Cortical gyrification induced by fibroblast growth factor 2 in the mouse brain

Abstract

Gyrification allows an expanded cortex with greater functionality to fit into a smaller cranium. However, the mechanisms of gyrus formation have been elusive. We show that ventricular injection of FGF2 protein at embryonic day 11.5-before neurogenesis and before the formation of intrahemispheric axonal connections-altered the overall size and shape of the cortex and induced the formation of prominent, bilateral gyri and sulci in the rostrolateral neocortex. We show increased tangential growth of the rostral ventricular zone (VZ) but decreased Wnt3a and Lef1 expression in the cortical hem and adjacent hippocampal promordium and consequent impaired growth of the caudal cortical primordium, including the hippocampus. At the same time, we observed ectopic Er81 expression, increased proliferation of Tbr2-expressing (Tbr2(+)) intermediate neuronal progenitors (INPs), and elevated Tbr1(+) neurogenesis in the regions that undergo gyrification, indicating region-specific actions of FGF2 on the VZ and subventricular zone (SVZ). However, the relative number of basal radial glia-recently proposed to be important in gyrification-appeared to be unchanged. These findings are consistent with the hypothesis that increased radial unit production together with rapid SVZ growth and heightened localized neurogenesis can cause cortical gyrification in lissencephalic species. These data also suggest that the position of cortical gyri can be molecularly specified in mice. In contrast, a different ligand, FGF8b, elicited surface area expansion throughout the cortical primordium but no gyrification. Our findings demonstrate that individual members of the diverse Fgf gene family differentially regulate global as well as regional cortical growth rates while maintaining cortical layer structure.

Figure 1.

Intraventricular injection of FGF2 or FGF8b at E11.5 produces different effects on cortical surface expansion by E13.5. Embryos injected with FGF2 (B, E), showed different rostral and caudal effects compared with control littermates (A, D). Rostrally, the lateral ventricles were expanded and the perimeter of the cortical VZ (area between arrowheads) was substantially larger than controls (A, B, G). In contrast, the caudal cortical primordium appeared shrunken, but the diencephalon expanded, compared with control littermates or vehicle-injected controls (D, E, G). The rostral perimeter increase was compensated for by decreased caudal perimeter, and the total surface area of the FGF2-injected cortex was not significantly different from controls (I; n = 3 controls and n = 3 for 100 ng FGF2 embryos; p = 0.95). In contrast, embryos injected with FGF8b showed modest increases in cortical perimeter both rostrally and caudally, and the brain was substantially longer along the rostrocaudal axis (C, F, H). Overall, cortical surface area was significantly increased after FGF8b injection by 23.7% (J; n = 3 controls and n = 3 for 200 ng FGF8b-injected embryos; p = 0.002). LV, Lateral ventricle; CGE, caudal ganglionic eminence; SE, septum; DI, diencephalon. Scale bar, 1 mm. *p < 0.05; **p < 0.005.

Figure 2.

Intraventricular injection of FGF2 induces tangential expansion of rostral cortical VZ and early folding of the Pax6⁺/Emx1⁺ cortical wall by E13.5. After FGF2 injection at E11.5, embryonic brains are significantly larger than controls (A). Immunohistochemistry for Tbr2/PH3 (B, C) indicates the extent of the cortical domain (SVZ) and normal location of ventricular and ab-ventricular mitoses. The Emx1 domain, often considered the best neocortical marker, is shown by in situ hybridization (D, E; boundaries shown by arrowheads). Note that the Emx1 domain does not fully extend to the pallial–subpallial boundary in both control and FGF2-injected embryos (arrows). In FGF2-injected embryos, the Dlx2 domain (F, G, arrowheads) appeared to extend farther toward the morphological pallial–subpallial boundary (arrows). Total surface area of the Dlx2 domain in the rostral (black bars) and caudal (gray bars) telencephalon did not show significant differences (H). In the dorsal cortical domain, we consistently observed buckling of the cortical wall (asterisks in C, E, G). mes, mesencephalon; tel, telencephalon; np, nasal process. Scale bar: A, 1.8 mm; B–G, 1 mm.

Figure 3.

FGF2 injection alters canonical FGF-responsive gene expression within 8 h, downregulates Wnt3a in the cortical hem, and induces ectopic Er81 within the folding cortical region. Excess transcription of Spry1 mRNA was detected in both the rostral and caudal cortex 8 h after FGF2 injection (A–D). In contrast, CoupTF1 mRNA was severely downregulated (E, F) in the same time period. Downregulation of CoupTF1 mRNA persisted 48 h after FGF2 injection (G, H). Ectopic Er81 mRNA expression was found in the rostrolateral cortical primordium, within the SVZ region, coinciding with early cortical folding (I, J, asterisk; arrowheads in I, J indicate pallial–subpallial boundary). In this embryo, folding was observed unilaterally, coinciding with Er81 actopic expression. Wnt3a in the cortical hem was substantially reduced in E13.5 FGF2-injected embryos injected at E11.5 (K, K′, L, L′). M, Lef1 mRNA was significantly decreased in the caudomedial pallium, comprising the anlage of the hippocampus, by qRT-PCR. n = 6 controls and n = 3 FGF2 injected. Scale bar: I, J, 1.2 mm; all other panels, 1 mm. *p = 0.02.

Figure 4.

Decreased neurogenesis immediately after FGF2 injection followed by increased Tbr2⁺ intermediate progenitor proliferation and neurogenesis. BrdU was injected 4 h after intraventricular FGF2 injection to label cells affected by excess FGF2 activity and follow their fate. Tbr1/BrdU colabeling analysis at E13.5 A–H, indicated that more Tbr1⁺ cells in the cortical primordium were BrdU⁺ (I). However, fewer of these cells appeared to show the bright, full labeling indicative of immediate neurogenesis (C–H, arrows). Stereological estimates of BrdU⁺ cell density in the cortical wall appeared to be slightly higher, but this difference was not statistically significant (n = 3 control embryos and n = 3 FGF2-injected embryos; p = 0.239) (J). However, densitometry of the VZ versus non-VZ cortical wall showed that significantly more of the BrdU label was retained in the VZ of FGF2-injected embryos compared with controls (n = 3 controls and n = 3 FGF2 injected; p = 0.021) (K). The number of Tbr2⁺ cells in the lateral SVZ and VZ was increased nearly twofold in FGF2-injected embryos at E13.5 (n = 3 controls and n = 4 FGF2 injected; p = 0.0358) (L–Q, R), correlating with increased appearance of PH3⁺ cells in the lateral SVZ (R, S). The ratio of SVZ/VZ PH3⁺ cells in the GF region; GF was greatly increased in FGF2-injected embryos (n = 3) compared with control embryos (n = 3) (p = 0.024), whereas this ratio in the dorsal cortical primordium (NGF region) was not statistically different (S). PP, Preplate. Scale bar: A, B, L, M, 1 mm; C–H, N–Q, 250 μm.

Figure 5.

Induction of cortical gyri and sulci by E18.5 after FGF2 injection at E11.5. Immunohistochemistry for Tbr1/Ctip2 (A, A′, B, B′) and Cux1/Zfpm2 (C, D), demonstrated foldings of the cortical wall at E18.5. The mouse cortical plate typically has a smooth (lissencephalic) appearance (A, A′, C), but the cortical layered structure demonstrated peaks and troughs (B, B′, D, arrows) consistently in both hemispheres. At the depth of induced sulci, deep layers (marked by Zfpm2, Ctip2, and Tbr1) appeared much thinner than in non-gyrified regions (B, B′, D). In situ hybridization for Cdh6 and Cdh8 demonstrated that the induction of gyri and sulci (indicated by arrows) occurs in the parietal, putative somatosensory neocortex, as well as the putative insular cortex (E–H). neo, Neocortex; ins, insular cortex; lv, Lateral ventricle; pir, piriform cortex; par, parietal cortex; st, striatum. Scale bar: A, B, E–H, 1 mm; C, D, 500 μm.

Figure 6.

FGF2 injection does not alter rostrocaudal patterning of Cadherin 6/8. In situ hybridization for Cdh6 and Cdh8 at E18.5 (A, B, D, E). The length of the cadherin expression domain, relative to the major dimensions of the cortex, was measured in FGF2-injected embryos and compared with that of control embryos. The average length of each segment was computed and is shown in C. No significant difference was observed. Scale bar, 1 mm.

Figure 7.

FGF2 injection induces fully layered cortical gyri and sulci that persist until adulthood. FGF2-injected adults showed a raised forehead (A, arrowhead). Whole-mount dorsal or ventral views of adult brains injected with FGF2 (C, E) compared with control littermates (B, D). Cresyl violet staining demonstrated deep bilateral sulci in the rostrolateral cortex in two different FGF2-injected brains (F–I, arrows), as well as accentuated piriform gyrification (G, I, arrowheads). Cortical lamination, as shown by Cux1 (green) and Smi32 (red), appeared organized around the deep fissure; neocortical layer structure ended at the sulcus depth, giving way to insular cortex (J, K, same brain shown in H, I). The lateral gyri and sulci were sometimes visible from the exterior surface of the brain (L, M, asterisk). High-magnification views of the brain sections depicted in F and G are shown in N and O. Also in this brain, cortical layers 2/3 and 5/6 can be seen wrapping around the deep cortical sulcus (O, arrow), and a short white matter radiation can be seen protruding into the new gyrus from the intermediate zone (G, O, blue asterisk). 3D reconstructions of the surface of the brain shown in F, G, and L–O are rendered in side views in P and rostral views in Q, highlighting the new neocortical sulcus (arrow) and deepened rhinal sulcus (arrowhead) and gyrus (asterisk). cb, Cerebellum; iz, intermediate zone; ins, insular cortex; pir, piriform cortex; RS, Rhinal sulcus; ctx, cerebral cortex. Scale bar: A, 1 cm; B–E, 5 mm; F, G, 1.8 mm; H, I, 2 mm; J, K, N, O, 350 μm; L, M, 3 mm; P, Q, 1.5 mm.

Figure 8.

Impaired hippocampal growth in FGF2-injected mice. Although the rostral cortex was enlarged, the hippocampus was severely reduced in size in adult FGF2-injected brains, as shown by cresyl violet staining (A–D). The brains shown in A–D are the same as in Figure 8, A–E and H–K. The CA1, CA3, and dentate gyrus regions were clearly identifiable in FGF2-injected brains, indicating a high level of organization (C, D). In addition, the growth of the hippocampal primordium, labeled by Emx1, was drastically curtailed just 48 h after FGF2 injection at E11.5 (E, F). dg, Dentate gyrus; neo, neocortex; ic, internal capsule; th, thalamus; lv, lateral ventricle; cge, caudal ganglionic eminence. Scale bar: A, B, 2 mm; C–F, 1 mm.

Figure 9.

oRG and axonal connections do not contribute significantly to initial stages of gyrus formation after FGF2 injection. Staining for Pax6 and βIII tubulin (A–J) showed an orderly VZ in both control and FGF2-injected embryos at E13.5. C–J are high-magnification views of the boxed regions in A and B. Very rare Pax6⁺ cells were observed outside of the VZ proper in both control and FGF2-injected embryos (A–F). However, there was no apparent increase of these cells in the GF lateral cortical primordium versus NGF regions. Axons and neuropil stained for βIII tubulin showed the presence of very few laterally projecting corticofugal axons, with no hyperabundance in the gyrifying region (A, B, G, H, green arrows). I, J, DAPI counterstain. Quantification of adenoviral GFP labeling of >600 cortical progenitors at the time of FGF2 injection at E11.5 K–O revealed no significant difference in the proportions of RG (K; Pax6⁺/GFP⁺), intermediate neuronal precursors (L; Tbr2⁺/GFP⁺), or oRG (M; Tbr2⁻/Pax6⁺/GFP⁺ cells in the outer VZ or SVZ lacking an apical process, arrow) in both gyrifying (N; n = 3 controls and n = 4 FGF2-injected brains; p = 0.90, p = 0.81, and p = 0.53 for RGs, INPs, and oRGs, respectively) and non-gyrifying (O; p = 0.63, p = 0.72, and p = 0.79 for RGs, INPs, and oRGs, respectively) regions. Single-channel GFP, Pax6, and Tbr2 z-stack images for the oRG cell in M (arrow) are shown in M′, M″, and M‴, respectively. MGE, Medial ganglionic eminence; PP, preplate. Scale bar: A, B, 1 mm; C–J, 250 μm; K–M, 125 μm.