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Xenopus as a model system for the study of GOLPH2/GP73 function: Xenopus GOLPH2 is required for pronephros development.

Li L, Wen L, Gong Y, Mei G, Liu J, Chen Y, Peng T - PLoS ONE (2012)

Bottom Line: The morpholino-mediated knockdown of golph2 results in edema formation.Expression patterns of the transcription factors WT1, Pax2, Pax8, Lim1, GATA3, and HNF1β are also examined in the golph2 knockdown embryos, the expression of WT1 is increased in the glomus and expanded laterally in the pronephric region.We conclude that the deletion of golph2 causes an increase in the expression of WT1, which may promote glomus formation and inhibit pronephric tubule differentiation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

ABSTRACT
GOLPH2 is a highly conserved protein. It is upregulated in a number of tumors and is being considered as an emerging biomarker for related diseases. However, the function of GOLPH2 remains unknown. The Xenopus model is used to study the function of human proteins. We describe the isolation and characterization of Xenopus golph2, which dimerizes and localizes to the Golgi in a manner similar to human GOLPH2. Xenopus golph2 is expressed in the pronephros during early development. The morpholino-mediated knockdown of golph2 results in edema formation. Additionally, Nephrin expression is enhanced in the glomus, and the expression of pronephric marker genes, such as atp1b1, ClC-K, NKCC2, and NBC1, is diminished in the tubules and duct. Expression patterns of the transcription factors WT1, Pax2, Pax8, Lim1, GATA3, and HNF1β are also examined in the golph2 knockdown embryos, the expression of WT1 is increased in the glomus and expanded laterally in the pronephric region. We conclude that the deletion of golph2 causes an increase in the expression of WT1, which may promote glomus formation and inhibit pronephric tubule differentiation.

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The inhibition of golph2 expression causes edema formation. A.Sequences of the two golph2 pseudoalleles and the mRNA used for the rescue experiment. The arrow heads indicates the mismatch in the golph2-MO-targeting site of the second golph2 allele. B. The expression of endogenous golph2 was inhibited by injection with morpholino into the vegetal pole of fertilized eggs. Western blotting analysis was performed at stage 33. Line 1∶56 ng of Control-MO; line 2∶16 ng of golph2-MO; line 3∶32 ng of golph2-MO; line 4∶48 ng of golph2-MO; line 5∶56 ng of golph2-MO. C. The specificity of golph2-MO was examined; 32 ng of golph2-MO was injected along with either 0.5 ng of rescue mRNA (line 2) or 0.5 ng of allele mRNA (line 4). Xenopus golph2 expression was detected in embryos of stage 10 using the antibody 10F12. D. Morphology of Control-MO-injected embryos. E. Morphology of golph2-MO-injected embryos displaying edema at the tadpole stage. F. Quantification of edema formation in embryos injected either with Control-MO (56 ng), golph2-MO (56 ng), golph2-MO (56 ng) with golph2 rescue mRNA (1 ng) or golph2-MO (56 ng) with human GOLPH2 mRNA (1 ng).
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pone-0038939-g005: The inhibition of golph2 expression causes edema formation. A.Sequences of the two golph2 pseudoalleles and the mRNA used for the rescue experiment. The arrow heads indicates the mismatch in the golph2-MO-targeting site of the second golph2 allele. B. The expression of endogenous golph2 was inhibited by injection with morpholino into the vegetal pole of fertilized eggs. Western blotting analysis was performed at stage 33. Line 1∶56 ng of Control-MO; line 2∶16 ng of golph2-MO; line 3∶32 ng of golph2-MO; line 4∶48 ng of golph2-MO; line 5∶56 ng of golph2-MO. C. The specificity of golph2-MO was examined; 32 ng of golph2-MO was injected along with either 0.5 ng of rescue mRNA (line 2) or 0.5 ng of allele mRNA (line 4). Xenopus golph2 expression was detected in embryos of stage 10 using the antibody 10F12. D. Morphology of Control-MO-injected embryos. E. Morphology of golph2-MO-injected embryos displaying edema at the tadpole stage. F. Quantification of edema formation in embryos injected either with Control-MO (56 ng), golph2-MO (56 ng), golph2-MO (56 ng) with golph2 rescue mRNA (1 ng) or golph2-MO (56 ng) with human GOLPH2 mRNA (1 ng).

Mentions: To examine the role of golph2 during Xenopus development, a morpholino was designed to specifically block golph2 translation (Fig. 5A). To test its efficiency, 16 ng to 56 ng of golph2-MO was injected into the vegetal pole of the fertilized eggs. As shown in Fig. 5B, golph2-MO inhibited endogenous expression of golph2 to an undetectable level, even at lower dosages, relative to the Control-MO. Because Xenopus laevis is a pseudotetraploid, a gene may exist as two alleles. A second golph2 allele (NCBI accession number BP696966) that contained two nucleotide mutations in the golph2-MO-targeting site was identified (Fig. 5A, indicated by an arrowheads), and golph2-MO could also efficiently target this second allele (Fig. 5C, compare lanes 3 and 4) but could not target the rescue mRNA (Fig. 5C, compare lanes 1 and 2).


Xenopus as a model system for the study of GOLPH2/GP73 function: Xenopus GOLPH2 is required for pronephros development.

Li L, Wen L, Gong Y, Mei G, Liu J, Chen Y, Peng T - PLoS ONE (2012)

The inhibition of golph2 expression causes edema formation. A.Sequences of the two golph2 pseudoalleles and the mRNA used for the rescue experiment. The arrow heads indicates the mismatch in the golph2-MO-targeting site of the second golph2 allele. B. The expression of endogenous golph2 was inhibited by injection with morpholino into the vegetal pole of fertilized eggs. Western blotting analysis was performed at stage 33. Line 1∶56 ng of Control-MO; line 2∶16 ng of golph2-MO; line 3∶32 ng of golph2-MO; line 4∶48 ng of golph2-MO; line 5∶56 ng of golph2-MO. C. The specificity of golph2-MO was examined; 32 ng of golph2-MO was injected along with either 0.5 ng of rescue mRNA (line 2) or 0.5 ng of allele mRNA (line 4). Xenopus golph2 expression was detected in embryos of stage 10 using the antibody 10F12. D. Morphology of Control-MO-injected embryos. E. Morphology of golph2-MO-injected embryos displaying edema at the tadpole stage. F. Quantification of edema formation in embryos injected either with Control-MO (56 ng), golph2-MO (56 ng), golph2-MO (56 ng) with golph2 rescue mRNA (1 ng) or golph2-MO (56 ng) with human GOLPH2 mRNA (1 ng).
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pone-0038939-g005: The inhibition of golph2 expression causes edema formation. A.Sequences of the two golph2 pseudoalleles and the mRNA used for the rescue experiment. The arrow heads indicates the mismatch in the golph2-MO-targeting site of the second golph2 allele. B. The expression of endogenous golph2 was inhibited by injection with morpholino into the vegetal pole of fertilized eggs. Western blotting analysis was performed at stage 33. Line 1∶56 ng of Control-MO; line 2∶16 ng of golph2-MO; line 3∶32 ng of golph2-MO; line 4∶48 ng of golph2-MO; line 5∶56 ng of golph2-MO. C. The specificity of golph2-MO was examined; 32 ng of golph2-MO was injected along with either 0.5 ng of rescue mRNA (line 2) or 0.5 ng of allele mRNA (line 4). Xenopus golph2 expression was detected in embryos of stage 10 using the antibody 10F12. D. Morphology of Control-MO-injected embryos. E. Morphology of golph2-MO-injected embryos displaying edema at the tadpole stage. F. Quantification of edema formation in embryos injected either with Control-MO (56 ng), golph2-MO (56 ng), golph2-MO (56 ng) with golph2 rescue mRNA (1 ng) or golph2-MO (56 ng) with human GOLPH2 mRNA (1 ng).
Mentions: To examine the role of golph2 during Xenopus development, a morpholino was designed to specifically block golph2 translation (Fig. 5A). To test its efficiency, 16 ng to 56 ng of golph2-MO was injected into the vegetal pole of the fertilized eggs. As shown in Fig. 5B, golph2-MO inhibited endogenous expression of golph2 to an undetectable level, even at lower dosages, relative to the Control-MO. Because Xenopus laevis is a pseudotetraploid, a gene may exist as two alleles. A second golph2 allele (NCBI accession number BP696966) that contained two nucleotide mutations in the golph2-MO-targeting site was identified (Fig. 5A, indicated by an arrowheads), and golph2-MO could also efficiently target this second allele (Fig. 5C, compare lanes 3 and 4) but could not target the rescue mRNA (Fig. 5C, compare lanes 1 and 2).

Bottom Line: The morpholino-mediated knockdown of golph2 results in edema formation.Expression patterns of the transcription factors WT1, Pax2, Pax8, Lim1, GATA3, and HNF1β are also examined in the golph2 knockdown embryos, the expression of WT1 is increased in the glomus and expanded laterally in the pronephric region.We conclude that the deletion of golph2 causes an increase in the expression of WT1, which may promote glomus formation and inhibit pronephric tubule differentiation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Respiratory Disease, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.

ABSTRACT
GOLPH2 is a highly conserved protein. It is upregulated in a number of tumors and is being considered as an emerging biomarker for related diseases. However, the function of GOLPH2 remains unknown. The Xenopus model is used to study the function of human proteins. We describe the isolation and characterization of Xenopus golph2, which dimerizes and localizes to the Golgi in a manner similar to human GOLPH2. Xenopus golph2 is expressed in the pronephros during early development. The morpholino-mediated knockdown of golph2 results in edema formation. Additionally, Nephrin expression is enhanced in the glomus, and the expression of pronephric marker genes, such as atp1b1, ClC-K, NKCC2, and NBC1, is diminished in the tubules and duct. Expression patterns of the transcription factors WT1, Pax2, Pax8, Lim1, GATA3, and HNF1β are also examined in the golph2 knockdown embryos, the expression of WT1 is increased in the glomus and expanded laterally in the pronephric region. We conclude that the deletion of golph2 causes an increase in the expression of WT1, which may promote glomus formation and inhibit pronephric tubule differentiation.

Show MeSH
Related in: MedlinePlus