Thanks to all authors for collaborating! @gvalentinepfl.bsky.social ntinepfl.bsky.social Lucille Lopez-Delisle @madeline-ryan.bsky.social Sandra Gitto, Paula Barrera Gómez, Bénédicte Mascrez, Thomas Mansour @nakamuralab.bsky.social Matthew Harris @neilshubin.bsky.social

Nature creates novelty from pre-existing systems! We propose that the regulatory system active in distal limbs was reused in tetrapods from a preexisting cloacal regulatory machinery. Importantly, this involved the co-option of the complete regulatory landscape rather than individual enhancers.

We show that distal hox genes are essential for correct cloacal formation in zebrafish, as hox13 loss results in terminal tract fusion, similar to mouse mutants.

Instead, we found that deleting the 5DOM completely ablates expression in the cloaca, a structure related by ancestry to the mammalian urogenital sinus, where the terminal regions of the intestinal, excretory and reproductive systems come together.

Deleting the 5DOM in mice ablates Hoxd expression in the digits. To our shock, we found that deleting this region in zebrafish does not disrupt Hoxd expression in fins! This indicates that Hoxd expression in fins is controlled by a regulatory mechanism independent of the one active in limbs.

The presence of a syntenic region in zebrafish, which lacks digits, suggests deep homology of Hoxd gene regulation in vertebrate appendages. But is this region really activating Hoxd transcription in fins? We tested this hypothesis by deleting the entire 5DOM (~400 kb!) in zebrafish.

Analysis of Hox gene expression has been critical to explain the origin of novel features such as the limb. Hoxd gene transcription in the digits of terrestrial vertebrates depends on a large regulatory landscape (5DOM). However, how this regulatory region evolved has remained unresolved.

I hope you got a good grade 😜