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Evidence for a role of vertebrate Disp1 in long-range Shh signaling.

ABSTRACT: Dispatched 1 (Disp1) encodes a twelve transmembrane domain protein that is required for long-range sonic hedgehog (Shh) signaling. Inhibition of Disp1 function, both by RNAi or dominant-negative constructs, prevents secretion and results in the accumulation of Shh in source cells. Measuring the Shh response in neuralized embryoid bodies (EBs) derived from embryonic stem (ES) cells, with or without Disp1 function, demonstrates an additional role for Disp1 in cells transporting Shh. Co-cultures with Shh-expressing cells revealed a significant reduction in the range of the contact-dependent Shh response in Disp1(-/-) neuralized EBs. These observations support a dual role for Disp1, not only in the secretion of Shh from the source cells, but also in the subsequent transport of Shh through tissue.

SUBMITTER: Etheridge LA

PROVIDER: S-EPMC2796928 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Evidence for a role of vertebrate Disp1 in long-range Shh signaling.

Etheridge L Alton LA Crawford T Quinn TQ Zhang Shile S Roelink Henk H

Development (Cambridge, England) 20100101 1

Dispatched 1 (Disp1) encodes a twelve transmembrane domain protein that is required for long-range sonic hedgehog (Shh) signaling. Inhibition of Disp1 function, both by RNAi or dominant-negative constructs, prevents secretion and results in the accumulation of Shh in source cells. Measuring the Shh response in neuralized embryoid bodies (EBs) derived from embryonic stem (ES) cells, with or without Disp1 function, demonstrates an additional role for Disp1 in cells transporting Shh. Co-cultures wi ...[more]

PMID: 20023168

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

Project description:OBJECTIVE:To investigate the regulatory role of classical Shh signaling pathway in the development of the epithelium and mesenchyme (bronchial cartilage and smooth muscles) during lung development in fetal mice. METHODS:Immunohistochemical technique was used to detect the expression of Shh signaling pathway receptor Smo and Pdgfr-? in murine fetal lungs to explore the spatial and temporal characteristics of their expression. Based on the interstitial specificity of Pdgfr-? expression, we constructed a Pdgfr-?-cre to establish a E12.5 - E16.5 transgenic mice with specific knockout of the key Shh signaling molecule Smo in the pulmonary interstitium with tamoxifen induction. Immunofluorescence technique was used to observe the epithelium and mesenchyme (bronchial cartilage and smooth muscle) during fetal lung development in the transgenic mice to assess the role of Shh signaling pathway in the epithelial-to-mesenchymal (EMT) transition during the lung development. RESULTS:Smo was highly expressed in the epithelial and stromal lung tissues in the pseudoglandular stage and was gradually lowered over time with its distribution mainly in the interstitial tissues. Pdgfr-? was enriched in the distal lung epithelial and mesenchy tissues in early embryonic lungs and gradually migrated to the proximal stroma until becoming concentrated around the main bronchial proximal stroma. We successfully specific established mouse models of specific mesenchymal Smo knockout. Compared with the control group, the transgenic mice during E12.5-E16.5 showed significantly reduced lung the volume and bronchial branching with also decreased expression of the proximal epithelial P63 (P<0.05). The transgenic mice exhibited alterations in the expression of ?-smooth muscle actin with delayed bronchial cartilage development and decreased expression of mucoprotein. CONCLUSION:The temporospatial specific expression of Shh signaling pathway plays an important role in developmental regulation of mouse embryonic lung epithelium and mesenchyme (bronchial cartilage and smooth muscle).

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Evidence for a role of vertebrate Disp1 in long-range Shh signaling.

Publications

Evidence for a role of vertebrate Disp1 in long-range Shh signaling.

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