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New study links cloaca regulatory DNA to digit formation
Recent comparative genomics and functional experiments indicate that the regulatory DNA controlling the development of human fingers and toes has deep evolutionary roots in a structure found in fish: the cloaca. A team of US and Swiss researchers report that the regulatory landscape adjacent to Hoxd genes — the DNA switches that help pattern limbs — originally operated in the cloacal region of fish around 380 million years ago, and was later co-opted during the emergence of tetrapod digits.
Scientific background: Hoxd genes and regulatory landscapes
Hoxd genes are part of the Hox gene family, a set of developmental regulators that determine body plan and appendage identity across animals. In tetrapods, Hoxd genes help control digit number and morphology, but how fin structures in ancestral fish became distinct digits has been debated. Instead of arising from entirely new genes, the new study suggests evolution repurposed pre-existing regulatory elements — enhancers and other noncoding DNA — that were initially active in the cloaca, a multipurpose terminal orifice used for excretion and reproduction in many vertebrates.
Key experiments and findings
A comparative analysis of mouse and zebrafish genomes showed that although zebrafish lack some Hoxd genes present in tetrapods, they retain the broader regulatory landscape around remaining Hox loci. To determine the activity and function of these elements, researchers used fluorescent reporter tagging in embryos: when the candidate regulatory sequences were linked to fluorescent markers, mouse embryos showed activation in developing digits, while zebrafish showed activation in the cloacal region.
A zebrafish embryo with the cloacal region lit with Hox activity.
Functional tests used CRISPR–Cas9 to delete the conserved regulatory region. In mice with that region removed, digits failed to form properly, producing clear limb-development defects. In zebrafish, deletion disrupted cloacal development without altering fin formation. The pattern — regulatory activity in terminal structures and parallel phenotypic effects when removed — supports the hypothesis that the regulatory landscape’s ancestral role was cloaca-related and that it was later redeployed to pattern distal limb structures in tetrapods.
Implications for evolutionary developmental biology
These results illustrate a common evolutionary mechanism: exaptation, in which existing genetic modules are repurposed for new functions. As developmental geneticist Denis Duboule of the University of Geneva observed, "The fact that these genes are involved is a striking example of how evolution innovates, recycling the old to make the new." Geneticist Aurélie Hintermann added that cloaca and digits share a conceptual similarity as terminal structures — ends of tubes or limbs — which may have facilitated regulatory reuse.
Beyond clarifying a long-standing question about the origin of digits, the work highlights how noncoding regulatory DNA can drive major morphological transitions. It also suggests new avenues for studying congenital limb malformations: mutations in enhancers, rather than in coding genes themselves, may underlie some developmental disorders.
Methods and related technologies
The study combined comparative genomics, enhancer-reporter assays, fluorescent in vivo imaging, and targeted CRISPR deletions. These techniques are core to modern evo-devo research: genome comparisons identify conserved noncoding elements, reporter constructs reveal spatiotemporal activity, and genome editing tests function directly in developing embryos. Continued advances in single-cell transcriptomics and chromatin conformation capture will allow finer mapping of regulatory interactions in both fish and tetrapod embryos.
Expert Insight
Dr. Lena Morales, evolutionary developmental biologist at the Salk Institute (fictional), commented: "This work elegantly demonstrates that innovation in evolution often derives from regulatory rewiring rather than invention of new proteins. The functional deletion experiments are particularly persuasive because they show parallel developmental consequences in two very different vertebrate models. Future comparative work across additional fish and early tetrapod lineages will be valuable to trace the timing and sequence of these regulatory shifts."
Future directions
Researchers will next test regulatory activity across a broader set of species and developmental stages to reconstruct the evolutionary trajectory more precisely. Integrating paleontological data, gene regulatory network models, and functional assays will help determine when and how the shift from cloacal to distal limb regulation occurred. Such multidisciplinary approaches will refine our understanding of how complex structures like fingers emerge from ancient genetic toolkits.
Conclusion
This study provides compelling evidence that the DNA regulatory landscape now guiding digit formation was originally active in fish cloacas. By demonstrating enhancer activity and necessity across species, the research underscores regulatory repurposing as a driver of morphological innovation. The findings deepen our understanding of limb evolution and highlight the functional importance of noncoding DNA in shaping major evolutionary transitions.
Source: sciencealert
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