In situ mutational screening and CRISPR interference definecis-regulatory inputs during compartment boundary formation
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
The establishment of tissue axes is fundamental during embryonic development. In thewing, the anterior/posterior (AP) and the dorsal/ventral (DV) compartment boundaries provide the basic coordinates around which the tissue develops. These boundaries arise as a result of two lineage decisions, the acquisition of posterior fate by the selector gene() and dorsal fate by the selector gene(). While theexpression domain is set up during embryogenesis,expression begins only during early wing development. Thus, the correct establishment of theexpression pattern relative tomust be tightly controlled. Here, we functionally investigate the transcriptional inputs integrated by the earlyenhancer (apE) and their requirement for correct boundary formation. Detailed mutational analyses using CRISPR/Cas revealed a role for apE in positioning the DV boundary relative to the AP boundary, with apE mutants often displaying mirror-image anterior wing duplications. We then designed and applied methods to accomplish tissue-specific enhancer disruption via dCas9 expression. This approach allowed us to dissect the spatiotemporal requirement for apE function, clarifying the mechanism by which apE misregulation leads to AP defects. Base-pair-resolution analyses of apE uncovered a single HOX-binding site essential for wing development that, when mutated, led to wingless flies. We demonstrated that the transcription factors Pointed (Pnt), Homothorax (Hth), and Grain (Grn) are required for apE function, an
Abstract
The establishment of tissue axes is fundamental during embryonic development. In thewing, the anterior/posterior (AP) and the dorsal/ventral (DV) compartment boundaries provide the basic coordinates around which the tissue develops. These boundaries arise as a result of two lineage decisions, the acquisition of posterior fate by the selector gene() and dorsal fate by the selector gene(). While theexpression domain is set up during embryogenesis,expression begins only during early wing development. Thus, the correct establishment of theexpression pattern relative tomust be tightly controlled. Here, we functionally investigate the transcriptional inputs integrated by the earlyenhancer (apE) and their requirement for correct boundary formation. Detailed mutational analyses using CRISPR/Cas revealed a role for apE in positioning the DV boundary relative to the AP boundary, with apE mutants often displaying mirror-image anterior wing duplications. We then designed and applied methods to accomplish tissue-specific enhancer disruption via dCas9 expression. This approach allowed us to dissect the spatiotemporal requirement for apE function, clarifying the mechanism by which apE misregulation leads to AP defects. Base-pair-resolution analyses of apE uncovered a single HOX-binding site essential for wing development that, when mutated, led to wingless flies. We demonstrated that the transcription factors Pointed (Pnt), Homothorax (Hth), and Grain (Grn) are required for apE function, and the HOX gene Antennapedia (Antp) contributes to early wing development. Together, our results provide a comprehensive molecular basis of earlyactivation and the developmental consequences of its misregulation, shedding light on how compartmental boundaries are set up during development.