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 of Nkx3.2 that 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 zebrafish Danio rerio, and was thus designated as jaw joint regulatory sequence 1 (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 of Nkx3.2 and leads to transient jaw joint deformation and partial fusion. The emergence of this Nkx3.2 enhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws.

Dataset: Optical Projection Tomography Raw Image Stacks

A custom-built Optical Projection Tomography (OPT) (Sharpe et al., 2002; Zhang et al., 2020) system was used for imaging of 9 dpf skeletal stained zebrafish larvae. The OPT system, reconstruction algorithms, and alignment workflow were based on the previously described method (Allalou et al., 2017). All larvae were kept in 99% glycerol before they were loaded into the system for imaging. The rotational images were acquired using a 3X telecentric objective with a pixel resolution of 1.15 μm/pixel. The tomographic 3D reconstruction was done using a filtered back projection (FBP) algorithm in MATLAB (Release R2015b; MathWorks, Natick, MA) together with the ASTRA Toolbox (Palenstijn et al., 2013). For the data alignment, the registration toolbox elastix (Klein et al., 2010; Shamonin et al., 2014) was used. To reduce the computational time all 3D volumes in the registration were down-sampled to half the resolution.

The registration workflow was similar to the methods described by Allalou et al. (2017) where the wild-type fish were initially aligned and used to create an average reference fish using an Iterative Shape Averaging (ISA) algorithm (Rohlfing et al., 2001). All wild-type (n=12), nkx3.2^+/DJRS1 (n=10), and nkx3.2^DJRS1/DJRS1 (n=8) zebrafish were then aligned to the reference.

OPT Image Stacks include

1. Average Stack Dataset

2. Individual Fish Joint Stack Dataset

WT- wild-type, Het-  nkx3.2^+/DJRS1, Mut-nkx3.2^DJRS1/DJRS1, L-left joint, R-right joint