Sox9a, not sox9b is required for normal cartilage development in zebrafish

Qiaohong Lin, Yan He, Jian-Fang Gui, Jie Mei

a College of Fisheries, Huazhong Agricultural University, Wuhan, 430070, China

b State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, University of the Chinese Academy of Sciences,Wuhan, 430072, China

Keywords:sox9a sox9b Cartilage development Zebrafish

ABSTRACT

1. Introduction

SOX family transcription factors are characterized by the evolutionarily conserved high-mobility-group (HMG) box, and play diverse roles in developmental processes, cell proliferation and differentiation.Based on sequence similarities of the HMG box, the Sox family members can be subdivided into ten groups (named A-J), and Sox8, Sox9, and Sox10 are assigned into group E (Kamachi & Kondoh, 2013). The Sox family, especially group E has been shown to play crucial roles in sex differentiation and gonad development (Jiang, Hou, She, & Yang, 2013;Li & Gui, 2018a, 2018b; Mei & Gui, 2015). Sox9 activation was associated with testis development in the XX transgenic mice (Bishop et al.,2000), while Sox9 mutation resulted in complete sex reversal and produced partially fertile XY oocytes (Lavery et al., 2011). In zebrafish, sox9 axis signaling could induce juvenile ovary-testis transformation (Sun et al., 2013).

Sox9 has been identified as a regulator of the chondrocyte lineage and to be required for cartilage formation in mammals (Bi, Deng, Zhang,Behringer, & de Crombrugghe, 1999; Umeda et al., 2015). There are two sox9 paralogs, sox9a and sox9b in teleosts due to genome duplication(Cresko et al., 2003; Zhou & Gui, 2017). Sox9 played multiple roles in zebrafish organ development, including neural crest, craniofacial, pectoral fin, eye and pancreaticobiliary ductal system development(Cheung & Briscoe, 2003; Delous et al., 2012; Yan et al., 2005; Yokoi et al., 2009). Similar to morpholino produced phenotype, both retroviral insertion induced sox9a mutant (hi1134Tg) and ENU induced sox9a point mutant (tw37) zebrafish displayed severe defects in chondrogenesis and cartilage morphogenesis (Yan et al., 2002). Moreover, in ENU induced sox9b point mutant (fh313) zebrafish, malformations of pancreaticobiliary duct and hepatopancreatic duct development were detected (Delous et al., 2012; Manfroid et al., 2012a). The b971 mutant zebrafish had a terminal deletion of about 10 cM in the lower tip of LG3 where sox9b and sox8 are located. This zebrafish mutant showed a curly-down body axis, reduced first and second pharyngeal arches and neurocranial cartilages (Yan et al., 2005).

It is a major challenge to detect a specific mutation which is expected to occur only once in the zebrafish after N-ethyl-N-nitrosourea (ENU)treatment, because of pre-existing polymorphisms and errors introduced during PCR and sequencing (Pan et al., 2015). Recently, several gene editing approaches, such as TALEN and CRISPR/Cas9, have made it easier to create mutation or deletion of a specific gene in zebrafish and other organisms (Dan et al., 2018; Jao, Wente, & Chen, 2013; Zhang &Zhou, 2014). In this study, CRISPR/Cas9 technology was performed to disrupt the function of either sox9a or sox9b, to further confirm the phenotype induced by ENU treatment or morpholino injection. Then,these available targeting sites of sox9a and sox9b for CRISPR/Cas9 willbe applied in tissue-specific gene disruption of these two genes in zebrafish, according to a previous report (Ablain, Durand, Yang, Zhou, &Zon, 2015).

Table 1 Primers used in this study (5′- 3′).

2. Material and methods

2.1. Zebrafish lines and maintenance

AB line zebrafish used in this study were reared in the zebrafish facility of Institute of Hydrobiology, Chinese Academy of Sciences. The maintenance, breeding and staging of zebrafish were performed according to the standard methods (Kimmel, Ballard, Kimmel, Ullmann, &Schilling, 1995; Westerfield, 2000). All animal experiments were conducted in accordance with the guidelines and approval of the respective Animal Research and Ethics Committees of the Institute of Hydrobiology.

2.2. Establishment of sox9a and sox9b mutant zebrafish

Fig.1. Targeted disruption of sox9a gene by CRISPR/Cas9 in zebrafish. (A) Schematic diagram of the genomic structure of zebrafish sox9a and genotypes of the mutation. Target sequence is blue, mutations are in red and the PAM is underlined. WT, wild-type. (B) Sequencing results of the targeted region from the WT,heterozygotes and homozygotes of sox9a mutants. (C) Schematic representation of the protein functional domains of sox9a in WT and two kinds of mutants. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig.2. Sox9a is essential for zebrafish chondrogenesis. (A-C) lateral views of live larvae. (D-F)dorsal views of live larvae. (G-I) lateral views of head of the Alcian-stained larvae. (J-L) pharyngeal cartilages stained with Alcian, showing deletion or reducing of mandible and most pharyngeal cartilages.(M-O) Alcian-stained skeletons of pectoral fin, scapulocoracoid deleted. All animals at 5 dpf. Both sox9aΔ10 and sox9aΔ67 represent the homozygous sox9a mutants that are obtained as progenies of heterozygous parents. Abbreviations: bh, basihyal; cb,ceratobranchials; ch, ceratohyal; cl, cleithrum; co,scapulocoracoid; ed, endoskeletal disk; ep, ethmoid plate; hs, hyosymplectic; mk, Meckel’s cartilage; no,notochord; pq, palatoquadrate; tr, trabecular; mk?,putative Meckel’s cartilage; ch?, presumed ceratohyal; ?, uncertainty; cb?, presumed ceratobranchials; bh?, presumed basihyal. Scale bars: in F, 150 μm for A-F; in L, 75 μm for G-L; in O, 75 μm for M-O.

Sox9a and sox9b mutant zebrafish were generated by CRISPR/Cas9 technology. The DNA template preparation, gRNA and Cas9 mRNA in vitro transcription and purification were performed as previously described (Lin et al., 2017; Zhang et al., 2018). In brief, the gRNA target sites were designed with an online tool, ZIFIT Targeter (http://zifit.par tners.org/ZiFiT/CSquare9Nuclease.aspx). A BLAST search was performed in the zebrafish genome to avoid off-targets, according to the principles reported previously (Cong et al., 2013). And gRNA was transcribed with the TranscriptAid T7 High Yield Transcription Kit(Thermo). For Cas9 mRNA synthesis, pCS2-Cas9 expression vector was linearized with XbaI, purified and transcribed using the mMESSAGE mMACHINE T7 ULTRA kit (Ambion) according to the manufacturer’s instructions. Cas9 mRNA (300 ng/μl) and gRNA (20 ng/μl) were co-injected into one-cell-stage zebrafish embryos, and the mutations were analyzed by genomic PCR and sequencing as described (Lin et al.,2017). Primers used in this study are shown in Table 1.

2.3. Cartilage staining and analysis

Alcian blue staining was performed to visualize the cartilage development of zebrafish as previously described (Walker & Kimmel, 2007).Generally, 5-day-old zebrafish larvae were fixed in 4% paraformaldehyde for 24 h and then stained with 0.1% Alcian blue (Sigma-Aldrich, USA) for 24 h, followed by washes with 90%, 70%, 40%,15% ethanol and water for 2-3 h, respectively. Then, the larvae were bleached with 3% HO/1.5% KOH for 1 h and digested with 1% trypsin dissolving in saturated sodium borate for 1 h to remove the muscle.Finally, the samples were stored in 50% glycerol/50% PBS for following photographing.

3. Results

3.1. Efficient disruption of sox9a gene by CRISPR/Cas9 technology

To investigate the functions of sox9a, CRISPR/Cas9 was performed to generate sox9a mutant zebrafish. Sox9a contains three exons and the targeting site was chosen in the first exon. Finally, 10-bp deletions(named sox9aΔ10) and 67-bp deletions (10-bp insertions plus 77-bp deletions, named sox9aΔ67) were identified to establish sox9a mutant lines (Fig.1A). The genotypes of wild-type (WT), heterozygotes and homozygotes were confirmed by genomic sequencing (Fig.1B). Zebrafish sox9a encodes a protein with 462 amino acids, containing HMG domain near the N terminus and the Gln- and Ser-rich (Q/S) domain at the C terminus (Fig.1C). All mutations in sox9a resulted in the open reading frame (ORF) shift and caused premature termination codons on the first exon. Both sox9aΔ10 and sox9aΔ67 mutants lost the HMG domain and Q/S domain (Fig.1C).

3.2. Sox9a is essential for cartilage development in zebrafish

Fig.3. Targeted disruption of sox9b gene by CRISPR/Cas9 in zebrafish. (A) Schematic diagram of the genomic structure of zebrafish sox9b and genotypes of the mutation. Target sequence is blue, mutations are in red and the PAM is underlined. WT, wild-type. (B) Sequencing results of the targeted region from the WT,heterozygotes and homozygotes of sox9b mutants. (C) Schematic representation of the protein functional domains of sox9b from WT and two kinds of mutants. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

The WT and sox9a heterozygotes were normal and viable, while all sox9a homozygous mutants died before 14 dpf (days post fertilization),presumably due to the inability to take food. Gross morphology comparisons revealed that maxilla and underjaw were absent in both sox9aΔ10 and sox9aΔ67 mutants, compared to WT at 5 dpf (Fig.2A-F).Further, to examine the defects in cartilage development, we stained mutants and WT zebrafish with Alcian blue. In WT larvae, we observed the cartilages in the neurocranium, pharyngeal arches and pectoral appendages, while homozygous mutant larvae of both sox9aΔ10 and sox9aΔ67 lacked all neurocranial cartilage and most cartilage elements of the pharyngeal arches (Fig.2G-L). Additionally, the mutant larvae had mis-shaped pectoral fins and lacked the scapulocoracoid cartilage,but the cleithrum bone and endoskeletal disk cartilage appeared normal,compared to the normal larvae with fin buds supported by cartilage(Fig.2M-O). These results indicated that sox9a is essential for the formation of cartilages in the neurocranium, pharyngeal arches and pectoral girdle.

3.3. Efficient disruption of sox9b gene by CRISPR/Cas9 technology

To investigate the functions of sox9b in zebrafish, the CRISPR/Cas9 system was utilized to generate sox9b-mutated zebrafish. Sox9b contains three exons and the targeting site was chosen in the first exon, closing to the start codon, to disrupt both the HMG domain and Q/S domain(Fig.3A). Finally, 11-bp deletions (named sox9bΔ11) and 20-bp deletions (named sox9bΔ20) were identified to establish sox9b mutant lines (Fig.3A). The genotypes of WT, heterozygotes and homozygotes were confirmed by genomic sequencing (Fig.3B). Zebrafish sox9b encodes a protein of 407 amino acids, with HMG domain near the N terminus and Q/S domain at the C terminus (Fig.3C). The mutations in sox9bΔ11 and sox9bΔ20 resulted in the ORF shift and caused premature termination codons. Both mutants lost the HMG domain and Q/S domain (Fig.3C).

3.4. Sox9b is not required for cartilage development in zebrafish

All the homozygous sox9b mutants were normal and viable as the heterozygotes and WT zebrafish throughout the lifespan. Gross morphology comparisons showed the normal maxilla, underjaw and pectoral fins in all sox9b mutants as WT at 5 dpf (Fig.4A-F). We further confirmed the cartilage development by staining mutants and WT larvae with Alcian blue at 5 dpf. Results exhibited the normal cartilages of neurocranium, pharyngeal arches and pectoral appendages in both sox9bΔ11 and sox9bΔ20 mutants, which were fully consistent with the WT phenotypes (Fig.4G-O). These results demonstrated that sox9b is not required for the cartilage development in the neurocranium,pharyngeal arches and pectoral girdle.

Fig.4. Sox9b is not required for zebrafish cartilage development. (A-C) lateral views of live larvae;(D-F) dorsal views of live larvae; (G-I) lateral views of head of the Alcian-stained larvae; (J-L) pharyngeal cartilages stained with Alcian; (M-O) Alcian-stained skeletons of pectoral fin. All animals at 5 dpf. Both sox9bΔ11 and sox9bΔ20 represent the homozygous sox9b mutants that are obtained as progenies of heterozygous parents. Abbreviations: bh, basihyal; cb,ceratobranchials; ch, ceratohyal; cl, cleithrum; co,scapulocoracoid; ed, endoskeletal disk; ep, ethmoid plate; hs, hyosymplectic; mk, Meckel’s cartilage; no,notochord; pq, palatoquadrate; tr, trabecular. Scale bars: in F, 150 μm for A-F; in L, 75 μm for G-L; in O,75 μm for M-O.

4. Discussion

As one member of the SoxE subfamily, sox9 is crucial for development in vertebrates. At very early stage, sox9 is one of the key regulators of neural crest cell development, which gives rise to pigment cells, peripheral neurons, glia, craniofacial cartilage, and bone (e.g. Eisen &Weston, 1993). Then sox9 plays important roles in chondrogensis, and subsequent development of cartilage-replacement bones (Bi et al., 2001;Kist, Schrewe, Balling, & Scherer, 2002; Wagner et al., 1994; Yan et al.,2002). Besides, sox9 is involved in testis determination (Vidal, Chaboissier, de Rooij, & Schedl, 2001; Wagner et al., 1994). Thus, sox9 is a multifunctional gene in mammals.

In teleosts, due to the genome duplication event, there are two coorthologs sox9a and sox9b (Cresko et al., 2003; Postlethwait, Amores,Cresko, Singer, & Yan, 2004; Yan et al., 2002, 2005). Many studies suggested that ancestral sox9 gene functions partitioned between the two paralogs (Chiang et al., 2001; Cresko et al., 2003; Li, Zhao, Wang,Zhao, & Meng, 2002; Yan et al., 2002), which was not according to initial thought that duplicated genes may be redundant in function. For instance, loss of sox9a in zebrafish led to loss of craniofacial cartilage elements, while loss of both sox9a and sox9b led to more severe cartilage defects (Lynch & Force, 2000; Yan et al., 2002, 2005). Besides, sox9a and sox9b genes were regulated by different pathways. During zebrafish fin regeneration, sox9a was downregulated in the absence of BMP (bone morphogenetic family of proteins) signaling, but BMP signaling inhibition did not affect sox9b expression (Smith, Avaron, Guay, Padhi, &Akimenko, 2006). The location of ancestral sox9 gene expression was also partitioned by the paralogs. Sox9 played a vital function in pancreas in mouse (Seymour et al., 2007; Seymour & Sander, 2011), while only sox9b expressed in pancreas. In addition, sox9b was an upstream gene of pthlha (Parathyroid-like hormone a) in zebrafish pancreas development(Manfroid et al., 2012b; Yan et al., 2012).

Chondrocyte differentiation was disturbed in sox9b morphants,sox9a mutants and sox9b mutants, as well as in double mutants (Yan et al., 2005). However, when sox9a and sox9b were disrupted by CRISPR/Cas9 technology in this study, the results were different.Missing of sox9a function indeed led to severe the cartilage defects(Fig.2), whereas loss of sox9b function had no effect on cartilage development (Fig.4). The results suggested that sox9a is required for zebrafish chondrogensis, but sox9b is not necessary for the cartilage development in maxilla, underjaw and pectoral fin in zebrafish. The results suggest that when sox9b was disrupted, some other genes might have compensatory effect on zebrafish chondrogensis. In addition, sox9a had high expression in the scapulocoracoid, but with low expression level in the endochondral disk and cleithrum (Chiang et al., 2001; Yan et al., 2005). Consequently, lack of sox9a function impaired scapulocoracoid development, but had no effect on cleithrum and endochondral disk (Fig.2M-O). In contrast, sox9b was not expressed in the scapulocoracoid (Chiang et al., 2001; Yan et al., 2002), and this skeletal element was normal in the sox9b mutants (Fig.4M-O). As a kind of new gene editing approaches, CRISPR/Cas9 technology can be used to create mutation or deletion of a specific gene more efficiently and accurately in zebrafish and other organisms (Jao et al., 2013; Zhang & Zhou, 2014),compared with ENU or gamma radiation induced mutation. This study confirmed ancestral sox9 gene functions partitioned between the two paralogs in zebrafish.

Declaration of competing interest

All authors declare that they have no conflict of interest.

Acknowledgements