| In vitro: |
| Dev Biol. 2015 Feb 15;398(2):163-76. | | Ecdysone regulates morphogenesis and function of Malpighian tubules in Drosophila melanogaster through EcR-B2 isoform.[Pubmed: 25476260] | Malpighian tubules are the osmoregulatory and detoxifying organs of Drosophila and its proper development is critical for the survival of the organism. They are made up of two major cell types, the ectodermal principal cells and mesodermal stellate cells. The principal and stellate cells are structurally and physiologically distinct from each other, but coordinate together for production of isotonic fluid. Proper integration of these cells during the course of development is an important pre-requisite for the proper functioning of the tubules.
METHODS AND RESULTS:
We have conclusively determined an essential role of ecdysone hormone in the development and function of Malpighian tubules. Disruption of ecdysone signaling interferes with the organization of principal and stellate cells resulting in malformed tubules and early larval lethality. Abnormalities include reduction in the number of cells and the clustering of cells rather than their arrangement in characteristic wild type pattern. Organization of F-actin and β-tubulin also show aberrant distribution pattern. Malformed tubules show reduced uric acid deposition and altered expression of Na(+)/K(+)-ATPase pump.
CONCLUSIONS:
B2 isoform of ecdysone receptor is critical for the development of Malpighian tubules and is expressed from early stages of its development. | | Insect Mol Biol. 2014 Jun;23(3):341-56. | | Ecdysone response elements in the distal promoter of the Bombyx Broad-Complex gene, BmBR-C.[Pubmed: 24576019] |
METHODS AND RESULTS:
As determined by a luciferase assay, the transcriptional activity of Pdist, but not Pprox, was activated by Ecdysone. Further analyses using reporters driven by sequential deletion Pdist mutants indicated that two regions, Ecdysone responsive element (EcRE)-D and EcRE-P, -4950 bp and -3480 bp upstream from the distal transcription start site, respectively, were important in the responsiveness of Pdist to 20-hydroxyEcdysone (20E); however, no significant sequence similarities were found between the canonical EcRE and the EcRE-D or EcRE-P regions. Electrophoretic mobility shift assays showed that both the EcRE-D and -P sequences specifically bound to Bombyx protein(s). Sequence analyses and competition assays suggested that the protein(s) bound to EcRE-P might include components other than the Ecdysone receptor (EcR), suggesting that BmBR-C transcription was indirectly activated by Ecdysone through the EcRE-P. Remarkably, protein binding to the mid-region of the EcRE-D, EcRE-Db, was competitively inhibited by an oligonucleotide containing the Drosophila hsp27 EcRE sequence. Furthermore, an anti-EcR antibody interfered with the formation of the protein-EcRE-Db complex.
CONCLUSIONS:
These results indicated that a functional Bombyx Ecdysone receptor binds to EcRE-D and activates the expression of BmBR-C. |
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