CRX/NRL/NR2E3-regulated tbx2b integrates BMP2 signaling to control opsin expression and larval feeding in fish

The T-box transcription factor tbx2b , containing a highly conserved DNA-binding domain, plays a pivotal role in vertebrate visual development, yet its transcriptional regulation remains poorly understood. In this study, we characterized the tbx2b promoter in mandarin fish ( Siniperca chuatsi ), identifying a functional −1189/+269 bp regulatory region. Through comprehensive molecular analyses, we demonstrate that while photoreceptor transcription factors crx , nrl , and nr2e3 all enhance tbx2b promoter activity, only nr2e3 directly binds to the promoter, as confirmed by site-directed mutagenesis and EMSA assays. Intriguingly, BMP2 signaling exhibited dual regulatory effects: while it suppressed tbx2b promoter activity in vitro , in vivo treatment from embryogenesis through 3 days post-hatching (dph) significantly upregulated tbx2b mRNA expression. This treatment also enhanced expression of short-wavelength opsins ( sws1 , sws2Aβ , sws2Aα ) and photoreceptor determinants ( nrl , nr2e3 ), without altering retinal morphology. Beyond photoreceptor development, BMP2 treatment markedly improved first-feeding success, as evidenced by increased food intake, enhanced gut filling, and stimulated locomotor activity with distinct behavioral patterns. Our findings establish a novel regulatory network connecting tbx2b with key photoreceptor transcription factors, and reveal BMP2 as a multifunctional modulator coordinating visual system development with feeding initiation in larval fish. These results provide new insights into the molecular mechanisms governing visual pathway development and its integration with early life history strategies in teleosts. • A tiered regulatory mechanism involves direct tbx2b binding by nr2e3 and indirect enhancement by crx and nrl . • BMP2 signaling upregulates tbx2b and short-wavelength opsins, enhancing photoreceptor differentiation in vivo . • BMP2 boosts larval first-feeding success by stimulating gut filling, food intake, and locomotor activity.