NK2.2/Vnd acts as a transcriptional activator of deadpan to maintain neuroblast identity in Drosophila embryos.

Abstract

Background: Drosophila ventral nervous system defective (vnd/NK2.2) encodes a homeodomain (HD) transcription factor essential for establishing neuroblast (NB) identity in the ventral nerve cord. However, its direct downstream targets during embryonic neurogenesis remain largely unknown.

Objective: This study aimed to identify target genes directly regulated by Vnd and elucidate the molecular mechanisms underlying their regulation during the formation of embryonic NBs.

Methods: Expression profiles and embryonic phenotypes were analyzed in either vnd⁶/Y or dpn¹/dpn¹ single mutants and vnd⁶/Y; dpn¹/dpn¹ double mutants using immunofluorescence. Direct binding of the Vnd HD to the deadpan (dpn) promoter was assessed via EMSA, and functional binding sites were validated using dual-luciferase reporter assays in S2 cells. The dosage-dependent relationship between vnd and dpn was characterized in primary neural cultures using real-time PCR and quantitative immunofluorescence.

Results: Both vnd⁶/Y and dpn¹/dpn¹ single mutants exhibited similar developmental defects, including NB mis-specification and central/peripheral nervous system disorganization. Consistently, vnd⁶/Y; dpn¹/dpn¹ double mutants displayed severely aggravated, overlapping neurogenic phenotypes, suggesting their functional convergence in a combinatorial regulatory mechanism. EMSA and reporter assays demonstrated that Vnd HD specifically binds to the distal V2 site (- 810 to - 803 bp from the dpn transcription start site) containing a 5'-AAGT-3' core motif. In primary cultures, vnd overexpression significantly elevated dpn transcripts and signal intensities, whereas vnd knockdown markedly reduced dpn expression.

Conclusion: Our findings demonstrate that Vnd directly binds to a specific cis-regulatory motif within the dpn promoter, functioning as a transcriptional activator to maintain NB identity and architectural integrity during Drosophila embryogenesis.