Dissection of the Fgf8 regulatory landscape by in vivo CRISPR-editing reveals extensive intra- and inter-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 poorly characterized. An example of this phenomenon is that distinct sets of enhancers have been proposed to direct Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary. Using in vivo CRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the apical ectodermal ridge, the control of Fgf8 expression appears distributed between different enhancers. In contrast, we find that in the midbrain-hindbrain boundary, one of the three active enhancers is essential while the other two are dispensable. We further dissect the essential midbrain-hindbrain boundary enhancer to reveal that it is also composed by a mixture of essential and dispensable modules. Cross-species transgenic analysis of this enhancer suggests that its composition may have changed in the vertebrate lineage.

Cellular mechanisms of chick limb bud morphogenesis

SummaryAlthough some of the molecular pathways involved in limb bud morphogenesis have been identified, the cellular basis of the process is not yet understood. Proposed cell behaviours include active cell migration and oriented cell division, but ultimately, these questions can only be resolved by watching individual mesenchymal cells within a completely normal developmental context. We developed a minimally-invasive in ovo two-photon technique, to capture high quality time-lapse sequences up to 100 microns deep in the unperturbed growing chick limb bud. Using this technique, we characterized cell shapes and other oriented behaviours throughout the limb bud, and found that cell intercalation drives tissue movements, rather than oriented cell divisions or migration. We then developed a 3D cell-based computer simulation of morphogenesis, in which cellular extensions physically pull cells towards each other, with directional bias controlled by molecular gradients from the ectoderm (Wnts) and the Apical Ectodermal Ridge (FGFs). We defined the initial and target shapes of the chick limb bud in 3D by OPT scanning, and explored which orientations of mesenchymal intercalation correctly explain limb morphogenesis. The model made a couple of predictions: Firstly, that elongation can only be explained when cells intercalate along the direction towards the nearest ectoderm. This produces a general convergence of tissue towards the central proximo-distal (PD) axis of the limb, and a resultant extension of the tissue along the PD axis. Secondly, the correct in silico morphology can only be achieved if the contractile forces of mesenchymal cells in the very distal region (under the Apical Ectodermal Ridge) have shorter life times than in the rest of the limb bud, effectively making the tissue more fluid by augmenting the rate of cell rearrangement. We argue that this less-organised region of mesenchyme is necessary to prevent PD-oriented intercalation events in the distal tip that would otherwise inhibit outgrowth.

Dissection of the Fgf8 regulatory landscape by in vivo CRISPR-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 direct Fgf8 in the limb apical ectodermal ridge (AER) and the midbrain-hindbrain boundary (MHB). Using in vivo CRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the AER, the control of Fgf8 expression 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 for Fgf8 expression 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.