Limits...
T-box3 is a ciliary protein and regulates stability of the Gli3 transcription factor to control digit number.

Emechebe U, Kumar P P, Rozenberg JM, Moore B, Firment A, Mirshahi T, Moon AM - Elife (2016)

Bottom Line: In contrast, loss of anterior T-box3 results in preaxial polydactyly, as seen with dysfunction of primary cilia or Gli3-repressor.T-box3 interacts with Kif7 and is required for normal stoichiometry and function of a Kif7/Sufu complex that regulates Gli3 stability and processing.Thus, T-box3 controls digit number upstream of Shh-dependent (posterior mesenchyme) and Shh-independent, cilium-based (anterior mesenchyme) Hedgehog pathway function.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, United States.

ABSTRACT
Crucial roles for T-box3 in development are evident by severe limb malformations and other birth defects caused by T-box3 mutations in humans. Mechanisms whereby T-box3 regulates limb development are poorly understood. We discovered requirements for T-box at multiple stages of mouse limb development and distinct molecular functions in different tissue compartments. Early loss of T-box3 disrupts limb initiation, causing limb defects that phenocopy Sonic Hedgehog (Shh) mutants. Later ablation of T-box3 in posterior limb mesenchyme causes digit loss. In contrast, loss of anterior T-box3 results in preaxial polydactyly, as seen with dysfunction of primary cilia or Gli3-repressor. Remarkably, T-box3 is present in primary cilia where it colocalizes with Gli3. T-box3 interacts with Kif7 and is required for normal stoichiometry and function of a Kif7/Sufu complex that regulates Gli3 stability and processing. Thus, T-box3 controls digit number upstream of Shh-dependent (posterior mesenchyme) and Shh-independent, cilium-based (anterior mesenchyme) Hedgehog pathway function.

No MeSH data available.


Related in: MedlinePlus

Tbx3 immunoreactivity in limb cilia is also detected by a commercial anti-Tbx3 antibody against the N-terminus of Tbx3.(A) Maximum image projection of Arl13b channel from control forelimb z-stack. (B) Calculated digital image overlap (see Methods section) of Arl13b (cilia) and Tbx3 positive pixels in control limb bud shown above using Abcam (Abcam ab99302) anti-Tbx3 antibody to the N-terminus of mouse Tbx3. 27/97 (28%) of mesenchymal cilia are Tbx3+. (C) Maximum image projection of Arl13b channel from Tbx3;PrxCre mutant forelimb z-stack. (D) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in Tbx3;PrxCre forelimb bud shown above. 2/52 mesenchymal cilia are Tbx3+. (E) Scatter plot obtained using ImageJ comparing Tbx3 and Arl13b intensities from control and mutant anterior forelimb buds stained with Abcam anti-Tbx3 antibody shown in A–D.DOI:http://dx.doi.org/10.7554/eLife.07897.024
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fig5s2: Tbx3 immunoreactivity in limb cilia is also detected by a commercial anti-Tbx3 antibody against the N-terminus of Tbx3.(A) Maximum image projection of Arl13b channel from control forelimb z-stack. (B) Calculated digital image overlap (see Methods section) of Arl13b (cilia) and Tbx3 positive pixels in control limb bud shown above using Abcam (Abcam ab99302) anti-Tbx3 antibody to the N-terminus of mouse Tbx3. 27/97 (28%) of mesenchymal cilia are Tbx3+. (C) Maximum image projection of Arl13b channel from Tbx3;PrxCre mutant forelimb z-stack. (D) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in Tbx3;PrxCre forelimb bud shown above. 2/52 mesenchymal cilia are Tbx3+. (E) Scatter plot obtained using ImageJ comparing Tbx3 and Arl13b intensities from control and mutant anterior forelimb buds stained with Abcam anti-Tbx3 antibody shown in A–D.DOI:http://dx.doi.org/10.7554/eLife.07897.024

Mentions: Kif7 and other proteins required for Gli3 processing and function are present in, or translocate to, primary cilia in response to Hedgehog pathway activity (Goetz and Anderson, 2010; Ryan and Chiang, 2012). Dual immunostaining for Tbx3 and Arl13b on whole mount optically sectioned E10.5 forelimbs shows that Tbx3 is present in control limb anterior mesenchymal cilia (Figure 5 A-E, Figure 5—source data 1). Specificity of Tbx3 staining was confirmed by loss of signal in mesenchymal cilia of Tbx3;PrxCre mutant limbs (Figure 5F-J, Figure 5—source data 2). The digital image overlap calculator in Zen software showed that 18/50 anterior mesenchymal cilia were Tbx3+ (36%, Figure 5—figure supplement 1A,B). Of note, no epithelial cilia were Tbx3+ in control limb epithelium, providing an internal negative control for the signal in mesenchymal cilia. This same calculation in Tbx3;PrxCre mutant anterior mesenchyme showed only 2/54 (<4%) of mesenchymal cilia had background Tbx3 signal (Figure 5—figure supplement 1, C, D). These findings were reproduced with a commercially available anti-Tbx3 antibody (Abcam ab99302) which also showed Tbx3 ciliary staining on control limb mesenchymal cilia (24/87, 28%) that was virtually absent in Tbx3 mutant limbs (2/52, <4%; Figure 5—figure supplement 2).10.7554/eLife.07897.020Figure 5.Tbx3 localizes to the primary cilia in limb mesenchyme.


T-box3 is a ciliary protein and regulates stability of the Gli3 transcription factor to control digit number.

Emechebe U, Kumar P P, Rozenberg JM, Moore B, Firment A, Mirshahi T, Moon AM - Elife (2016)

Tbx3 immunoreactivity in limb cilia is also detected by a commercial anti-Tbx3 antibody against the N-terminus of Tbx3.(A) Maximum image projection of Arl13b channel from control forelimb z-stack. (B) Calculated digital image overlap (see Methods section) of Arl13b (cilia) and Tbx3 positive pixels in control limb bud shown above using Abcam (Abcam ab99302) anti-Tbx3 antibody to the N-terminus of mouse Tbx3. 27/97 (28%) of mesenchymal cilia are Tbx3+. (C) Maximum image projection of Arl13b channel from Tbx3;PrxCre mutant forelimb z-stack. (D) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in Tbx3;PrxCre forelimb bud shown above. 2/52 mesenchymal cilia are Tbx3+. (E) Scatter plot obtained using ImageJ comparing Tbx3 and Arl13b intensities from control and mutant anterior forelimb buds stained with Abcam anti-Tbx3 antibody shown in A–D.DOI:http://dx.doi.org/10.7554/eLife.07897.024
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Related In: Results  -  Collection

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fig5s2: Tbx3 immunoreactivity in limb cilia is also detected by a commercial anti-Tbx3 antibody against the N-terminus of Tbx3.(A) Maximum image projection of Arl13b channel from control forelimb z-stack. (B) Calculated digital image overlap (see Methods section) of Arl13b (cilia) and Tbx3 positive pixels in control limb bud shown above using Abcam (Abcam ab99302) anti-Tbx3 antibody to the N-terminus of mouse Tbx3. 27/97 (28%) of mesenchymal cilia are Tbx3+. (C) Maximum image projection of Arl13b channel from Tbx3;PrxCre mutant forelimb z-stack. (D) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in Tbx3;PrxCre forelimb bud shown above. 2/52 mesenchymal cilia are Tbx3+. (E) Scatter plot obtained using ImageJ comparing Tbx3 and Arl13b intensities from control and mutant anterior forelimb buds stained with Abcam anti-Tbx3 antibody shown in A–D.DOI:http://dx.doi.org/10.7554/eLife.07897.024
Mentions: Kif7 and other proteins required for Gli3 processing and function are present in, or translocate to, primary cilia in response to Hedgehog pathway activity (Goetz and Anderson, 2010; Ryan and Chiang, 2012). Dual immunostaining for Tbx3 and Arl13b on whole mount optically sectioned E10.5 forelimbs shows that Tbx3 is present in control limb anterior mesenchymal cilia (Figure 5 A-E, Figure 5—source data 1). Specificity of Tbx3 staining was confirmed by loss of signal in mesenchymal cilia of Tbx3;PrxCre mutant limbs (Figure 5F-J, Figure 5—source data 2). The digital image overlap calculator in Zen software showed that 18/50 anterior mesenchymal cilia were Tbx3+ (36%, Figure 5—figure supplement 1A,B). Of note, no epithelial cilia were Tbx3+ in control limb epithelium, providing an internal negative control for the signal in mesenchymal cilia. This same calculation in Tbx3;PrxCre mutant anterior mesenchyme showed only 2/54 (<4%) of mesenchymal cilia had background Tbx3 signal (Figure 5—figure supplement 1, C, D). These findings were reproduced with a commercially available anti-Tbx3 antibody (Abcam ab99302) which also showed Tbx3 ciliary staining on control limb mesenchymal cilia (24/87, 28%) that was virtually absent in Tbx3 mutant limbs (2/52, <4%; Figure 5—figure supplement 2).10.7554/eLife.07897.020Figure 5.Tbx3 localizes to the primary cilia in limb mesenchyme.

Bottom Line: In contrast, loss of anterior T-box3 results in preaxial polydactyly, as seen with dysfunction of primary cilia or Gli3-repressor.T-box3 interacts with Kif7 and is required for normal stoichiometry and function of a Kif7/Sufu complex that regulates Gli3 stability and processing.Thus, T-box3 controls digit number upstream of Shh-dependent (posterior mesenchyme) and Shh-independent, cilium-based (anterior mesenchyme) Hedgehog pathway function.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, United States.

ABSTRACT
Crucial roles for T-box3 in development are evident by severe limb malformations and other birth defects caused by T-box3 mutations in humans. Mechanisms whereby T-box3 regulates limb development are poorly understood. We discovered requirements for T-box at multiple stages of mouse limb development and distinct molecular functions in different tissue compartments. Early loss of T-box3 disrupts limb initiation, causing limb defects that phenocopy Sonic Hedgehog (Shh) mutants. Later ablation of T-box3 in posterior limb mesenchyme causes digit loss. In contrast, loss of anterior T-box3 results in preaxial polydactyly, as seen with dysfunction of primary cilia or Gli3-repressor. Remarkably, T-box3 is present in primary cilia where it colocalizes with Gli3. T-box3 interacts with Kif7 and is required for normal stoichiometry and function of a Kif7/Sufu complex that regulates Gli3 stability and processing. Thus, T-box3 controls digit number upstream of Shh-dependent (posterior mesenchyme) and Shh-independent, cilium-based (anterior mesenchyme) Hedgehog pathway function.

No MeSH data available.


Related in: MedlinePlus