Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 31 (43), 15604-17

FGF Signaling Expands Embryonic Cortical Surface Area by Regulating Notch-dependent Neurogenesis

Affiliations

FGF Signaling Expands Embryonic Cortical Surface Area by Regulating Notch-dependent Neurogenesis

Brian G Rash et al. J Neurosci.

Abstract

The processes regulating cortical surface area expansion during development and evolution are unknown. We show that loss of function of all fibroblast growth factor receptors (FgfRs) expressed at the earliest stages of cortical development causes severe deficits in surface area growth by embryonic day 12.5 (E12.5) in the mouse. In FgfR mutants, accelerated production of neurons led to severe loss of radial progenitors and premature termination of neurogenesis. Nevertheless, these mutants showed remarkably little change in cortical layer structure. Birth-dating experiments indicated that a greater proportion of layer fates was generated during early neurogenic stages, revealing that FgfR activity normally slows the temporal progression of cortical layer fates. Electroporation of a dominant-negative FgfR at E11.5 increased cortical neurogenesis in normal mice--an effect that was blocked by simultaneous activation of the Notch pathway. Together with changes in the expression of Notch pathway genes in FgfR mutant embryos, these findings indicate that Notch lies downstream of FgfR signaling in the same pathway regulating cortical neurogenesis and begin to establish a mechanism for regulating cortical surface expansion.

Figures

Figure 1.
Figure 1.
Impaired surface area expansion in Emx1;TKO embryos. A, B, Recombination of Emx1Cre in R26R mice; green shows β-Gal+ cells within the Pax6+ cortical VZ (red) at E10.0. B is a higher-magnification view of the boxed region in A. C–F, Emx1;TKO embryos show a near complete loss of Pea3 mRNA expression in the cortical primordium (C, D), while CoupTF1 is upregulated (E, F). G–I, Developmental analysis from E11.5 to E18.5 showing severely reduced cortical volume and surface area after E11.5 (G). Cortical wall surface area and volume estimates are plotted in H and I. For cortical plate surface area across all ages from E11.5 to E18.5, a factorial ANOVA showed a main effect of genotype (F = 16.94; p = 0.0021) and age (F = 25.49; p < 0.0001) (n = 10 Emx1;TKOs and 11 littermate controls). For cortical wall volume a factorial ANOVA showed a main effect of genotype (F = 109.5; p < 0.0001), age (F = 395.8; p < 0.0001), and interaction of genotype * age (F = 50.2; p < 0.0001). LV, Lateral ventricle; NP, nasal process; CP, cortical primordium; MGE, medial ganglionic eminence; LGE, lateral ganglionic eminence; SE, septum; STR, striatum. Scale bar, 1 mm.
Figure 2.
Figure 2.
Substantial rescue of the Emx1;TKOs phenotype by restoration of one allele of FgfR2 or FgfR3. A–K, Tbr1-immunostained E18.5 coronal sections (A–C, E–G) (insets in E–G show whole brains; dorsal view) and cresyl violet-stained E15.5 sections (I–K) showing severely reduced cortical surface area in Emx1;TKOs compared with control littermates, and extent of rescue by restoration of one copy of FgfR3. The septum and striatum were similar in size in all embryos examined. D, H, Total cortical surface area (D) and rostral vs caudal section perimeter measurements (H) showed that restoration of one copy of FgfR2 or FgfR3 rescued the Emx1;TKO phenotype to a similar extent both rostrally and caudally. L–Q, Radial glia completely lacked BLBP expression in Emx1;TKOs (L, M), with rescue by the presence of a single copy of FgfR3 (N), although the radial glial scaffold appeared intact as assessed by Nestin staining (O–Q). ctx, Cerebral cortex; mb, midbrain; ST, striatum; CP, cortical plate; CC, corpus callosum; PB, Probst bundle; HhH, Emx1+/c; FgfR1f/f; FgfR2+/f; FgfR3−/−; HHh, Emx1+/c; FgfR1f/f; FgfR2f/f; FgfR3+/−. Scale bars: A–K, 1 mm; L–Q, 250 μm.
Figure 3.
Figure 3.
Caspase 3-mediated cell death and proliferation appear unchanged in Emx1;TKOs at E11.5. A–D, Isolated Caspase 3 (Casp-3)+ cells (green) were detectable in control (A, C) and Emx1;TKO embryos (B, D); however, they were extremely rare. C and D are high-power images of the boxed regions in A and B. In these brains we estimated 952 Casp-3+ cells per cortical hemisphere (Emx1;TKO) vs 624 cells in wild type. E–H, Positive cells were often found far from the lateral ventricles, in the SVZ or preplate, but some were also observed in the VZ. PH3 (red) labels mitotic cells primarily at the VZ surface at E11.5 in both control and Emx1;TKO embryos, and the number was only modestly different (G). BrdU incorporation at E10.5 was imaged and quantified after 1 d survival (E, F, H), and was nearly identical in both control and Emx1;TKO embryos (H). EC, ectoderm. Scale bars: A, B, 500 μm; C–F, 62.5 μm.
Figure 4.
Figure 4.
Early excess of cortical neurons and later deficit in Emx1;TKOs. βIII tubulin (A–D) and Tbr1 (G–N) immunostaining showing excess early cortical neurons at E12.5 (G, H) and E13.5 (I, J), and increased preplate/cortical plate thickness (arrowheads in A, B) in mutants compared with control littermates. By E15.5 (K, L) thinning of the cortical plate begins and the final number of Tbr1+ neurons is decreased by 51.7% in Emx1;TKOs at E18.5 (M, N). Stereological estimates of Tbr1+ cell density and total number are plotted in E and F, respectively. A factorial ANOVA showed a main effect of genotype (F = 45.7; p < 0.0001), age (F = 245.1; p < 0.0001), and interaction of genotype * age (F = 29.6; p < 0.0001) (n = 10 Emx1;TKOs and 11 littermate controls) on the total number of Tbr1+ neurons, and a main effect of genotype (F = 7.88; p < 0.017) and age (F = 16.18; p = 0.0001) on Tbr1+ cell density. PP, Preplate; MZ, marginal zone; IZ, intermediate zone. Scale bars: A–D, 1 mm; G–N, 125 μm.
Figure 5.
Figure 5.
Emx1;TKO embryos show precocious production of INPs followed by SVZ depletion. A–H, Tbr2 immunohistochemistry revealing overproduction of INPs at E12.5–E13.5 (A–D) and increased Tbr2+ cell density in cingulate (inset in A, B) and lateral cortex (C, D) in Emx1;TKOs compared with littermate controls. Tbr2+ cells began to be depleted at E15.5 (E, F), showing an 84.2% deficit by E18.5 (G, H). Stereological quantification in A, J. A factorial ANOVA showed a main effect of genotype (F = 25.27; p = 0.0015), age (F = 9.78; p = 0.0067, n = 7), and an age * genotype interaction (F = 9.07; p = 0.0083) on the total number of Tbr2+ cells (n = 7 Emx1;TKOs and 8 littermate controls). Scale bars: A, B, 1 mm; C–H, 125 μm.
Figure 6.
Figure 6.
Increased cortical neurogenesis and decreased progenitor self-renewal in Emx1;TKO embryos. Dotted lines demarcate the cortical wall; analysis excluded the overlying head ectoderm and mesoderm. Tbr1/BrdU immunostaining (A–F) and Ki67/BrdU immunostaining (G–L) at E11.5, following BrdU incorporation at E10.5, with stereological quantification (M, N). The fraction of BrdU+ cells that colabeled for Tbr1 (E, F, yellow cells) was significantly increased in Emx1;TKOs as plotted in (M) for E11.5 through E13.5. A factorial ANOVA revealed effects of genotype (F = 53.898; p < 0.0001), age (F = 45.91; p < 0.0001) and interaction of genotype*age (F = 4.69; p = 0.045, n = 7 Emx1;TKOs and 7 control littermates). Green arrows in A and B indicate Tbr1+ cells in the VZ; PP, preplate. The fraction of BrdU+ cells that colabeled for Ki67, representing cells that reenter the cell cycle (K, L, yellow cells) was significantly decreased in Emx1;TKOs across age as plotted in (N) for E11.5–E13.5. A factorial ANOVA showed a main effect of genotype (F = 17.06; p = 0.006) and age (F = 21.90; p = 0.0017, n = 5 Emx1;TKOs, 6 control littermates). The number of cells that stained for BrdU only, representing cells that are likely postmitotic, was increased in Emx1;TKOs (compare I, J with K, L, arrows). Asterisks, Green and yellow cells in the ectoderm/mesenchyme are mainly red blood cells that cross-react with the Tbr1 antibody. Scale bar, 125 μm.
Figure 7.
Figure 7.
Depletion of VZ progenitors leads to an early termination of cortical neurogenesis. Pax6 immunostaining in control (A, C, E) or Emx1;TKO (B, D, F) embryos showing a progressive decrease in VZ thickness in Emx1;TKOs, confirmed by stereological estimates of the total number of Pax6+ cells in the dorsal telencephalon (neocortex and hippocampus) (M). A factorial ANOVA showed a main effect of genotype for decreased Pax6+ cells: F = 21.24; p = 0.0025 (n = 7 Emx1;TKOs and 8 littermate controls). VZ surface area, estimated using Neurolucida, was also strongly reduced as plotted in N (factorial ANOVA: F = 36.21; p = 0.0005). The fraction of Pax6+ cells in the DAPI total measured in 250-μm-wide sectors of dorsolateral neocortex showed a more rapid decrease in Emx1;TKOs than in controls (factorial ANOVA: genotype, F = 301.86; p < 0.0001) (O). Ngn2 in situ hybridization (G–L) labels the cortical VZ, showing an increase in staining at E13.5 and severe depletion by E18.5. Radial glia electroporated in utero at E11.5 with pBLBP-eGFP alone (P) or together with dominant-negative FgfR (SW2) (Q) and cultured for 3 d in vitro showed that attenuated FgfR signaling increases the number of eGFP+/βIII tubulin+ neurons (R) as well as the proportion of clones exclusively composed of eGFP+/βIII tubulin+ cells (S) (t test; p = 0.003), reduces clone size (T) and decreases the proportion of clones composed only of eGFP+/Nestin+ cells (S) (t test; p = 0.005). A two-way ANOVA revealed a statistically significant interaction of neurogenesis rate and clone size; F = 5.12, p = 0.0378 (n = 4 control eGFP; n = 6 SW2). Arrowheads indicate thickness of the VZ in C and D. Scale bars: A–F, P, Q, 125 μm; G–L, 500 μm.
Figure 8.
Figure 8.
Cortical layer development in Emx1;TKO embryos. Control (A, D, G, J) or Emx1;TKO (B, E, H, K) embryos processed for the indicated layer markers show similar layer structure. Overall layer order appeared normal, as was the ratio of Cux1 and ZFPM2+ cells (A–C) (n = 3 Emx1;TKOs and 3 littermate controls; t test: p = 0.92). There was a modest imbalance toward an increase in Ctip2 in Emx1;TKO embryos, reflecting greater early (deep layer) cortical neurogenesis (D–F) (n = 3 controls, 3 Emx1;TKOs; two-way ANOVA assessing interaction of genotype * layer population; p = 0.05). However, both Cux1+ (G–I) and ZFPM2+ (J–L) cells were more likely to have been born on E12.5 (CldU+) than E16.5 (IdU+) in Emx1;TKO embryos, whereas fewer Cux1+ cells were born at E16.5 in Emx1;TKO embryos compared with littermate controls (G–I) (two-way ANOVA assessing interaction of genotype * Cux1+ birthdate: F = 33.49; p = 0.0004; n = 3 Emx1;TKOs, n = 3 control littermates). As expected, of over 7500 cells counted, no ZFPM2+ cell was IdU+ in either controls or Emx1;TKOs (J–L). SP, Subplate; 2/3, cortical layers 2/3; 6, cortical layer 6. Scale bar, 125 μm.
Figure 9.
Figure 9.
FgfR signaling regulates Notch and proneural gene expression in cortical neurogenesis. NICD staining of intracellular puncta was reduced in Emx1;TKO embryos (A, B), suggesting that Notch production and/or cleavage is impaired. Insets (A, B) are high-magnification images of the boxed regions. In situ hybridization for Notch1 demonstrated decreased expression in the cortical VZ of Emx1;TKO embryos at E13.5 (C–F). E, F, High-power images of the boxed regions in C, D. Analysis of Notch downstream targets showed decreased Hes1 (G, H), increased Ngn2 (I, J), and increased Dll1 (K, L) mRNA production as early as E11.5 in the cortical VZ (arrowheads) during the period of increased early neurogenesis in Emx1;TKOs. (M, N). Scale bars: A, B, E, F, 125 μm; C, D, G, H, 2 mm; I–L, 1 mm.
Figure 10.
Figure 10.
Notch is downstream of FgfR signaling in cortical neurogenesis. A–E, In utero electroporation at E11.5 of pBLBP-eGFP, alone (A, D) or together with a dominant-negative FgfR (SW2) (Werner et al., 1993; Shin et al., 2004) (B, E), was coupled with immunocytochemical detection and densitometric measurements of NICD (A, B) or Hes1 (D, E) proteins to determine cell autonomous effects of attenuating FgfR signaling. a′, b′, d′, and e′ are higher-magnification views of A, B, D, and E, respectively. Both NICD and Hes1 protein levels were significantly reduced in eGFP+ cells compared with nearby cells (C, F). G–L, Tbr1 immunostaining in eGFP+ cells electroporated with pBLBP-eGFP alone (G) or together with SW2 (H), constitutively active FgfR (FgfR3E) (I), constitutively active Notch (pUB-NICD) (J), or SW2 + NICD (K), demonstrating that SW2 increased cortical neurogenesis and NICD virtually abolished it. However, SW2 was unable to reverse the effects of NICD, suggesting that FgfR signaling is upstream of NICD in cortical neurogenesis. Overexpression of NICD was confirmed in NICD-electroporated eGFP+ cells by double immunostaining (inset in J). The number of pBLBP-eGFP+ cells (radial glia and their progeny) coexpressing the neuronal marker, Tbr1 is quantified in (L). *p < 0.05, **p < 0.0005 compared with pBLBP-eGFP alone, Student's t test. Scale bars: A, B, D, E, 125 μm; a′, b′, d′, e′, 40 μm; G–K, 250 μm; J inset, 400 μm.

Similar articles

See all similar articles

Cited by 37 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback