A novel cis-regulatory element drives early expression of Nkx3.2 in the gnathostome primary jaw joint

  1. Jake Leyhr
  2. Laura Waldmann
  3. Beata Filipek-Górniok
  4. Hanqing Zhang
  5. Amin Allalou
  6. Tatjana Haitina

  • Curated by eLife

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    Evaluation Summary:

    In this elegant study, Leyhr et al. identify the first potent nkx3.2 jaw joint enhancer, which they show to be deeply conserved across gnathostomes and likely to be absent from jawless fishes. The data support the hypothesis that this enhancer arose with the origin of hinged jaws during vertebrate evolution and is required for some aspects of early joint development in zebrafish. The work has important implications both for our basic understanding of enhancer function and evolution as well as potential genetic causes of craniofacial defects in humans.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

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 a transient jaw joint deformation and partial fusion. Emergence of this Nkx3.2 enhancer in early gnathostomes may have contributed to the origin and shaping of the articulating surfaces of vertebrate jaws.

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  1. Version published to 10.7554/elife.75749 on eLife
  2. eLife

    Evaluation Summary:

    In this elegant study, Leyhr et al. identify the first potent nkx3.2 jaw joint enhancer, which they show to be deeply conserved across gnathostomes and likely to be absent from jawless fishes. The data support the hypothesis that this enhancer arose with the origin of hinged jaws during vertebrate evolution and is required for some aspects of early joint development in zebrafish. The work has important implications both for our basic understanding of enhancer function and evolution as well as potential genetic causes of craniofacial defects in humans.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    In this interesting study, which focuses on evolution of jointed jaws, the authors identified a conserved non-coding sequence motif JRS1 downstream of the Nkx3.2 gene, which has an established role in patterning joints. JRS1 is present in most vertebrates with jointed jaws, but apparently absent in jawless hagfish. The authors show that the enhancer has likely binding sites for transcription factors that are known players in skeletal patterning. This section could be improved by validation to demonstrate that one or more of the putative TFs does indeed bind the JRS1.

    The authors then take the JRS1 element from various species and use these to drive fluorescent proteins in the zebrafish. Using beautiful imaging, they show that the sequence derived from all species tested leads to expression that is restricted to the joint site. This is very compelling.

    The authors also delete JRS1 from the zebrafish genome and show that while nx3.2 expression levels are comparable, the larvae have transient abnormalities in joint patterning, including partial joint fusions. These experiments are compelling, but would be enhanced by characterisation of local changes to chondrocyte maturation.

  4. eLife

    Reviewer #2 (Public Review):

    Extending their recent study of nkx3.2 function in the developing zebrafish jaw (Waldmann et al., 2021), the authors here set out to identify regulatory sequences that drive expression of this gene in the jaw joint region. Through sequence conservation analysis, they successfully identified an enhancer for Nkx2.3 (JRS1) that proved to be deeply conserved in gnathostomes but absent in the jawless hagfish. This is the first such jaw joint enhancer to be identified for this gene, despite the link between Nkx3.2 and the jaw joint having been known for several decades. Given how important the mobile jaw has been in vertebrate evolution, this is a significant and impactful find. The authors identify multiple conserved binding motifs known to be preferred by transcription factors active in the pharyngeal arches, suggesting a wealth of potential upstream regulators. The authors then present a series of beautiful JRS1:mCherry reporter lines showing restricted expression in and around the jaw joint, with expression declining coincident with chondrocyte differentiation. Homologous JRS1 sequences from a wide range of gnathostomes drove essentially identical patterns in zebrafish transgenesis assays, even when extracted from mammalian species that have evolved a new secondary jaw joint between the mandible and squamosal bone. These data support the specificity of this enhancer as well as highly conserved interactions with upstream activators and/or repressors.

    To test the contribution of the JRS1 enhancer to nkx3.2 expression in zebrafish, the authors deleted the entire enhancer sequence using CRISPR/Cas9. Homozygous mutants presented early deformation of the jaw joint and transient partial fusion, though not the complete fusion observed in previously published null nkx3.2 mutant and morphant lines (Waldmann et al. 2021, Smeeton et al. 2021, Miyashita et al. 2020, Miller et al. 2003). The mutant jaw later resolved to resemble that of control siblings. The authors checked nkx3.2 expression levels by qPCR at 6 dpf and saw no significant differences. Mild phenotypes upon enhancer deletion are common in the literature, and are usually attributed to the activity of shadow enhancers. The authors did well to evaluate the phenotype at multiple stages of development, revealing robustness in the developmental process that would have been overlooked if the assessment had been limited to early larval stages. One weakness, however, is that they only evaluated endogenous expression of nkx3.2 in the enhancer mutants at a single stage and with a method that could mask local changes in expression. The true contribution of this enhancer to early nkx3.2 expression thus remains unclear.

    Overall, this straightforward study fills a gap in knowledge about regulation of the best known "jaw joint" gene. The sequence can be used to generate and test hypotheses in future studies on potential upstream regulators as well as evolutionary divergence in jaw joint morphology.

  5. eLife

    Reviewer #3 (Public Review):

    The Nkx3.2 transcription factor is both necessary and sufficient for jaw joint development. In this paper the authors use comparative genomics to identify a conserved Nkx3.2 enhancer that they call a jaw regulatory sequence (JRS). With transgenic zebrafish they show that JRSs from multiple species of gnathostomes, including humans, can drive reporter expression in the larval jaw joint. Transgene expression in each case includes the joint interzone as well as adjacent cartilage and perichondrium. With CRISPR-targeted deletion they show requirements for the zebrafish JRS in larval jaw joint development. Analyses of additional gnathostome genomic sequence near Nkx3.2 suggest that jawless hagfish lack JRS-like sequences, consistent with an important role for this enhancer and Nkx3.2 in the evolutionary origin of jaws. Despite a growing recognition of the importance of enhancers in development and disease, very few have been shown to have essential functions in vivo.

    The first part of the paper is straightforward, with clear evidence that the JRSs tested act as functional enhancers, since they drive reporter expression in very specific and similar subsets of skeletal cells. The images are high quality and the cellular resolution is impressive. There are also several moderate/major weaknesses that should be addressed. The experiments showing larval joint defects following JRS deletion in zebrafish are less clear, since the phenotypes are subtle in mutants and later recover to resemble wild type siblings. One major weakness is that there is no evidence that JRS deletion alters Nkx3.2 expression at any stage. In the absence of these data, it is possible that the JRS acts on other nearby genes. Another weakness is a lack of quantification of variation in this phenotype in the JRS mutants. Though the images of larval joint defects in the JRS mutants are clear, they are single examples. Finally, the apparent absence of an obvious JRS in the region adjacent to Nkx3.2 in hagfish is used to argue that it arose together with the origin of jaws during vertebrate evolution. Alternatively, this may instead reflect a unique loss of this element in hagfish.

  6. Version published to 10.1101/2021.11.26.470082v1 on bioRxiv