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Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway

C. Brian Bai^1,2, Wojtek Auerbach^1,*, Joon S. Lee¹, Daniel Stephen¹ and Alexandra L. Joyner^1,2,3,

¹ Howard Hughes Medical Institute and Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA
² Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA
³ Department of Physiology and Neuroscience, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA
^* Present address: Regeneron Pharmaceutical, 777 Old Saw Mill River Rd, Tarrytown, NY 10591, USA

View larger version (23K): [in a new window]   Fig. 1. Gli1 gene targeting strategy and ES cell screening. (A, top) The Gli1 genomic locus and targeting construct. White boxes indicate Gli1 exons. Black boxes indicate Gli1 zinc-finger domains 1-5. B, BamHI; RV, EcoRV; H, HindIII; Hs, Hsp921; Xb, XbaI. (A, middle) Targeted allele with neo cassette. The N-terminal and zinc-finger domains are replaced by nuclear lacZ and a neo cassette. The neo cassette is in the opposite orientation relative to Gli1 transcription. (A, bottom) Targeted allele without neo allele. Cre recombinase was used to remove the loxP-neo-loxP cassette. (B) ES cell Southern blot hybridization using 5' and 3' external probes indicated in A following EcoRV and XbaI digestion, respectively. The 5' probe identifies a 9 kb mutant fragment and a >20 kb wild-type fragment, whereas the 3' probe identifies a 4.5 kb mutant fragment and a 9 kb wild-type fragment.

View larger version (53K): [in a new window]   Fig. 2. Gli1lz/lz;Gli2zfd/+ embryos have defects similar to Gli1zfd/zfd;Gli2zfd/+ embryos. In E10.5 Gli1lz/lz;Gli2zfd/+ embryos, there is a variable loss of floor-plate cells. In the most extreme mutant embryos, HNF3ß- and Nkx2-2-expressing cells are greatly reduced (compare E and F with A and B), such that Isl1/2-expressing cells occupy the ventral midline of the spinal cord (compare G and H with C and D). (C,G) Sections at forelimb level; (D,H) sections at hindlimb level. Arrowhead indicates ventral midline of spinal cord. (I) At E12.5, Gli1lz/lz;Gli2zfd/+ lungs are smaller than wild-type lungs, in addition, Gli1lz/lz;Gli2zfd/zfd lungs have only two lobes and are much smaller than wild-type lungs. At E18.5, a postaxial nubbin was found in the limbs of Gli1lz/lz;Gli2zfd/zfd embryos (K), similar to Gli1zfd/zfd;Gli2zfd/zfd embryos. (J) E18.5 wild-type limb. Scale bar: 0.1 mm in A-H.

View larger version (69K): [in a new window]   Fig. 3. Shh is required for transcription of Gli1. (A-D) RNA in situ hybridization of Shh at E7.5, E8.0, E8.5 and E9.5 in wild-type embryos. (E-H) X-gal staining of lacZ in Gli1lz/+ embryos at E7.5, E8.0, E8.5 and E9.5. Note the brownish weak X-gal staining of Gli1-lacZ at E7.5. The embryo in E is slightly older than the embryo in A. (I-N) X-gal staining of Gli2-lacZ from E7.5 to E9.5. Note, one embryo in I is at mid-streak stage (ms), and the other embryo is at the late-streak stage (ls). (L) Spinal cord section of an E8.5 embryo showing Gli2-lacZ in the ventral midline. (M,N) Spinal cord section of an E9.5 embryo at an anterior (M) or posterior (N) position. Arrowhead indicates floorplate cells. Spinal cord is outlined by broken red lines. a, anterior; hp, head process; fb, forebrain; fg, foregut; ls, late-streak; mb, midbrain; ms, mid-streak; nd, notochord; p, posterior. (O-R) X-gal staining of lacZ in Gli1lz/+ embryos (O,Q) or Gli1lz/+;Shh–/– embryos (P,R) at E8.5 (O,P) and E10.5 (Q,R). Limb buds in Q and R are outlined by broken white lines. At E8.5, Gli1-lacZ expression can be detected in the CNS and the gut (indicated by an arrow) in wild-type embryos (O), whereas it can only be detected weakly in the gut in Shh mutant embryos (P). At E10.5, Gli1-lacZ is strongly expressed in the gut (arrow), posterior limb bud (red arrowhead) and CNS (black arrowhead) in wild-type embryos (Q). In Shh mutant embryos, the expression in the posterior limb bud and the CNS cannot be detected (R).

View larger version (95K): [in a new window]   Fig. 4. Gli2, but not Gli1, is required for Shh signaling in Ptc mutants. X-gal staining of Ptc-lacZ in six- to eight-somite E8.5 embryos. In wild-type embryos, Ptc-lacZ is expressed in the ventral CNS and somites (A). The expression of Ptc-lacZ is not altered in Gli1zfd/zfd;Ptc+/– embryos (B), but the expression is downregulated in Gli2zfd/zfd;Ptc+/– embryos (C). Loss of Ptc function results in upregulation of Ptc-lacZ throughout the embryos (D). Removal of Gli1 function in Ptc–/– embryos does not rescue the overexpression of Ptc-lacZ (E). However, removal of Gli2 function in Ptc–/– embryos (F) reduces the overexpression of Ptc-lacZ in the head (arrow) and the trunk mesoderm (arrowhead). Scale bar: 0.5 mm.

View larger version (56K): [in a new window]   Fig. 5. Gli2 can only partially rescue the Ptc mutant phenotype at E10.5. Ptc+/– (A,D), Ptc+/–;Gli2zfd/zfd(B,E) and Ptc–/–;Gli2zfd/zfd(C,F) embryos. Ptc–/–;Gli2zfd/zfd embryos survive beyond E10.5, whereas Ptc–/– embryos die by E9.5. At E10.5, even though the spinal cords are closed, Ptc–/–;Gli2zfd/zfd embryos show exencephaly (indicated by a white arrow). (D-F) X-gal staining of Ptc-lacZ. The expression of Ptc-lacZ is reduced in Ptc+/–;Gli2zfd/zfd embryos (E, indicated by an arrow), when compared with Ptc+/– embryos (D). In Ptc–/–;Gli2zfd/zfd embryos at E10.5, Ptc-lacZ expression is upregulated in the brain.

View larger version (87K): [in a new window]   Fig. 6. The ventral spinal cord phenotype of Ptc mutants can be partially rescued by removing Gli2 function. In Ptc mutants that survive to E9.5, ventral spinal cord markers expand dorsally (A-D) compared with wild-type embryos a day later at E10.5 (E-H). Insert in A is a more anterior spinal cord section. In Ptc;Gli2 double homozygous mutants, Shh expression is lost in the spinal cord (I), Nkx2-2 expression is greatly reduced in the ventral midline (J), and the expansion of Isl1/2 and Pax6 seen in Ptc–/– mutants are shifted more ventrally (K,L). White arrowhead indicates ventral limit of Pax6 domain. Scale bar: 78 µm in A-D; 100 µm in E-L.

View larger version (107K): [in a new window]   Fig. 7. Endogenous Gli1 is not required when the Shh pathway is activated by ectopic Gli1. In Gli2 mutant spinal cords (A-D), Shh expression cannot be detected, Nkx2-2 expression is greatly reduced in the ventral midline, and the expression domains of Isl1/2 and Pax6 are shifted ventrally. When Gli1 is expressed from the endogenous Gli2 locus in Gli21ki/1ki embryos, the Gli2 mutant defects are rescued and Shh signaling is restored (E-H). Removal of endogenous Gli1 does not alter ventral spinal cord patterning in Gli1lz/lz;Gli21ki/1ki embryos (I-L). Arrowhead indicates ventral limit of Pax6 expression domain. Scale bar: 50 µm.