The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

Biological Sciences Graduate Program University of California San Diego La Jolla CA USA

Cluster of Excellence Physics of Life Technische Universität Dresden Dresden Germany

Comparative Biochemistry Program University of California Berkeley CA 94720 USA

Department for BioMedical Research University of Bern Bern Switzerland

Department of Anatomy University of California San Francisco San Francisco CA USA

Department of Cardiology Bern University Hospital Bern Switzerland

Department of Integrative Biology and Ecology Evolution and Behavior Program Michigan State University East Lansing MI USA

Department of Medicine Health Sciences University of California San Diego La Jolla CA USA

Department of Molecular Biology Biological Sciences University of California San Diego La Jolla CA USA

Department of Orofacial Sciences University of California San Francisco San Francisco CA USA

Environmental Genomics and Systems Biology Division Lawrence Berkeley National Laboratory Berkeley CA USA

Institute for Human Genetics University of California San Francisco San Francisco CA USA

Institute of Molecular Genetics of the ASCR v v i Prague Czech Republic

Institute of Molecular Life Sciences University of Zurich Zurich Switzerland

Max Planck Institute for Molecular Cell Biology and Genetics Dresden Germany

Program in Craniofacial Biology University of California San Francisco San Francisco CA USA

School of Natural Sciences University of California Merced Merced CA USA

Section of Developmental Biology Department of Pediatrics University of Colorado Anschutz Medical Campus Aurora CO USA

Technische Universität Dresden CRTD Center for Regenerative Therapies Dresden Dresden Germany

US Department of Energy Joint Genome Institute Lawrence Berkeley National Laboratory Berkeley CA USA

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bioRxiv. 2023 Apr 20:2023.04.20.536761. doi: 10.1101/2023.04.20.536761. PubMed

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