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First published online 24 July 2008
doi: 10.1242/dev.021618

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Fgf8 controls regional identity in the developing thalamus

RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-Shi, Saitama 351-0198, Japan.

View larger version (65K): [in this window] [in a new window]   Fig. 1. Pattern of expression of Fgf8 in developing diencephalon. The spatial distribution of Fgf8 and its activity are shown by whole-mount in situ hybridization at E9.5 in mice. Strong expression of Fgf8 is observed at the midbrain/hindbrain boundary (MHB, red arrow) and in the anterior telencephalon (black arrow) (A). Strong expression of Shh is observed in the basal region of the diencephalon (black arrow) (B). (C-F) Whole-mount in situ hybridization of the dissected brain at E10.5 is shown. Lateral is towards the left. In addition to Fgf8 expression in MHB and anterior telencephalon (red arrow and black arrow, respectively), expression in the dorsal part of diencephalon (green arrow) and dorsal to ZLI (orange arrow) is detected. One side of the hemisphere has been removed for a better view of Fgf8 expression in the diencephalon (C). Shh expression in ZLI is shown (blue arrow) (D). Two-color in situ hybridization for Fgf8 (blue) and Shh (brown) shows the spatial relationships of Fgf8 and Shh expression (arrow) (E). FGF activity, shown by Sprouty2 (Spry2, blue), is detected in both p2 and p3 (arrows), which are divided by Shh (brown) (F). (G-I) Section in situ hybridization shows the detailed patterns of expression of Fgf8 and Shh. Estimated section positions are shown in E and F (broken line). Fgf8 expression is restricted to p3 (arrow) (G) and a high-magnification view is shown (I). Fgf8 activity shown by Spry2 reached a greater distance on both sides of ZLI marked by Shh (H) at E10.5. di, diencephalon; H, hindbrain; M, midbrain; tel, telencephalon. Scale bar in F: 500 µm for A-F; 200 µm for G,H; 100 µm for I.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Fgf8 electroporation expands the specific population posterior to ZLI in the early embryonic stage. (A) The selected angles of section and plane are illustrated, in sagittal view, with anterior towards the left. Brains were embedded and sectioned perpendicular to ZLI as indicated by Shh expression (pink). The section at the halfway point of the diencephalic tube along the D/V axis, which is also at the end of Spry2 expression (purple) is selected (arrow). The section contains p1 (yellow), p2 (orange) and p3 (green). (B) Scheme of the semi-coronal section used in this study at the position indicated in A. The dorsal (pallium) and ventral (subpallium) telencephalon are divided by the cortical hem (purple) and pallium/subpallium boundary (antihem, red). The diencephalon contains p1, p2 and p3. Fgf8 and EGFP constructs are electroporated close to ZLI at E10.5 (C,E,G,I), and sFgfr3 and EGFP constructs are electroporated at E9.5 (D,F,H,J). Brains were collected at E12.5, and only those exhibiting strong EGFP fluorescence were processed for in situ hybridization. The electroporated side is to the left. No shift is observed with p3 markers Dlx1 (C, blue) and Dlx2 (D, blue) and the ZLI marker Shh (brown) in Fgf8-overexpressing (C) or -inhibited brain (D). Two-color fluorescent in situ hybridization with Ngn2 (green) and Mash1 (red) reveals an alternating pattern of expression in the diencephalic VZ. The Mash1+ Rim VZ is expanded (red bracket), whereas Ngn2+ VZ is shrunken (green bracket) in Fgf8-overexpressing brain (E). The site of electroporation is shown by GFP expression (blue) by in situ hybridization (inset). In Fgf8-inhibited brain, Ngn2+ VZ (green bracket) is expanded and Mash1+ Rim VZ (red arrow) is shrunken (F). Nkx2.2 (brown), expressed in the region flanking ZLI (arrow), is expanded by Fgf8 electroporation, without expansion of Shh (blue) (G). A specific marker of the Rim, Sox14 (blue), is also expanded by Fgf8 electroporation (arrow) (I). Expression of Nkx2.2 and Sox14 is shrunken in Fgf8-inhibited brain (H,J, arrow). Scale bar in J: 500 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 3. Gene expression posterior to ZLI enables further subdivision. Six3 (A), Arx (B) and Gad67 (C) expression (blue) and Shh expression (brown) are compared at E12.5. Besides strong expression of Six3, Arx and Gad67 in p3, there is distinctive expression of Six3+ close to Shh (A, arrow), Arx+ close to pial (B, arrow), and Gad67+ close to Shh (C, arrow). Expression of Gad67 is observed only in the medial part of the Rim, which is marked by Nkx2.2 shown by fluorescent in situ hybridization (D, arrow). Six3 expression close to Shh overlaps with that of Nkx2.2 completely (E, inset). No part of the Arx expression domain overlaps with that of Nkx2.2 (F, inset). Two-color in situ hybridization of Tal2 (blue) and Nkx2.2 (brown) reveals that Tal2 is expressed in the Rim VZ (G, inset). Tal2 expression in the Rim VZ overlaps with that of Mash1 but not that of Shh, as shown by triple fluorescent in situ hybridization. A closer view of ZLI is shown (H). Pitx2 (blue) is expressed lateral to Shh (brown) (I, arrow). Triple fluorescent in situ hybridization demonstrates no overlap of Six3 (green), Pitx2 (red) and Shh (blue) (J). Scale bar in A: 500 µm for A-G,I; 250 µm for H,I,E-G, inset.

View larger version (67K): [in this window] [in a new window]   Fig. 4. Fgf8 controls the size of the Rim VZ and medial Rim. Diagram shows genes expressed around the Rim and the Stream. Electroporated brains were collected at E12.5 and examined by two-color in situ hybridization. Only the experimental side of the diencephalon is shown at high magnification around ZLI (VZ is on the right side). (A-C) The Rim VZ, labeled by Tal2 (blue), is enlarged in Fgf8-overexpressing brain (B) and shrunken in Fgf8-inhibited brain (C) (black arrow), whereas Shh expression (brown) is unchanged. (D-F) The medial Rim, shown by Six3 (blue), is enlarged in Fgf8-overexpressing brain (E) and shrunken in Fgf8-inhibited brain (F) (green arrow), while expression of Arx in lateral diencephalon (orange arrow) is unchanged. (G-I) The medial Rim, shown by Gad67 (blue), is specifically expanded by Fgf8 electroporation and shrunken in Fgf8-inhibited brain (I) (pink arrow) while expression of Pitx2-labeled cells is unchanged (blue arrow). Scale bar in C: 125 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 5. Fgf8 electroporation expands the posterior vLGN and EML. Electroporated brains were collected at E15.5, sectioned coronally and examined by two-color in situ hybridization. Anterior is towards the bottom and posterior is towards the top according to the longitudinal axis (A,E). A closer view of vLGN and the EML region is shown (B-D,F-H). Nkx2.2 (blue) marks the posterior vLGN and EML, while Arx (brown) marks the anterior vLGN and EML (blue and yellow arrows, respectively, B). Gad67 (blue) labels all of PTh but not the small medial population in vLGN, which is stained by Pitx2 (brown) (green arrow, F). The posterior vLGN is expanded posteriorly and position of posterior EML is abnormal in Fgf8-overexpressing brain (arrows in C,G) but without alteration of anterior structures labeled by Arx (brown staining, B,C) or the small medial population of vLGN labeled by Pitx2 (brown staining, F,G). Inhibition of Fgf8 decreased the size of the posterior vLGN (D,H, arrows), but no change was observed in the anterior vLGN (D) or Pitx2+ population (H). Scale bar in E: 500 µm for A,E; 200 µm for B-D,F-H.

View larger version (94K): [in this window] [in a new window]   Fig. 6. Thalamic nuclei patterning is controlled by Fgf8 activity. Brains were collected at P6 and examined with X-gal histochemistry (A), cytochrome oxidase (CO) histochemistry (B,E,G-I) and Nissl staining (C,F). Fgf8 and Cre constructs were co-electroporated at E10.5 into R26R mice. Brains, which have restricted staining of X-gal at the thalamus/prethalamus (Th/PTh) border (A), exhibit a shift of the barreloid closer to the pretectum (PTc) (B,C, bracket). Position of the mammillothalamic tract (MMT) is indicated as a landmark. sFgfr3 and YFP constructs were co-electroporated at E9.5. YFP fluorescence is detectable (D), and brains exhibit slight anterior shift of the barreloid (E,F, bracket). (G-I) Higher-magnification views of VP show structural change in each brain. The ventral posterior medial (VPM) and ventral posterior lateral (VPL) subfields and their position are compared with MMT in control brain (G, double-headed arrow). Fgf8 overexpression shifts the VPM and VPL posteriorly and compresses them (H, double-headed arrow). In Fgf8-inhibited brain, VPM is closer to MML and VPL is stretched (l, double-headed arrow and arrowheads). Scale bar in C: 500 µm in A-I; 200 µm in J-L.

View larger version (62K): [in this window] [in a new window]   Fig. 7. Area marker genes are shifted posteriorly by Fgf8 overexpression. (A) Steel is specifically expressed in the Th and cerebral peduncle in PTh (arrow). The border of dLGN and vLGN is distinguished by Steel expression (red arrow). The dorsal lateral geniculate nucleus (dLGN), mammilothalamic tract (MMT) and hippocampus (Hi). (B) The entire PTh is labeled by Gad67, and structures such as vLGN are easily distinguished (red arrow). (C) VP is specifically labeled by Cdh6. (D) Fgf8 electroporation shifts Steel expression posteriorly (arrowheads). The gap between the cerebral peduncle (arrow) and Steel-expressing border is expanded. (E) The region of Gad67 expression in PTh is enlarged in Fgf8-electroporated brain (arrow). However, it remains unclear which specific prethalamic nuclei are expanded. (F) Specific Cdh6 expression is shifted posteriorly and compressed in Fgf8 electroporated brain. Scale bar in D: 0.75 mm.

View larger version (49K): [in this window] [in a new window]   Fig. 8. Schematic representation of a model of A/P patterning of the diencephalon by Fgf8 activity.

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