Proneural gene function : Modeling olfactory placode development

Previously, we have studied regulatory element required for the expression of the zebrafish proneural gene neurogenin1 (neurog1) (Blader et al., 2004). We are now addressing how this bHLH transcription factor regulates its targets. Indeed, we have shown that Neurog1 regulates its target deltaA via three non-redundant E-boxes (Madelaine and Blader, 2011).

More recently, we have shown that, neurog1 and a second zebrafish Atonal genes neurod4, are redundantly required for development of both early-born olfactory neurons (EON) and later olfactory sensory neurons (OSN). We show that neurod4 expression is initially regulated by Neurog1 but recovers in the neurog1 mutant and is sufficient for the delayed development of OSN. In contrast, EON numbers are significantly reduced in neurog1 mutant embryos despite the recovery of neurod4 expression. Our results suggest that a shortened time window for EON development causes this reduction ; EON leave the cell cycle later in neurog1 mutant embryos than in siblings but are all post-mitotic at the same stage as wild type embryos. Finally, we show that the expression of the EON specific gene robo2 is never detected in neurog1 mutant embryos. Failure of robo2 expression to recover correlates with defects in the fasciculation of neurog1 mutant olfactory axonal projections and the organisation of proto-glomerulii when projections arrive at the olfactory bulb that are comparable to those seen in robo2/astray mutant embryos. We conclude that the duration of proneural expression in EON progenitors is critical for correct development of the zebrafish olfactory system (Madelaine et al., 2011).

In the zebrafish olfactory system, dispersed precursors of olfactory neurons differentiate while concomitantly assembling into a compact placode on either side of the brain. We have already shown that the bHLH proneural Neurogenin1 (Neurog1) plays a key role in olfactory neuron differentiation (Madelaine et al., 2011). How morphogenesis of the olfactory placode is coordinated with neurogenesis, however, remains unclear. In the lab, we aim to decipher how these processes are coupled to build a functional sensory organ using both live imaging and molecular/transcriptomic analysis.

We have quantitatively described the early steps of olfactory placode morphogenesis and can show that these parameters are disrupted in neurog1 mutants. This process seems also affected in some cell-migration molecules/ guidance cues mutants, which in this system could act as Neurog1 direct target genes. We plan to unravel Neurog1 direct target genes involved in this process, by identifying E-boxes’s cluster within regulatory elements that could control their expression in a Neurog1-dependant manner.

We are using both conventional candidate promoter analyses and CRISPR/Cas9 genome editing tools to investigate the function of these E-boxes in controlling candidate gene expression in the olfactory placode.

Finally, in collaboration with the IMT, we are building a theoretical mathematical model of olfactory organ formation to refine the hypotheses that can be tested in the embryo.  In collaboration with the LAAS-CNRS, we are developing microfluidic chips that mimic olfactory morphogenesis in order to gain a better understanding of this process.

Altogether, our results could reveal a parsimonious mechanism for coordinating neurogenesis and morphogenesis in which proneural genes control both processes via distinct sets of targets.

Funding