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First published online October 27, 2004
doi: 10.1242/10.1242/dev.01428

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Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex

Department of Neurobiology, Pharmacology and Physiology, Committees on Neurobiology and Developmental Biology, University of Chicago, 947 E 58th Street, MC0926, Chicago, IL 60637, USA

View larger version (83K): [in a new window]   Fig. 1. Excess anterior FGF8 downregulates WNT gene expression in the hem. (A) E11.5 cerebral hemisphere viewed from the medial face, anterior towards the left, processed for two color in situ hybridization. The cortical hem is marked by Wnt3a expression (blue); Fgf8 expression (brown) is separated from the hem by a short gap (arrow). (B-L) Brains electroporated anteriorly at E11.5 with Ap or Fgf8 and analyzed at E13.5. B-D and K,L are coronal sections; E-J are hemispheres viewed from the medial face. (B,C) In adjacent sections from the same brain, ectopic expression of FGF8 (B) obliterates Wnt3a expression in the hem (C, black arrow indicates normal Wnt3a expression site). (Note endogenous Fgf8 expression ventrally in B.) Wnt2b expression fills the cortical hem of an Ap electroporated brain (E) but is lost anteriorly in a brain electroporated with Fgf8 (arrow, F). Wnt5a shows a similar loss of expression (I, arrow in J). In contrast to WNT gene expression, Bmp4 expression is not downregulated at this age by excess FGF8 (D, white arrow indicates site of excess FGF8), nor is expression of Msx1, a indicator of BMP activity (G,H). Overexpression of FGF8 (L) has no apparent effect on Foxg1 expression at this age, indicating that the experimental protocol does not cause general damage. The boundary between the hem and the rest of the cortical neuroepithelium not obviously affected (K). Scale bar: 0.8 mm for A,E-J; 0.17 mm for B-D; 0.12 mm for K,L.

View larger version (124K): [in a new window]   Fig. 2. Anterior overexpression of FGF8 causes prolonged downregulation of WNT expression in the hem, and a defective hippocampus. (A-L) Coronal sections of E15.5 brains processed for in situ hybridization. Medial is towards the left. Brains are electroporated with Ap (A-C,G-I) or Fgf8 (D-F,J-L) at E11.5. Each row illustrates three near-adjacent sections from the same brain. (A-C) A brain electroporated with Ap (transgene in B) shows normal expression of Wnt3a at the hem remnant (arrow, A), and a dense patch of Ka1 in the developing hippocampal CA3 field (arrow, C). A brain electroporated with Fgf8 (E) shows a little Wnt3a expression (D), and diffuse Ka1 expression in the hippocampus (asterisk, F). (G-L) Similarly a brain electroporated with Ap displays robust Wnt5a expression in the distal region of the hippocampus (G, asterisk), compared with very sparse and diffuse expression in a brain electroporated with Fgf8 (J, asterisk). Prox1 marks the developing DG in I, but not in L. Scale bar: 0.25 mm.

View larger version (95K): [in a new window]   Fig. 3. Similar changes in Fgf8 and Wnt3a expression by BMP inhibition or loss of EMX2. (A-I) E10.5 forebrains viewed dorsally, anterior towards the top, processed for whole-mount in situ hybridization. EGFP alone (A,D,G) or EGFP and Nog together (B,E,H) were electroporated into the telencephalic vesicle at E9.5. EGFP fluorescence indicates the position of the electroporated site (insets). Brains electroporated with EGFP alone show wild-type expression patterns of Fgf8, Wnt3a and Msx2 (a reporter of BMP activity). Co-electroporation of Nog results in inhibition of BMP activity, reflected in decreased Msx2 expression (arrow in H), increased and ectopic Fgf8 expression in the cortical primordium (B), and lowered expression of Wnt3a in the cortical hem (arrow in E). The forebrains of Emx2 homozygote mutant mice are not identical to Nog-electroporated brains, but, like the latter, show expanded Fgf8 expression (C, arrow) and reduced expression of Wnt3a in the hem (F, arrow). A slight decrease in Msx2 expression is most evident in the cortical hem (I, arrow). The latter region is buried in a dense mass of Msx2 expression in control forebrains (G). Scale bar: 0.4 mm.

View larger version (43K): [in a new window]   Fig. 4. Noggin induces ectopic Fgf8 expression throughout the depth of the cortical neuroepithelium. (A) E10.5 forebrain viewed from the dorsal side. Noggin misexpression has induced a patch of ectopic Fgf8 (arrowhead). In addition, the normal domain of Fgf8 expression (asterisk) is expanded along the two sides of the dorsal midline. (B) Coronal section through a different cortical hemisphere, processed in the same way. Fgf8 expression is evident at the midline (arrowhead) posterior to its normal limit. A patch of ectopic expression extends further laterally, filling the width of the neuroepithelium. (C) A higher magnification of Fgf8 expression in B. Typical of dividing neuroepithelial cells, labeled cell bodies lie near both the pial and ventricular surfaces (arrows in C) and show polarized processes. Scale bar: 0.35 mm for A; 0.1 mm for B; 0.03 mm for C.

View larger version (92K): [in a new window]   Fig. 5. Decreased BMP activity in the Emx2 mutant forebrain. (A-K) E8.5 forebrains viewed from the lateral side just after closure of the neural tube, anterior is towards the left. At this stage of telencephalic development in wild-type mice, Nog expression appears in a restricted domain at the anterior dorsal midline (A,I, arrows). In three Emx2 homozygous mutant mice (Emx2–/–), this domain of Nog expression extends about three times as far posteriorly along the midline (B,J,K). In addition, Nog expression appears stronger than in wild type (arrows in L,M). Mice represented in A and B, I and J, and L and M are paired littermates. No detectable differences were seen in Bmp4 or7 expression (C-F), but BMP activity appears deficient in the Emx2 mutant, assessed by the loss of Msx2 expression (G,H, asterisks). Scale bar: 0.3 mm for A-H; 0.17 mm for I-J; 0.06 mm for L,M.

View larger version (66K): [in a new window]   Fig. 6. Substantial rescue of WNT gene expression in the Emx2 mutant cortical hem. (A-F) E13.5 hemispheres viewed from the medial side. Wild-type expression of Wnt3a and Wnt2b (A,D). (B,C,E,F) Hemispheres electroporated anteriorly with sFGFR3c at E.9.5. Each brain was co-electroporated with EGFP. WNT gene expression remains depleted in Emx2 mutant brains with sparse or undetected EGFP (inefficiently transfected) (B,E), but expression is substantially restored in two brains that were efficiently transfected with sFGFR3c, assessed by high-density EGFP fluorescence (C,F). Scale bar: 0.4 mm.

View larger version (110K): [in a new window]   Fig. 7. Partial rescue of the dentate gyrus in Emx2 mutant hippocampus. (A-I) Coronal sections through the hippocampus of E18.5 wild-type mice (A,D,G), Emx2 homozygote mice (B,E,H) and Emx2 homozygotes electroporated at E9.5 with sFGFR3c (C,F,I). (D-F) High-power micrographs of a different set of brains from those in (A-C). (A,D,G) Prox1 expression reveals the developing V-shaped DG in the hippocampus (A,D, arrow in A); EphB1 expression marks the distal end of the hippocampus, including the DG (G, arrow). (B,E,H) Emx2 homozygote mutants show no (B, arrow) or only tiny patches of (E) Prox1-expressing cells in the region of the presumptive DG, and greatly reduced EphB1 expression (arrow in H). (C,F,I) When excess FGF8 is reduced in Emx2 mutant brains, the DG expands, forming a dense band not unlike the ventral DG blade (C, arrow, F). Distal EphB1 expression is also partially restored (arrow, I). Scale bar: 0.75 mm for A-C,G-I; 0.125 mm for D-F.

View larger version (50K): [in a new window]   Fig. 8. Preliminary model of signaling interactions that pattern the mouse cerebral cortex. Transcription factors EMX2, LHX2 and FOXG1 expressed in the early cortical primordium regulate Bmp or Nog expression and thereby influence BMP4/7 activity. BMP signaling is active early in choroid plexus development, perhaps specifying the choroid plexus epithelial cell fate. BMP4 signaling further regulates expression of Fgf8/17. FGF8/17 signaling is crucial for neocortical area patterning by regulating the AP axis of the cortical primordium, and at least indirectly, for hippocampal development. FGF8/17 inhibits WNT gene expression; canonical WNT signaling at the cortical hem is required for hippocampal development (Boncinelli et al., 1993; Bulchand et al., 2001; Dou et al., 1999; Fukuchi-Shimogori and Grove, 2001; Fukuchi-Shimogori and Grove, 2003; Galceran et al., 2000; Garel et al., 2003; Hebert et al., 2002; Lee et al., 2000; Mallamaci et al., 1998; Monuki et al., 2001; Panchision et al., 2001; Shimamura et al., 1995; Shimamura and Rubenstein, 1997; Storm et al., 2003).