In situ mutational screening and CRISPR interference define apterous cis-regulatory inputs during compartment...

Area of Science:

• Developmental Biology
• Genetics
• Molecular Biology

Background:

• Tissue axes establishment is crucial for embryonic development.
• In Drosophila, anterior/posterior (AP) and dorsal/ventral (DV) boundaries define wing coordinates.
• Selector genes engrailed (en) and apterous (ap) establish these boundaries, with ap expression initiated during early wing development.

Purpose of the Study:

• Investigate the transcriptional inputs of the early apterous enhancer (apE).
• Determine the role of apE in correctly establishing the DV boundary relative to the AP boundary.
• Clarify the mechanism of apE misregulation and its impact on AP axis defects.

Main Methods:

• CRISPR/Cas9 for detailed mutational analyses of apE.
• dCas9 expression for tissue-specific enhancer disruption.
• Base-pair-resolution analyses to identify functional elements within apE.

Main Results:

• Mutations in apE caused mirror-image anterior wing duplications, indicating a role in DV/AP boundary positioning.
• Disruption of apE function revealed spatiotemporal requirements for its activity.
• A single HOX-binding site in apE was essential for wing development; its mutation resulted in wingless phenotypes.
• Transcription factors Pointed (Pnt), Homothorax (Hth), Grain (Grn), and HOX gene Antennapedia (Antp) were identified as critical for early apE function.

Conclusions:

• This study provides a molecular basis for early apterous activation and the consequences of its misregulation.
• The findings elucidate how compartmental boundaries are established during Drosophila wing development.
• apE plays a critical role in integrating positional information to ensure proper wing patterning.