Real-time single-molecule imaging in zebrafish embryos uncovers noncanonical translation.

Precise spatiotemporal control of protein synthesis is essential during embryogenesis, yet directly measuring translation kinetics in vivo remains challenging in vertebrates. In particular, it remains unclear how translation efficiency is determined for key developmental regulators and which kinetic steps limit their production. Here, we used ALFA array-based nascent chain labeling combined with lattice light-sheet microscopy to visualize bmp2b translation in real time and at single-molecule resolution in early zebrafish embryos. When combined with MS2/MCP labeling to visualize all bmp2b messenger RNA (mRNAs), we found that only some of them are being actively translated, suggesting that limited mRNA translation competence contributes to overall translation efficiency. We found that bmp2b translation operates below a maximal initiation regime, as replacement of its untranslated regions (UTRs) with viral UTRs increases ribosome loading. Furthermore, the bmp2b, but not actb2, 5'UTR supports cap-independent translation with ribosome loading comparable to cap-dependent initiation. Together, this approach provides a quantitative in vivo framework to dissect translation kinetics during early vertebrate development.