Loss of <i>Usp9x</i> disrupts TGF-β signalling in hippocampal neurons.

<p>(a–b) TGF-β luciferase reporter assays conducted in either wildtype (Nes-<i>Usp9x^+/Y</i>) or null (Nes-<i>Usp9x^−/Y</i>) embryonic hippocampal neuronal cultures. Hippocampal neurons were isolated and transfected with both renilla control and pGL3-TGF-β luciferase reporter plasmids. (a) Cells were grown for 3 days before analysis using dual-luciferase reporter assays and data normalised relative to wildtype readings. (b) Luciferase reporter activity in response to increasing concentrations of TGF-β. Data normalised to controls in the absence of TGF-β. All luciferase data from 6 biological replicates (i.e. cultures isolated from 6 <i>Usp9x^+/y</i> and 6 <i>Usp9x^−/Y</i> embryos). (c) Response of established TGFβ target genes in presence or absence of <i>Usp9x</i>, analysed by RT-qPCR. Isolated hippocampal neurons grown for 2 days prior to the addition of 1 ng/ml TGF-β. (d–e). Morphological analysis of hippocampal neurons exposed to 1 ng/ml TGF-β in the presence or absence of Usp9x. (d) Comparison of mean primary axonal length. (e). Comparison of number of neurite termini.</p

Loss of <i>Usp9x</i> reduces neuronal outgrowth.

<p>Embryonic hippocampal neurons were isolated, transfected with a plasmid encoding Enhanced Green Fluorescent Protein, and grown in-vitro for 3, 5 or 7 days. (a) Example immunofluorescent images of wildtype (Nes-<i>Usp9x^+/Y</i>) and null (Nes-<i>Usp9x^−/Y</i>) neurons resolved using GFP expression (Green) and co-stained with the axonal and dendritic specific antibodies, TAU1 (cyan) and MAP2 (red) respectively. (b–c) Morphometric analysis was employed to record mean primary axonal length (b) and number of neurite termini (c). *p<0.05 by student 2-tailed t-test.</p

<i>Usp9x</i> loss affects neuronal and astrocytic projections.

<p>NF160 antibodies decorate axonal projection from the neocortex (Nc) to the hippocampus (Hp) in E18.5 Nes-<i>Usp9x^+/Y</i> mice (A). These projections were absent in Nes-<i>Usp9x^−/Y</i> mice (B). GFAP staining is reduced in both the hippocampus and neocortex of E18.5 <i>Usp9x ^−/Y</i> embryos (D) compared with littermate controls (C). In the hippocampus GFAP-labeled projections extended toward the CA3 region in control embryos (arrowhead in E) but not in the absence of <i>Usp9x</i> (F). Scale bar = 20 µm (A,B), 160×µm (C,D), 80×µm (E,F).</p

Loss of <i>Usp9x</i> disrupts the architecture of the embryonic neocortex.

<p>The Nestin-cre mediated deletion of Usp9x (B,D) results in loss of demarcation between the cells of the ventricular and sub-ventricular zones (VZ/SVZ), the more disperse cellular density of the intermediate zone (IZ) and the neurons of the cortical plate (CP) seen in control littermates (A,C). C and D are higher magnification images of A and B, respectively. Nestin (E,F) and BLBP (G,H) staining in E18.5 embryos indicated that neural progenitors were more loosely organized in the VZ/SVZ. Neurons of the cortical plate were disorganized in the absence of <i>Usp9x</i> (J) compared with littermates (I). Nissl stain of <i>Usp9x^+/Y</i> (A,C,G) and <i>Usp9x^−/Y</i> (B,D,H) in E16.5 embryos (A–D, G–H). V = ventricle. Scale bar = 100 µm (A), 50 µm (C), 40 µm (E), 100 µm (G), 40 µm (I).</p

<i>Usp9x</i> is required for hippocampal development.

<p>The hippocampi of adult Emx1-<i>Usp9x^−/Y</i> mice (B) were reduced in area compared with <i>Usp9x^+/Y</i> littermates (A). Nissl stain of 7–8week old mice. (C) Quantification of hippocampal area of <i>Usp9x^+/Y</i> adult males (Control, n = 4) compared with <i>Usp9x^−/Y</i> (cKO, n = 4) (** p<0.01). (D–F) Higher magnification identifying disruption and reduction of the CA3 region in <i>Usp9x ^−/Y</i> males. (D - <i>Usp9x^+/Y</i>; E,F independent <i>Usp9x^−/Y</i> males) Scale bar = 100 µm (A,B), 150 µm (D–F).</p

Allele frequencies of the FRA2A associated CGG repeat in a population of 100 control individuals.

<p>PCR amplification of the repeat and subsequent sequencing in 200 control chromosomes revealed that it is highly polymorphic with a length ranging from 3 to 20 copies. The most commonly found allele contains eight repeated units.</p

Pyrosequencing reliably quantified the methylation of four consecutive CpG dinucleotides per individual (chr2:100721843–100721885; hg19).

<p>A fifth CpG site is preceded by a 7 base pair T stretch (after bisulfite conversion), making it impossible to assess the exact percentage of cytosines compared to thymines for that particular site due to the limitations of the pyrosequencing technique. For each individual at least three separate analysis of different bisulfite conversions were performed. Average methylation percentages and confidence intervals are presented in this table. Methylation cut-off value was set at 10% according to the manufacturers guidelines.</p

Analysis of rs4851214 which maps to coding exon 14 of <i>AFF3</i> using paired genomic DNA and cDNA templates from the unaffected carrier mother BI.2 and the affected carrier son BII.1 from family B.

<p><b>A:</b> Both BI.2 and BII.1 are heterozygous for SNP rs4851214 in genomic DNA as T and C peaks are visible at this site. <b>B:</b> Analysis of cDNA from subject BII.1, shows that only the C allele could be detected in this patient, indicating monoallelic expression, while in cDNA of subject BI.2 the heterozygous T/C signal was observed, indicating transcription of both alleles.</p

AFF3 protein-domain and gene structure.

<p><b>A.</b> diagrammatic representation of the <i>AFF3</i> protein, showing the N-terminal homology domain (NHD), C-terminal homology domain (CTHD) and two predicted nuclear localisation signals. The 18 amino acids encoded by the alternatively spliced exon 4 are represented as an “insertion” at position 18 of isoform 1. The 6-bp in-frame deletion we identified in exon 14 of subjects AI.1 and AII.4, removing two amino acids (position 619 and 621 respectively) is indicated in red. This deletion was predicted to be benign. <b>B.</b> Genomic structure of <i>AFF3</i> with the alternately used spliced exon 4 shown in red with asterisk. AFF3 exons 2, 3 and the alternate first exon are not separately resolvable at this resolution but shown in detail in panel C. Transcription left to right is shown in blue, right to left in brown. CAGE tag defined transcription start sites are shown aligned with annotated gene structure (blue forward strand transcription, brown reverse strand transcription shown with negative counts). Y-axis values show average tags per million (TPM) from CAGE libraries from the indicated tissue groups. <b>C.</b> Finer details of the <i>AFF3</i> TSS regions are shown along with histograms of human fetal brain RNA-seq read coverage; three replicates are coloured separately. Splicing of the intron between the alternate first exon and exon 3 (blue asterisk) was supported by 9 independent RNA-seq reads and found in all three replicates. The CGG repeat and abnormally methylated region (AMR) are shown in red and green respectively, The major transcription start sites are shown as black arrowed lines. There is no supporting evidence for the RefSeq TSS represented by the pink arrowed line in our data. <b>D.</b> Alignment of human CGG repeat region and associated TSS with the orthologous mouse region. Nucleotides are color coded (A = green, G = yellow, C = blue, T = red, alignment gaps are grey). Orange histograms show the predicted G-quadruplex forming potential of the human and mouse sequences. Outer histograms show CAGE tag 5′ ends at single nucleotide resolution in both human (top) and mouse (bottom). TPM counts shown are the average from brain derived CAGE libraries in each species and represent the precise location of transcription initiations.</p

Sizing the repeat in all available family members with gene specific repeat primed PCR.

<p>Alleles were sized by gene specific repeat primed PCR. As expansions containing over 300 repeated units cannot be reliably sized using this technique, we indicated these alleles as “>300”. Subject A.II.4 is marked with an asterisk as she is mosaic for a 134-bp deletion taking away the entire CGG repeat in combination with a largely expanded allele, and this in addition to a normal sized allele.</p