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A sonic hedgehog-dependent signaling relay regulates growth of diencephalic and mesencephalic primordia in the early mouse embryo

Makoto Ishibashi^* and Andrew P. McMahon

Department of Molecular and Cellular Biology, The Biolabs, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
^* Present address: Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan

View larger version (57K): [in a new window]   Fig. 1. A failure in growth of diencephalic and midbrain primordia in Shh mutants. Morphology of wild-type (A,C) and Shh mutant (B,D) brains at 8.5 dpc (13 somite; A,B) and 9.5 dpc (26 somite; C,D). At 8.5 dpc, the presumptive diencephalon is recognized as a narrow region between two constrictions (Region I). Region II corresponds to the midbrain and Region III to the anterior hindbrain between the isthmus and otic vesicle (ot). A sharp ventral constriction was observed in Shh mutants (arrows in B,D). At 9.5 dpc, regions I and II of Shh mutant were smaller than those of wild type, but Region III of the mutants was still comparable with that of wild type (see text, Table1). (E-J) Expression patterns of Emx2, En1 and Pax6. A gap between the Emx2 and En1 expression domains represents the anterior midbrain and P1/2 (brackets in E-H). No difference was observed in this region at the 14-somite stage (E,F) but by the 21-somite stage, the gap was greatly reduced in Shh mutants (G H). The anterior midbrain, a gap between the Pax6 and En1 expression domains, was also reduced in Shh mutants (I,J). Arrowheads in I,J indicate the forebrain-midbrain boundary. Scale bars: 200 µm.

View larger version (91K): [in a new window]   Fig. 2. Expression of region-specific markers in the forebrain and midbrain at 9.5 dpc. Bf1 (A,B), Emx2 (C,D), Pax6 (E,F), Wnt3a (G,H), Wnt7b (I,J) and Dbx1 (K,L) were examined in wild-type (A,C,E,G,I,K) and Shh mutants (B,D,F,H,J,L). Expression of Bf1 was weaker in Shh mutants than in wild type. Emx2 expression was essentially the same between wild-type and Shh mutants, although the telencephalon of Shh mutants was smaller than that of wild-type embryos at this stage. Pax6 expression in the forebrain terminates at the forebrain-midbrain boundary (arrowhead) in both wild type and Shh mutants. Wnt3a expression undergoes a wedge-shaped lateral expansion in P2 of wild-type embryos at this stage (G, arrow). This expansion was detected in the mutant, although expression was reduced (H, arrow). Wnt7b was expressed in the anterior diencephalon (I, arrow) as well as the telencephalon of wild type. Wnt7b expression in the anterior diencephalon was also detectable in Shh mutants (J, arrow). Dbx1 was expressed in the basal plate of P3 (K, arrow) and alar plates of P1/2 and midbrain (K). Dbx1 expression was almost at background levels in Shh mutants (L). All arrowheads indicate the boundary between the diencephalon and midbrain. hs, hemispheric sulcus (the boundary between the telencephalon and diencephalon). Scale bars: 200 µm.

View larger version (69K): [in a new window]   Fig. 3. Cell proliferation defects in Shh mutants. (A-G) BrdU incorporation analysis at the 15- to 16-somite stages. (A,B) The plane of sections examined. BrdU was detected by immunostaining with Alexa 568-conjugated secondary antibody [yellow because of overlap with YoPro1 (green) labeled nuclei]. In wild type, the telencephalon (C), diencephalon (D) and midbrain (E) showed similar incorporation rates (G, black square). In Shh mutants (F), only the telencephalon showed a comparable rate with that of wild-type embryos, while the diencephalon and midbrain showed significantly decreased rates of BrdU incorporation (G, circles). (H,I) Ccnd1 (cyclin D1) expression at the 17-somite stage. In wild type (H), Ccnd1 expression was detected in all three brain regions. The anterior midbrain showed a higher level of expression than others. In Shh mutants (I), its expression was absent from the diencephalon and weaker in the anterior midbrain. Note that the strong expression in the tail was maintained in Shh mutants (I, asterisk). Arrowhead in H and I indicates the diencephalon-midbrain boundary. t, telencephalon; d, diencephalon; m, midbrain.

View larger version (61K): [in a new window]   Fig. 4. Expression analysis of Wnt signaling components. Tcf4 expression was examined at the 12- (A), 14- (B,C) and 16-somite (D,E) stages. At the 12-somite stage, Tcf4 expression was not detectable in the diencephalon (A). Expression here was first observed at the 14-somite stage in wild type (B), but not in Shh mutants (C). r5 expression of Tcf4 was observed in both wild-type and Shh mutants at this stage (B,C, arrow). At the 16-somite stage, Tcf4 expression became stronger in the wild-type diencephalon (D), but was still not detectable in the diencephalon of Shh mutants (E). Wnt1 (F,G) and Wnt3a (H,I) were also examined at the 14- and 16-somite stages, respectively. Their expression in the dorsal midline of the diencephalon (*) was maintained in Shh mutants (G,I). Arrowhead indicates the diencephalon-midbrain boundary. Scale bars: 100 µm.

View larger version (77K): [in a new window]   Fig. 5. Increased cell death in Shh mutant brains. TUNEL assay was performed on wild-type (A) and Shh mutant (B) sections at the 15-somite stage. The plane of sections is shown in Fig. 4A,B. Few cells were positive for TUNEL (orange dot) in wild-type embryos, while Shh mutants exhibited increased cell death especially in the diencephalon and anterior midbrain. Bmp4 expression was examined at the 15-somite (C,D) and 19-somite (E,F) stages. At the 15-somite stage, there was no Bmp4 expression in the dorsal midline of the diencephalon and midbrain. At the 19-somite stage, robust expression of Bmp4 was observed in this region in Shh mutants (F). Arrowhead indicates the diencephalon-midbrain boundary. t, telencephalon; d, diencephalon; m, midbrain.

View larger version (116K): [in a new window]   Fig. 6. Shh signaling is confined to ventral brain regions. Shh (A,C) and Ptch (B,D) expression were examined at the 14- (A, B), 24- (C) and 21-somite (D) stages. Ptch was not detectable in any dorsal parts of the brain. Note that lateral expansion of Shh expression in the ZLI initiates at the 24-somite stage (C, arrow). Arrowhead indicates the diencephalon-midbrain boundary.

View larger version (101K): [in a new window]   Fig. 7. Fgf15 and Fgf receptor expression in the diencephalon and midbrain of wild-type (A,B,D,F,H,J,L,N) and Shh (C,E,G,I,K,M,O) mutant embryos. (A) Fgf15 and Shh expression. (B-G) Fgf15, (H-K) Fgfr2 and (L-O) Fgfr3 expression. (A) At the 12-somite stage, Fgf15 (purple) was strongly expressed in the ventral regions of the diencephalon (white arrow), dorsal to the Shh expression domain (white arrowhead, brown). Black arrows indicate the ventral Shh expression domain in the midbrain. (B) A lateral view reveals the extent of ventral expression of Fgf15 in the brain. Note that Fgf15 expression was undetectable in dorsal regions of the diencephalon and midbrain at this stage. (D,F), Fgf15 shows a ventral to dorsal expansion from the 12- to 16-somite stage in diencephalic and midbrain primordia of wild-type embryos. (C,E,G), Shh mutants showed no expression of Fgf15 in the caudal diencephalon and anterior midbrain at all stages examined, whereas expression at the midbrain/hindbrain isthmus (arrow) remains in Shh mutants. Expression of Fgfr2 in the diencephalon and anterior midbrain in wild type (H,J) extends ventrally in Shh mutants (I,K). Downregulation in the expression of Fgfr3 (compare L,N with M,O) correlates with the growth defect in these regions of Shh mutants. Arrowhead indicates the diencephalon-midbrain boundary. Scale bars: 100 µm.

View larger version (99K): [in a new window]   Fig. 8. Fgf15 induces an expansion of Tcf4 expression in brain explants. (A) GFP activity in the right side of the brain explant shown in B after electroporation with pCIG-F15. The broken line in both panels indicates the ventral midline. Explants were cultured for 40 hours after electroporation and examined for Tcf4 expression. In the case of the control vector expressing only GFP (C), there was no significant difference in Tcf4 expression on the electroporated (right, indicated by arrow) and non-electroporated sides (left). By contrast, explants electroporated with the Fgf15-expressing vector (pCIG-F15) showed an expansion of the Tcf4 expression domain on the electroporated side (D, arrow). Rostral is towards the top in all panels.