Abstract
The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these 'in locus' mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis.
Highlights
The Drosophila wing provides an excellent model system for studying the relationship between gene expression and morphogenesis since it is a well-studied, two-dimensional, developmental system that generates invariant morphological features, such as veins and sensory organs (Sturtevant et al, 1993)
As summarized in the introductory section, well-validated CRISPR/Cas9 genome editing tools make it routine to generate genome alterations via random mutagenesis near guide RNA cleavage sites or to create targeted edits based on templates carrying homologous sequences on either side of the gRNA-directed cut site
We utilized two gRNAs directing Cas9 cleavage on each side of a specific region to be deleted, as well as single-stranded oligonucleotides donors with 60 bp of homology to either DNA end flanking the region targeted for deletion
Summary
The Drosophila wing provides an excellent model system for studying the relationship between gene expression and morphogenesis since it is a well-studied, two-dimensional, developmental system that generates invariant morphological features, such as veins and sensory organs (Sturtevant et al, 1993). Secreted morphogens, including Hedgehog (Hh) and Decapentaplegic (Dpp), activate the expression of primary response genes, such as spalt (sal = salm + salr) and optomotor-blind (omb, known as bifid), in broad domains. These patterning genes have been proposed to subsequently induce the formation of vein primordia along their borders by ‘forexport-only-signaling’ mechanisms (reviewed in [Bier, 2000]) and cross-repressive interactions (Al Khatib et al, 2017; Martın et al, 2017)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
