Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation

Letelier, Joaquin et al.Developmental programs often rely on parallel morphogenetic mechanisms that guarantee precise tissue architecture. While redundancy constitutes an obvious selective advantage, little is known on how novel morphogenetic mechanisms emerge during evolution. In zebrafish, rhombomeric boundaries behave as an elastic barrier, preventing cell intermingling between adjacent compartments. Here, we identify the fundamental role of the small-GTPase Rac3b in actomyosin cable assembly at hindbrain boundaries. We show that the novel rac3b/rfng/sgca regulatory cluster, which is specifically expressed at the boundaries, emerged in the Ostariophysi superorder by chromosomal rearrangement that generated new cis-regulatory interactions. By combining 4C-seq, ATAC-seq, transgenesis, and CRISPR-induced deletions, we characterized this regulatory domain, identifying hindbrain boundary-specific cis-regulatory elements. Our results suggest that the capacity of boundaries to act as an elastic mesh for segregating rhombomeric cells evolved by cooption of critical genes to a novel regulatory block, refining the mechanisms for hindbrain segmentation.This work was supported by La Marató-TV3 Grant 345/C/2014 (to C.P.), “Equipe FRM” (to S.R.), and the Spanish Ministry of Economy and Competitiveness [MINECO-FEDER; Grants BFU2012-31994 and BFU2015-67400-P (to C.P.)], Unidad de Excelencia María de Maetzu [Grants MDM-2014-0370 to DCEXS-UPF (Department of Experimental and Health Sciences-Universitat Pompeu Fabra); BFU2011-22916, P11-CVI-7256, BFU2014-53765, and BFU2014-55738-REDT (to J.R.M.-M.); and BFU2016-81887-REDT/AEI (to J.R.M.-M. and C.P.)]. J.L. and C.A.U. were funded by postdoctoral fellowships from the Becas Chile program (CONICYT, Chile). J.T. was a recipient of a postdoctoral Beatriu de Pinos fellowship (AGAUR, Generalitat de Catalunya), A.V. is a recipient of a predoctoral fellowship from LaCaixa, and I.B. holds a predoctoral Fromació d’Investigadors (FI) fellowship (AGAUR, Generalitat de Catalunya). C.P. is a recipient of an ICREA Academia Award (Institució Catalana per la Recerca i Estudis Avançats, Generalitat de Catalunya). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Grant 658521 (to I.M.).Peer reviewe