Transcriptome analyses of E10.5 <i>Magoh</i>, <i>Rbm8a</i>, and <i>Eif4a3</i> haploinsufficient cortices reveal common downstream pathways.

<p>(A) Diagrammatic overview of RNA sequencing analysis of E10.5 neocortices from indicated genotypes. (B) qPCR showing expression of <i>Magoh</i>, <i>Rbm8a</i>, and <i>Eif4a3</i> in their respective mutant E10.5 cortices. (C) Heatmaps showing z-score transformed normalized expression for all affected transcripts with an FDR corrected p-value, <i>q</i>< 0.05. Genes and samples were clustered using correlation distance with complete linkage. (D) Scatter plots of transcripts significantly upregulated (green dots) and downregulated (red dots) in E10.5 <i>Emx1</i>-Cre;<i>Magoh</i>^<i>lox/+</i>, <i>Emx1</i>-Cre;<i>Rbm8a</i>^<i>lox/+</i>, and <i>Emx1</i>-Cre;<i>Eif4a3</i>^<i>lox/+</i> cortices (q<0.05). (E, F) qRT-PCR validation at E11.5 compared to relative RNA-seq values of <i>Tbr2</i> (E) and <i>Ngn2</i> (F) in the indicated genotypes. For RNA-seq and qPCR, each control was normalized to 1.0 and compared to mutants. (G) Graph depicting common KEGG terms identified by GSEA analysis that were significant in all 3 EJC mutants, showing corresponding enrichment score. Student’s <i>t</i> test (B,E,F), Error bars, S.D. *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>p</i><0.001.</p

Haploinsufficiency for Core Exon Junction Complex Components Disrupts Embryonic Neurogenesis and Causes p53-Mediated Microcephaly

<div><p>The exon junction complex (EJC) is an RNA binding complex comprised of the core components Magoh, Rbm8a, and Eif4a3. Human mutations in EJC components cause neurodevelopmental pathologies. Further, mice heterozygous for either <i>Magoh</i> or <i>Rbm8a</i> exhibit aberrant neurogenesis and microcephaly. Yet despite the requirement of these genes for neurodevelopment, the pathogenic mechanisms linking EJC dysfunction to microcephaly remain poorly understood. Here we employ mouse genetics, transcriptomic and proteomic analyses to demonstrate that haploinsufficiency for each of the 3 core EJC components causes microcephaly via converging regulation of p53 signaling. Using a new conditional allele, we first show that <i>Eif4a3</i> haploinsufficiency phenocopies aberrant neurogenesis and microcephaly of <i>Magoh</i> and <i>Rbm8a</i> mutant mice. Transcriptomic and proteomic analyses of embryonic brains at the onset of neurogenesis identifies common pathways altered in each of the 3 EJC mutants, including ribosome, proteasome, and p53 signaling components. We further demonstrate all 3 mutants exhibit defective splicing of RNA regulatory proteins, implying an EJC dependent RNA regulatory network that fine-tunes gene expression. Finally, we show that genetic ablation of one downstream pathway, p53, significantly rescues microcephaly of all 3 EJC mutants. This implicates p53 activation as a major node of neurodevelopmental pathogenesis following EJC impairment. Altogether our study reveals new mechanisms to help explain how EJC mutations influence neurogenesis and underlie neurodevelopmental disease.</p></div

Loss of <i>p53</i> partially rescues microcephaly of <i>Magoh</i>, <i>Rbm8a</i>, and <i>Eif4a3</i> haploinsufficient mutants.

<p>(A-D, F-I, and K-N) Whole mount brains of E18.5 embryos with indicated genotypes. (E, J, O) Quantification of cortical area in E18.5 embryonic brains with indicated genotypes. Dotted lines demarcate the dorsal cortical areas measured. The surface area of littermate control brains was set to 100. ANOVA with Tukey posthoc, **, <i>p</i><0.01, ***, <i>p</i><0.001, NS, not significant. Error bars, S.D. n = 3–9 biological replicates each. Scale bars, A-D, E-I, and K-N, 1 mm.</p

Haploinsufficiency for EJC components alters mRNA splicing of splicing regulators.

<p>(A) Bar graph showing alternative splicing events for each mutant relative to the control. (B-D) Clustered column graphs of the distribution of ψ values of all identified intron retention (RI) events in E10.5 dorsal cortices for each EJC mutant, using a threshold of 20 for Bayes factor. Ψ<0 indicates higher probability for the mutant to have intron retention when compared to the control. (E) Top: IGV view of increased <i>Mapk13</i> intron 6–7 reads in red frame. Primers indicated as arrows. Bottom: RT-PCR showing increased <i>Mapk13</i> RI isoforms in <i>Emx1</i>-Cre;<i>Rbm8a</i>^<i>lox/+</i> E11.5 dorsal cortices compared to the control. (F) Bar graph of common KEGG terms that were significant in all 3 EJC mutants, showing corresponding enrichment score. ANOVA (A), Modified fisher’s exact test (F), *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>p</i><0.001.</p

EJC components are co-expressed in neurogenesis.

<p>(A) Schematic of embryonic neurogenesis of the dorsal telencephalon. NSC, neural stem cell; IP, intermediate progenitor. (B) Two main questions posed in this study. 1. Does <i>Eif4a3</i> haploinsufficiency cause microcephaly? 2. Do EJC components regulate common pathways during neurogenesis? (C) qPCR of <i>Magoh</i>, <i>Eif4a3</i>, and <i>Rbm8a</i> mRNA levels in developing neocortices of indicated ages. qPCR was performed using a standard curve, with <i>Magoh</i> relative expression at E10.5 set to 1.0, and all expression levels normalized to <i>Gapdh</i>. (D-I) Immunofluorescence of E10.5 dorsal neocortices for Hoechst (blue), Magoh (D, E), Rbm8a (F, G), and Eif4a3 (H, I). (E, G, I) are high magnification images of D, F, H, respectively. Student’s <i>t</i> test, Error bars, S.D., **, <i>p</i><0.01, ns = not significant. n = 3 biological replicates each age. Scale bars, D, F, H; 50 μm; E, G, I, 25 μm.</p

Proteomic analysis of E11.5 EJC mutant brains reveals alterations in levels of ribosome-associated proteins and ribonucleoproteins.

<p>(A) Diagrammatic representation of workflow to perform proteomic analysis of E11.5 dorsal cortices. (B, C) Bar graph of all common enriched KEGG terms (B) and top common GO terms (C) among all 3 EJC mutants showing corresponding fold enrichment and <i>P</i> values. (D) STRING network analysis of proteins within the broadest GO category, “Ribonucleoprotein Complex” altered in any of the 3 EJC mutants. Stronger associations are represented by thicker lines, and circles are colored based upon alteration in 1 or more mutants and level of significance. Two networks of splicing regulators and ribosome-associated proteins are detected. Modified fisher’s exact test, *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>P</i><0.001.</p

Loss of <i>p53</i> partially rescues neuron number and distribution associated with <i>Rbm8a</i> haploinsufficiency.

<p>(A-D) Coronal sections of E18.5 brains of indicated genotypes stained with Hoechst (white). (E-L) Regions of coronal sections indicated in (A-D, red dotted square) stained for Cux1 (E-H) and Tbr1 (I-L). (M, N) Quantification of Cux1+ (M) and Tbr1+ (N) density within a 250 μm radial column for indicated genotypes. (O, P) Bar graphs depicting density of Tbr1+ neurons in VZ/SVZ (bin 1–2, O) and cortical plate (bin 3–5, P) of indicated genotypes. Bins were quantified as indicated in I. Brackets denote general boundaries of Cux1 and Tbr1 layers. ANOVA with Tukey posthoc *, <i>p</i><0.05, **, <i>p</i><0.01, ***, <i>p</i><0.001, ns, not significant. Error bars, S.D. n = 2–3 biological replicates each. Scale bars, A-L, 50 μm.</p

Transcriptome analyses of E10.5 <i>Magoh</i> germline haploinsufficient brains identifies alterations in ribosome and p53 signaling pathways.

<p>(A) Diagrammatic overview of RNA sequencing analysis of E10.5 neocortices (dotted lines) from indicated genotypes. (B) Heatmaps showing z-score transformed normalized expression for control and <i>Magoh</i>^{<i>Mos2/+</i>}. Genes and samples were clustered using correlation distance with complete linkage. (C) Scatter plot of transcripts significantly upregulated (green dots) and downregulated (red dots) in E10.5 <i>Magoh</i>^{<i>Mos2/+</i>} cortices (<i>q</i><0.05), n = 4 biological replicates each. (D) Validation and RNA-seq values for <i>Dclk1</i> and <i>Tbr2</i> in indicated E11.5 mutant dorsal neocortices. Controls were normalized to 1.0. (E) Graph depicting top ranked KEGG terms by GSEA analysis in <i>Magoh</i>^{<i>Mos2/+</i>} showing corresponding fold enrichment. Student’s <i>t</i> test (D), Error bars, S.D. *, <i>p</i><0.05, ***, <i>p</i><0.001.</p

<i>Eif4a3</i> is required for embryonic neurogenesis and brain size.

<p>(A) Top, <i>Eif4a3</i> genomic mouse locus. Middle, targeted allele with 2 loxp sites (black arrowheads), <i>Neo</i> cassette, and 2 <i>FRT</i> sites (white arrowheads). Genotyping primers are indicated. Bottom, the conditional allele following FLP- and Cre-mediated recombination. (B) qPCR quantification of <i>Eif4a3</i> mRNA levels in E10.5 neocortices, following normalization using <i>Gapdh</i>. <i>Eif4a3</i> mRNA level of <i>Emx1</i>-Cre samples was set to 1.0. (C) Quantification of Eif4a3 protein levels in E11.5 dorsal cortices by densitometry of western blots, following normalization with α-Tubulin for loading. (D,E) Whole mount E12.5 <i>Emx1</i>-Cre and <i>Emx1</i>-Cre;<i>Eif4a3</i>^<i>lox</i>/+ brains. Note the forebrain (dotted lines) is noticeably smaller in the <i>Eif4a3</i> mutant. (F) Quantification of cortical thickness of E12.5 <i>Emx1</i>-Cre and <i>Emx1</i>-Cre;<i>Eif4a3</i>^<i>lox</i>/+ dorsal neocortices. (G-J) 4 different coronal sections from E12.5 <i>Emx1</i>-Cre (G,H) and <i>Emx1</i>-Cre;<i>Eif4a3</i>^<i>lox</i>/+ (I,J) neocortices stained for Hoechst (blue), Pax6 (green, G,I) or Tuj1 (green, H,J). (K) Density of Pax6+ cells within 200 μm wide radial columns spanning the E12.5 cortices of indicated genotypes. (L, M) Images of E11.5 <i>Emx1</i>-Cre (L) or <i>Emx1</i>-Cre;<i>Eif4a3</i>^<i>lox</i>/+ (M) cortices stained for PH3 (green). (N) Graph depicting percentage of all cells which are PH3-positive for indicated genotypes at E11.5. (O-T) E12.5 coronal sections from <i>Emx1</i>-Cre (O) and <i>Emx1</i>-Cre;<i>Eif4a3</i>^<i>lox</i>/+ brains (P-T) stained for Hoechst (blue), CC3 (red), Pax6 (green, Q, S), and Tuj1 (green, R,T). S and T are high-magnification views of Q and R, respectively, as indicated. Arrowheads depict cells co-labeled for apoptotic and cell fate markers. (U-X) Coronal sections of E14.5 <i>Emx1</i>-Cre (U,W) and <i>Emx1</i>-Cre;<i>Eif4a3</i>^<i>lox</i>/+ (V,X) cortices stained for Hoechst (white or blue) and Tuj1 (green). W and X are high-magnification images of U and V, respectively as indicated. Red brackets denote cortical thickness. Vent, ventricle. Student’s <i>t</i> test, *, <i>p</i><0.05, ***, <i>p</i><0.001. Error bars, S.D. n = 3 biological replicates each. Scale bars, D, E, 1 mm; G-J, L,M,O-R, W, X, 50 μm; S,T, 20 μm; U,V, 200 μm.</p

Water-Resistant Efficient Stretchable Perovskite-Embedded Fiber Membranes for Light-Emitting Diodes

Cesium lead halide perovskite nanocrystals (NCs) with excellent intrinsic properties have been employed universally in optoelectronic applications but undergo hydrolysis even when exposed to atmospheric moisture. In the present study, composite CsPbX₃ (X = Cl, Br, and I) perovskite NCs were encapsulated with stretchable (poly­(styrene-butadiene-styrene); SBS) fibers by electrospinning to prepare water-resistant hybrid membranes as multicolor optical active layers. Brightly luminescent and color-tunable hydrophobic fiber membranes (FMs) with perovskite NCs were maintained for longer than 1 h in water. A unique remote FMs packaging approach was used in high-brightness perovskite light-emitting diodes (PeLEDs) for the first time