Temporal cell fate determination in the spinal cord is mediated by the duration of Notch signalling

During neural development, progenitor cells generate different types of neurons in specific time windows. Despite the characterisation of many of the transcription factor networks involved in these differentiation events, the mechanism behind their temporal regulation is poorly understood. To address this question, we studied the temporal differentiation of the simple lateral floor plate (LFP) domain in the zebrafish spinal cord. LFP progenitors generate both early-born Kolmer-Agduhr" (KA") interneuron and late-born V3 interneuron populations. Analysis using a Notch signalling reporter demonstrates that these cell populations have distinct Notch signalling profiles. Not only do V3 progenitors receive higher total levels of Notch response, but they collect this response over a longer duration compared to KA" progenitors. To test whether the duration of Notch signalling determines the temporal cell fate specification, we combined a transgene that constitutively activates Notch signalling in the ventral spinal cord with a heat shock inducible Notch signalling terminator to switch off Notch response at any given time. Sustained Notch signalling results in expanded LFP progenitors while KA" and V3 interneurons fail to specify. Early termination of Notch signalling leads to exclusively KA" cell fate, despite the high level of Notch signalling, whereas late attenuation of Notch signalling drives only V3 cell fate. This suggests that the duration of Notch signalling, not simply the level, mediates cell fate specification. Interestingly, knockdown experiments reveal a role for the Notch ligand Jag2b in maintaining LFP progenitors and limiting their differentiation into KA" and V3 interneurons. Our results indicate that Notch signalling is required for neural progenitor maintenance while a specific attenuation timetable defines the fate of the postmitotic progeny.