Journal: Development (Cambridge, England)

Article Title: Interplays of Gli2 and Gli3 and their requirement in mediating Shh-dependent sclerotome induction.

doi: 10.1242/dev.00851

Figure Lengend Snippet: Fig. 1. All three Gli genes are expressed in the ventral somite. Gene expression within the PSM and early somites was examined by radioactive section in situ hybridization on sagittal (A-C′) and transverse (D- F′) sections through E9.5 embryos. Bright-field images (A-F) are shown with corresponding dark field images (A′-F′). Radioactive section in situ hybridization signal corresponds to silver granules highlighted by dark-field illumination. Dotted lines indicate the boundary between the PSM and first fully formed somite (A-C′). Gli1 is expressed upon somite formation (A,A′) in the sclerotome (D,D′). Gli2 is expressed in the anterior PSM and the somites (B,B′,E,E′). Gli3 is expressed in the PSM (C,C′) and the somites (F,F′). nt, neural tube; dm, dermomyotome; scl, sclerotome.

Article Snippet: Production of fusion proteins was confirmed by western blot using anti-EGFP (Molecular probes) or anti-Gli1, Gli3 and Smo antibodies (Santa Cruz).

Techniques: Gene Expression, In Situ Hybridization

Journal: Development (Cambridge, England)

Article Title: Interplays of Gli2 and Gli3 and their requirement in mediating Shh-dependent sclerotome induction.

doi: 10.1242/dev.00851

Figure Lengend Snippet: Fig. 2. Gli2–/–Gli3–/– mutant mice exhibit a severe loss of sclerotomal gene expression. Expression of Pax1, Pax9 and Sox9 was assessed by whole-mount in situ hybridization in wild type (A,A′,A′′), Gli2–/– (B,B′,B′′), Gli3–/– (C,C′,C′′), Gli2–/–Gli3+/– (D,D′,D′′) and Gli2–/–Gli3–/– (E,E′,E′′) E9.5 embryos. Pax1 and Pax9 are expressed normally in all allelic combinations (A-D,A′-D′) except for Gli2–/–Gli3–/– embryos, which exhibit a severe reduction and delay in Pax1 and Pax9 (between arrowheads in E and E′) expression. In Gli3–/– embryos Sox9 expression is reduced in the anterior somites, with the dorsal region most affected (compare arrowheads in inset of A′′ and C′′). Gli2–/– and Gli2–/–Gli3+/– embryos exhibit reduced Sox9 in both ventral and dorsal domains of anterior somites (arrowheads in B′′ and D′′). Sox9 appears normal in the posterior somites of these embryos (indicated by the black line A′′-D′′). Sox9 is undetectable in Gli2–/–Gli3–/– embryos. Scale bar: 0.5 mm.

Article Snippet: Production of fusion proteins was confirmed by western blot using anti-EGFP (Molecular probes) or anti-Gli1, Gli3 and Smo antibodies (Santa Cruz).

Techniques: Mutagenesis, Gene Expression, Expressing, In Situ Hybridization

Journal: Development (Cambridge, England)

Article Title: Interplays of Gli2 and Gli3 and their requirement in mediating Shh-dependent sclerotome induction.

doi: 10.1242/dev.00851

Figure Lengend Snippet: Fig. 3. Dorsoventral patterning of the somite is abnormal in Gli2–/–Gli3–/– embryos. Transverse sections through the trunk of wild-type (A), and Gli2–/–Gli3–/– (B) embryos at E10.5 were stained to reveal tissue morphology. In Gli2–/–Gli3–/– embryos, ectopic epithelium extends ventromedially (open arrowheads), the sclerotome is reduced and myotome is not distinguishable. dm dermomyotome, scl sclerotome, my myotome. Expression of Pax3 and Myf5 were assessed by whole-mount in situ hybridization in wild-type (C,E) and Gli2–/–Gli3–/– (D,F) E9.5 embryos. Pax3 in Gli2–/–Gli3–/– embryos appeared normal in wholemount. Myf5 expression in Gli2–/–Gli3–/– somites was diffuse and expanded laterally. Additionally, dorsomedial-specific expression (arrowhead in E) was absent (arrowhead in F). Scale bar: 0.5 mm. (G-R) Expression of Pax3, Sim1, Pax1 and Myf5, were assessed by whole- mount in situ hybridization and sectioned. Transverse sections though the trunks of E.9.5 wild- type (G,I,K,O), Gli2–/–Gli3–/– (H,J,L,P), Shh–/– (M,Q) and Shh–/–Gli3–/– (N,R) embryos were collected. Broken lines outline ectopic epithelium in H,J,L,P. In Gli2–/–Gli3–/– embryos Pax3 is expressed in the ectopic ventromedial epithelium of Gli2–/–Gli3–/– somites (arrowhead in H). Sim1 expression is normal in wild-type (arrowhead in I) and Gli2–/–Gli3–/– mutants (arrowhead in J). Pax1 expression is weak in Gli2–/–Gli3–/– embryos (L), undetectable in the trunk of Shh–/– embryos (M), but restored in Shh–/–Gli3–/– embryos (N). In wild type, Myf5 is restricted to the layer of myotome (O), whereas in Gli2–/–Gli3–/– somites, Myf5 is expressed throughout the mesenchymal cells within the epithelial spheres (P). In Shh–/– somites, dorsomedial expression of Myf5 is reduced (arrowhead in Q), whereas in Shh–/–Gli3–/– somites Myf5 is restored in the dorsomedial lip (R). Scale bar: 50 µm.

Article Snippet: Production of fusion proteins was confirmed by western blot using anti-EGFP (Molecular probes) or anti-Gli1, Gli3 and Smo antibodies (Santa Cruz).

Techniques: Staining, Expressing, In Situ Hybridization

Journal: Development (Cambridge, England)

Article Title: Interplays of Gli2 and Gli3 and their requirement in mediating Shh-dependent sclerotome induction.

doi: 10.1242/dev.00851

Figure Lengend Snippet: Fig. 4. One copy of either Gli2 or Gli3 is required for tissue autonomous Shh responsiveness. (A) Shh expression was assessed by whole-mount in situ hybridization at E9.5. Transverse sections through the trunk reveal dramatically reduced Shh in the notochord and floorplate of Gli2–/–Gli3–/– embryos (compare arrowheads in A). (B) PSM tissue was isolated from E9.5 wild-type, Gli3–/–, Gli2–/–, Gli2–/–Gli3+/– and Gli2–/–Gli3–/– embryos and cultured in the presence (+) or absence (–) of Shh-N for 24 hours. Induction of target genes was assessed by RT-PCR. β-Actin serves as a control for normalization. Compared with wild-type PSM, Gli3–/– PSM shows Shh-responsiveness with an increase in Pax9 expression in the absence of Shh-N (n=4). Gli2–/– PSM shows reduced responsiveness in activating Ptch and Pax9, but normal induction of other targets (n=4). In Gli2–/–Gli3+/– PSM, all targets are induced with some reduction in Pax9 induction (n=2). In Gli2–/– Gli3–/– PSM, all targets tested are expressed identically in the presence and absence of Shh-N. There is increased basal expression of Ptch and Pax9 in Gli2–/–Gli3–/– PSM independent of Shh-N (n=3).

Article Snippet: Production of fusion proteins was confirmed by western blot using anti-EGFP (Molecular probes) or anti-Gli1, Gli3 and Smo antibodies (Santa Cruz).

Techniques: Expressing, In Situ Hybridization, Isolation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Development (Cambridge, England)

Article Title: Interplays of Gli2 and Gli3 and their requirement in mediating Shh-dependent sclerotome induction.

doi: 10.1242/dev.00851

Figure Lengend Snippet: Fig. 5. ∆NG2 can mimic Shh and Smo* induction of targets in presomitic mesoderm. (A) PSM explants were cultured alone (–Shh), with Shh-N (+Shh) or infected with adenoviruses carrying EGFP, Smo or Smo* for 24 hours and analyzed for induction of Shh target genes by RT-PCR. Shh-N induces target genes, while infection with EGFP virus does not. Expression of Smo* induces all Shh targets tested (n=8). (B) PSM explants were infected with adenoviruses carrying either EGFP, full-length Gli1, Gli2, Gli3 or ∆NG2 for 24 hours and analyzed for induction of Shh targets by RT-PCR. Gli1 induced Ptch, Hhip and moderate levels of Ccnd2. Gli2 induced Ccnd2, and moderate levels of Ptch. Gli3 did not induce any Shh targets, while ∆NG2 activated all targets tested (n=8). (C) PSM was cultured for 24 hours in the absence (–) or presence (+) of Shh-N and adenovirus as indicated. Expression of Ptch and Pax1, was assessed by RT-PCR. Infection with EGFP and Gli1 does not affect induction of target genes by Shh, while infection with Gli2 does seem to have a moderate inhibitory affect specifically on Pax1. Infection with Gli3 acts to repress Shh induction of both Ptch and Pax1 (n=3). (D) PSM infected with Gli2 and ∆NG2 induce Gli1 at 24 hours (n=8). (E) PSM explants cultured alone, with Shh-N, with 1 µg/ml cycloheximide (CHX) or the combination for 9 hours were analyzed for Gli1, Ccnd2 and Hhip by RT-PCR. In the presence of CHX, Shh activates only Ccnd2 and Gli1, but not Hhip (n=2). CHX (1 µg/ml) effectively inhibits over 90% of new protein synthesis under similar culture conditions (Fu et al., 2002).

Article Snippet: Production of fusion proteins was confirmed by western blot using anti-EGFP (Molecular probes) or anti-Gli1, Gli3 and Smo antibodies (Santa Cruz).

Techniques: Cell Culture, Infection, Reverse Transcription Polymerase Chain Reaction, Virus, Expressing

Journal: Development (Cambridge, England)

Article Title: Interplays of Gli2 and Gli3 and their requirement in mediating Shh-dependent sclerotome induction.

doi: 10.1242/dev.00851

Figure Lengend Snippet: Fig. 6. Gli1 cannot restore Shh responsiveness in Gli2–/–Gli3–/– PSM. PSM from wild-type and Gli2–/–Gli3–/– embryos was cultured with Shh-N in the presence (+) or absence (–) of Gli1 adenovirus for 24 hours. Induction of Shh target genes was assessed by RT-PCR. Infection with Gli1 adenovirus increased the expression of Ptch and Hhip but not other Shh target genes in wild-type PSM. In Gli2–/–Gli3–/– PSM infection with Gli1 adenovirus does not restore responsiveness of any Shh targets nor increase the expression of Ptch and Hhip (n=3).

Article Snippet: Production of fusion proteins was confirmed by western blot using anti-EGFP (Molecular probes) or anti-Gli1, Gli3 and Smo antibodies (Santa Cruz).

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Infection, Expressing

Journal: Development (Cambridge, England)

Article Title: Interplays of Gli2 and Gli3 and their requirement in mediating Shh-dependent sclerotome induction.

doi: 10.1242/dev.00851

Figure Lengend Snippet: Fig. 7. A model for Gli-mediated Shh target gene induction in the somite. (A) In wild type, Shh signaling modifies Gli2 into a strong activator, Gli2A, to activate target genes, including Gli1. Gli1 acts secondarily to activate Ptch and Hhip. In the absence of Shh, or when overexpressed, Gli3 acts as a repressor, Gli3R. In Gli2–/– and Gli2–/–Gli3+/–, an activator function of Gli3 is revealed (in blue), to compensate for loss of Gli2. In Gli3–/– and Gli2+/–Gli3–/–, a repressor function of Gli2 is revealed (in blue), to compensate for loss of Gli3. In wild-type embryos Gli2R and Gli3A may be present but play minor roles. This possibility is not illustrated for simplicity. In Gli2–/–Gli3–/– embryos, both activator and repressor functions of Gli2 and Gli3 are lost resulting in a low level of Shh-independent expression of target genes. The low level of Gli1 in these embryos is non-functional (X). (B) The diagram illustrates somite morphology and gene expression in wild type and Gli mutants. Somite patterning and gene expression is largely normal in Gli2–/–, Gli3–/– and Gli2–/–Gli3+/– embryos (left). In Gli2–/–Gli3–/– embryos, there is ventromedial expansion of Pax3-positive cells (Pax3+), mixing of Pax1+ and Myf5+ cells, and expression of Myf5 in the medial lip is lost (right).

Article Snippet: Production of fusion proteins was confirmed by western blot using anti-EGFP (Molecular probes) or anti-Gli1, Gli3 and Smo antibodies (Santa Cruz).

Techniques: Expressing, Functional Assay, Gene Expression