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Yeast-based assay identifies novel Shh/Gli target genes in vertebrate development.

Milla LA, Cortés CR, Hodar C, Oñate MG, Cambiazo V, Burgess SM, Palma V - BMC Genomics (2012)

Bottom Line: Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates.Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches.We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Sciences, Universidad de Chile, Santiago, Chile.

ABSTRACT

Background: The increasing number of developmental events and molecular mechanisms associated with the Hedgehog (Hh) pathway from Drosophila to vertebrates, suggest that gene regulation is crucial for diverse cellular responses, including target genes not yet described. Although several high-throughput, genome-wide approaches have yielded information at the genomic, transcriptional and proteomic levels, the specificity of Gli binding sites related to direct target gene activation still remain elusive. This study aims to identify novel putative targets of Gli transcription factors through a protein-DNA binding assay using yeast, and validating a subset of targets both in-vitro and in-vivo. Testing in different Hh/Gli gain- and loss-of-function scenarios we here identified known (e.g., ptc1) and novel Hh-regulated genes in zebrafish embryos.

Results: The combined yeast-based screening and MEME/MAST analysis were able to predict Gli transcription factor binding sites, and position mapping of these sequences upstream or in the first intron of promoters served to identify new putative target genes of Gli regulation. These candidates were validated by qPCR in combination with either the pharmacological Hh/Gli antagonist cyc or the agonist pur in Hh-responsive C3H10T1/2 cells. We also used small-hairpin RNAs against Gli proteins to evaluate targets and confirm specific Gli regulation their expression. Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates. Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches. We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo.

Conclusion: A recently described yeast-based screening allowed us to identify new Hh/Gli target genes, functionally important in different contexts of vertebrate embryonic development.

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sfrp2 RNA levels are disturbed in Hh pathway mutants and after pharmacological treatments. (A, A'') In WT embryos, sfrp2 expression is evident in adaxial cells. This expression is significantly down-regulated in yot embryos (B, B''). Pharmacological treatments with pur and cyc show altered expression in pharyngeal arches (asterisk in E, compare C, D and E for different levels of RNA). Anterior is towards left, lateral views are shown. Full lateral views of embryos are shown in A' and B'.
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Figure 6: sfrp2 RNA levels are disturbed in Hh pathway mutants and after pharmacological treatments. (A, A'') In WT embryos, sfrp2 expression is evident in adaxial cells. This expression is significantly down-regulated in yot embryos (B, B''). Pharmacological treatments with pur and cyc show altered expression in pharyngeal arches (asterisk in E, compare C, D and E for different levels of RNA). Anterior is towards left, lateral views are shown. Full lateral views of embryos are shown in A' and B'.

Mentions: The sfrp2 gene encodes a protein that binds Wnt ligands through a cystein-rich domain (CRD). It has been implicated in both antagonism of Wnt signaling [20] and beta-catenin stabilization [21]. ISH analysis verified that sfrp2 transcripts can be detected in adaxial cells, pectoral fins and branchial arches by the 48 hpf stage, as previously reported [22]. Adaxial cell expression is absent in yot mutants, and expression is also regionally lost in the CNS (Figure 6B, B'''). Pectoral fins do not display any changes in sfrp2 expression. This is consistent with the reported alterations in myogenesis in yot mutants, where slow muscle genesis is affected [23]. While it is known that a gradient of Wnt signaling opposes the Shh signaling gradient in the neural tube, regulation of sfrp2 in the brain by Shh has not previously been demonstrated.


Yeast-based assay identifies novel Shh/Gli target genes in vertebrate development.

Milla LA, Cortés CR, Hodar C, Oñate MG, Cambiazo V, Burgess SM, Palma V - BMC Genomics (2012)

sfrp2 RNA levels are disturbed in Hh pathway mutants and after pharmacological treatments. (A, A'') In WT embryos, sfrp2 expression is evident in adaxial cells. This expression is significantly down-regulated in yot embryos (B, B''). Pharmacological treatments with pur and cyc show altered expression in pharyngeal arches (asterisk in E, compare C, D and E for different levels of RNA). Anterior is towards left, lateral views are shown. Full lateral views of embryos are shown in A' and B'.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285088&req=5

Figure 6: sfrp2 RNA levels are disturbed in Hh pathway mutants and after pharmacological treatments. (A, A'') In WT embryos, sfrp2 expression is evident in adaxial cells. This expression is significantly down-regulated in yot embryos (B, B''). Pharmacological treatments with pur and cyc show altered expression in pharyngeal arches (asterisk in E, compare C, D and E for different levels of RNA). Anterior is towards left, lateral views are shown. Full lateral views of embryos are shown in A' and B'.
Mentions: The sfrp2 gene encodes a protein that binds Wnt ligands through a cystein-rich domain (CRD). It has been implicated in both antagonism of Wnt signaling [20] and beta-catenin stabilization [21]. ISH analysis verified that sfrp2 transcripts can be detected in adaxial cells, pectoral fins and branchial arches by the 48 hpf stage, as previously reported [22]. Adaxial cell expression is absent in yot mutants, and expression is also regionally lost in the CNS (Figure 6B, B'''). Pectoral fins do not display any changes in sfrp2 expression. This is consistent with the reported alterations in myogenesis in yot mutants, where slow muscle genesis is affected [23]. While it is known that a gradient of Wnt signaling opposes the Shh signaling gradient in the neural tube, regulation of sfrp2 in the brain by Shh has not previously been demonstrated.

Bottom Line: Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates.Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches.We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Sciences, Universidad de Chile, Santiago, Chile.

ABSTRACT

Background: The increasing number of developmental events and molecular mechanisms associated with the Hedgehog (Hh) pathway from Drosophila to vertebrates, suggest that gene regulation is crucial for diverse cellular responses, including target genes not yet described. Although several high-throughput, genome-wide approaches have yielded information at the genomic, transcriptional and proteomic levels, the specificity of Gli binding sites related to direct target gene activation still remain elusive. This study aims to identify novel putative targets of Gli transcription factors through a protein-DNA binding assay using yeast, and validating a subset of targets both in-vitro and in-vivo. Testing in different Hh/Gli gain- and loss-of-function scenarios we here identified known (e.g., ptc1) and novel Hh-regulated genes in zebrafish embryos.

Results: The combined yeast-based screening and MEME/MAST analysis were able to predict Gli transcription factor binding sites, and position mapping of these sequences upstream or in the first intron of promoters served to identify new putative target genes of Gli regulation. These candidates were validated by qPCR in combination with either the pharmacological Hh/Gli antagonist cyc or the agonist pur in Hh-responsive C3H10T1/2 cells. We also used small-hairpin RNAs against Gli proteins to evaluate targets and confirm specific Gli regulation their expression. Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates. Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches. We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo.

Conclusion: A recently described yeast-based screening allowed us to identify new Hh/Gli target genes, functionally important in different contexts of vertebrate embryonic development.

Show MeSH