Gene regulation is directed by several layers of control including 3D architecture, epigenetic modifications and gene transcription. The Pitx1 gene is an important marker for hindlimb development and expression relies on the ability of its promoter to contact the pan-limb enhancer Pen in the 3D space of the nucleus, whereas this contact is absent in transcriptionally inactive forelimbs. Pitx1 misregulation leads to skeletal malformations in mammals. To understand the molecular drive behind gene activation and repression, we study chromatin changes within specific cell populations during limb development in a high throughput fashion and the contribution of single enhancers to morphogenesis.