Hdac4 mediates perichondral ossification and pharyngeal skeleton development in the zebrafish.

April Delaurier,Cynthia Lizzet Alvarez,Kali J Wiggins

doi:10.7717/peerj.6167

Abstract

BackgroundHistone deacetylases (HDACs) are epigenetic factors that function to repress gene transcription by removing acetyl groups from the N-terminal of histone lysines. Histone deacetylase 4 (HDAC4), a class IIa HDAC, has previously been shown to regulate the process of endochondral ossification in mice via repression of Myocyte enhancer factor 2c (MEF2C), a transcriptional activator of Runx2, which in turn promotes chondrocyte maturation and production of bone by osteoblasts.Methods & MaterialsIn this study, we generated two zebrafish lines with mutations in hdac4 using CRISPR/Cas9 and analyzed mutants for skeletal phenotypes and expression of genes known to be affected by Hdac4 expression.ResultsLines have insertions causing a frameshift in a proximal exon of hdac4 and a premature stop codon. Mutations are predicted to result in aberrant protein sequence and a truncated protein, eliminating the Mef2c binding domain and Hdac domain. Zygotic mutants from two separate lines show a significant increase in ossification of pharyngeal ceratohyal cartilages at 7 days post fertilization (dpf) (p < 0.01, p < 0.001). At 4 dpf, mutant larvae have a significant increase of expression of runx2a and runx2b in the ceratohyal cartilage (p < 0.05 and p < 0.01, respectively). A subset of maternal-zygotic (mz) mutant and heterozygote larvae (40%) have dramatically increased ossification at 7 dpf compared to zygotic mutants, including formation of a premature anguloarticular bone and mineralization of the first and second ceratobranchial cartilages and symplectic cartilages, which normally does not occur until fish are approximately 10 or 12 dpf. Some maternal-zygotic mutants and heterozygotes show loss of pharyngeal first arch elements (25.9% and 10.2%, respectively) and neurocranium defects (30.8% and 15.2%, respectively). Analysis of RNA-seq mRNA transcript levels and in situ hybridizations from zygotic stages to 75–90% epiboly indicates that hdac4 is highly expressed in early embryos, but diminishes by late epiboly, becoming expressed again in larval stages.DiscussionLoss of function of hdac4 in zebrafish is associated with increased expression of runx2a and runx2b targets indicating that a role for hdac4 in zebrafish is to repress activation of ossification of cartilage. These findings are consistent with observations of precocious cartilage ossification in Hdac4 mutant mice, demonstrating that the function of Hdac4 in skeletal development is conserved among vertebrates. Expression of hdac4 mRNA in embryos younger than 256–512 cells indicates that there is a maternal contribution of hdac4 to the early embryo. The increase in ossification and profound loss of first pharyngeal arch elements and anterior neurocranium in a subset of maternal-zygotic mutant and heterozygote larvae suggests that maternal hdac4 functions in cartilage ossification and development of cranial neural crest-derived structures.

Highlights

The majority of the vertebrate skeleton including the axial, limb, and pharyngeal elements form as cartilaginous elements that grow rapidly during early development through the proliferation of matrix-secreting chondrocytes (Karsenty & Wagner, 2002)
Frameshifts were induced by insertion of nucleotides into exon 5, resulting in aberrant amino acids being added to the protein sequence (Fig. 1C). In both mutant lines the frameshift is predicted to cause the loss of the Mef2c binding domain and premature stop codons resulting in truncated proteins 174 aa and 181 aa in length (Fig. 1D)
We observed significantly increased expression of runx2a and runx2b in the ceratohyal of hdac4-mutant zebrafish compared to wild-types, which we speculate is due to a de-repression of Mef2c function leading to increased transcription of runx2 genes

Summary

Introduction

The majority of the vertebrate skeleton including the axial, limb, and pharyngeal elements form as cartilaginous elements that grow rapidly during early development through the proliferation of matrix-secreting chondrocytes (Karsenty & Wagner, 2002). The timing of the transition between endochondral growth and ossification is important in the determining size and shape of skeletal elements (Nagata et al, 2011; Eames et al, 2011; Grünbaum, Cloutier & Vincent, 2012; Harrington, Harrison & Sheil, 2013; Arenas-Rodríguez, Rubiano Vargas & Hoyos, 2018) This process is precisely regulated by expression of factors in chondrocytes and osteoblasts including Ihh, Pthrp, Runx (Cbfa1), and Sp7 (Osx) (Vortkamp et al, 1996; Komori et al, 1997; Nakashima et al, 2002; Maeda et al, 2007). The increase in ossification and profound loss of first pharyngeal arch elements and anterior neurocranium in a subset of maternal-zygotic mutant and heterozygote larvae suggests that maternal

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Conclusion