and the subtlety of the in vivo phenotype, it has remained unclear whether the neuronal phenotype in the absence of MeCP2 function is due to lack of Mecp2 in the affected neurons themselves (cell-autonomous mechanisms) and/or due to lack of Mecp2 in surrounding cells (non-cell-autonomous mechanisms).

In the experiments reported here, we investigate whether loss of Mecp2 function affects neuronal maturation largely cell-autonomously or non-cell-autonomously. Previous studies from our lab and others have shown that MeCP2 functions in neocortical neuronal maturation and dendritic development (Fukuda et al., 2005, Kishi and Macklis, 2004). In particular, Golgi staining, which allows visualization of the morphology of individual neurons, showed that Mecp2-null layer 2/3 pyramidal neurons (predominantly interhemispheric CPN) specifically exhibit reduced dendritic complexity and smaller somata than those in wt mice (Kishi and Macklis, 2004). Similar findings have been reported in human postmortem Rett syndrome brains (Armstrong et al., 1995, Bauman et al., 1995, Schule et al., 2008). Appropriate callosal connectivity is thought to underlie associative aspects of higher cognition, and has been centrally implicated in autism spectrum disorders (Egaas et al., 1995). To investigate whether these deficits in CPN development are due to MeCP2 transcriptional dysregulation within the neurons themselves, we performed a series of neuronal transplantation experiments using eGFP-labeled wt and Mecp2-null neurons, generating physical chimeras in both wt and Mecp2-null recipients. Our results demonstrate that transplanted Mecp2-null layer 2/3 pyramidal neurons are smaller than and have reduced dendritic complexity compared with transplanted wt neurons in the wt environment, indicating that the mechanisms underlying abnormal dendritic development of CPN in Mecp2-null mice are largely cell-autonomous.

Section snippets

Animals

All mouse experimental protocols were approved by the institutional animal care and use committee, and adhere to NIH guidelines. Wild-type C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA). GFP-expressing transgenic mice (C57BL/6-Tg(ACTB-EGFP)1Osb/J) (Okabe et al., 1997) were purchased from the Jackson Laboratories (Bar Harbor, ME). This transgenic mouse line with an "enhanced" GFP (eGFP) cDNA under the control of a chicken beta-actin promoter and cytomegalovirus

Results

Our previous data showed that layer 2/3 pyramidal neurons (predominantly interhemispheric CPN; Molyneaux et al., 2009) in Mecp2-null mice have reduced dendritic complexity and smaller somata than those in wt mice (Kishi and Macklis, 2004). These results indicate that Mecp2 is involved in neuronal maturation and/or maintenance of CPN. The specific CPN phenotype indicates that layer 2/3 pyramidal/callosal projection neurons are an ideal target cell population in which to investigate MeCP2

Discussion

These experiments using neuroblast transplantation to produce physical chimeras demonstrate that the decreased dendritic arborization that we previously observed in Mecp2-null callosal projection neurons is largely due to cell-autonomous mechanisms, indicating that Mecp2 acts predominantly cell-autonomously during maturation of these neurons.

Multiple studies using Mecp2 heterozygous female mice are consistent with our results and interpretation (Belichenko et al., 2009, Braunschweig et al., 2004

Acknowledgments

We thank Dr. Bartley Mitchell for his critical advice on cellular transplantation, and his helpful comments on this study. We thank Eiman Azim, Mollie Woodworth and Drs. Tina Lai and Jessica MacDonald for critical reading of the manuscript. We also thank Kyle MacQuarrie, Alexander Eswar, Kathryn Quinn, Karen Billmers, and Ashley Palmer for excellent technical assistance. We thank Dr. Rudolf Jaenisch for generously sharing the Mecp2 mutant mice generated in his laboratory. This work was

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