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First published online 15 September 2004
doi: 10.1242/dev.01349

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Temporal requirement for hedgehog signaling in ventral telencephalic patterning

¹ Developmental Genetics Program and the Department of Cell Biology, The Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA
² Harvard University, Department of Molecular and Cellular Biology, Cambridge, MA 02138, USA

View larger version (58K): [in a new window]   Fig. 1. Foxg1Cre removes the Hedgehog responsiveness of telencephalic progenitors in conditional Smo mice. (A-C) ß-gal expression of Foxg1Cre;ROSA26R mice shows the extent of Cre-mediated recombination at E7.5 (A), 10-16 somite stage (B), and E9.5 (C). Note that A is a flattened dissection of the anterior neural plate as diagrammed in the inset, with the anterior neural ridge at the top and the broken line representing the approximate telencephalic primordium. The arrow in B is marking the lacZ staining in the anterior neural ridge. (C) Foxg1Cre-mediated recombination extends throughout the anterior-posterior extent of the E9.5 telencephalon. (D,E) Coronal section of an E10.5 Smoc/– (D) and Smoc/–;Foxg1Cre mutant (E) telencephalon showing the absence of Gli1 transcripts in the latter, a sensitive readout of active Hedgehog signaling. (F) Comparison of E18.5 wild-type and Smoc/–;Foxg1Cre mutant embryos. (G) Wholemount dorsal view of wild-type and Smoc/–;Foxg1Cre mutant brains shows severe size reduction of the telencephalic vesicles and olfactory bulbs. The rudimentary olfactory bulb of the mutants is marked with an asterisk. Scale bars: 100 µm in A; 200 µm in D,E.

View larger version (110K): [in a new window]   Fig. 2. Altered levels of cell death in Smoc/–;Foxg1Cre mutants. (A-D) E10.5 coronal sections of the telencephalon stained with antibodies against BrdU (A,B) or caspase and TuJ1 (C,D). Smoc/–;Foxg1Cre mutants have a general increase in cell death at E10.5 and an absence of the earliest differentiating neurons (D) as compared with wild-type littermates (C). (E,F) E12.5 coronal sections of the telencephalon stained with antibodies against caspase and TuJ1. Smoc/–;Foxg1Cre mutants show continued widespread cell death with the exception of the dorsal cortical midline (F). Note that the pattern of TuJ1 expression in mutant embryos (F) resembles that seen in wild-type LGE (E). Scale bars: 200 µm in A-D; 300 µm in E,F.

View larger version (49K): [in a new window]   Fig. 3. Early ventral patterning is never established in Smoc/–;Foxg1Cre mutants. (A-H) E10 coronal sections of the telencephalon stained by in situ hybridization with probes against Foxg1 (A,B), Pax6 (C,D), Nkx2.1 (E,F) and Gsh2 (G,H). The expression of Pax6 (C), which is normally restricted to the dorsal telencephalon (arrowhead at dorsal-ventral boundary), is found throughout the Smoc/–;Foxg1Cre mutant telencephalon (D). The wild-type ventral expression of Nkx2.1 (E) is absent from the mutant, with the exception of a small region of Nkx2.1 staining (F, arrow) that is of diencephalic origin (compare with arrow in B). Gsh2, which marks the intermediate telencephalic ventricular zone at this stage (G), is never observed in Smoc/–;Foxg1Cre mutants (H). Scale bar in A: 200 µm for A-H.

View larger version (125K): [in a new window]   Fig. 4. Smoc/–;Foxg1Cre mutants show complete dorsalization. (A-L) E12.5 coronal sections of the wild-type (A,C,E,G,I,K) and Smoc/–;Foxg1Cre mutant (B,D,F,H,J,L) telencephalon stained by in situ hybridization. Foxg1 staining of the mutant telencephalon shows the morphological absence of all three ventral eminences (B). Both Ngn2 and Emx2, which are normally restricted to the developing cortical fields (C,E), are expanded throughout the entire ventral extent of the mutant telencephalon (D,F). Nkx2.1, Gsh2 and Mash1 are all expressed within overlapping domains of the ventral telencephalon (G,I,K). These three markers are absent from the Smoc/–;Foxg1Cre mutant telencephalon (H,J,L). Note that the ventral staining for Nkx2.1 and Mash1 is not telencephalic (asterisks, compare with asterisk in B). Scale bars: 300 µm in A-L.

View larger version (83K): [in a new window]   Fig. 5. Most cortical interneuron precursors are dependent on early Hedgehog signaling. (A-F) Coronal sections of an E12.5 wild-type (A,C,E) and Smoc/–;Foxg1Cre mutant (B,D,F) telencephalon, stained by in situ hybridization. The Dlx2, Lhx6 and GAD67 genes have been implicated in various aspects of interneuron development, and are expressed strongly in the basal E12.5 telencephalon (A,C,E). These genes are absent from the Smoc/–;Foxg1Cre mutant telencephalon (B,D,F). (G-J) Coronal sections of an E18.5 wild-type (G,I) and Smoc/–;Foxg1Cre mutant telencephalon (H,J), stained with antibodies against Calbindin and GABA. Both Calbindin and GABA are expressed by cortical interneurons throughout the layers of the wild-type cortical plate (G,I, see insets). In Smoc/–;Foxg1Cre mutants, Calbindin and GABA staining is absent from the cortical plate and is restricted to a ventral-lateral region of the cortex (H,J, see insets). Scale bars: 300 µm in A-F; 500 µm in G-J for low-power field, and 125 µm for high-power inset.

View larger version (50K): [in a new window]   Fig. 6. Oligodendrocyte development is severely perturbed in the Smoc/–;Foxg1Cre mutant. (A-D) Coronal sections of an E12.5 wild-type (A,C) and Smoc/–;Foxg1Cre mutant (B,D) telencephalon. Expression of Olig2 and Pdgfra (A,C), both markers of oligodendrocyte precursors, was absent from the mutant telencephalon (B,D). Note that the anterior entopeduncular area, where markers of oligodendrocyte precursors are typically first seen, is not present in Smoc/–;Foxg1Cre mutants. (E,F) Representative low-power fields of cortical cultures derived from E16.5 wild-type (E) and mutant (F) cortex, stained four days later with O4, TuJ1 and DAPI. Smoc/–;Foxg1Cre mutant cultures show a 25-fold reduction in O4-positive oligodendrocyte precursors (G). Scale bars: 300 µm in A-D.