Dissection of theFgf8regulatory landscape byin vivoCRISPR-editing reveals extensive inter- and intra-enhancer redundancy

Developmental genes are often regulated by multiple elements with overlapping activity. Yet, in most cases, the relative function of those elements and their contribution to endogenous gene expression remain uncharacterized. Illustrating this situation, distinct sets of enhancers have been proposed to directFgf8in the limb apical ectodermal ridge (AER) and the midbrain-hindbrain boundary (MHB). Usingin vivoCRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the AER, the control ofFgf8expression appears extremely distributed between different enhancers. In contrast, in the MHB, one of the three active enhancers is essential while the other two are dispensable. Further dissection of the essential MHB enhancer revealed another layer of redundancy and identified two sub-parts required independently forFgf8expression and formation of midbrain and cerebellar structures. Interestingly, cross-species transgenic analysis of this enhancer suggests changes of the organisation of this essential regulatory node in the vertebrate lineage.