TC014939
Fly Ortholog:
Lim1
Student Name:
Bridget Dames and Sarah Kosse
School Name:
Miami University
Year of Study:
2018
Gene Information:
Lim1 is a member of the LIM- homeodomain (LIM-HD) family and controls differentiation of neural and lymphoid cell. It has been shown in Drosophila melanogaster that Lim1 transcription factor works together with a Chip co-factor to prevent ey expression in the anterior eye disc from spreading to the ventral head and antenna tissue. In Tribolium castaneum, Lim1 has been shown to be involved in the development of the scape-pedicle joint of the antenna and the leg.
NCBI Link:
iBeetle Link:
Tribolium Transcript: 5' UTR:
TTCGGTTGGCAACATTTTTTTCCATGACGTTCTTCATTTGTTTTTGAATCAAGCAAAGTGAGGCGTCCTTTTTAAAACCAAGGTTTTTGTGTAAAAAAACGGTGATTAGAGTGTTTGTAACAATGCGTAGGTGTTAAAATATTAATTATCGAACGATCGCAATGTGTTTTTTGTGAAATACGTCGCAATCTTGCGTTTTTTGTGCACGATAGAAGAGGCAAGGGATGGTCGCTCGAAGTGGAAAAAATAAGGTCTGCCATCGAACCGTCTATTATAAAGAAGCAGAGGCGGTGCTGGGACGAAAACAGAAGAACGTCGGGGAGTTTGGCCATTATCTCTCATGGTCTGAACGCATCAAGACGGCCGGGCACCGGAAAAATCGCCACCGACCGTCACAGAAATCACAAACTCTATAGATAGCTCCAACGTTCCTCAACCAAAAACTTGAAACCATGAGAAAAAATCAACGGTCGGTGCGAGCCAGCCGCCGGT
Tribolium Transcript: CDS:
ATGTTGATATCCTGCGCGGCCTGCGACAAACCAATCCTGGATAAATTCCTGCTAAACGTCCTGGAAAGGACGTGGCATGCGGACTGCGTCCGCTGTTTCGACTGTCACGCCCCCCTAACCGATAAGTGCTTCTCAAGGGAAAACAAACTATTCTGCAGGAACGATTTCTTCAGGCGGTACGGTACAAAGTGCGGTGGTTGTGGGCAAGGCATATCGCCTAGTGACTTAGTGCGAAAAGCGCGCGACAAAGTGTTCCATCTCAATTGTTTCACCTGTCTCGTGTGCAGGAAACAGCTTTCGACCGGTGAAGAACTTTACGTTTTAGACGATAACAAGTTTATTTGCAAAGACGATTACCTCAACGGAAACAAGACGGCCGCCATTACCGGACACCACCACGGTGGGTCCAGCAGTTCCGAATTATCGATATTTAAAGGCCTCGCACCCGTCGCTAGCGTATCGCCGACGTTCAATTTCAAATCAAATTCGTTGATGGGCTCAGCGTCGGAGGACGAAGAAGACGACGACAACAGCACGGCCTCCTCGGTACTAAAACATCACACGTTGCACGGTTCCTTACCCCCAGCCCCCGGGGAAACAAACAATAATTCTTTGCCCCATTCAGACATTAGCAGTTCGATAGGACAGGATTCAAAAACCGGTCAAGACGACTCCGAAGACCAAAACTCCTTCGACGGTGATGCCGAAATGCGGGATTCGCAAACAGAGAATAAAAGTCCTGATGATAGCAGCGCCGGCTCGAAACGTCGCGGCCCTCGAACGACGATCAAAGCGAAACAATTAGAAATACTAAAGACGGCCTTTTCGCAAACGCCGAAACCCACAAGACACATCAGGGAGCAGCTGGCGAAAGAAACAGGCCTGCCTATGAGGGTGATACAGGTGTGGTTCCAGAACAAGCGGTCGAAAGAGCGCCGCTTGAAGCAGCTCACCTCGATGGGGCGCGGCCCCTTTTTCGGGGGCTCGCGCAAAATGCGGGGCTTCCCCATGAACCTGTCCCCCGGCGGCCTCGAAGACGGCCCTCCCGGCTTCCCGTACTTCGCGACCGCCGACGGAAAATTCGAGTTCGGCTACGGGGGGCCCCCGTTCCATCCGCACGACGGCCCCTTCTTCTCCGGCCACCACCCCGGGGGCGGCGGGCCCGGGGGCCAAGGCATGCCCTTCAACCAAGGAGGTGGACCCATGGACCACGGCGGCCCCATGAACATGGCCGGCGATTTCGGGACGATGCCGAACGAGCAAGGCCAGTTCATGCCTCAGCAGCCGGGCCCCGAACAGATGATGCCGGGGAACGGGGGCAGGGGGGGCAGTCCGGAGTTTATGTCGCCGGGGTCCTTCTCCGACAACCCCCAGCAGCTCAACGAAGGGCTCGTTTGGTAG
Tribolium Transcript: 3' UTR:
CTGGCGAAGGCTCTAGCCAAAAGGGCTAGAGAATGATTTATGGTTTGAGTGGTGTAAATAGTGACGAGGACCGGTTGTGACTAAAAACGTACCTCGGTTTTGTGCAATTTTTATTCATAACGTGTGGTTTATTAACGTTCATGTCTTTGCTTTCGGAACACAAAAATTAACACCCTGTGTACATAATTTATTTGTTAACACAAATTTCTGAAAAGTGCCAAGTTGTAGTACAGTGTTGGGCGGGATCGTCGAAGGACAAACCCTTCACCTCCTATCATAACACTTTCCATCCAACACTGTACGATTTTTTTCGTTAATTGTTTTATTTATGTTACACCACAATGTACTATTTATAAATGTATAAAAAACAAATCCTAGCTTCTAAACTGCTTGTACATATTATAAATTCTCGAAGGACGGTTATTATATGTTATGTTTTGACTAGGCCTAGCTGTCGTACAAAATGTTTGTACAGGGACTTGACACGCTGTATAAAAAGCCATCCGATTATATAATTAGTGTATATTGTTAATTTAGGTGTACTATCCAATAAAACAAATTCTCTAAG
Tribolium Protein:
MLISCAACDKPILDKFLLNVLERTWHADCVRCFDCHAPLTDKCFSRENKLFCRNDFFRRYGTKCGGCGQGISPSDLVRKARDKVFHLNCFTCLVCRKQLSTGEELYVLDDNKFICKDDYLNGNKTAAITGHHHGGSSSSELSIFKGLAPVASVSPTFNFKSNSLMGSASEDEEDDDNSTASSVLKHHTLHGSLPPAPGETNNNSLPHSDISSSIGQDSKTGQDDSEDQNSFDGDAEMRDSQTENKSPDDSSAGSKRRGPRTTIKAKQLEILKTAFSQTPKPTRHIREQLAKETGLPMRVIQVWFQNKRSKERRLKQLTSMGRGPFFGGSRKMRGFPMNLSPGGLEDGPPGFPYFATADGKFEFGYGGPPFHPHDGPFFSGHHPGGGGPGGQGMPFNQGGGPMDHGGPMNMAGDFGTMPNEQGQFMPQQPGPEQMMPGNGGRGGSPEFMSPGSFSDNPQQLNEGLVW
Forward Primer 1:
AGGCGGTACGGTACAAAGTG
Reverse Primer 1:
CCGTGCAACGTGTGATGTTT
Forward Primer 2:
CTTCCCCATGAACCTGTCCC
Reverse Primer 2:
ATGAACTGGCCTTGCTCGTT
Gel Picture:
Cloned Fragment:
AGGCGGTACGGTACAAAGTGCGGTGGTTGTGGGCAAGGCATATCGCCTAGTGACTTAGTGCGAAAAGCGCGCGACAAAGTGTTCCATCTCAATTGTTTCACCTGTCTCGTGTGCAGGAAACAGCTTTCGACCGGTGAAGAACTTTACGTTTTAGACGATAACAAGTTTATTTGCAAAGACGATTACCTCAACGGAAACAAGACGGCCGCCATTACCGGACACCACCACGATTCGTTGATGGGCTCAGCGTCGGAGGACGAAGAAGACGACGACAACAGCACGGCCTCCTCGGTACTAAAACATCACACGTTGCACGG
Injection concentration:
1 µg/µl
Number Injected:
15
Number Survived to Adult:
11
Number w/ RNAi Phenotype:
11
Percent Penetrance:
100% for the beetles that survived to adults
Assay Description:
We observed the beetles under light microscope to observe our beetle’s eye, antenna, and leg morphology. We also observed images of the beetles with high resolution microscopy to see detailed changes in the leg and antenna morphology.
RNAi Phenotype Description:
There was no observable change in the eye. However, the RNAi beetles showed a reduced, almost absent coxa, reduced/absent segmentation between the femur and proximal tibia, and only one pre tarsus. The most distal segment of antenna is fused (positive control).
RNAi Phenotype/Assay Images:
Discussion:
In Drosophila melanogaster, Lim1 is involved in restricting the expression of key genes involved in eye tissue development, eyeless (ey) and twin of eyeless (toy), in the correct regions during head development. However, it is important to study Lim1 in other model organisms to provide insight into the possible role of the Lim1 human orthologue in eye tissue development. The role of Lim1 has been studied in the development of the Tribolium castreum antenna, The loss of Lim1 in Tribolium led to fused antenna development at the scape-pedicle joint. Therefore, this phenotype acted as a positive control in our experiments.. Knocking down Lim1 in Tribolium using RNAi Tribolium castaneum demonstrated that there was no observable change in eye development like we saw in Drosophila. However, the antenna was fused at the distal end which indicated that the RNAi was successful. In the RNAi beetles, there were several defects in morphology that differed from that in Drosophila. In the leg, we found a reduced, almost absent coxa, reduced/absent segmentation between the femur and proximal tibia, and only one pretarsus. We later found a paper that had described the function of Lim1 in intermediate leg development which explains the fused femur and tibia. However, the coxa and pretarsus, or claw, were unaffected in their study. For future research we would be particularly interested in analyzing the effect of Lim1 in leg development. For example, understanding why knocking down Lim1 results in loss of one of the pretarsus. Many primitive insects without wings have only one claw/pretarsus. This could indicate that the knockdown of Lim1 reverts the Tribolium phenotype back to the an ancestral state. In addition, since we did not see ectopic eye development, there are other ways we could observe the role of Lim1 in eye development. First, we could analyze the eye function of our RNAi beetle from this study, Second, we could observe the role of Chip in eye development for future research. Chip and Lim1 work closely together in Drosophila to localize eyeless (ey) for eye development. Therefore we could knockdown Chip in Tribolium castaneum and observe eye morphology.
Notebook:
Annotated Genbank File:
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