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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

Digital image overlap of Tbx3 and Arl13b in limb bud anterior mesenchyme.(A) Maximum image projection of Arl13b channel from control limb z-stack shown in Figure 5—source data 1. Both mesenchymal and epithelial cilia are apparent in the maximum projection. (B) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in control limb bud. Note that all epithelial cilia in the stack are Tbx3 negative and of the 50 mesenchymal cilia, 18 (36%) are Tbx3 positive. Please see Experimental Procedures for use of Zen and Image J software to calculate pixel overlap in separate channels. (C) Maximum image projection of Arl13b channel from control limb z-stack shown in Figure 5—source data 2. Both mesenchymal and epithelial cilia are apparent in the maximum projection. (D) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in Tbx3;PrxCre limb bud. Note that all epithelial cilia in the stack are Tbx3 negative and of the 54 mesenchymal cilia, 2 (4%) are Tbx3 positive consistent with low level of background antibody staining in mutant (Figure 5, panels G, I).DOI:http://dx.doi.org/10.7554/eLife.07897.023
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fig5s1: Digital image overlap of Tbx3 and Arl13b in limb bud anterior mesenchyme.(A) Maximum image projection of Arl13b channel from control limb z-stack shown in Figure 5—source data 1. Both mesenchymal and epithelial cilia are apparent in the maximum projection. (B) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in control limb bud. Note that all epithelial cilia in the stack are Tbx3 negative and of the 50 mesenchymal cilia, 18 (36%) are Tbx3 positive. Please see Experimental Procedures for use of Zen and Image J software to calculate pixel overlap in separate channels. (C) Maximum image projection of Arl13b channel from control limb z-stack shown in Figure 5—source data 2. Both mesenchymal and epithelial cilia are apparent in the maximum projection. (D) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in Tbx3;PrxCre limb bud. Note that all epithelial cilia in the stack are Tbx3 negative and of the 54 mesenchymal cilia, 2 (4%) are Tbx3 positive consistent with low level of background antibody staining in mutant (Figure 5, panels G, I).DOI:http://dx.doi.org/10.7554/eLife.07897.023

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)

Digital image overlap of Tbx3 and Arl13b in limb bud anterior mesenchyme.(A) Maximum image projection of Arl13b channel from control limb z-stack shown in Figure 5—source data 1. Both mesenchymal and epithelial cilia are apparent in the maximum projection. (B) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in control limb bud. Note that all epithelial cilia in the stack are Tbx3 negative and of the 50 mesenchymal cilia, 18 (36%) are Tbx3 positive. Please see Experimental Procedures for use of Zen and Image J software to calculate pixel overlap in separate channels. (C) Maximum image projection of Arl13b channel from control limb z-stack shown in Figure 5—source data 2. Both mesenchymal and epithelial cilia are apparent in the maximum projection. (D) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in Tbx3;PrxCre limb bud. Note that all epithelial cilia in the stack are Tbx3 negative and of the 54 mesenchymal cilia, 2 (4%) are Tbx3 positive consistent with low level of background antibody staining in mutant (Figure 5, panels G, I).DOI:http://dx.doi.org/10.7554/eLife.07897.023
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fig5s1: Digital image overlap of Tbx3 and Arl13b in limb bud anterior mesenchyme.(A) Maximum image projection of Arl13b channel from control limb z-stack shown in Figure 5—source data 1. Both mesenchymal and epithelial cilia are apparent in the maximum projection. (B) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in control limb bud. Note that all epithelial cilia in the stack are Tbx3 negative and of the 50 mesenchymal cilia, 18 (36%) are Tbx3 positive. Please see Experimental Procedures for use of Zen and Image J software to calculate pixel overlap in separate channels. (C) Maximum image projection of Arl13b channel from control limb z-stack shown in Figure 5—source data 2. Both mesenchymal and epithelial cilia are apparent in the maximum projection. (D) Calculated digital image overlap of Arl13b (cilia) and Tbx3 positive pixels in Tbx3;PrxCre limb bud. Note that all epithelial cilia in the stack are Tbx3 negative and of the 54 mesenchymal cilia, 2 (4%) are Tbx3 positive consistent with low level of background antibody staining in mutant (Figure 5, panels G, I).DOI:http://dx.doi.org/10.7554/eLife.07897.023
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