Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Aug 5;35(31):10911-26.
doi: 10.1523/JNEUROSCI.0601-15.2015.

Thalamic WNT3 Secretion Spatiotemporally Regulates the Neocortical Ribosome Signature and mRNA Translation to Specify Neocortical Cell Subtypes

Affiliations
Free PMC article

Thalamic WNT3 Secretion Spatiotemporally Regulates the Neocortical Ribosome Signature and mRNA Translation to Specify Neocortical Cell Subtypes

Matthew L Kraushar et al. J Neurosci. .
Free PMC article

Abstract

Neocortical development requires tightly controlled spatiotemporal gene expression. However, the mechanisms regulating ribosomal complexes and the timed specificity of neocortical mRNA translation are poorly understood. We show that active mRNA translation complexes (polysomes) contain ribosomal protein subsets that undergo dynamic spatiotemporal rearrangements during mouse neocortical development. Ribosomal protein specificity within polysome complexes is regulated by the arrival of in-growing thalamic axons, which secrete the morphogen Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3). Thalamic WNT3 release during midneurogenesis promotes a change in the levels of Ribosomal protein L7 in polysomes, thereby regulating neocortical translation machinery specificity. Furthermore, we present an RNA sequencing dataset analyzing mRNAs that dynamically associate with polysome complexes as neocortical development progresses, and thus may be regulated spatiotemporally at the level of translation. Thalamic WNT3 regulates neocortical translation of two such mRNAs, Foxp2 and Apc, to promote FOXP2 expression while inhibiting APC expression, thereby driving neocortical neuronal differentiation and suppressing oligodendrocyte maturation, respectively. This mechanism may enable targeted and rapid spatiotemporal control of ribosome composition and selective mRNA translation in complex developing systems like the neocortex.

Significance statement: The neocortex is a highly complex circuit generating the most evolutionarily advanced complex cognitive and sensorimotor functions. An intricate progression of molecular and cellular steps during neocortical development determines its structure and function. Our goal is to study the steps regulating spatiotemporal specificity of mRNA translation that govern neocortical development. In this work, we show that the timed secretion of Wingless-related MMTV (mouse mammary tumor virus) integration site 3 (WNT3) by ingrowing axons from the thalamus regulates the combinatorial composition of ribosomal proteins in developing neocortex, which we term the "neocortical ribosome signature." Thalamic WNT3 further regulates the specificity of mRNA translation and development of neurons and oligodendrocytes in the neocortex. This study advances our overall understanding of WNT signaling and the spatiotemporal regulation of mRNA translation in highly complex developing systems.

Keywords: Wnt; mRNA translation; neocortex; ribosome; thalamocortical.

Figures

Figure 1.
Figure 1.
Neocortical 40S-60S-80S and polysomes show temporal specificity in R-protein composition. A, Schematic of the experimental approach used throughout our study to identify proteins and mRNAs differentially associated with 40S-60S-80S and polysomes during neocortical development. Representative density gradient curves monitored during fractionation for the conditions and stages analyzed are presented. B, KEGG pathway analysis of proteins differentially expressed in WT 40S-60S-80S and polysome fractions between the E13, E16, and P0 neocortex measured by mass spectrometry with p < 0.01. The mmu03010: Ribosome pathway represents the largest temporally dynamic group, which included predominantly ribosomal proteins. C, Simplified schematic of polysome biogenesis with R-protein incorporation in the nucleus and cytoplasm. D, Western blot (left) and quantification (right) of E13, E16, and P0 total neocortical lysates for candidate R-proteins measured by mass spectrometry as changed in 40S-60S-80S and polysomes (RPL7) versus an unchanged control (RPL34; n = 3 per stage; t test, p < 0.05). E, Western blot analysis of nuclear-cytoplasmic fractionations from E13, E16, and P0 total neocortical lysates for R-protein subcellular distribution. Confirmation of nuclear-cytoplasmic separation by histone 3 (H3) and GAPDH, respectively. F, Western blot analysis of E13, E16, and P0 total neocortex gradient fractionated lysates for R-protein distribution in 40S-60S-80S and polysomes. G, Quantification of F (multivariate ANOVA, followed by ANOVA and then Tukey's HSD post hoc, p < 0.05).
Figure 2.
Figure 2.
R-protein polysome distribution is regionally and layer specific during neocortical development. A, Schematic (left) of anterior sensorimotor versus posterior auditory-visual neocortex subdissection at P0. qRT-PCR analysis of 18S rRNA in anterior versus posterior P0 neocortex gradient fractionations demonstrating equivalent fractionation curves (top right; Bonferroni-corrected t test, p ≥ 0.185). Western blot analysis of RPL7 40S-60S-80S and polysome gradient distribution in the P0 anterior versus posterior neocortex (bottom right). B, Schematic of LCM method to analyze the layer-specific R-protein composition in E16 and P0 gradient fractionated sensorimotor neocortices. Coronal sections processed by LCM within the rostrocaudal sensorimotor neocortex boundaries at E16 and P0 are denoted by the Electronic Prenatal Mouse Brain Atlas (EPMBA; http://www.epmba.org) and Allen Brain Atlas (ABA; http://developingmouse.brain-map.org) positions, respectively. C, Representative E16 (left) and P0 (right) sensorimotor neocortex coronal sections with LCM regions collected in parallel outlined by dotted yellow lines, and corresponding Western blot measurement for layer-specific markers (SATB2, TLE4, PAX6) and total RPL7 levels at right. D, qRT-PCR analysis of 18S rRNA levels in gradient fractionations of E16 and P0 LCM isolated layers from inputs in C. E, Western blot analysis of RPL7 polysome specificity in E16 VZ/SVZs versus CP on left, and P0 LLs versus ULs on right.
Figure 3.
Figure 3.
Timed thalamocortical WNT3 secretion regulates the R-protein composition of polysomes in the developing sensorimotor neocortex. A, Schematic of the spatiotemporal innervation of the neocortex beginning at midneurogenesis E15 by thalamic axons. B, Wnt3 qRT-PCR of the developing neocortex and thalamus (t test, p < 0.05). C, Immunohistochemistry and coronal imaging of Wnt3-Gfp mouse neocortical and subcortical structures at E15. GFP (green) in thalamus and thalamocortical axons reaching the neocortex (arrowheads; DAPI in blue). D, WNT3 immunohistochemistry (red) in coronal sections of the developing neocortex. LLs, ULs, intermediate zone (IZ) white matter, and VZ/SVZs (DAPI in blue). E, GFP immunohistochemistry (green) in coronal sections of Kcnc2-Cre/Gfp transgenic mice at E16. Thalamocortical axons reaching the neocortex are denoted by white arrowheads (DAPI in blue). Kcnc2 in situ hybridization in E14.5 neocortical sagittal sections (inset; from the Eurexpress Database, www.eurexpress.org), with thalamic expression denoted by black arrowhead. F, Western blot analysis of P0 Wnt3-cKO vs WT lysates confirming thalamic WNT3 depletion, and levels of RPL7 in total and nuclear-cytoplasmic fractionated neocortices (balanced to H3 and GAPDH). G, Western blot analysis of RPL7 in P0 Wnt3-cKO vs WT fractionated total neocortices (left). Quantification of RPL7 40S-60S-80S and polysome distribution (right; Mann–Whitney U test, p = 0.012). H, Western blot analysis of RPL7 levels in P0 Wnt3-cKO vs WT anterior sensorimotor and posterior auditory-visual fractionated neocortices. I, Western blot analysis of RPL7 levels in neuronal cell line (N2a) cultures exposed to mock, WNT3, or WNT3 plus SFRP1 inhibitor conditions; total levels (left); and nuclear-cytoplasmic fractionated levels (right) balanced to GAPDH and H3. J, Western blot analysis for RPL7 levels in polysome fractionations of N2a cultures exposed to mock, WNT3, or WNT3 plus SFRP1 inhibitor conditions.
Figure 4.
Figure 4.
mRNAs temporally regulated in neocortical 40S-60S-80S and polysomes are functionally related and layer specific. A, Volcano plot showing distinct mRNAs revealed by RNAseq measurement as higher in total neocortical levels at E13 (yellow) or higher at P0 (green). Those not significantly changed (p > 0.05) are shaded black. B, Volcano plot showing the distribution of mRNAs measured by RNAseq that are significantly different in 40S-60S-80S-associated (red) or polysome-associated (blue) fractions but unchanged in total levels, compared with those changed in total levels (black) between E13 and P0. C, Venn diagram summary of RNAseq showing the number of mRNAs that changed significantly in total, 40S-60S-80S-associated levels between E13 and P0, and/or polysome-associated levels between E13 and P0, compared with those unchanged in total levels (false discovery rate, ≤5%). D, RNAseq GO (left) and KEGG (right) pathway analysis of mRNAs significantly changed in 40S-60S-80S or polysomes between E13 and P0, but unchanged in total levels. E, Bioinformatics analysis of layer-specific mRNAs dynamic in 40S-60S-80S (left) or polysomal (right) levels, but unchanged in total levels.
Figure 5.
Figure 5.
Thalamic WNT3 regulates the translation of Foxp2 and Apc in the neocortex. A, Immunohistochemistry and coronal imaging of general differentiated neuron marker MAP2 (green), axonal marker L1 (red), differentiated projection neuron markers FOXP1 (green) and FOXP2 (red), and oligodendrocyte markers APC-CC1 (green) and OLIG2 (red) in P0 Kcnc2-Wnt3-cKO vs WT neocortices (4× left, inset right, DAPI in blue). L5, Layer 5; BG, basal ganglia; CC, corpus callosum. B, Immunohistochemistry and coronal imaging of FOXP2 (green), APC (red), and OLIG2 (red) in P0 Emx1-Wnt3-cKO vs WT neocortices (DAPI in blue). C, qRT-PCR analysis of Foxp2 and Apc mRNA total levels in P0 Kcnc2-Wnt3-cKO vs WT neocortices relative to Gapdh. D, qRT-PCR analysis of Foxp2 and Apc mRNA in polysome fractionations of E13, E16, and P0 WT vs P0 Kcnc2-Wnt3-cKO neocortices. Quantification of mRNA distribution on top, statistical analysis on bottom, significant comparisons in red (multivariate ANOVA, ANOVA, Games–Howell post hoc, p < 0.05).
Figure 6.
Figure 6.
WNT3 regulates Foxp2 mRNA translation via its 3′UTR. A, FOXP2 (blue) and RFP (red) immunocytochemistry in primary neocortical neuronal cultures after Rfp IUEP at E13 in the sensorimotor neocortex, then cultured in mock, WNT3, or WNT3 plus SFRP1 inhibitor conditions for 48 h (images and inset on left; arrowheads indicate FOXP2 RFP-positive neurons). Quantification (right) of the fold change in the percentage of FOXP2 RFP-positive neurons among all RFP-positive neurons when normalized to mock levels (t test, p < 0.05). B, Western blot analysis of FOXP2 protein expression in primary neuronal cultures exposed to mock, WNT3, or WNT3 plus SFRP1 inhibitor conditions. C, Schematic (left) and quantification (right) of luciferase measurement in E13 neocortex slices IUEP with Foxp1-3UTR-Luciferase or Foxp2-3UTR-Luciferase constructs in the sensorimotor region, then cultured in mock, WNT3, or WNT3 plus SFRP1 inhibitor conditions (shown relative to mock; ANOVA, Games–Howell post hoc, p ≤ 0.009).
Figure 7.
Figure 7.
Model for the spatiotemporal regulation of mRNA translation during neocortical development. A, Small (40S) and large (60S) ribosomal subunits combined contain ∼80 ribosomal proteins (R-proteins). R-proteins associated with the small (RPS, pink) and large (RPL, blue) subunits may differentially associate with these subunits in response to timed signals during neocortical development, coordinated with the dynamic translation of subsets of mRNAs. B, Schematic of how timed WNT3 secretion from thalamocortical axons regulates mRNA translation in the developing neocortex. This may influence the spatiotemporal translation of specific mRNAs, such as Foxp2, that drive neocortical projection neuron differentiation.

Similar articles

See all similar articles

Cited by 16 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback