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The acquisition of movable jaws was a major event during vertebrate evolution. The role of NK3 homeobox 2 (Nkx3.2) transcription factor in patterning the primary jaw joint of gnathostomes (jawed vertebrates) is well known, however knowledge about its regulatory mechanism is lacking. In this study, we report a proximal enhancer element ofiNkx3.2/ithat is deeply conserved in most gnathostomes but undetectable in the jawless hagfish and lamprey. This enhancer is active in the developing jaw joint region of the zebrafishiDanio rerio/i, and was thus designated asijaw joint regulatory sequence 1/i(JRS1). We further show that JRS1 enhancer sequences from a range of gnathostome species, including a chondrichthyan and mammals, have the same activity in the jaw joint as the native zebrafish enhancer, indicating a high degree of functional conservation despite the divergence of cartilaginous and bony fish lineages or the transition of the primary jaw joint into the middle ear of mammals. Finally, we show that deletion of JRS1 from the zebrafish genome using CRISPR/Cas9 results in a significant reduction of early gene expression ofinkx3.2/iand leads to a transient jaw joint deformation and partial fusion. Emergence of thisiNkx3.2/ienhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws.

Here, we describe an automated platform suitable for large-scale deep-phenotyping of zebrafish mutant lines, which uses optical projection tomography to rapidly image brain-specific gene expression patterns in 3D at cellular resolution. Registration algorithms and correlation analysis are then used to compare 3D expression patterns, to automatically detect all statistically significant alterations in mutants, and to map them onto a brain atlas. Automated deep-phenotyping of a mutation in the master transcriptional regulator fezf2 not only detects all known phenotypes but also uncovers important novel neural deficits that were overlooked in previous studies. In the telencephalon, we show for the first time that fezf2 mutant zebrafish have significant patterning deficits, particularly in glutamatergic populations. Our findings reveal unexpected parallels between fezf2 function in zebrafish and mice, where mutations cause deficits in glutamatergic neurons of the telencephalon-derived neocortex., National Institutes of Health (U.S.) (Director’s Pioneer Award DP1-NS082101), David & Lucile Packard Foundation. Award in Science and Engineering, Broad Institute of MIT and Harvard (SPARC Award), Epilepsy Foundation of America (Postdoctoral Fellowship)