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Loss of Tgif function causes holoprosencephaly by disrupting the SHH signaling pathway.

Taniguchi K, Anderson AE, Sutherland AE, Wotton D - PLoS Genet. (2012)

Bottom Line: Early anterior patterning is relatively normal, but expression of Shh is reduced in the forebrain, and Gli3 expression is up-regulated throughout the neural tube.Tgif1 and Tgif2 are transcriptional repressors that limit Transforming Growth Factor β/Nodal signaling, and we show that reducing Nodal signaling in embryos lacking both Tgifs reduces the severity of HPE and partially restores the output of Shh signaling.Together, these results support a model in which Tgif function limits Nodal signaling to maintain the appropriate output of the Shh pathway in the forebrain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics and Center for Cell Signaling, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT
Holoprosencephaly (HPE) is a severe human genetic disease affecting craniofacial development, with an incidence of up to 1/250 human conceptions and 1.3 per 10,000 live births. Mutations in the Sonic Hedgehog (SHH) gene result in HPE in humans and mice, and the Shh pathway is targeted by other mutations that cause HPE. However, at least 12 loci are associated with HPE in humans, suggesting that defects in other pathways contribute to this disease. Although the TGIF1 (TG-interacting factor) gene maps to the HPE4 locus, and heterozygous loss of function TGIF1 mutations are associated with HPE, mouse models have not yet explained how loss of Tgif1 causes HPE. Using a conditional Tgif1 allele, we show that mouse embryos lacking both Tgif1 and the related Tgif2 have HPE-like phenotypes reminiscent of Shh embryos. Eye and nasal field separation is defective, and forebrain patterning is disrupted in embryos lacking both Tgifs. Early anterior patterning is relatively normal, but expression of Shh is reduced in the forebrain, and Gli3 expression is up-regulated throughout the neural tube. Gli3 acts primarily as an antagonist of Shh function, and the introduction of a heterozygous Gli3 mutation into embryos lacking both Tgif genes partially rescues Shh signaling, nasal field separation, and HPE. Tgif1 and Tgif2 are transcriptional repressors that limit Transforming Growth Factor β/Nodal signaling, and we show that reducing Nodal signaling in embryos lacking both Tgifs reduces the severity of HPE and partially restores the output of Shh signaling. Together, these results support a model in which Tgif function limits Nodal signaling to maintain the appropriate output of the Shh pathway in the forebrain. These data show for the first time that Tgif1 mutation in mouse contributes to HPE pathogenesis and provide evidence that this is due to disruption of the Shh pathway.

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Defective separation of facial features.(A) Frontal forebrain images of stage matched control, cdKO and Gli3+/r;cdKO embryos analyzed by in situ hybridization for Pax7. (B) Side and ventral views of embryos analyzed for Pax2 expression are shown. The Gli3+/r;cdKO embryos shown in A and B are representative of 7 and 4 embryos respectively, other images are representative of at least 3. Scale bars: 250 µm for Pax2 and Pax7 side view, and 200 µm for Pax2 ventral view.
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pgen-1002524-g006: Defective separation of facial features.(A) Frontal forebrain images of stage matched control, cdKO and Gli3+/r;cdKO embryos analyzed by in situ hybridization for Pax7. (B) Side and ventral views of embryos analyzed for Pax2 expression are shown. The Gli3+/r;cdKO embryos shown in A and B are representative of 7 and 4 embryos respectively, other images are representative of at least 3. Scale bars: 250 µm for Pax2 and Pax7 side view, and 200 µm for Pax2 ventral view.

Mentions: To further characterize ventral structure, we analyzed the expression pattern of Pax7, a nasal field marker, as well as the eye field marker, Pax2[61]. Normally by 10.0 dpc, the nasal field is well separated as evidenced by the position of the ventral neuroepithelium clearly separating the facial field (see Figure 1D, for example). In Shh embryos, Pax7 expression is present in a single central region suggesting that the nasal field is not fully separated, whereas when the dose of Gli3 is reduced in Shh embryos Pax7 expression becomes separated to the two nasal fields [61]. In cdKO embryos, Pax7 expression was observed as a single continuous band, suggesting that nasal field separation is defective (Figure 6A). In Gli3+/r;cdKO embryos, Pax7 expression was clearly well separated and was more similar to that seen in controls, suggesting that the nasal field separation defect is partially rescued in Gli3 heterozygous cdKO embryos (Figure 6A). Similarly, Pax2 expression was reduced and was seen as a single continuous band in cdKO embryos, suggesting that eye field separation is defective (Figure 6B). In Gli3+/r;cdKO embryos, the Pax2 expression level was increased, and appeared as less of a continuous band with distinct eye fields on both sides of the forebrain (Figure 6B). These results suggest that the increase in Gli3 expression, and the altered balance between Gli3 and Shh contribute to the HPE phenotype seen in cdKO embryos resulting in a disruption of the separation of facial primordia.


Loss of Tgif function causes holoprosencephaly by disrupting the SHH signaling pathway.

Taniguchi K, Anderson AE, Sutherland AE, Wotton D - PLoS Genet. (2012)

Defective separation of facial features.(A) Frontal forebrain images of stage matched control, cdKO and Gli3+/r;cdKO embryos analyzed by in situ hybridization for Pax7. (B) Side and ventral views of embryos analyzed for Pax2 expression are shown. The Gli3+/r;cdKO embryos shown in A and B are representative of 7 and 4 embryos respectively, other images are representative of at least 3. Scale bars: 250 µm for Pax2 and Pax7 side view, and 200 µm for Pax2 ventral view.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285584&req=5

pgen-1002524-g006: Defective separation of facial features.(A) Frontal forebrain images of stage matched control, cdKO and Gli3+/r;cdKO embryos analyzed by in situ hybridization for Pax7. (B) Side and ventral views of embryos analyzed for Pax2 expression are shown. The Gli3+/r;cdKO embryos shown in A and B are representative of 7 and 4 embryos respectively, other images are representative of at least 3. Scale bars: 250 µm for Pax2 and Pax7 side view, and 200 µm for Pax2 ventral view.
Mentions: To further characterize ventral structure, we analyzed the expression pattern of Pax7, a nasal field marker, as well as the eye field marker, Pax2[61]. Normally by 10.0 dpc, the nasal field is well separated as evidenced by the position of the ventral neuroepithelium clearly separating the facial field (see Figure 1D, for example). In Shh embryos, Pax7 expression is present in a single central region suggesting that the nasal field is not fully separated, whereas when the dose of Gli3 is reduced in Shh embryos Pax7 expression becomes separated to the two nasal fields [61]. In cdKO embryos, Pax7 expression was observed as a single continuous band, suggesting that nasal field separation is defective (Figure 6A). In Gli3+/r;cdKO embryos, Pax7 expression was clearly well separated and was more similar to that seen in controls, suggesting that the nasal field separation defect is partially rescued in Gli3 heterozygous cdKO embryos (Figure 6A). Similarly, Pax2 expression was reduced and was seen as a single continuous band in cdKO embryos, suggesting that eye field separation is defective (Figure 6B). In Gli3+/r;cdKO embryos, the Pax2 expression level was increased, and appeared as less of a continuous band with distinct eye fields on both sides of the forebrain (Figure 6B). These results suggest that the increase in Gli3 expression, and the altered balance between Gli3 and Shh contribute to the HPE phenotype seen in cdKO embryos resulting in a disruption of the separation of facial primordia.

Bottom Line: Early anterior patterning is relatively normal, but expression of Shh is reduced in the forebrain, and Gli3 expression is up-regulated throughout the neural tube.Tgif1 and Tgif2 are transcriptional repressors that limit Transforming Growth Factor β/Nodal signaling, and we show that reducing Nodal signaling in embryos lacking both Tgifs reduces the severity of HPE and partially restores the output of Shh signaling.Together, these results support a model in which Tgif function limits Nodal signaling to maintain the appropriate output of the Shh pathway in the forebrain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics and Center for Cell Signaling, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT
Holoprosencephaly (HPE) is a severe human genetic disease affecting craniofacial development, with an incidence of up to 1/250 human conceptions and 1.3 per 10,000 live births. Mutations in the Sonic Hedgehog (SHH) gene result in HPE in humans and mice, and the Shh pathway is targeted by other mutations that cause HPE. However, at least 12 loci are associated with HPE in humans, suggesting that defects in other pathways contribute to this disease. Although the TGIF1 (TG-interacting factor) gene maps to the HPE4 locus, and heterozygous loss of function TGIF1 mutations are associated with HPE, mouse models have not yet explained how loss of Tgif1 causes HPE. Using a conditional Tgif1 allele, we show that mouse embryos lacking both Tgif1 and the related Tgif2 have HPE-like phenotypes reminiscent of Shh embryos. Eye and nasal field separation is defective, and forebrain patterning is disrupted in embryos lacking both Tgifs. Early anterior patterning is relatively normal, but expression of Shh is reduced in the forebrain, and Gli3 expression is up-regulated throughout the neural tube. Gli3 acts primarily as an antagonist of Shh function, and the introduction of a heterozygous Gli3 mutation into embryos lacking both Tgif genes partially rescues Shh signaling, nasal field separation, and HPE. Tgif1 and Tgif2 are transcriptional repressors that limit Transforming Growth Factor β/Nodal signaling, and we show that reducing Nodal signaling in embryos lacking both Tgifs reduces the severity of HPE and partially restores the output of Shh signaling. Together, these results support a model in which Tgif function limits Nodal signaling to maintain the appropriate output of the Shh pathway in the forebrain. These data show for the first time that Tgif1 mutation in mouse contributes to HPE pathogenesis and provide evidence that this is due to disruption of the Shh pathway.

Show MeSH
Related in: MedlinePlus