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Gli1 can rescue the in vivo function of Gli2

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 and Physiology and Neuroscience, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA

View larger version (28K): [in a new window]   Fig. 1. Knock-in gene targeting strategy. (A) The Gli2 locus, targeting vector and knock-in alleles (third and fourth diagrams) with and without neo. The first three exons of Gli2 are shown as boxes, with white boxes representing untranslated exons and black boxes representing translated exons. The cDNA represents either the 1ki, 2ki or lzki (shown in B). CRE-mediated recombination was used to remove the neo cassette in the knock-in alleles in mice. P1-P4 represent primers for PCR genotyping. (B) Knock-in cDNA constructs. All cDNAs contain the Gli2 3' UTR followed by three SV40 polyA signals. (C) Typical ES cell Southern blot analysis. Genomic DNA from ES cells was digested with BamHI and probed with 5' and 3' probes. Different knock-in constructs gave different hybridization bands. Size in kb is shown on the left.

View larger version (73K): [in a new window]   Fig. 2. Homozygous Gli2lzki/lzki embryos have defects in ventral spinal cord development at E10.5, similar to Gli2zfd/zfd mutants. In Gli2lzki mutant spinal cords (G-L), the floor plate cells that express Shh and HNF3ß are lost (G,H). Nkx2.2-expressing V3 interneurons are greatly reduced in number(I). Loss of floor plate also results in a ventral expansion of Islet1/2- (J) and Pax6- (K) expressing cells. Pax7 expression is unaffected (L). White arrowhead represents ventral limit of Pax6 or Pax7 expression domain. (M,N) E10.5 spinal cord stained with an Isl1/2 antibody for motoneurons. The floor plate fails to develop in Gli2lzki/zfd embryos, and motoneurons occupy the ventral midline. (O,P) Hematoxylin and Eosin staining of E12.5 spinal cord. The notochord (indicated by black arrow) fails to regress in Gli2lzki/zfd embryos. (Q,R) E12.5 Gli2lzki/zfd embryos have smaller lungs without an accessory lobe. The margins of the spinal cord and spinal canal are outlined by broken white lines. The margin of lungs is outlined by broken black lines. Scale bar: 100 µm in A-E,D-K; 78 µm in F,L; 130 µm in M,N.

View larger version (52K): [in a new window]   Fig. 3. Gli1 is expressed in the Gli2 domains from the knock-in allele. (A,C,E,G) Whole embryos and (B,D,F,H) high-power images of the limbs of the same embryos. (A,B) Wild-type E10.5 embryo stained with a Gli1 probe. (C,D) Wild-type embryo stained with a Gli2 probe. (E,F) Gli21ki/+ embryo stained with a Gli1 probe. (G,H) Gli21ki/1ki embryo stained with a Gli1 probe. Gli1 is expressed in the Gli1 (white arrows in F,H) and Gli2 (between red arrowheads in F,H) expression domains. Note the elevated Gli1 expression in H compared with F.

View larger version (92K): [in a new window]   Fig. 4. Expression of one copy of Gli1 can rescue the Gli2 mutant floor plate defects. In Gli2 mutant embryos, the floor plate cells are missing (Fig. 2G-L). At E10.5, ectopic expression of one copy of Gli1 in the Gli2 domain results in rescue of the ventral spinal cord defect, as assessed by immunohistochemical staining for Shh, Nkx2.2 and Islet1/2 (A-C) or by X-gal staining in Gli21ki/lzki embryos (D-F). (A-C,F) Gli21ki/lzki embryos, (D) Gli2lzki/+ embryo and (E) Gli2lzki/lzki embryo. Red asterisk indicates floor plate that does not express lacZ from the Gli2 allele. The margins of the spinal cord and spinal canal are outlined by broken lines. Scale bar: 100 µm.

View larger version (86K): [in a new window]   Fig. 5. Loss of Gli2 does not rescue the Shh mutant defects and expression of Gli1 from the Gli2 allele can only partially rescue the Shh forebrain mutant defects at E10.5. (A,E) Wild-type embryos. (B,F) Shh–/– embryos. (C,G) Gli2lzki/lzki;Shh–/– embryos. (D,H) Gli21ki/+;Shh–/– embryos. In Shh–/– embryos, the telencephalic vesicles are fused (arrows in B,F). In half the Gli21ki+/–;Shh–/– embryos, the telencephalic vesicles are partially separated. The embryo in D represents the most fully rescued embryo. (E-H) Higher magnification views of the telencephalic regions of the same embryos as in (A-D), shown in frontal view. F is a frontal view, in order to visualize the lack of separation of the two vesicles, and the broken red lines in E,H indicate the midline. (I-L) lacZ is expressed in Gli2lzki/+ and Gli2lzki/+;Shh–/– embryos at E8.5 (I,J) and sections at E10.5 (K,L). DRG, dorsal root ganglion. The margins of the spinal cord are outlined by broken red lines. Scale bar: 50 µm.

View larger version (92K): [in a new window]   Fig. 6. Loss of Gli2 does not rescue the Shh mutant defects and expression of Gli1 from the Gli2 allele in the neural plate does not rescue the ventral spinal cord defects in Shh mutant embryos. Antibody staining of E10.5 embryos for Isl1/2, HNF3ß and Nkx2.2. In wild-type embryos, Nkx2.2-expressing interneurons and Isl1/2-expressing motoneurons are expressed in the ventralateral spinal cord (A,E), and HNF3ß is expressed in the floor plate (I). These markers are not expressed in Shh–/– (B,F,J) or Gli2–/–;Shh–/– embryos (C,G,K). In addition, expression of one copy of Gli1 in the place of Gli2 in Shh mutant embryos does not rescue these ventral cell types (D,H,L). Scale bar: 50 µm.

View larger version (68K): [in a new window]   Fig. 7. Expression of two copies of Gli1 from the Gli2 allele does not result in spinal cord patterning defects. Immunoflurorescent staining of Shh, HNF3ß, Nkx2.2, Isl1/2, Pax6 and Pax7 in E10.5 Gli21ki/+ embryos (A-F), or Gli21ki/1ki embryos (G-L). The size of each mutant used for the analysis was slightly different. White arrowhead indicates the ventral limit of the Pax6 or Pax7 expression domains. Margins of the spinal cord and spinal canal are outlined by broken lines. Scale bar: 100 µm.

View larger version (80K): [in a new window]   Fig. 8. Gli2 knock-in mice expressing Gli1 develop hair defects. (A) Gli21ki/+ mice develop hair defects at three months of age. (B) Gli2n1ki/+ mice are normal, whereas Gli2n1ki/n1ki mice develop hair defects and are much smaller than Gli2n1ki/+ littermates. Shown at 3 months of age, the Gli2n1ki/n1ki mouse weighed 15 g and the Gli2n1ki/+ mouse weighed 29 g. (C) Removal of one copy of Gli3 enhances the hair loss defect in Gli21ki/+;Gli3+/– mice at 6 weeks of age. (D) Expression of one copy of Gli1 from the Gli2 allele enhances the polydactyly in Gli21ki/+;Gli3+/– mice.