Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Room: Virginia A
Teomie Rivera-Miranda - University of Puerto Rico Rio Piedras
Co-Author(s): Joseph Hanly, George Washington University, Washington, DC; Jose Juan Hermina-Perez, George Washington University, Washington, DC; Christopher R. Day, George Washington University, Washington, DC; Riccardo Papa, University of Puerto Rico Rio Piedras, San Juan, PR; Arnaud Martin, George Washington University, Washington, DC
Frizzled (fz) genes encode receptors of the Wnt signaling pathway and play important developmental roles, such as head and leg formation, in all animals. In insects, both frizzled receptors and Wnt ligands have been shown to pattern embryos, and the Wnt pathway is also associated with the formation of color patterns at various life stages. We chose the butterfly Vanessa cardui to study the expression and function of frizzled receptors during various developmental processes. The roles of fz1, fz2, and fz4 have been specified for Tribolium, but no specific function for fz3 has been described (Beerman et al., 2011). We hypothesized, that fz1, fz2, and fz4 in butterflies will share function and expression domains with Tribolium at the embryo stage. To address this, we used CRISPR somatic mutagenesis to test the roles of four frizzled receptors fz1-fz4, in developing embryos (26 hours old) and 5th instar larval wing disks of V. cardui and compare their expression patterns using in situ hybridizations (ISH) of wild-type individuals. In situ hybridization results indicated that fz1 is ubiquitously expressed in the embryos, fz2 is segmentally expressed at 26 hours, fz3 seemed weakly expressed on 26 hours embryos but showed a pattern-related expression in the 5th instar caterpillar hindwing, where it marks the preliminary location of blue chevrons. No embryonic expression for fz4 was observed on embryos. CRISPR knockouts of the four fz receptors revealed interesting results even though fz1 and fz3 did not show any apparent effects on embryonic development. fz2 knockout effects ranged from head and mouthpart deformation to completely impeding embryo development. On the other hand, fz4 knockouts interfered with head and limb development. ISH results observed for fz1 coincide with what has been seen in Tribolium and fz4 results are still not clear for both embryos and 5th instar wing disks. As for the knockouts, there are some similarities to other insects? frizzled functions but there were major differences observed, such as the effect of fz4 knockout in V. cardui compared to RNAi results observed in Tribolium. To validate these results and further work will be done to study these four genes on different embryonic stages to see if there is a relation between time and where the gene is expressed. We would also like to work with other butterfly species to compare results, and test the effects of knocking out different combinations of the fz genes: what would happen if we knock out a combination of fz1+fz2 or fz2+fz4 at 22-, 24- and 28-hour embryos? The methods used in this study have been proven to be both important and efficient in understanding gene involvement in developing organisms not only insects, and by further studying these genes, such as frizzled, we will achieve a deeper understanding of their functions and importance to then be able to apply such knowledge to species that impact our lives in a more direct way.
Funder Acknowledgement(s): This study was supported, in part, by the NSF-IOS Award number 1656389 'cis-Regulatory Basis of Butterfly Wing Pattern Evolution' to Riccardo Papa.
Faculty Advisor: Riccardo Papa, firstname.lastname@example.org
Role: For this research, I performed dissections and fixations for in situ hybridizations on two butterfly stages: 26 hours old embryos and 5th instar caterpillar in Vanessa cardui. For the in situ hybridizations, I did the 4-day protocol on both stages. In addition, I performed the CRISPR injections to knockout the genes of interest, frizzled 1-4; I also was involved in the imaging process.