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T396I mutation of mouse Sufu reduces the stability and activity of Gli3 repressor.

Makino S, Zhulyn O, Mo R, Puviindran V, Zhang X, Murata T, Fukumura R, Ishitsuka Y, Kotaki H, Matsumaru D, Ishii S, Hui CC, Gondo Y - PLoS ONE (2015)

Bottom Line: Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro.In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation.Taken together, the results of this study show that the Thr396 residue of Sufu is specifically required for regulation of Gli3 but not Gli2.

View Article: PubMed Central - PubMed

Affiliation: Mutagenesis and Genomics Team, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.

ABSTRACT
Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3FL) to an N-terminal repressor (Gli3REP). Recent studies using a Sufu knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that Thr396 residue of Sufu played a key role in regulation of Gli3 activity. SufuT396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of SufuT396I/T396I were rescued by a constitutive Gli3REP allele (Gli3∆699), strongly suggesting that SufuT396I reduced the truncated Gli3 repressor. In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr396 residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated.

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SufuT396I/T396I shows reduction of Gli3 activity but not Gli2 activity.(A–H) Immunofluorescence images of transverse sections at thoracic level. Sections from wild-type (A and E), SufuT396I/T396I (B and F), and SmoG457X/G457X; SufuT396I/T396I (C and G) embryos at E10.5 and SmoG457X/G457X embryos (D and H) at E9.5 were immunostained with anti-Olig2 (A–D, magenta), anti-Nkx2.2 (A–D, green), and anti-FoxA2 (E–H, green) antibodies. Dashed lines outline the neural tubes. Scale bar, 100 μm. Images with nuclear staining are shown in S7 Fig.
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pone.0119455.g004: SufuT396I/T396I shows reduction of Gli3 activity but not Gli2 activity.(A–H) Immunofluorescence images of transverse sections at thoracic level. Sections from wild-type (A and E), SufuT396I/T396I (B and F), and SmoG457X/G457X; SufuT396I/T396I (C and G) embryos at E10.5 and SmoG457X/G457X embryos (D and H) at E9.5 were immunostained with anti-Olig2 (A–D, magenta), anti-Nkx2.2 (A–D, green), and anti-FoxA2 (E–H, green) antibodies. Dashed lines outline the neural tubes. Scale bar, 100 μm. Images with nuclear staining are shown in S7 Fig.

Mentions: To investigate whether the SufuT396I mutation also affects GliACT as well as GliREP, we analyzed development of the ventral neural tube, given that the generation of neural progenitor cells along its D-V axis is dependent on the Shh signaling gradient and GliACT activity [3]. In particular, activation of Gli2 is essential to generate floor plate (FoxA2 positive cells) and p3 progenitors (Nkx2.2 positive cells) in response to high Shh signaling (Fig. 4A, green and 4E) [35]. Motor neuron progenitor cells (Olig2 positive cells) are generated dorsal to p3 progenitors in response to intermediate Shh signaling and a balance between Gli2ACT and Gli3REP (Fig. 4A, magenta) as has been reported before [11,36]. Previous studies [37–39] showed that loss of Smo, a core pathway regulator upstream of Sufu, leads to a decreased level of GliACT and excess Gli3REP, resulting in a failure of ventral specification. A double homozygous mutation of Smo and Gli3 restores expression of Olig2 in the ventral neural tube [36,39], indicating that Gli3REP represses Olig2 in the Smo−/− genetic background.


T396I mutation of mouse Sufu reduces the stability and activity of Gli3 repressor.

Makino S, Zhulyn O, Mo R, Puviindran V, Zhang X, Murata T, Fukumura R, Ishitsuka Y, Kotaki H, Matsumaru D, Ishii S, Hui CC, Gondo Y - PLoS ONE (2015)

SufuT396I/T396I shows reduction of Gli3 activity but not Gli2 activity.(A–H) Immunofluorescence images of transverse sections at thoracic level. Sections from wild-type (A and E), SufuT396I/T396I (B and F), and SmoG457X/G457X; SufuT396I/T396I (C and G) embryos at E10.5 and SmoG457X/G457X embryos (D and H) at E9.5 were immunostained with anti-Olig2 (A–D, magenta), anti-Nkx2.2 (A–D, green), and anti-FoxA2 (E–H, green) antibodies. Dashed lines outline the neural tubes. Scale bar, 100 μm. Images with nuclear staining are shown in S7 Fig.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356511&req=5

pone.0119455.g004: SufuT396I/T396I shows reduction of Gli3 activity but not Gli2 activity.(A–H) Immunofluorescence images of transverse sections at thoracic level. Sections from wild-type (A and E), SufuT396I/T396I (B and F), and SmoG457X/G457X; SufuT396I/T396I (C and G) embryos at E10.5 and SmoG457X/G457X embryos (D and H) at E9.5 were immunostained with anti-Olig2 (A–D, magenta), anti-Nkx2.2 (A–D, green), and anti-FoxA2 (E–H, green) antibodies. Dashed lines outline the neural tubes. Scale bar, 100 μm. Images with nuclear staining are shown in S7 Fig.
Mentions: To investigate whether the SufuT396I mutation also affects GliACT as well as GliREP, we analyzed development of the ventral neural tube, given that the generation of neural progenitor cells along its D-V axis is dependent on the Shh signaling gradient and GliACT activity [3]. In particular, activation of Gli2 is essential to generate floor plate (FoxA2 positive cells) and p3 progenitors (Nkx2.2 positive cells) in response to high Shh signaling (Fig. 4A, green and 4E) [35]. Motor neuron progenitor cells (Olig2 positive cells) are generated dorsal to p3 progenitors in response to intermediate Shh signaling and a balance between Gli2ACT and Gli3REP (Fig. 4A, magenta) as has been reported before [11,36]. Previous studies [37–39] showed that loss of Smo, a core pathway regulator upstream of Sufu, leads to a decreased level of GliACT and excess Gli3REP, resulting in a failure of ventral specification. A double homozygous mutation of Smo and Gli3 restores expression of Olig2 in the ventral neural tube [36,39], indicating that Gli3REP represses Olig2 in the Smo−/− genetic background.

Bottom Line: Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro.In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation.Taken together, the results of this study show that the Thr396 residue of Sufu is specifically required for regulation of Gli3 but not Gli2.

View Article: PubMed Central - PubMed

Affiliation: Mutagenesis and Genomics Team, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.

ABSTRACT
Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3FL) to an N-terminal repressor (Gli3REP). Recent studies using a Sufu knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that Thr396 residue of Sufu played a key role in regulation of Gli3 activity. SufuT396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of SufuT396I/T396I were rescued by a constitutive Gli3REP allele (Gli3∆699), strongly suggesting that SufuT396I reduced the truncated Gli3 repressor. In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr396 residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated.

Show MeSH
Related in: MedlinePlus