Foxp-mediated suppression of N-cadherin regulates neuroepithelial character and progenitor maintenance in the CNS - PubMed (original) (raw)

Foxp-mediated suppression of N-cadherin regulates neuroepithelial character and progenitor maintenance in the CNS

David L Rousso et al. Neuron. 2012.

Abstract

Neuroepithelial attachments at adherens junctions are essential for the self-renewal of neural stem and progenitor cells and the polarized organization of the developing central nervous system. The balance between stem cell maintenance and differentiation depends on the precise assembly and disassembly of these adhesive contacts, but the gene regulatory mechanisms orchestrating this process are not known. Here, we demonstrate that two Forkhead transcription factors, Foxp2 and Foxp4, are progressively expressed upon neural differentiation in the spinal cord. Elevated expression of either Foxp represses the expression of a key component of adherens junctions, N-cadherin, and promotes the detachment of differentiating neurons from the neuroepithelium. Conversely, inactivation of Foxp2 and Foxp4 function in both chick and mouse results in a spectrum of neural tube defects associated with neuroepithelial disorganization and enhanced progenitor maintenance. Together, these data reveal a Foxp-based transcriptional mechanism that regulates the integrity and cytoarchitecture of neuroepithelial progenitors.

Copyright © 2012 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Elevated Foxp4 Expression Coincides with Neuronal Differentiation and Neuroepithelial Detachment

(A–E) Foxp2, Foxp4, and Foxp1 are successively expressed by differentiating MN progenitors and other spinal NPCs. Foxp4 levels increase during this transition. (F–K) Foxp4low cells express Sox2; Foxp4high cells express the proneural proteins Ngn2 and NeuroM, and display cytoplasmically localized Numb. Foxp2 and Foxp4 are absent from mature NeuN+ Isl2+ MNs. (L–P) Neuroepithelial attachment, cell cycle exit, and neuronal maturation are distinguished by HRP injections into the ventricle, rhodamine-dextran injections into the ventral roots, and BrdU labeling. (Q) Quantification of Foxp4 levels in HRP+ (neuroepithelial) and HRP− (detached) cells. Mean pixel intensities ± SEM calculated from at least 25 cells per group from multiple embryos are shown. ***p < 0.001. (R–V) The sequential pattern of Foxp expression is conserved in the mouse spinal cord. (U) At e11.5, Foxp4 is also observed in a putative LMC motor pool, and in numerous interneurons (INs). (W) Summary of Foxp4 and Foxp2 expression during MN differentiation. AJs, adherens junctions. See also Figure S1.

Figure 2

Figure 2. Foxp2 and Foxp4 Promote Neurogenesis and Suppress Neuroepithelial Character

(A, C, and D) Analysis of spinal cords transfected with _CMV::IRES-nEGFP_-control or (B, E, and F) _CMV::Foxp4_-_IRES_-nEGFP expression vectors. Brackets with asterisks indicate areas of ectopic neurogenesis. (G and H) Quantification of changes in neurogenesis. NPC, Sox2+; Neuron, NeuN+; pMN, Olig2+; MN, Isl1/2+. Mean ± SEM from multiple sections taken from 5 embryos for each condition are shown. ***p < 0.001. (J–U) Analysis of spinal cords transfected with Foxp2 and Foxp4 shRNA-IRES-nEGFP vectors or a nontargeting control shRNA-IRES-nEGFP vector. Most double-knockdown (dKD) cells express Sox2 and do not form NeuN+ neurons (asterisk in K and M). Some dKD cells differentiate within the neuroepithelium (arrows in O). Asterisk in (Q) indicates a dorsal expansion of Olig2+ MN progenitors; brackets denote a decrease in MNs. (R and S) Foxp2/4 dKD MNs extend Hb9::LacZ+ axons toward the periphery (arrows), but their cell bodies fail to migrate laterally (arrowheads). (T and U) Foxp2/4 dKD MNs retain apical attachments (open arrowhead) and radial morphology. (I) Summary of the effects of Foxp2 and Foxp4 manipulation on neurogenesis and apical attachment. See also Figures S2, S3, and S4.

Figure 3

Figure 3. Foxp4 Represses N-Cadherin and Influences Adherens Junction Ultrastructure

(A–E) Transverse and open book views of the spinal cord reveal differential expression of Foxp4 and N-cadherin in distinct progenitor (p) domains. d, dorsal; v, ventral; m, medial; l, lateral; r, rostral; c, caudal. (F) N-cadherin and Foxp4 expression are inversely correlated [f(x) f _x_−0.75, R2 = 0.722]. (G–R) Analysis of spinal cords transfected with (G-L) _CMV::IRES-nEGFP_-control or (M-R) _CMV::Foxp4_-_IRES_-nEGFP vectors. + indicates the transfected side of the spinal cord. Arrows and arrowheads indicate apical membranes and radial processes, respectively. Asterisks denote disruptions in neuroepithelial cytoarchitecture. Arrows in (J) and (P) indicate the intact pial surface. (K and Q) Transmission electron micrograph of normal (arrowheads) and disrupted AJs. N, nucleus. (L and R) Open book view of Numb anchored to the apical membrane and its redistribution after Foxp4 misexpression. (S and T) N-cadherin and Numb accumulate at the apical surface of the neuroepithelium upon Foxp2/4 double knockdown. (U) Summary of the suppressive effects of Foxp4 and Foxp2 on N-cadherin and AJ maintenance. See also Figures S2, S3, S4, and S5.

Figure 4

Figure 4. N-Cadherin Is a Primary Target of Foxp4

(A–O) Analysis of spinal cords transfected at e3 with a _CMV::Foxp4_-IRES-nEGFP expression vector and collected 12, 24, and 36 hr later. N-cadherin protein declines (asterisk) between 12 and 24 hr posttransfection while Sox2 perdures. By 24 hr, Numb accumulates in the cytoplasm (bracket). At R 36 hr, Sox2 is abolished and cytoplasmic Numb is associated with ectopic NeuN+ neurons. (P) Quantitative PCR analysis of gene expression 6 and 12 hr after Foxp4 transfection relative to control electroporations. Mean _Gapdh_-normalized mRNA expression levels ± SEM from at least 3 embryos per condition are shown. *p < 0.05; ***p < 0.001. (Q) Chromatin immunoprecipitation analysis of Foxp4 and Sox2 at evolutionarily conserved regions of the Cdh2 (N-cadherin) locus indicated by the orange lines. Foxp4 shows significant binding to an element in intron 3 [i3a] relative to other regions in the Cdh2 locus and unrelated genes such as Gapdh. Sox2 binds to an element in intron 2 [i2a] but not to regions where Foxp4 is bound. Mean binding activity ± SEM from four experiments is plotted. **p < 0.01; ****p < 0.0001. See also Figure S6.

Figure 5

Figure 5. Sox2 and N-Cadherin Promote Neuroepithelial Character and Oppose Foxp4 Function

(A, F, K, and P) Analysis of spinal cords transfected with a CMV::IRES-nEGFP control vector compared to equivalent vectors encoding: (B, G, L, and Q) dominant-negative N-cadherin at high (dnN-cadhigh) or (C, H, M, and R) low (dnN-cadlow) dosage; (D, I, N, and S) full-length N-cadherin; (E, J, O, and T) Sox2. Most cells transfected with dnN-cadhigh or dnN-cadlow express NeuN and lack neuroepithelial characteristics. Full-length N-cadherin maintains cells in an NPC-like state within the VZ (D), similar to Sox2 (E). Arrows and brackets with asterisks denote the normal and altered distribution of proteins, respectively. + indicates the transfected side of the spinal cord. (U–Y) Summaries of the effects of manipulating N-cadherin or Sox2 on neurogenesis and apical attachment. (Z–AE) Cotransfection of Foxp4 with full-length N-cadherin or Sox2 restores neuroepithelial character. Asterisk in (AC) indicates region of Foxp4-induced neuroepithelial disruption. Arrowheads in (AD)–(AE) indicate restoration of radial fibers. (AF) Quantitation of Sox2+ NPCs and NeuN+ neurons in the ventral VZ under the indicated conditions. Plots show mean ± SEM from multiple sections collected from 3–5 embryos for each condition. ***p < 0.001. (AG) Summary of the opposing actions of Foxp4 and Sox2 in regulating N-cadherin expression and progenitor maintenance.

Figure 6

Figure 6. Proneural Gene Activity Requires Foxp2/4 Function to Promote Neurogenesis

(A) Outline of the double transfection protocol. CMV::nuclear-tagged LacZ (nLacZ) and CMV::nuclear Myc-tags (nMyc) distinguish the first and second transfections, respectively. (B) Model of the neural tube showing the region analyzed. Costaining with β-gal, Myc, and markers for Sox2+ NPCs or NeuN+ neurons results in “white” triple-labeled cells in (H)–(Q). (C, H, and M) Cells doubly transfected with control vectors comprise NPCs and neurons equally. (D, I, and N) Cells doubly transfected with control vectors followed by Ngn2 predominantly form neurons in the MZ. Cells doubly transfected with Foxp2/4 shRNA followed by control (E, J, and O) or Ngn2 vectors (F, K, and P) do not form neurons and remain in the VZ. (G, L, and Q) Cells doubly transfected with Foxp2/4 shRNA followed by low amounts of the dnN-cad vector detach and differentiate. (R–V) Summary of double transfection results. (W) Quantification of progenitor versus neuronal fates of doubly transfected cells. Plots display mean ± SEM for 250–500 β-gal+Myc+ cells from multiple sections taken from at least 3 embryos for each vector combination. *p < 0.05; ***p < 0.001; n.s., not significant. (X) Summary of relationships between proneural genes, Foxp activity, and neuroepithelial adhesion. See also Figure S7. (C) Plot of the frequency of neural tube defects (NTD) seen in Foxp4 mutant embryos. (D and E) Example of intermediate holoprosencephaly at e12.5 resulting from Foxp4 deletion. (F–I) Analysis of e10.5 control and (J-M) Foxp4LacZ/LacZ mutant spinal cords. (N–Q) Analysis of e12.5 control and (R–U) Foxp4Neo/LacZ mutant cortex. N-cadherin is inversely correlated with Foxp2 and Foxp4 expression in control mice and upregulated in Foxp4 mutants. (Q and U) NeuN+ neurons form inappropriately within the VZ of the Foxp4 mutant cortex (arrowheads in U). (V–Y) In utero electroporation of CMV::IRES-nEGFP control and (Z–AC) _CMV::Foxp4-IRES_-nEGFP vectors in the e14.5 cerebral cortex suppresses N-cadherin, Sox2, and β-catenin, leading to ectopic neuronal differentiation. (AD) Quantitation of Foxp2and N-cadherin protein as a function of mediolateral position in controland _Foxp4Neo/LacZ_mutant cortices averaged across multiple sections. (AE) Quantitation of the total number of spinal and cortical NPCs (Sox2+) and neurons (NeuN+) in Foxp4 mutants relative to littermate controls. Mean ± SEM for multiple sections taken from 5–10 embryos for each genotype is shown. *p < 0.05; ***p < 0.001; n.s., not significant. (AF) Quantitation of the effects of Foxp4 misexpression on cortical neurogenesis. Plots show mean ± SEM for multiple sections taken from 5 embryos for each condition. **p < 0.01; ***p < 0.001. See also Figure S8.

Figure 7

Figure 7. _Foxp4_-Deficient Mice Exhibit Defects in Neural Tube Morphology and Enhanced Progenitor Maintenance

(A) Diagram of Foxp4 mutant alleles. (B) Early lethality occurs by e11.5 in Foxp4LacZ/LacZ mutants relative to Foxp4Neo/Neo mice. *p < 0.05; c2 = 6.39.

Figure 8

Figure 8. Opponent Activities of Foxp and Sox2 Regulate Neuroepithelial Niche Maintenance by Modulating N-Cadherin Expression

(A) Model depicting how Foxp-mediated suppression of N-cadherin offsets the progenitor maintenance activity of Sox2 and promotes neuroepithelial delamination. Sox2 provides positive inputs onto N-cadherin gene expression to maintain the neuroepithelial niche and ensure the apical localization of AJ components such as Numb. (B) Elevated Foxp expression levels within neural progenitors eliminate N-cadherin-based AJs and promote ectopic neurogenesis. (C) Loss of Foxp2 and Foxp4 function enhances apical AJs and maintenance of neuroepithelial progenitors.

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