Rbm8a haploinsufficiency disrupts embryonic cortical development resulting in microcephaly

Abstract

The cerebral cortex is built during embryonic neurogenesis, a period when excitatory neurons are generated from progenitors. Defects in neurogenesis can cause acute neurodevelopmental disorders, such as microcephaly (reduced brain size). Altered dosage of the 1q21.1 locus has been implicated in the etiology of neurodevelopmental phenotypes; however, the role of 1q21.1 genes in neurogenesis has remained elusive. Here, we show that haploinsufficiency for Rbm8a, an exon junction complex (EJC) component within 1q21.1, causes severe microcephaly and defective neurogenesis in the mouse. At the onset of neurogenesis, Rbm8a regulates radial glia proliferation and prevents premature neuronal differentiation. Reduced Rbm8a levels result in subsequent apoptosis of neurons, and to a lesser extent, radial glia. Hence, compared to control, Rbm8a-haploinsufficient brains have fewer progenitors and neurons, resulting in defective cortical lamination. To determine whether reciprocal dosage change of Rbm8a alters embryonic neurogenesis, we overexpressed human RBM8A in two animal models. Using in utero electroporation of mouse neocortices as well as zebrafish models, we find RBM8A overexpression does not significantly perturb progenitor number or head size. Our findings demonstrate that Rbm8a is an essential neurogenesis regulator, and add to a growing literature highlighting roles for EJC components in cortical development and neurodevelopmental pathology. Our results indicate that disruption of RBM8A may contribute to neurodevelopmental phenotypes associated with proximal 1q21.1 microdeletions.

Keywords: cortical development; embryonic; microcephaly; neurogenesis; radial glia; rbm8a.

Figure 1.

Rbm8a is expressed in the developing cerebral cortex. A, Graph depicting relative Rbm8a mRNA levels in dorsal telencephalon at three developmental stages. Values were calculated using a standard curve, normalized to Gapdh, and the E10.5 value was set to 1.0. B, C, In situ hybridization for Rbm8a in sagittal E14.5 mouse sections (B) and higher magnification view (for region boxed in B) of the neocortex (C). Note high expression (*) in the VZ/SVZ, relative to the CP. Images are from www.genepaint.org (Visel et al., 2004). D, Top, Diagram representation of a coronal section from an E14.5 brain. Box highlights region depicted in diagram schematic at bottom. Bottom, Diagram representation of major cell types examined in this study including radial glial progenitors (red), postmitotic neurons (blue), and intermediate progenitors (green). Radial glia undergo proliferative self-renewal divisions (curved arrow) or neurogenic divisions giving rise to a new radial glia and another daughter cell (orange), which is either a neuron or intermediate progenitor. E–H, Images of immunofluorescence staining of Rbm8a (green) in E10.5 (E), E12.5 (F), and E14.5 (G, H) coronal sections of dorsolateral neocortex. The dotted lines denote the boundary between neocortex and overlying basal epithelium. H, Image of the E14.5 coronal section in G costained with Rbm8a (green) and Tuj1 (red). Note Rbm8a is expressed throughout the neocortex in the VZ/SVZ, IZ, and CP, and is coexpressed with Tuj1 in the CP. I, J, High-magnification images for regions boxed in E (I) and F (J) showing nuclear enrichment of Rbm8a (green), evidenced by colocalization with Hoechst (blue). Dotted circles denote nuclei. K–P, Images of E14.5 primary cell cultures stained for Pax6 (red; K), TuJ1 (red; N), and Rbm8a (green; L, O) and merged with Hoechst (blue; M, P). Student's t test, *p = 0.050, **p < 0.001, ns, p = 0.1074. Error bars indicate SD, n = 3 biological replicates each. Scale bars: E–H, 50 μm; I, J, 10 μm; K–P, 20 μm.

Figure 2.

Conditional Rbm8a haploinsufficiency in the dorsal telencephalon causes severe microcephaly. A, Top, Schematic of wild-type genomic locus of Rbm8a with six exons (black boxes) and introns (lines, not to scale). Middle, Targeted allele with two loxp sites (black arrowheads), neo cassette, and two FRT sites. Primers for genotyping are shown as arrows (LoxF1, LoxR1). Bottom, Conditional allele following Cre-mediated recombination. B, Representative PCR genotyping result from Emx1-Cre (control) and Emx1-Cre;Rbm8a^loxp/+ mice. Note a single band (550 bp) in control and two bands (550 and 584 bp) in Emx1-Cre;Rbm8a^loxp/+. C, qRT-PCR quantification of Rbm8a mRNA levels in E10.5 neocortices and E12.5 dorsal neocortices of indicated genotypes, following normalization using Gapdh. Rbm8a mRNA levels of Emx1-Cre samples were set to 1.0. D, Representative Western blot of E11.5 dorsal neocortical lysates probed for anti-Rbm8a or anti-α-tubulin. Note a single band is evident in Emx1-Cre;Rbm8a^loxp/+. Protein samples were run to the bottom of the gel without running off to allow transfer of all molecular weights. E, Quantification of Rbm8a protein levels by densitometry of Western blots, following normalization with α-tubulin for loading. F–I, Images of primary E12.5 cortical cells from Emx1-Cre (F, G) or Emx1-Cre;Rbm8a^loxp/+ (H, I) dorsal neocortices, stained for RBM8A (white) or Hoechst (blue). Images were captured at identical exposure settings. Note lower immunofluorescence Rbm8a staining in Rbm8a mutant cells. J, K, Images of whole-mount brains at P12 from Emx1-Cre (J) and Emx1-Cre;Rbm8a^loxp/+ (K) mice. Dotted circles denote dorsal cortex. L, Quantification of relative cortical area in P12 brains of indicated genotypes. Student's t test, *p ≤ 0.05, ***p < 0.0001. Error bars indicate SD, n = 3 biological replicates each. Scale bars: F–I, 20 μm; J, K, 2 mm.

Figure 3.

Rbm8a haploinsufficiency results in a thinner neocortex. A–H, Cortical sections from Emx1-Cre (A, C, E, G) and Emx1-Cre;Rbm8a^loxp/+ (B, D, F, H) brains from E11.5 (A, B), E12.5 (C, D), and E13.5 (E–H) embryos stained for Hoechst. Images in A–D were from lateral regions at medial rostrocaudal positions. G, H, High-magnification images of regions indicated in E and F. I, Quantification of cortical thickness in Emx1-Cre and Emx1-Cre;Rbm8a^loxp/+ dorsal neocortices of indicated ages. Note measurements were made at both dorsal (dor) and more lateral (lat) regions as indicated in E. J, K, Whole-mount E14.5 brains from indicated genotypes. L, M, Hoechst-stained coronal neocortical sections from indicated genotypes at rostrocaudal regions indicated by dotted lines in J and K. N–U, High-magnification images of coronal neocortical sections from regions indicated in L and M. Images were stained for Hoechst (white or blue), Tbr2 (green; P, Q), Tuj1 (green; R, S), and Pax6 (green; T, U). Images in N, P, R, and O, Q, S are from identical sections, and images in T and U are from different sections. Note the markedly reduced ventricle and dorsal telencephalon in Emx1-Cre;Rbm8a^loxp/+. V, ventricle; GE, ganglionic eminence. All images show sections from medial rostrocaudal positions. Student's t test, ns, p > 0.80, **p < 0.01, ***p < 0.0001. Error bars indicate SD, n = 3 biological replicates each. Scale bars: A–D, G, H, N–U, 50 μm; E, F, L, M, 200 μm; J, K, 1 mm.

Figure 4.

Rbm8a is required for proper number of radial glia and intermediate progenitors. A–H, Coronal sections from Emx1-Cre (A, C, E, G) and Emx1-Cre;Rbm8a^loxp/+ (B, D, F, H) brains from E11.5 (A, B), E12.5 (C, D), and E13.5 (E–H) embryos, and stained for Hoechst (blue; A–H), Pax6 (green; A–F), and Tbr2 (green; G, H). I, Pax6-positive cells within a 35 × 10³ μm² field of view in cortices of indicated genotypes and ages. J, Tbr2-positive cells within a 55 × 10³ μm² field of view in cortices of indicated genotypes at E13.5. All images were taken from dorsolateral regions at the medial rostrocaudal position represented in Figure 1D. Student's t test, **p < 0.01, ***p < 0.0001, ns, p = 0.435. Error bars indicate SD, n = 3 biological replicates each. Scale bars: A–H, 50 μm.

Figure 5.

Rbma8a-deficient brains contain ectopic neurons. A–F, Coronal sections from Emx1-Cre (A, C, E) and Emx1-Cre;Rbm8a^loxp/+ (B, D, F) brains from E11.5 (A, B), E12.5 (C, D), and E13.5 (E, F) embryos, and stained for Hoechst (blue) and Tuj1 (green). Note the expanded domain of Tuj1-positive cells in E12.5 and E13.5 Rbm8a-deficient brains. G, H, E12.5 coronal sections from Emx1-Cre (G) and Emx1-Cre;Rbm8a^loxp/+ (H) brains stained for Hoechst (blue) and Tbr1 (green). Note the disorganized distribution of Tbr1-positive neurons in the Rbm8a-deficient brains. I, Tbr1-positive cells within a 10 × 10³ μm² cortical area of indicated genotypes at E12.5. J, K, Primary dissociated cells from E12.5 dorsal neocortices from indicated genotypes and stained for Pax6 (red), Tuj1 (green), and Hoechst (blue). L, Bar graph depicting fraction of positive cells for indicated markers. All images were taken from dorsolateral regions at the medial rostrocaudal position represented in Figure 1D. I, Student's t test. L, χ² analysis followed by post hoc two-tailed Student's t test, **p < 0.007, ***p < 0.0001, ns, p > 0.16. Error bars indicate SD, n = 3 biological replicates each. Scale bars: A–K, 50 μm.

Figure 6.

Rbm8a regulates cell-cycle exit of neural progenitors. A–F, Primary dissociated cells from E11.5 dorsal neocortices from Emx1-Cre (A–C) or Emx1-Cre;Rbm8a^loxp/+ (D–F) were cultured for 1 d following a 30 min EdU pulse and then stained for Tuj1 (red), Ki67 (green), and EdU (purple). Arrowheads, EdU+Ki67+ cells; arrows, EdU+Tuj1+ cells. G, H, Percentages of all EdU-positive cells that are Ki67+ (G) or Tuj1+ (H). I, J, Images of E11.5 Emx1-Cre (I) or Emx1-Cre;Rbm8a^loxp/+ (J) brains stained for PH3 (green) and Hoechst (blue). Note the basal surface is autofluorescent. K, Graph depicting percentage of PH3-positive cells for indicated genotypes. Student's t test, *p < 0.05, **p < 0.01. Error bars indicate SD, n = 3–6 cultures or brains from independent biological replicates each genotype. Scale bars: A–F, 20 μm; I, J, 50 μm.

Figure 7.

Rbm8a deficiency induces dramatic apoptosis of neurons and radial glia. A–F, Coronal sections from Emx1-Cre (A, C, E) and Emx1-Cre;Rbm8a^loxp/+ (B, D, F) dorsal neocortices from E11.5 (A, B) and E12.5 (C–F) embryos and stained for Tuj1 (green) and Hoechst (blue),CC3 (red), or TUNEL (red). G, A higher magnification image of framed region in F showing costaining of TUNEL with Hoechst. H–O, Coronal sections from Emx1-Cre;Rbm8a^loxp/+ dorsal neocortices stained for Tuj1 (green; H–K), Pax6 (green; L–O), and CC3 (red). The fractions of total Tuj1+ (J) or Pax6+ (N) cells that are CC3+ (apoptotic) are listed. Note a higher fraction of neurons are CC3+ compared with radial glia. K, O, High-magnification images of the regions boxed in J (K) and in N (O). Note images representing the X-Z (left–right) and Y-Z (top–bottom) planes are shown to demonstrate colocalization. All images were taken from dorsolateral regions at the medial rostrocaudal position represented in Figure 1D. n = 3 biological replicates each, representative images were shown. Scale bars: A–J, L–N, 50 μm; K, O; 10 μm.

Figure 8.

Rbm8a haploinsufficiency disrupts cortical lamination. A, B, Whole-mount P0 brains from indicated genotypes. Dotted circles denote dorsal cortices. C, D, Coronal sections of Hoechst-stained P0 brains from indicated genotypes (plane indicated by arrowheads in A and B). E–J, Coronal sections of P0 Emx1-Cre (E, G, I) and Emx1-Cre;Rbm8a^loxp/+ (F, H, J) stained for Cux1 (E, F), Foxp1 (G, H), and Tbr1 (I, J) at regions indicated in C and D. Dotted lines demarcate dorsal cortex. Neuronal layers (layers II–V) are indicated. Arrowheads indicate Foxp1+ cells in the dorsal cortices of Emx1-Cre;Rbm8a^loxp/+ brains (H). K, Quantification of Cux1+, Foxp1+, and Tbr1+ cells in Emx1-Cre and Emx1-Cre;Rbm8a^loxp/+ brain sections. Note the significant reduction of Cux1+ and Foxp1+ cells observed in Emx1-Cre;Rbm8a^loxp/+ brains. L, Fraction of Tbr1+ cells located in bins (as indicated in Fig. 8I,J) with bin 5 most pial and bin 1 adjacent to the ventricle. Note the distribution of Tbr1+ is significantly different between Emx1-Cre and Emx1-Cre;Rbm8a^loxp/+ brains. K, Student's t test. L, χ² analysis, ns, p = 0.517, ***p < 0.0001. Error bars indicate SD, n = 3 biological replicates each. Scale bars: A–D, 1 mm; E–J, 50 μm.

Figure 9.

Overexpression of RBM8A does not impact distribution of cells in the developing neocortex. A–C, Coronal sections of E16.5 brains, in utero electroporated at E13.5 with representative electroporated region (A), with pCAGGS-EGFP plus either pCAGGS-EX (B) or pCAGGS-Flag-RBM8A (C). Numbers indicate bins for quantitation in D and brackets indicate CP, IZ, and SVZ/VZ. D, Quantitation of distribution of EGFP-positive cells in five bins for cortices; with bin 5 most pial and bin 1 adjacent to the ventricle. Note there was no significant difference in the distribution of GFP-positive cells between control and RBM8A electroporated brains. E, Coronal section of an E16.5 brain electroporated with pCAGGS-Flag-RBM8A (RBM8A overexp.). F–K, Higher magnification images of region 1 (F–H) and region 2 (I–K) stained for anti-FLAG (red; F, I), EGFP (green), and Hoechst (blue; G, J) and merge (H, K). Arrowheads, electroporated EGFP cells coexpressing FLAG (yellow). Note high FLAG expression in electroporated cells. L–Q, Coronal sections of E16.5 electroporated with pCAGGS-EGFP and either pCAGGS-EX (L–M, control) or pCAGGS-Flag-RBM8A (O–Q, RBM8A overexp.) and stained for Rbm8a (red; L, O), EGFP (green; M, P), and merge (N, Q). Arrowheads, electroporated EGFP cells showing high Rbm8a staining in pCAGGS-Flag-RBM8A cells (yellow), but not in pCAGGS-EX transfected cells. R, S, Coronal sections of E16.5 brains electroporated with pCAGGS-EGFP and either pCAGGS-EX (R, control) or pCAGGS-Flag-RBM8A (S, RBM8A) and stained for anti-Pax6 (red) and anti-Tbr2 (gray). T–W, High-magnification images of regions indicated in R (T, V) and S (U, W) and stained with either anti-Pax6 (red; T, U) or anti-Tbr2 (red; V, W). Arrowheads, EGFP+ cells (yellow) costained with either Pax6 (T, U) or Tbr2 (V, W). X, Y, Coronal sections of E16.5 brains electroporated with pCAGGS-EGFP (green) and either pCAGGS-EX (X, control) or pCAGGS-Flag-RBM8A (Y, RBM8A) and stained for anti-PH3 (red). Z, AA, High-magnification images of regions indicated in X (Z) and Y (AA), and stained for anti-PH3 (red). Arrowheads, EGFP+ cells costained with PH3 (yellow). BB, Fractions of EGFP+ cells costained with Pax6, Tbr2, or PH3. Note there was no significant difference between control and RBM8A electroporated brains. D, χ² analysis. BB, Student's t test, ns, p > 0.2. Error bars indicate SD, n = 3 biological replicates each condition. For each biological replicate, three to four sections were quantified. Scale bars: A, 200 μm; B, C, E, 100 μm; F–AA, 50 μm.

Figure 10.

RBM8A-injected embryos have normal head size and HuC/D expression in the forebrain. A, B, Representative images show dorsal views of a sham-injected control (A) and an embryo injected with human RBM8A RNA (B). C, Quantification of head size by measuring the distance across the convex tip of the eyecups (yellow arrows) at 4.5 dpf. Note RBM8A RNA-injected embryos have normal head size, p = 0.46, Student's t test. D, E, Representative images showing HuC/D-antibody staining and ventral views of a sham-injected control (D) and RBM8A-injected embryo at 2 dpf (E). Note HuC/D expression is bilateral and restricted to the roof plate of the neural tube in the forebrain at 2 dpf in both RBM8A overexpressants and controls. F, Percentage of embryos with normal (bilateral) or abnormal (unilateral) HuC/D staining in the anterior forebrain. Note embryos injected with RBM8A exhibit normal bilateral expression of HuC/D compared with controls (p = 0.75, Fisher exact test,). G, RT-PCR using human RBM8A and zebrafish β-actin primers on RNA extracted from control and RBM8A mRNA-injected embryos at 2 dpf. M, marker (1 kb plus ladder). Error bars indicate SEM, n = 60 embryos per group; experiments repeated three times. Scale bars: 200 μm.