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Figure 1 Phenotype of conditional Sp8 mutants. (a, b) WMISH of Sp8 in E8.5 and E9.5 embryos. Sp8 is strongly expressed in the anterior neural ridge and in the forebrain neuroepithelium at E8.5 (a). At E9.5, Sp8 expression covers the putative forebrain vesicle (b). (c) Foxg1-Cre activity, visualized by X-Gal staining, is evident throughout the telencephalon at E10.5. (d) Cre recombination ablates Sp8 expression in the telencephalon and olfactory placode of cKO at E10.5. (e, f") Histological (nissl stained) coronal sections at E18.5. (e', f') Mutant brains miss the septum and reveal a reduced size of the telencephalon. (e', e") Callosal fibers do not cross the midline and form probst bundles unilaterally. (g, g') On (nissl stained) sagittal sections, a strongly reduced cortical diameter is characteristic for cKO at E18.5. With 15% penetrance, cKO brains show an enhanced phenotype, highlighted by the complete absence of midline derivates. These mutants were termed 'cKO no midline' (e", f"). cKO and 'cKO no midline' only differ at rostral levels of the forebrain. In 'cKO no midline' specimens, a delamination of the cortex from the basal telencephalon is apparent medially, as a visible hole (asterisk in e"). Caudally in the brain, the difference between low and high penetrance of the phenotypes is not significant (f', f"). AC, anterior commissure; ANR, anterior neural ridge; CC, corpus callosum; CP, cortical plate; CTX, cortex; DI, diencephalon; FB, forebrain; HC, hippocampus; IZ, intermediate zone; LGE, lateral ganglionic eminence; MB, midbrain; MZ, marginal zone; OP, olfactory placode; PB, probst bundles; POA, preoptic area; SE, septum; SP, subplate; SVZ, subventricular zone; TH, thalamus; VZ, ventricular zone.
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Figure 2 Early patterning of the cKO forebrain. (a-f') WMISH and (g, g') ISH on sections. Shh (a, a') and Fgf8 expression (e, e') at E9.5 is unchanged in cKO. The septum and large parts of the MGE anlage (red and white arrowheads in (b, b')) are free of Nkx2.1 activity. Pax6 activity is diminished in cKO (c, c'). Emx2 is slightly up-regulated in mutants (d, d'). At E10.5, Fgf8 is expressed in the telencephalic midline and septum anlage in both genotypes (f, f'). At E12.5, Fgf8 mRNA is specifically lost in the septum (red arrow in (g, g')). The Fgf8 positive domain in cKO at E12.5 matches the midline of the POA (black arrow in (g')).
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Figure 3 D/V patterning defects at the medial pallial-subpallial boundary (mPSB). ISH on E12.5 (coronal) forebrain sections. Images show the right brain hemisphere. (c-k') Blow-up images of the medial/ventral part of each boxed section. In cKO, the pallial markers (a, a', c, c') Emx2, (b, b', d, d') Pax6, and (e, e') Ngn2 expand into the ventral midline. Conversely, ventral markers (g, g') Dlx1, (h, h') Gsh2, and (j, j') Mash1 are not expressed in the dorsal septum. (f, f') Nkx2.1 activity is lost in the septum and rostral MGE of Sp8 mutants. (k, k') The Nkx6.2+ domain reflects a rudimentary MGE territory in cKO and contacts the Emx2+, Pax6+ and Ngn2+ midline. Note the (l, l') expansion of the Ngn2+ domain (arrows) and the (m, m') reduction of the Dlx1+ domain (arrows) around the midline of the Sp8 deficient telencephalon at E15.5. (n, n') Nkx2.1 (arrow) remains absent in the septum and rostral MGE of cKO at this stage. (o, o') Depletion of (Gad67+) interneurons in the Sp8 mutant cerebral cortex (arrow) at E18.5. For a better visualization of the shift in gene expression pattern, the arrowheads in (e, e') point at the constriction between the septum anlage and the LGE. ISH for Emx2/Pax6 (a, a', b, b', c, c', d, d') and Ngn2/Mash1 riboprobes (e, e', j, j') were performed on adjacent sections.
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Figure 4 Caudalized gene expression and thalamic innervation in the brain of Sp8 mutants. (a, a', b, b') WMISH on dissected E12.5 forebrains, using Emx2 and Pax6 riboprobes. The Emx2 gradient is up-regulated in cKO (arrows in (a, a')). The expression level of Pax6 is diminished (arrowheads in (b, b')) in Sp8 mutants. Analysis of the area specific marker genes (c, c') EphrinA5, (d, d') EphA7, (e, e') Coup-TF1 and (f, f') ID-2 on E18.5 sagittal sections using ISH (rostral is to the left). The visual cortex area in cKO cortices appears expanded towards the rostral brain, as demonstrated by EphA7, ID-2 and Coup-TF1 expression (strong rostral domain of EphA7 in (d'), strong ID-2 domain in (f'), strong domain of Coup-TF1 in (e')). The somatosensory cortex (area between the red arrows in (c, d') and between the black and red arrows in (f)) shifts rostrally in Sp8 conditional mutants (compare (c, d) with (c', d'), and (f) with the area indicated by the asterisk in (f')). The motor cortex expression area appears condensed (compare rostral to left arrow in (c, c', d, d')) in Sp8 deficient specimens. (g, g') Coronal sections of E18.5 brains labeled with DiI and DiO and counterstained with DAPI. In controls, DiI, placed in the visual cortex (inset in (g)), retrogradelly labels only cells in the dLGN (g). Placing DiO in the somatosensory cortex (inset in (g)) marks only cells in the VP (g). In the mutants the green dye, placed into somatosensory cortex (inset in (g')) labels cells in the VP and the dLGN (g'). (h) GST-pull down reveals that Emx2 lacking the homeobox (Emx2ΔHox) does not bind GST, GST-Sp8 (GST-Sp8FL) or GST-Sp8 lacking zinc fingers (GST-Sp8ΔZn) (lanes 2–4). Full-length Emx2 protein (Emx2FL) does not bind GST, but interacts with GST-Sp8 (GST-Sp8FL) and GST-SP8 lacking zinc fingers (GST-Sp8ΔZn) (lanes 6–8). Lanes 1 and 5 show 10% of the radiolabeled Emx2 isoforms, used as input for the binding assays in lanes 2–4 and 6–8.
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Figure 5 Apoptotic cell death in Sp8 deficient brains. TUNEL staining on (a, a', c) E12.5 (coronal) and (b, b', d) E15.5 forebrain sections (sagittal; rostral is to the left). The arrow in (c) demarcates a typical cluster of apoptotic cells, found in cKO, and found from E12.5 to E18.5. Clusters typically contained five to seven apoptotic cells (blow-up in (c)). (d) Tuj/TUNEL double staining in cKO reveals TUNEL+ cells within the putative CP (blue arrow) and presumptive proliferative zones and the IZ (white arrows). (e) Quantification of TUNEL+ nuclei on E10.5, E12.5, E15.5 and E18.5 forebrain sections (n = 3–4 for each stage) shows an increased cell death without Sp8 function. Fate mapping of (f, f') early-born and (g, g') late-born pallial neurons (using BrdU injection at E12.5 or E15.5) on E18.5 sagittal sections. Putative early-born neurons migrate through the CP (f, f'). Their number is diminished in mutant cortices (f') compared to controls (f). In cKO, BrdU+ neurons populate ectopic positions in the upper CP, when compared to the relative position of Tbr1 immunoreactive cells (white arrow in (g')), possibly reflecting the thinned cortex. BrdU labeling at E15.5 appears reduced in the SVZ of mutants (yellow arrow in (g')).
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Figure 6 Defective preplate splitting and subplate development. (a, a') ISH for Tbr1 transcript on E15.5 coronal sections (medial is to the right). (b, b') Immunohistochemical co-detection of Tbr1 and BrdU on E15.5 sagittal sections (rostral is to the left). Mutants lack well separated/positioned Tbr1+, putative subplate cells at E15.5 (arrow and asterisk in (a, a')). At this stage, BrdU+ (injected E11) cells co-labeled with Tbr1 form a superplate-like structure by populating the upper CP/MZ in cKO (arrow in (b)). In controls, those cells settle in the subplate area (arrow in (b)). Arrows indicate the dislocation of BrdU+/Tbr1+ cells in mutants (b'). Co-detection of Reelin- and Tbr1 protein in the MZ on coronal sections at E18.5 (c, c') (medial is to the right) cKO showed more Reelin+/Tbr1+ (putative Cajal-Retzius cells) in the MZ (c'). The PPL does not split properly in cKO; additionally Gap43+ axons (assayed on E18.5 coronal sections) form bundles around the internal capsule (arrowheads in (d, d') and some project ectopically into the MZ (arrow in (d, d')).
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Figure 7 Specification of individual cortical layer neurons. Coronal (E18.5) sections were double labeled with the Cux2 riboprobe and the Tbr1 antibody. (a, a') Cux2+ and Tbr1+ cell populations are separated from each other, and the generation of intra-/supragranular layers seems preserved in Sp8 mutants. ISH of layer specific marker genes on (c, c', f, f', j, j') E18.5 coronal (medial is to the right) and (b, b', d, d', e, e', h, h', k, k') sagittal (rostral is to the left) forebrain sections. (g, g', l, l') Detection of Tbr1/2 protein on E18.5 coronal sections (medial is to the right). A reduced expression of Cux2 (d, d'), Lhx2 (e, e'), Robo1 (h, h') and Tbr1 (k, k') is visible in mutant cortices. In Sp8cKO, the subpopulations of Cux1+ (c, c'), RzR-β+ (f, f'), and ER81+ (j, j') cortical neurons are not molecularly specified. Tbr2 immunoreactive progenitors are diminished in the proliferative compartment of the Sp8 mutant cortex (box in (g, g')). Immunohistochemistry reveals that some Tbr1+ cells ectopically populated the upper CP and MZ in Sp8 mutants (arrow in (l')).
..................................................Figure 8 Role of Sp8 in the forebrain. (a) A scheme illustrating the genetic interplay between Sp8 and Emx2. Emx2 and Pax6 mutually affect their graded expression characteristics along the A/P axis (red and blue arrows). Sp8 acts as a repressor of Emx2 to pattern the forebrain along the A/P axis. In cKO, the mutant Emx2 expression domain (red) is expanded rostrally. Accordingly, Pax6 expression territory (blue) is diminished. (b) Scheme recapitulating the gene expression of marker genes at the medial and lateral PSB. In conditional Sp8 mutant, a dorsalization of gene activity around the midline occurs, as highlighted by the ventral expansion of the domain marked in red. Dorsal: red domain, Pax6, Emx2, Ngn2; ventral: green and light-green domain, Gsh2, Dlx1, Mash1. Reciprocally, the expression area of ventral markers is lost in the dorsal septum (light green domain). Accordingly, Fgf8 and Nkx2.1 expression domains are not maintained in the mutant cortical midline. We favor a model in which Sp8 regulates the AP regionalization of cortex by directly suppressing Emx2, while the role of Sp8 in the maintenance of Nkx2.1 activity in SE and MGE could consist of controlling Fgf8.
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