DOI: 10.5061/dryad.p5hqbzkqw
title: OPT data from: A novel cis-regulatory element drives early expression of Nkx3.2 in the gnathostome primary jaw joint

description: 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.
description: 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
 <em>et al</em>., 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),
 <em>nkx3.2</em><sup>+/</sup><sup>D</sup><sup>JRS1</sup> (n=10), and <em>nkx3.2</em><sup>D</sup><sup>JRS1/</sup><sup>D</sup><sup>JRS1</sup> (n=8) zebrafish were then aligned to the reference.
description: OPT Image Stacks include 1. Average Stack
 Dataset 2. Individual Fish Joint Stack
 Dataset WT- wild-type, Het-  <em>nkx3.2</em><sup>+/</sup><sup>D</sup><sup>JRS1</sup>, Mut-<em>nkx3.2</em><sup>D</sup><sup>JRS1/</sup><sup>D</sup><sup>JRS1</sup>, L-left joint, R-right joint