TC012545
Fly Ortholog:
Expanded
Student Name:
Bethany Williams & Nelchi Prashali
School Name:
Miami University
Year of Study:
2018
Gene Information:
Promotes Hippo signaling by physically interacting with other components of the pathway, which inhibits growth. Independent of the pathway, it also controls growth through sequestration of Yorkie in the cytoplasm.
NCBI Link:
iBeetle Link:
Tribolium Transcript: 5' UTR:
ACCGCTCCAGAGTTGAAATAGGGAATTTAGTCGAAGAACACCTTTGCCGGTTGATAGAGACGGCGCTCTCTGGCTCTGCGAAAGCACGTGTGTGGTGCCTGTGGGTGCTCTGTGTTGTGAATTCACCCGAATTGGGAATTGCGCCGGAATGGGGCTGCGGAGGGACGGTTTTTCGGATTTTTCTTTCGGCACATGGTGAGCGGGCAACCCCCTTGAAGAGATGAGCGGGTC
Tribolium Transcript: CDS:
ATGCGCGCCGGCCTGTGCACGGTAAGCGCCCCTCTGGCCGCGTGCAGCCCGGCAGCAGCGCTACCCCCGTCGGCTCGCTTCTTGGCCCTGCATTTGCTGCACCAACAAACCCTCTACTTCATCGTACAGGCCAAATCCAGGGTCAGGGAACTGTACCAGCAGACATGCCTACACTTCGCCCAACAGGGGATGCTCGACACCGACTTGTTCGGGCTGGCCCTCATCTGCGATGGCGAGTACCTGTTCGCCGAGCTCGATAGTAAGCTCTCAAAGTACGCGCCCAAAAGTTGGCGTTCGTCGCACAGCCATGGCTTGGACGCAAACGGGCGTCCGGCCCTCGCCTTCTACTTCCGCGTCCAGTTCTACGTGGACAGCCCTCTCCTCCTGCGCGATGAAACGACCCGCCACCACTACTACCTTCAGCTCCGTCTTAACGCTTCTTCTGGAGCCGCTGGCGACGTCGCCATGCACCTGGCCGGTCTGGCCCTCCAGGCGGACCTCGGCGACCGAACCGACTCCACCACGTTCCGGCCCGAGGACTACGTCCCGCCGAACCTACGCGGGCCCGCTGCTCTGCGCACCATCCAGGTGCTGCACCGCTCCCACCGCGGGCTGTCCAAGACCGAGGCGCGGTCCCGGTTCATAACGGAGGCCTGCCAGCTGGAGGAGCCCGTCAACGCGCACGTCTTCCGCCTGCGGGCCTCCAAGGCCGAGCAGGCGCCCGGCTCCATCCTCCTGGCCGTCTGCGCCAAGGGGCTGAAGGTCTGCCCCGACAACAACCCGCCCTCGATCTTCCTCTGGAGCTCCATCGGCAAGCTCAGCTTCGAGCGCAAGAAGTTCGAGATACGGACGGGGCACGAGAAGCTGACGCTGTACACCAGCAGCGACGAGAAGAGCCGCCTGCTGCTGGCGCTGTGCAAGGCGGCGCATCTGTTCAGCATGGCCGTGGCACCGAGGCTCAGCGAGGCGCGCCGGGAGGAGGAGGAGAAGCAGCGATGGGACTGCGATCAGAGGATTTCGGTCATTTCGAGCACCTCGTCGAACACGACGTCGGGGATTGTCAGCGACCGGGTGCACAGCGAGGACGAGTTGGAGATTATGATCACGAGTCCGCCGGCACCTTCTACGGAGAGTTTGGCTCTGGCTCACCTGCTGGACAGTGCGCCAGCTAGCAGCCGAACCTCGGCGGCGTCCGCGACGGCTGCGCTTTCCGCACTCTCGCTCAAGGAGGACGAGGAAGAGGGCACCAAGCCGGCGTGCAGCGAGAGTTCGGCCAAAACGGCCGCGAGCGGGAACAAGTGTGCAGGATCGCAATGCAGCTCGTCGTGCAGTACGGTCGTCGTGACGCCGGTGCAAAATAAAACCAAAGGCCGCAGACCCTCAACCAGCAGCAGCCTCGAACTCGGTTACTCCCACACGGCCCAAAACTCAGCCGTCAGTGACGCCACCTGCATCGAGCTCGACACCCGCGAGCCCGTCTACACCTCGGGCCCGGCGCCCAGCAGCCACACGAGCGGCGTCTACACCCTCACCTCCAGCGACCAGTACACCCTAAGCTCGGACCAGTTCGCCAGTCAGAGCGACCAAGTGGACGGCCCCTTCAGAGACAGGTCCAACTCCAATGTGAGCAACTCGGGCTCGTTCCACGGCGATGGCAGCGACCCCACCGACAACAAACAAGCGCTGCTGTCGGCCGAGGAACTCTCCGACCTGATTGTGGGCCGCTACCCCAGCTGCAAAAGCGTCAGCCATACGCTGGATTCGGACTCTGATTACGTGACCCTTCCGTCGAGTTATCAGGGCTACGTCCCGGTGCCCCCTAAGCGGATCGACAGCGCGGCGGAGCAGAGATTCAGGCCCCCGCCGCCCCCATACGGGGCCCAATTGGTCAAGACGCCGCCAACTGAAGCCACAGAGTTTGACGACCAGAGTTTTGTCTCGGAGTTGTCGCGGGAGCCGCCCCCGTACCCCGAAAACCCGACCCCGGTACCACCCCCAGTGTACCCCTCCGAGGAGGCGGCAGCCCGGTTCATAACCACCCGATCCCCGAGTATCCTAACAGCGCATGCAAGCAGTGTTGGAGCGTCTTCAGCGAGTTATTCAACCCCAAGTTCCGTTCCTCCAATCCCCCCCAAAATCCCACGTGGGGTGCCTCCAATCAATGCCATTATCACACCAAATAATTATCTGGACGTTGCTGCTAGCAAAGCCAGTGTTTTGGTCCCTTGTACCTTCCTCCCACCTCCACCTCCTGCCCCCAGACAACCACCGCCGCCGCCCCCAGCCTTGGCCACGGTCTACACCAGCCAGCTGTCCCGCTCCCAAATCGAACAATACAAACAACAAATGTACAGTGACGTTGATTTCGTCATGTTCCCACTGAAGGAACCAGCAATCAGCAAACAGGAATATCTGGAAGCTAAGCTGGCAGCACTGGTCCAAACCCAACCGGTGTTTTACCGCAGCACCTCCTACTTGGGCCGTTACGCCTCCAGCCAGAACCTCTCCGACACTTACGTGCAGCTCCCGGTGTACGGGGGGCTGGAGCCGCCGCCGCCGCCGCTGCCCCCGAACCGCCCGCGGTTCATGCGGACGCGCTCCGACGACAATATCTTGAATTCGCTGGACGCGCCGCAGCCGCCGTTCCGGAGGCGGCTGCCGCCGCCGCCGCCCGTCGAGGAGAAGCCCAAGTGCGAGGCGAAAGCCGAGTGTGACAAGGGGTTGGATATTCGTACTTTGAGGGAAAAGAGCAAGAAAATGGACTTGCCGCTGATTTCCGCGTTGTGCAATGATCGGTCTCTGATAAAGCAGACGAAAGCGTTCGTGGTTCCGAAGCATCCGGGCGAGGTGACGCCGCGATCGTTGACTAGCACGCGAGCGAAGTACCCAGTGTCGGGCCTGAGCAGCACCAGGATCAGCAAGACCACGAGGAAGATGCCGTCGATCAGCCACCGGCACCCCGGGGACAAGCTGCCGGAGATCCCGAGGGCGCAGCCGAACAATTACGTGACGGATCCGCACGCCAAACACAAAACTATGCAGTCACATTCGTGA
Tribolium Transcript: 3' UTR:
ACTGAGCGTTCCAGTGCTAGAGTAGAGGAATTGTTATACTCAGGGTGCCATCATTGCAATATGTTAGGTTTAGGAAGGGGCCGATCTTTTGGGCAAACGAGGGCTGGTTACGCATTTTGACGAAATATCGGTGCCTTTTGCCATCCCTGTATGTACATAAATGGTCATTTTTTAATACCGCACGTAAGTTTACAGTACATAATGTATGTTTAGCAATAATGTATACACTTGCTCAACTTTTATTATTAAAATAATTATATTTTTAACTTTGTTTTATGTTTTTTTATTTATTTCAAGCATCCGAAATTCATTCAAAGCTTCAATCAAATGTACGGTGCTGCCTTCATTTTAAAAATAAAGCAATCTCCACAAAA
Tribolium Protein:
MRAGLCTVSAPLAACSPAAALPPSARFLALHLLHQQTLYFIVQAKSRVRELYQQTCLHFAQQGMLDTDLFGLALICDGEYLFAELDSKLSKYAPKSWRSSHSHGLDANGRPALAFYFRVQFYVDSPLLLRDETTRHHYYLQLRLNASSGAAGDVAMHLAGLALQADLGDRTDSTTFRPEDYVPPNLRGPAALRTIQVLHRSHRGLSKTEARSRFITEACQLEEPVNAHVFRLRASKAEQAPGSILLAVCAKGLKVCPDNNPPSIFLWSSIGKLSFERKKFEIRTGHEKLTLYTSSDEKSRLLLALCKAAHLFSMAVAPRLSEARREEEEKQRWDCDQRISVISSTSSNTTSGIVSDRVHSEDELEIMITSPPAPSTESLALAHLLDSAPASSRTSAASATAALSALSLKEDEEEGTKPACSESSAKTAASGNKCAGSQCSSSCSTVVVTPVQNKTKGRRPSTSSSLELGYSHTAQNSAVSDATCIELDTREPVYTSGPAPSSHTSGVYTLTSSDQYTLSSDQFASQSDQVDGPFRDRSNSNVSNSGSFHGDGSDPTDNKQALLSAEELSDLIVGRYPSCKSVSHTLDSDSDYVTLPSSYQGYVPVPPKRIDSAAEQRFRPPPPPYGAQLVKTPPTEATEFDDQSFVSELSREPPPYPENPTPVPPPVYPSEEAAARFITTRSPSILTAHASSVGASSASYSTPSSVPPIPPKIPRGVPPINAIITPNNYLDVAASKASVLVPCTFLPPPPPAPRQPPPPPPALATVYTSQLSRSQIEQYKQQMYSDVDFVMFPLKEPAISKQEYLEAKLAALVQTQPVFYRSTSYLGRYASSQNLSDTYVQLPVYGGLEPPPPPLPPNRPRFMRTRSDDNILNSLDAPQPPFRRRLPPPPPVEEKPKCEAKAECDKGLDIRTLREKSKKMDLPLISALCNDRSLIKQTKAFVVPKHPGEVTPRSLTSTRAKYPVSGLSSTRISKTTRKMPSISHRHPGDKLPEIPRAQPNNYVTDPHAKHKTMQSHS
Forward Primer 1:
GTTCTACGTGGACAGCCCTC
Reverse Primer 1:
CCTCCGTTATGAACCGGGAC
Forward Primer 2:
ATTACGTGACCCTTCCGTCG
Reverse Primer 2:
GAGGAACGGAACTTGGGGTT
Gel Picture:
Cloned Fragment:
ATTACGTGACCCTTCCGTCGAGTTATCAGGGCTACGTCCCGGTGCCCCCTAAGCGGATCGACAGCGCGGCGGAGCAGAGATTCAGGCCCCCGCCGCCCCCATACGGGGCCCAATTGGTCAAGACGCCGCCAACTGAAGCCACAGAGTTTGACGACCAGAGTTTTGTCTCGGAGTTGTCGCGGGAGCCGCCCCCGTACCCCGAAAACCCGACCCCGGTACCACCCCCAGTGTACCCCTCCGAGGAGGCGGCAGCCCGGTTCATAACCACCCGATCCCCGAGTATCCTAACAGCGCATGCAAGCAGTGTTGGAGCGTCTTCAGCGAGTTATTCAACCCCAAGTTCCGTTCCTC
Injection concentration:
1ug/ul
Number Injected:
18
Number Survived to Adult:
7
Number w/ RNAi Phenotype:
14 (including pupae)
Percent Penetrance:
82.4
RNAi Phenotype Description:
Pupae had large and/or misshapen wings. In the beetles that survived to adulthood, one had reduced segmentation on two of its tarsi and an extra claw. Femurs were larger in the RNAi beetles than in wild type beetles.
RNAi Phenotype/Assay Images:
Discussion:
Similar to the results seen in expanded mutants in Drosophila melanogaster, our beetles exhibited wing overgrowth, swollen legs, and reduced segmentation. Interestingly, we also observed an extra tarsal claw in one of the adults. In D. melanogaster, there was also loss of crossvein in the wings, hyperplasia in the wing discs, and misshapen wings due to elongated cells in the wings (Boedigheimer and Laughon 1993), however we did not examine wing morphology in Tribolium castaneum. Expectedly, the role of Expanded in both T. castaneum and D. melanogaster seems to be conserved. The Hippo pathway, which is responsible for controlling organ size and growth, is highly conserved in vertebrates and when deregulated can cause cancer (Halder and Johnson 2011). The Hippo pathway controls growth through its effector, Yorkie. A signal transducing complex comprised of Expanded, Merlin, and Kibra, promotes the Hippo pathway. Expanded also regulates organ size and growth independent of the Hippo pathway through directly sequestering Yorkie in the cytoplasm (Halder and Johnson 2011). When the Hippo pathway is on, Salvador activates Hippo and Hippo activates Mats which in turn activates Warts forming the core of the pathway. When the core kinase is on, Yorkie is phosphorylated and sequestered in the cytoplasm where it is degraded; this prevents it from entering the nucleus and transcribing its target genes such as: myc, cyclin E, diap1, all of which promote growth by forming complexes with transcription factors. In humans, overexpression of Yap/Taz, mammalian orthologs of Yorkie, is also observed in many cancers (Harvey et al 2013), suggesting that the Hippo pathway is conserved between arthropods and vertebrates. In conclusion, our hypothesis that ex is required for normal shape and size of the wings, elytra, and legs of the T. castaneum was correct. Studying in other arthropods and other invertebrates will shed more light on the functional conservation of Expanded and increase our knowledge of how organ size and development are controlled by this gene.
References
Boedigheimer M, Laughon A (1993) Expanded: a gene involved in the control of cell proliferation in imaginal discs Development 118:1291-1301
Halder G, Johnson R (2011) Hippo signaling: growth control and beyond Development 138:9-22
Harvey, K., Zhang, X. and Thomas, D. (2013) The Hippo pathway and human cancer Nature Reviews Cancer 13: 246-257
Notebook:
Annotated Genbank File:
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