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Identifier

000437584

Title

Characterization of Distal-less cis-regulatory sequences across pancrustaceans

Alternative Title

Χαρακτηρισμός των cis-ρυθμιστικών αλληλουχιών του γονιδίου Distal-less μεταξύ πανκαρκινοειδών

Author

Ράλλης, Ιωάννης Μ.

Thesis advisor

Παυλόπουλος, Αναστάσιος

Reviewer

Ηλιόπουλος, Ιωάννης
Νικολάου, Χριστόφορος

Abstract

Macroevolutionary comparisons between species which are separated by large evolutionary timescales have focused on addressing and highlighting similarities in developmental genetic programs. One such striking case is the finding that representatives of all Bilaterian clades, use a common genetic toolbox for patterning their outgrowing limbs along the three body axes [anterio-posterior (AP), dorso-ventral (DV), proximo-distal (PD)] (Pueyo and Couso, 2005; Tarazona et al., 2019). So far, comparative developmental studies in species that are not established as model systems were limited to expression analyses of several candidate genes whose role was extensively investigated in the major developmental genetic model organisms. Only rarely, unbiased functional studies have been conducted in a small number of emerging non-model species that have proved to be genetically tractable and amenable to an expanding palette of experimental manipulations. One of them is the crustacean amphipod Parhyale hawaiensis, which we use in the lab to get insights into the decades-old problem of the conservation and divergence of limb patterning mechanisms across the arthropods (Wolff et al., 2018; Pavlopoulos and Wolff, 2020) . During my Master’s thesis, I investigated the genetic basis of radical changes in the developmental timing of limb patterning mechanisms between direct and indirect developing pancrustacean limbs. In particular, I made a comparative study of the cis-regulatory sequences of a very important and highly conserved limb patterning gene, Distal-less (Dll), between the insect Drosophila melanogaster and the crustacean Parhyale hawaiensis. Parhyale has a direct embryonic limb development, whereas Drosophila limb development is indirect over embryonic, larval and pupal development. In this study, I identified putative Dll cis-regulatory elements in Parhyale using ATAC-seq data from Parhyale embryos and phylogenetic foot-printing between the Parhyale and the few other available amphipod genomes. Functional analysis of the identified cis-regulatory elements using reporter constructs revealed an enhancer mediating the early expression of Dll in the Parhyale limb buds. Bioinformatics analyses for overrepresented motifs in the Parhyale Dll enhancer led to the discovery of two putative binding sites for known Dll regulators in Drosophila. In a complementary approach, I tested in Parhyale previously characterized Drosophila Dll enhancers with reporter constructs to investigate their activity pattern and timing of expression. These functional cross-species comparisons of Dll cis-regulatory sequences and their upstream regulators provided some first insights into the conservation and divergence of patterning mechanisms between direct and indirect developing pancrustacean limbs.

Language

English

Subject

ATAC-SEQ

Appendage development

Crustacean

Enhancers

Ανάπτυξη άκρων

Ενισχυτές

Καρκινοειδή

Issue date

2020-11-27