Abstract
Topologically associating domains (TADs) have been proposed to both guide and constrain enhancer activity. Shh is located within a TAD known to contain all its enhancers. To investigate the importance of chromatin conformation and TAD integrity on developmental gene regulation, we have manipulated the Shh TAD - creating internal deletions, deleting CTCF sites, and deleting and inverting sequences at TAD boundaries. Chromosome conformation capture and fluorescence in situ hybridisation assays were used to investigate the changes in chromatin conformation that result from these manipulations. Our data suggest that these substantial alterations in TAD structure have no readily detectable effect on Shh expression patterns or levels of Shh expression during development - except where enhancers are deleted - and result in no detectable phenotypes. Only in the case of a larger deletion at one TAD boundary could ectopic influence of the Shh limb enhancer be detected on a gene (Mnx1) in the neighbouring TAD. Our data suggests that, contrary to expectations, the developmental regulation of Shh expression is remarkably robust to TAD perturbations.