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

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.