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 cilia increases in response to Hedgehog pathway stimulation without an overall increase in Tbx3 protein levels.(A) Quantitation of Tbx3+ cilia in wild type MEFS -/+ SAG shows marked increase in Tbx3 immunoreactive cilia in response to SAG. B) Western blot assaying Tbx3 and btubulin (loading control) protein levels in MEFs +/- SAG; the increase in number of Tbx3+ cilia occurs without an increase in amount of total Tbx3 protein. (C, D) Immunofluorescence images of SAG-treated (C) or SHH (D) MEFs assayed with a Santa Cruz commercial anti-Tbx3 antibody (A20) raised against an internal Tbx3 epitope (green) confirm colocalization with cilia/Arl13b (red). These merged images include DAPI in blue; white arrowheads highlight ciliary Tbx3. Please see Figure 6—source data 5,6 for z-stacks.DOI:http://dx.doi.org/10.7554/eLife.07897.032
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fig6s1: Tbx3 immunoreactivity in cilia increases in response to Hedgehog pathway stimulation without an overall increase in Tbx3 protein levels.(A) Quantitation of Tbx3+ cilia in wild type MEFS -/+ SAG shows marked increase in Tbx3 immunoreactive cilia in response to SAG. B) Western blot assaying Tbx3 and btubulin (loading control) protein levels in MEFs +/- SAG; the increase in number of Tbx3+ cilia occurs without an increase in amount of total Tbx3 protein. (C, D) Immunofluorescence images of SAG-treated (C) or SHH (D) MEFs assayed with a Santa Cruz commercial anti-Tbx3 antibody (A20) raised against an internal Tbx3 epitope (green) confirm colocalization with cilia/Arl13b (red). These merged images include DAPI in blue; white arrowheads highlight ciliary Tbx3. Please see Figure 6—source data 5,6 for z-stacks.DOI:http://dx.doi.org/10.7554/eLife.07897.032

Mentions: Murine embryonic fibroblasts (MEFs) are a robust system for studying ciliary proteins (Chen et al., 2009; Dorn et al., 2012; Liem et al., 2012; Ocbina and Anderson, 2008; Rohatgi et al., 2007), so we used them to further explore Tbx3 localization and trafficking. In untreated wild type MEFs, our custom C-terminal antibody detected Tbx3 in a subset of cilia (30%, N=56; Figure 6A and B, a1-a5, b1-b5; Figure 6—source data 1,2). No Tbx3+ cilia were detected in Tbx3 MEFS (Figure 6C–F; Figure 6—source data 3). Treatment of wild type MEFs with the smoothened agonist SAG increased the number of Tbx3+ cilia from 30% to 75% (Figure 6G-J p<0.005, Figure 6—source data 4, Figure 6—figure supplement 1A). Lysates from control and SAG-treated MEFs showed no detectable difference in Tbx3 protein levels indicating the increased Tbx3 signal in cilia was due to trafficking rather than increased protein levels (Figure 6—figure supplement 1B). The presence of Tbx3 in cilia and response to Hedgehog pathway activity were also detected with a commercially available anti-Tbx3 antibody with both SAG and Shh stimulation (Figure 6—figure supplement 1C,D and Figure 6—source data 5,6). In total, these data show that Tbx3 is present at baseline in cilia, and is trafficked to cilia in response to hedgehog signaling.10.7554/eLife.07897.025Figure 6.Tbx3 is present in some cilia at baseline in Murine Embryonic Fibroblasts and trafficks to cilia in response to hedgehog pathway activation.


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 cilia increases in response to Hedgehog pathway stimulation without an overall increase in Tbx3 protein levels.(A) Quantitation of Tbx3+ cilia in wild type MEFS -/+ SAG shows marked increase in Tbx3 immunoreactive cilia in response to SAG. B) Western blot assaying Tbx3 and btubulin (loading control) protein levels in MEFs +/- SAG; the increase in number of Tbx3+ cilia occurs without an increase in amount of total Tbx3 protein. (C, D) Immunofluorescence images of SAG-treated (C) or SHH (D) MEFs assayed with a Santa Cruz commercial anti-Tbx3 antibody (A20) raised against an internal Tbx3 epitope (green) confirm colocalization with cilia/Arl13b (red). These merged images include DAPI in blue; white arrowheads highlight ciliary Tbx3. Please see Figure 6—source data 5,6 for z-stacks.DOI:http://dx.doi.org/10.7554/eLife.07897.032
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fig6s1: Tbx3 immunoreactivity in cilia increases in response to Hedgehog pathway stimulation without an overall increase in Tbx3 protein levels.(A) Quantitation of Tbx3+ cilia in wild type MEFS -/+ SAG shows marked increase in Tbx3 immunoreactive cilia in response to SAG. B) Western blot assaying Tbx3 and btubulin (loading control) protein levels in MEFs +/- SAG; the increase in number of Tbx3+ cilia occurs without an increase in amount of total Tbx3 protein. (C, D) Immunofluorescence images of SAG-treated (C) or SHH (D) MEFs assayed with a Santa Cruz commercial anti-Tbx3 antibody (A20) raised against an internal Tbx3 epitope (green) confirm colocalization with cilia/Arl13b (red). These merged images include DAPI in blue; white arrowheads highlight ciliary Tbx3. Please see Figure 6—source data 5,6 for z-stacks.DOI:http://dx.doi.org/10.7554/eLife.07897.032
Mentions: Murine embryonic fibroblasts (MEFs) are a robust system for studying ciliary proteins (Chen et al., 2009; Dorn et al., 2012; Liem et al., 2012; Ocbina and Anderson, 2008; Rohatgi et al., 2007), so we used them to further explore Tbx3 localization and trafficking. In untreated wild type MEFs, our custom C-terminal antibody detected Tbx3 in a subset of cilia (30%, N=56; Figure 6A and B, a1-a5, b1-b5; Figure 6—source data 1,2). No Tbx3+ cilia were detected in Tbx3 MEFS (Figure 6C–F; Figure 6—source data 3). Treatment of wild type MEFs with the smoothened agonist SAG increased the number of Tbx3+ cilia from 30% to 75% (Figure 6G-J p<0.005, Figure 6—source data 4, Figure 6—figure supplement 1A). Lysates from control and SAG-treated MEFs showed no detectable difference in Tbx3 protein levels indicating the increased Tbx3 signal in cilia was due to trafficking rather than increased protein levels (Figure 6—figure supplement 1B). The presence of Tbx3 in cilia and response to Hedgehog pathway activity were also detected with a commercially available anti-Tbx3 antibody with both SAG and Shh stimulation (Figure 6—figure supplement 1C,D and Figure 6—source data 5,6). In total, these data show that Tbx3 is present at baseline in cilia, and is trafficked to cilia in response to hedgehog signaling.10.7554/eLife.07897.025Figure 6.Tbx3 is present in some cilia at baseline in Murine Embryonic Fibroblasts and trafficks to cilia in response to hedgehog pathway activation.

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