The Broad Role of Nkx3.2 in the Development of the Zebrafish Axial Skeleton

Zhang

et al. 2021

Preprint

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BackgroundThe development of the vertebrate limb skeleton requires a complex interaction of multiple factors to facilitate correct shaping and positioning of bones and joints. Growth and differentiation factor 5 (Gdf5), a member of the transforming growth factor-beta family (TGF-β) is involved in patterning appendicular skeletal elements including joints. Expression of gdf5 in zebrafish has been detected within the first pharyngeal arch jaw joint, fin mesenchyme condensations and segmentation zones in median fins, however little is known about the functional role of Gdf5 outside of Amniota.ResultsWe generated CRISPR/Cas9 knockout of gdf5 in zebrafish and analysed the resulting phenotype at different developmental stages. Homozygous gdf5 mutant zebrafish displayed changes in segmentation of the endoskeletal disc and, in consequence, loss of posterior radials in the pectoral fins. Mutant fish also displayed affected organisation and length of skeletal elements in the median fins, however joint formation and mineralisation process seemed unaffected.ConclusionsOur study demonstrates the importance of Gdf5 for the paired and median fin endoskeleton development in zebrafish and reveals that the severity of the effect increases from anterior to posterior side of the elements. Our findings are consistent with phenotypes observed in human and mouse appendicular skeleton in response to Gdf5 knockout, suggesting a broadly conserved role for Gdf5 in Osteichthyes.

Section: Discussioncontrasting

confidence: 87%

The role of Gdf5 in the development of the zebrafish fin endoskeleton

Zhang

et al. 2021

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“…If we assume elephant shark nkx3.2 gene expression mirrors that of elasmobranchs and that JRS1 drives nkx3.2 gene expression in all pharyngeal arches, our results would support the conclusion that much of the gene regulatory landscape of osteichthyan mandibular joints is found more broadly in the pharyngeal arches of chondrichthyans (Hirschberger et al, 2021). JRS1 knockout did not phenocopy the striking jaw joint fusion seen in zebrafish nkx3.2 gene knockout mutants (Miyashita et al, 2020;Waldmann et al, 2021), suggesting this enhancer is not essential for nkx3.2 expression at the time points analysed. However, as a subtle jaw joint phenotype was still evident in homozygous mutants, most notably the reduction of the RAP and partial fusion observed at 5-9 dpf, the loss of JRS1 must have some local effect on nkx3.2 expression, consistent with the specific jaw joint activity seen in transgenic reporter fish.…”

Section: Discussionsupporting

confidence: 57%

“…In order to characterize the nkx3.2 (JRS1):mCherry expressing cells in nkx3.2 gene mutants we incrossed previously reported nkx3.2 +/uu2803 mutant alleles (Waldmann et al, 2021) with nkx3.2 (JRS1):mCherry/ sox10 :egfp background and used these heterozygotes to generate homozygous nkx3.2 uu2803/uu2803 mutants (abbreviated to nkx3.2 -/- ). Notably, nkx3.2 (JRS1):mCherry expressing cells were detected lining the outside of the cartilage in the fused jaw joint region of nkx3.2 -/- mutants at 3-5 dpf (Figure 3E-F, E’-F’).…”

Section: Resultsmentioning

confidence: 99%

“…The sox10:egfp line labels neural crest cell derived populations comprising pharyngeal arch cartilages (Carney et al, 2006). At the onset of chondrogenesis (2 dpf), confocal live imaging revealed overlapping expression of nkx3.2(JRS1):mCherry and sox10:egfp in condensed mesenchyme cells in the jaw joint establishing zone (Figure 3A, A In order to characterize the nkx3.2(JRS1):mCherry expressing cells in nkx3.2 gene mutants we incrossed previously reported nkx3.2 +/uu2803 mutant alleles (Waldmann et al, 2021) with nkx3.2(JRS1):mCherry/sox10:egfp background and used these heterozygotes to generate homozygous nkx3.2 uu2803/uu2803 mutants (abbreviated to nkx3.2 -/-). Notably, nkx3.2(JRS1):mCherry expressing cells were detected lining the outside of the cartilage in the fused jaw joint region of nkx3.2 -/mutants at 3-5 dpf (Figure 3E-F, E'-F').…”

Section: Zebrafish Jrs1 Enhancer Drives Fluorescent Reporter Gene Expression Corresponding To Endogenous Nkx32 Expressionmentioning

confidence: 99%

“…The importance of Nkx3.2 for the development of the primary jaw joint has been shown in knock-down and knock-out experiments carried out in zebrafish and Xenopus . Reduction or loss of nkx3.2 expression led to absence of the joint and fusion of Meckel’s cartilage and the palatoquadrate (Lukas and Olsson, 2018a; Miller et al, 2003; Miyashita et al, 2020; Waldmann et al, 2021). Overexpression of nkx3.2 in Xenopus resulted in extra ectopic joints forming in the jaw cartilage (Lukas and Olsson, 2018b).…”

Section: Introductionmentioning

confidence: 99%

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An evolutionarily conserved cis-regulatory element of Nkx3.2 contributes to early jaw joint morphology in zebrafish

Filipek-Górniok

et al. 2021

Preprint

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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 of Nkx3.2 that is deeply conserved in gnathostomes but undetectable in the jawless hagfish. 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 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.

The role of Gdf5 in the development of the zebrafish fin endoskeleton

Zhang

et al. 2021

Developmental Dynamics

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Background: The development of the vertebrate limb skeleton requires a complex interaction of multiple factors to facilitate the correct shaping and positioning of bones and joints. Growth and differentiation factor 5 (Gdf5) is involved in patterning appendicular skeletal elements including joints. Expression of gdf5 in zebrafish has been detected in fin mesenchyme condensations and segmentation zones as well as the jaw joint, however, little is known about the functional role of Gdf5 outside of Amniota.Results: We generated CRISPR/Cas9 knockout of gdf5 in zebrafish and analyzed the resulting phenotype at different developmental stages. Homozygous gdf5 mutant zebrafish displayed changes in segmentation of the endoskeletal disc and, as a consequence, loss of posterior radials in the pectoral fins. Mutant fish also displayed disorganization and reduced length of endoskeletal elements in the median fins, while joints and mineralization seemed unaffected. Conclusions: Our study demonstrates the importance of Gdf5 in the development of the zebrafish pectoral and median fin endoskeleton and reveals that the severity of the effect increases from anterior to posterior elements. Our findings are consistent with phenotypes observed in the human and mouse appendicular skeleton in response to Gdf5 knockout, suggesting a broadly conserved role for Gdf5 in Osteichthyes.endoskeleton, fin radials, Gdf5 mutant, pectoral fin, zebrafish appendages 1 | INTRODUCTION Skeletal development requires a complex interaction of multiple factors to ensure correct positioning and shaping of cartilage elements and bones including the proper formation of joints articulating the skeleton. Skeletogenesis via endochondral ossification involves three major processes starting with mesenchyme condensation followed by chondrogenesis and finally the invasion of blood vessels and chondrocyte replacement by and transformation Laura Waldmann and Jake Leyhr contributed equally to this study.