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MIP/Aquaporin 0 represents a direct transcriptional target of PITX3 in the developing lens.

Sorokina EA, Muheisen S, Mlodik N, Semina EV - PLoS ONE (2011)

Bottom Line: In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation.Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter.Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Children's Research Institute, Medical College of Wisconsin and Children's Hospital of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
The PITX3 bicoid-type homeodomain transcription factor plays an important role in lens development in vertebrates. PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations.

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Analysis of mip1 expression in pitx3-mo and control embryos via in situ hybridization and RT-PCR.A, D, F-H. Normal mip1 expression in control-injected embryos at 29-, 34- and 48-hpf. B, C, E, I–K. Altered mip1 expression is observed in pitx3 morphants at 29-hpf with 64% of embryos demonstrating a complete absence of mip1 expression (B) and the remaining larvae showing markedly reduced mip1 expression (C and I). Reduced mip1 expression is also observed in 34- and 48-hpf embryos (E, J, K). Red arrows show sites of expected mip1 expression. Scale bars: A–E: 100 µM; F–L: 20 µM. L. Results of semi-quantitative RT-PCR showing reduced expression of mip1 in pitx3 morphants at early stages of development (red arrow).
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pone-0021122-g007: Analysis of mip1 expression in pitx3-mo and control embryos via in situ hybridization and RT-PCR.A, D, F-H. Normal mip1 expression in control-injected embryos at 29-, 34- and 48-hpf. B, C, E, I–K. Altered mip1 expression is observed in pitx3 morphants at 29-hpf with 64% of embryos demonstrating a complete absence of mip1 expression (B) and the remaining larvae showing markedly reduced mip1 expression (C and I). Reduced mip1 expression is also observed in 34- and 48-hpf embryos (E, J, K). Red arrows show sites of expected mip1 expression. Scale bars: A–E: 100 µM; F–L: 20 µM. L. Results of semi-quantitative RT-PCR showing reduced expression of mip1 in pitx3 morphants at early stages of development (red arrow).

Mentions: Examination of mip1 expression by in situ hybridization identified a specific and robust expression pattern in 100% of control-mo injected embryos (15/15), while a complete absence (9/14 or 64.3%) or a very low level (5/14 or 35.7%) of mip1 expression was seen in pitx3-mo embryos at 29-hpf (Figure 7A–C, F, I). mip1 expression is clearly observed in both control and pitx3-mo injected embryos at later stages but appears to be somewhat reduced in pitx3 morphants (Figure 7 D, E, G, H, J, K).


MIP/Aquaporin 0 represents a direct transcriptional target of PITX3 in the developing lens.

Sorokina EA, Muheisen S, Mlodik N, Semina EV - PLoS ONE (2011)

Analysis of mip1 expression in pitx3-mo and control embryos via in situ hybridization and RT-PCR.A, D, F-H. Normal mip1 expression in control-injected embryos at 29-, 34- and 48-hpf. B, C, E, I–K. Altered mip1 expression is observed in pitx3 morphants at 29-hpf with 64% of embryos demonstrating a complete absence of mip1 expression (B) and the remaining larvae showing markedly reduced mip1 expression (C and I). Reduced mip1 expression is also observed in 34- and 48-hpf embryos (E, J, K). Red arrows show sites of expected mip1 expression. Scale bars: A–E: 100 µM; F–L: 20 µM. L. Results of semi-quantitative RT-PCR showing reduced expression of mip1 in pitx3 morphants at early stages of development (red arrow).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3117865&req=5

pone-0021122-g007: Analysis of mip1 expression in pitx3-mo and control embryos via in situ hybridization and RT-PCR.A, D, F-H. Normal mip1 expression in control-injected embryos at 29-, 34- and 48-hpf. B, C, E, I–K. Altered mip1 expression is observed in pitx3 morphants at 29-hpf with 64% of embryos demonstrating a complete absence of mip1 expression (B) and the remaining larvae showing markedly reduced mip1 expression (C and I). Reduced mip1 expression is also observed in 34- and 48-hpf embryos (E, J, K). Red arrows show sites of expected mip1 expression. Scale bars: A–E: 100 µM; F–L: 20 µM. L. Results of semi-quantitative RT-PCR showing reduced expression of mip1 in pitx3 morphants at early stages of development (red arrow).
Mentions: Examination of mip1 expression by in situ hybridization identified a specific and robust expression pattern in 100% of control-mo injected embryos (15/15), while a complete absence (9/14 or 64.3%) or a very low level (5/14 or 35.7%) of mip1 expression was seen in pitx3-mo embryos at 29-hpf (Figure 7A–C, F, I). mip1 expression is clearly observed in both control and pitx3-mo injected embryos at later stages but appears to be somewhat reduced in pitx3 morphants (Figure 7 D, E, G, H, J, K).

Bottom Line: In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation.Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter.Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Children's Research Institute, Medical College of Wisconsin and Children's Hospital of Wisconsin, Milwaukee, Wisconsin, United States of America.

ABSTRACT
The PITX3 bicoid-type homeodomain transcription factor plays an important role in lens development in vertebrates. PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations.

Show MeSH
Related in: MedlinePlus