The diverse and often contradictory effects of MeCP2 mutation are not surprising given its complex regulatory network and multifunctional capacity. Expression levels of the two MeCP2 isoforms are non-homogeneous and vary with brain region and cortical layer [22,64–67]. MeCP2 is also subject to multiple transcriptional, post-transcriptional, and post-translational regula- tory influences [68–72]. Furthermore, the full functional repertoire of MeCP2 remains incompletely understood, but at present includes chromatin remodelling, gene silencing, transcriptional modulation, RNA splicing, and regulation of microRNAs [73–78]. Transcriptional regulation alone differentially affects thousands of genes and varies by neuroanatomical region [79–82]. Selectively labeled subpopulations like those in the YFP-H line present an attractive means to address questions about the context-dependent effects of different MeCP2 mutations. However, this advantage is complicated by the potential for interactions between the mutant protein and the reporter transgene. We found that in MeCP2J mutant male mice, the number of YFP- expressing cells was decreased by 65–70% in several brain regions, while the intensity of YFP fluorescence in individual cells was preserved. More extensive Thy-1-YFP silencing in mutant mice would appear at odds with the role of MeCP2 as a transcriptional repressor [73,83], but MeCP2 does influence the expression of other transcriptional regulators [84]. Furthermore, the Thy-1 promoter expression cassette is highly sensitive to chromosomal context, and identical constructs can produce highly variable expression patterns across transgenic lines [42,54]. To date, the Thy-1 gene has not emerged as significantly altered in any gene profiling studies, but effects on Thy-1 promoter-driven transgenes are not entirely surprising given the involvement of MeCP2 in chromatin remodelling. The sharply delineated, region-dependent decreases in YFP+ cell numbers that we observed in some cortical areas suggests that these changes are not a random consequence of altered chromatin architecture, but may instead reflect distinct changes in regulatory pathways for different cell populations. Our study does not rule out the possibility that the reduced number of YFP+ cells in MeCP2J mutants correlates with a selective alteration in the proportions of certain subclasses of Layer 5 pyramidal neurons. Although variegation between founders in transgenic mouse lines has been considered a stochastic effect arising from random chromosome insertion site and transgene copy number [85,86], several reports suggest that the pattern of YFP+ pyramidal cells in the YFP-H line is not random, but constitutes a limited set of distinct functional subclasses defined in part by their principal axonal targets [38,40,49]. Intriguingly, axonal targeting is disordered in MeCP2 mutant mice [31,33] and the developmental timing of axonal pruning partially overlaps with that of MeCP2 expression in cortex [22,66,87–91]. Establishing how the MeCP2J mutation affects factors driving Thy-1-YFP expression could reveal new roles for distinct functional domains of MeCP2 in establishing or maintaining subtle differences between closely related neuronal subpopulations. Methods Ethics Statement All procedures performed on animals were in compliance with the Canadian Council on Animal Care guidelines and were approved by the University of Victoria Animal Care Committee (Approval number 2008-021). Animal Breeding Mecp2 mutant mice (Mecp2tm1.1Jae/Mmcd) (MMRRC, UC Davis) [20] were maintained on a 129S2/SvPasCrl background Altered Dendrites in YFP-MeCP2 Mutant Mice (Charles River) and YFP-H transgenic mice (B6.Cg-Tg(Thy1- YFPH)2Jrs/J, Jackson Laboratory) [42] were independently maintained on a C57BL/6J background. Mecp2 heterozygous female mice were crossed with YFP-H male mice homozygous for the YFP transgene to generate experimental F1 animals. Mecp2 and YFP were genotyped by PCR of genomic DNA extracted from earclips. Mecp2 genotype was determined using primer sequences [59- CAC CAC AGA AGT ACT ATG ATC] and [ATG CTG ACA AGC TTT CTT CTA-39] which detect wildtype and mutant Mecp2 alleles as 3 kb and 250 bp bands respectively. Presence of the YFP transgene was determined using primers [59- TCT GAG TGG CAA AGG ACC TTA GG] and [CGC TGA ACT TGT GGC CGT TTA CG-39], detecting a 300 bp band. Histological Preparation Animals were anaesthetized using urethane (2 g/kg i.p.) (Sigma) and transcardially perfused with 10 ml 0.1 M phosphate-buffered saline (PBS, pH 7.4) and 40 ml room-temperature 4% formalde- hyde in 0.1 M phosphate buffer (PFA, pH 7.4). Brain tissue was dissected out and fixed in 4% PFA at 4uC overnight, washed three times in PBS, and sunk in 30% sucrose-PBS. Agar-embedded 200 mm coronal sections were cut on a Pelco Vibratome 1000 and coverslipped with Shandon Immunomount (Thermo Scientific). Imaging 102461024 pixel 3D confocal fluorescence image stacks were obtained using a Nikon Eclipse TE2000-U confocal microscope and Nikon EZ-C1 3.60 software or an Olympus Fluoview FV1000 with Fluoview software (v. 1.7c) and 488, 515 or 543 nm lasers. Multi-channel imaging was done sequentially. All images were averaged twice with scan speeds of 1.68–3.94 ms/pixel unless otherwise indicated. Image stacks were converted to 16-bit .tif files using ImageJ software for further analysis. Images for neuron density and percentage of YFP+ cells were obtained using a Nikon Plan APO 0.95 206 air objective, 1061 mm z-step and 30 mm pinhole. For reconstructions of apical and basal compartments of YFP-H neurons, images were obtained using a Nikon 406/1.3 S Fluor oil immersion objective, 400 nm z-step, and 30 mm pinhole. Laser power and gain were manually adjusted during imaging to maintain maximal subsat- uration pixel intensities at the apical dendrite. For dendritic spine counts of the apical and basal dendrites of YFP+ neurons, images were obtained using an Olympus UPlanFL N 1006/1.3 oil immersion objective, 56 zoom, 200 nm z-step, and 190 mm pinhole. For qualitative analysis of YFP expression patterns, 160061200 pixel widefield images of cerebral hemispheres were obtained using a 46/0.13 objective with a 0.56 projection lens on an Olympus IX70 inverted epifluorescence microscope. Images were captured with a Retiga 2000R digital CCD camera (QImaging). For the developmental staging of YFP expression, 256061920 pixel images were obtained using a Nikon 46/0.13 Plan Fluor objective with a Nikon DS-U1 camera and ACT-2U software. Images were adjusted for brightness and contrast and tiled using Adobe Photoshop CS4. Histology & Morphological Analysis L5 neuron density was measured by staining 200 mm coronal tissue sections with the neuron-specific fluorescent Nissl stain Neurotrace 530/615 (Invitrogen, 1:300 dilution). The upper boundary of L5 was determined with reference to the YFP+ cell somata. Only superficial (,40 mm depth) tissue was imaged due to limited dye penetration. Counts from five tissue sections per animal were averaged for each brain region analyzed. PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e31896