Hox genes are evolutionary conserved master regulators of patterning along the anterior-to-posterior axis during development. In most species, multiple Hox genes are found adjacent to one another at distinct genomic loci, a feature that facilitates coordinated expression. In vertebrates, the strict timing of Hox gene activation is required for proper patterning of the axis. Revealing the identity of the signals that control timely Hox gene activation, and the cis-regulatory elements through which they act, are fundamental to our understanding of developmental and evolutionary mechanisms. By targeted deletions we have previously shown that the microRNA-196 family control vertebral number and identity in mouse. We found that not only does miR-196 post-transcriptionally regulate its known direct Hox target genes, but remarkably also indirectly controls the timing of posterior Hox gene activation. Using mouse iPSCs & ESCs in combination with in vitro differentiation we have established that Gdf11 signalling is also required for activation of the Hox genes in a timely manner. We are now employing Next-Gen sequencing techniques (RNA-seq, ATAC-seq and ChIP-seq) to identify novel regulators that activate the 5’Hox genes, control axis elongation and vertebral number during mammalian development.