Xenopus laevis (age, ~3 years old; 20 females and 10 males) were obtained from the Korean Xenopus Resource Center for Research and housed with a 12-h light/dark cycle at 18°C in containers built specifically according to the requirements for maintenance of laboratory organisms by the Institutional Review Board of Ulsan National Institute of Science and Technology (ethical approval no. UNISTACUC-16-14). To induce ovulation, Xenopus females were injected with 1,000 IU/animal of human chorionic gonadotropin (Dae Sung Microbiological Labs., Co., Ltd., Seoul, Korea) into the dorsal lymph sac in the evening. The following day, the abdomens of female frogs were gently squeezed and eggs were placed in 60-mm petri dishes containing 1X Modified Barth's Saline (MBS; 88 mM NaCl, 5 mM Hepes, 2.5 mM NaHCO₃, 1 mM KCl, 1 mM MgSO₄, and 0.7 mM CaCl₂, pH 7.8). After washing three times with 0.1X MBS, eggs were fertilized using a suspension solution of sperm obtained from isolated testes of sacrificed males. Following successful fertilization, the embryos were swirled in a 2% L-cysteine solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) to remove the jelly coat and washed five times with 0.5X MBS. Live and healthy embryos were transferred to 0.5X MBS containing 2% Ficoll^® PM 400 (GE Healthcare Life Sciences, Little Chalfont, UK) while unfertilized eggs and non-viable embryos were removed by observation under a light microscope (magnification, ×1).

A full-length kdm3a cDNA clone (GeneBank ID: NM_001095502) was obtained from the American Type Culture Collection (Manassas, VA, USA). The amplification of Flag-tagged kdm3a was performed by polymerase chain reaction (PCR), as previously described (30). The amplified fragment was sub-cloned into a pCS107 vector (Laboratory of Protein Dynamics and Signaling, National Cancer Institute, Frederick, MD, USA). Tagged kdm3a was subsequently linearized with PvuII restriction endonucleases. SP6 mMes-sage mMachine kit (Ambion; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to synthesize capped mRNAs for microinjections. The kdm3a MOs and control MOs were synthesized by Gene Tools, LLC (Philomath, OR, USA; 5′-GTT CTC TTG CTG AGT GAG CAC CAT A-3′; Control MO, 5′-CCT CTT ACC TCA GTT ACA ATT TAT A-3′). Both blastomeres of two-cell staged embryos were microinjected with MOs (30 ng/blastomere) and/or mRNAs (1 ng) and incubated until the required stages (16, 22 and 32). For rescue experiments, a MO-resistant mRNA (kdm3a*) with seven point mutations in wobble codons followed by an ATG start codon (5′-ATG GTA CTG ACG CAA CAG GAA AAT-3′) was synthesized. For tissue-specific expression of neural crest-specific markers, kdm3a MO was microinjected along with β-galactosidase (Laboratory of Protein Dynamics and Signaling, National Cancer Institute; 300 pg) into one of the two blastomeres of two-cell stage embryos.

Xenopus embryos were collected at developmental stage 16, 22 and 32 and prefixed in MEMFA (4% parafor-maldehyde, 0.1 M MOPS pH 7.4, 1 mM MgSO₄ and 2 mM EGTA) for 2 h at room temperature. WISH was performed on fixed embryos by hybridization with neural crest-specific probes, including Twist-family bHLH transcription factor 1 (twist), snail family zinc finger 2 (snail2), transcription factor AP-2α (tfap2a), early growth response 2 (egr2) forkhead box d3 (foxd3), myc proto-oncogene, bHLH transcription factor (c-myc), msh homeobox 1 (msx1) and paired box 3 (pax3), as previously described (31). To distinguish the MO-injected side of the embryos, β-galactosidase staining was performed, as previously described (31).

Total RNA extraction was performed by lysing the embryos in Isol-RNA lysis reagent (5 Prime GmbH, Hamburg, Germany) and cDNA was prepared using the first strand cDNA synthesis kit (Takara Bio, Inc., Otsu, Japan) at 65°C for 5 min followed by 42°C for 1 h and 95°C for 5 min. qPCR reaction was performed using specific primers (Table I) and SYBR Premix Ex Taq, according to the manufacturer's protocol (Takara Bio, Inc.). Thermocycler was adjusted at 95°C for 30 sec to ensure denaturation, annealing temperature was set at 55°C for 30 sec followed by extension at 72°C for 1 min (30 cycles). The analysis was performed using StepOnePlus™ Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The relative expression levels of target genes were analyzed using the 2^-ΔΔCq method (32). All data are representative of at least three experiments. Primers were designed using Primer3 software (33) and ornithine decarboxylase was used as an internal control.

Xenopus embryos were microinjected with kdm3a MO and/or 2 ng of dominant negative bone morphogenetic protein 4 receptor (dnbr) mRNA (Laboratory of Protein Dynamics and Signaling, National Cancer Institute) which induced neural tissue formation from the prospective ectoderm. The animal caps were excised using forceps following removal of the chorion membrane from the vegetal side of late blastula (stage 8.5-9 embryos). Excised animal caps were cultured at 18°C in 0.5X MBS containing 2% Ficoll PM 400 until developmental stage 16 of Xenopus embryogenesis (34).

For alcian blue staining, Xenopus embryos were collected at the late tadpole stage (stage 43) and fixed in Bouin's solution for 2 h at room temperature. Following fixation, embryos were washed in 70% ethanol + 0.1% NH₄OH and stained with 0.05% Alcian Blue 8GX (Sigma-Aldrich; Merck KGaA) in 5% acetic acid for >2 h at room temperature. The embryos were subsequently washed in 5% acetic acid for 2 h at room temperature and cleared in 100% methanol. Following clearing, the embryos were kept in benzyl benzoate and benzyl alcohol at a ratio of 2:1, respectively.

The kdm3a MO-injected embryos were treated with the lysis buffer (137 mM NaCl, 20 mM Tris-HCl pH 8.0, 1% Nonidet-P40 and 10% glycerol) and 1 mM phenyl-methylsulfonyl fluoride (Amresco, LLC, Solon, OH, USA), 5 mM sodium orthovanadate (Sigma-Aldrich; Merck KGaA) and 1X protease inhibitor mix (Roche Diagnostics, Basel, Switzerland), followed by heating of lysates at 95°C in loading buffer for 5 min. Bradford assay was used for protein determination; lysates (40 µg/lane) were analyzed using SDS-PAGE (12% gel) followed by transfer to nitrocellulose membranes. The membranes were blocked with skim milk (5%) for 1 h at room temperature and proteins were detected with anti-Flag tag (cat. no. G188; Applied Biological Materials, Inc., Richmond, BC, Canada) for 1 h at room temperature. Subsequently, the membranes were incubated with goat anti-mouse (cat. no. sc2005; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and goat anti-rabbit (cat.no. sc2004; Santa Cruz Biotechnology, Inc.) horseradish peroxidase-conjugated antibodies by incubating for 1 h at room temperature. Primary and secondary antibodies were used at a dilution of 1:1,000. The immunoreactive bands were detected using an enhanced chemiluminescence kit (HyGLO™ kit; Denville Scientific, Inc., South Plainfield, NJ, USA). Histone H3 (cat. no. ab1791; Abcam, Cambridge, UK) antibody is used as a loading control.

Total RNA was harvested from each sample (3 embryos/sample) at tadpole stage (stage 32) and RNA sequencing library was constructed according to manufacturer's protocol (TruSeq RNA Sample Prep kit v.2; cat. nos. RS-122-2001 and RS-122-2002; Illumina, Inc., San Diego, CA, USA) using polyA enrichment. For estimation of mRNA abundance, the reads were mapped to the Xenopus laevis cDNA sequences from the genome project consortium (35) using BWA software (v.0.7.15) (36), and the significant differential expression of genes was estimated using edgeR software (v.3.3.1). Genes exhibiting >4-fold change and false discovery rate <0.01 were considered significantly differentially expressed. To determine biological processes in which the differentially expressed genes were enriched, Fisher's test provided by the PANTHER database (release 20171205) (37) was used with human orthologous genes based on the best-hit results using BLASTP search (38). RNA sequencing raw data are available at the National Center for Biotechnology Information Gene Expression Omnibus database (accession no. GSE117754).

ImageJ software (v.1.45; National Institutes of Health, Bethesda, MD, USA) was used to analyze the data from the WISH and PCR analyses, and the results are presented as the mean ± standard error of the mean. Results obtained from three independent experiments. The level of significance was calculated using an unpaired t-test or one-way analysis of variance followed by Tukey's post hoc test using GraphPad Prism software (v.7; GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To study the function of Kdm3a during Xenopus embryonic development, the present study investigated the expression pattern of kdm3a in early Xenopus embryos. qPCR analysis indicated that kdm3a was expressed at early stages of embryogenesis, ranging from the egg cell to the tadpole stage of development (Fig. 1A). However, markedly increased level of kdm3a expression was observed at the neurula stage (stage 17; Fig. 1A). The spatial expression of kdm3a was analyzed using WISH, and kdm3a mRNA localized to the CNS during the neurula stage of embryogenesis (stage 16; Fig. 1B). Furthermore, kdm3a was expressed at the anterior region, including the eye and the CNS at stage 22 (early tailbud stage), and was observed in the retina, otic vesicle and branchial arches at stage 32 (late tailbud stage) of Xenopus embryogenesis (Fig. 1B). The results of qPCR and WISH analysis indicate that kdm3a may serve roles during Xenopus embryogenesis and regulate neurogenesis during embryonic development.

To investigate the physiological functions of Kdm3a during Xenopus embryogenesis, loss-of-function experiments were performed using kdm3a MO to inhibit kdm3a translation. Embryos at the two-cell stage were injected with kdm3a MO to repress kdm3a expression.

Embryos injected with the kdm3a MO exhibited malformed phenotypes, including defective head formation and markedly smaller eyes compared with the control MO-injected embryos (Fig. 2A). Anterior defects were identified in >90% of kdm3a morphants (Fig. 2B). Furthermore, abnormal pigmentation was observed among kdm3a morphants (Fig. 2C). Alcian blue staining was performed to visualize the effects of kdm3a MOs on craniofacial development. kdm3a morphants exhibited markedly smaller cartilage compared with cartilage formation in control embryos (Fig. 2C).

To confirm whether the malformed phenotypes were specifically induced by the depletion of kdm3a, rescue experiments were performed by microinjecting kdm3a* into two-cell stage embryos. Injection of kdm3a* recovered the malformed phenotypes observed in kdm3a morphants (Fig. 2D). This validation experiment confirmed that the defects observed in kdm3a morphants were caused by kdm3a knockdown during Xenopus embryogenesis. Furthermore, western blot analysis was used to examine protein expression of kdm3a. No kdm3a expression was observed in the group treated with MO, confirming the efficiency of knockdown (Fig. 2E). In addition, protein expression in embryos injected with kdm3a* or kdm3a* + MO, verified that MO did not bind to kdm3a* (Fig. 2E). These results suggested that the malformed phenotypes including defective craniofacial formation were caused by the loss of kdm3a, validating the role of this gene during Xenopus embryonic development.

The cells of neural crest emanate from neural tube immediately after its closure, and subsequently, migrate to specific regions of the embryo where they differentiate to the peripheral nervous system, facial skeleton cells and pigment cells (39,40). As described above, kdm3a morphants exhibited defects in craniofacial formation, abnormal head development and dense pigmentation (Fig. 2C), and kdm3a expression was observed at the neural plate during the neural stage of development (Fig. 1B). The present study further investigated whether kdm3a may affect neural crest migration.

To examine the role of Kdm3a in neural crest migration, kdm3a MO and β-galactosidase mRNA were injected into one blastomere of two-cell stage embryos. The injected sides of the embryos were visualized by β-galactosidase assays and WISH analysis was performed using neural crest marker genes. twist, snail2, tfap2a, egr2 and foxd3 are widely used neural crest marker genes (41,42). WISH expression patterns of twist (stage 32; late tailbud), and snail2, foxd3 and tfap2a (all at stage 20; neurula) indicated that injection with kdm3a MO significantly reduced neural crest migration on the injected side of the embryos (Fig. 3). In addition, kdm3a MO-injected sides of the embryos exhibited abnormal expression patterns of egr2-a, a hindbrain-specific marker; however, normal development of rhombomere 3 and 5, and neural crest was observed in the uninjected sides of the embryos (Fig. 3). These genes serve important roles during development of progenitor cells of neural crest into bona fide neural crest cells (43). Furthermore, twist is involved in neural crest cell migration as a repressor of transcription (44). Injected embryos exhibited abnormal expression patterns of twist, snail2 and foxd3, compared with the uninjected control (Fig. 3B).

It has been previously demonstrated that Kdm3a may serve important roles in primary neuron formation; however, it was not required for neural induction (45). To verify these previous results, the present study observed the expression patterns of early markers of neural crest progenitor cells, including c-myc (46), msx1 and pax3 (47). Expression of c-myc was not affected by kdm3a knockdown (Fig. 3). Expression patterns of msx1 and pax3-neural crest induction markers (47,48) remained unaltered on the kdm3a MO-injected side (Fig. 3). Therefore, WISH analysis results together with the β-galactosidase assay demonstrated that depletion of kdm3a may affect the migration of neural crest cells by disturbing the expression of neural crest specifiers. Since facial cartilage is formed by the cranial neural crest and trunk neural crest cells differentiate into pigment cells (49,50); therefore, it may be hypothesized that the reduced size of cartilage and abnormal pigmentation in kdm3a morphants resulted from abnormal neural crest migration.

Kdm3a serves essential roles in primary neuron formation and its depletion downregulates the expression of neural-differentiation associated genes (45). Therefore, the present study aimed to investigate the effect of Kdm3a on neural-specific (anterior, posterior and pan) genes. For this purpose, kdm3a MO and dnbr mRNA were co-injected into the animal pole region of two-cell stage embryos. Animal caps were excised from the microinjected embryos at blastula stage (stage 8.5-9) and cultured until neurula stage (stage 16). Xenopus animal caps, equivalent to embryonic stem cells, develop into the ectoderm, however, these cells are pluripotent and can differentiate into neural, mesodermal and endo-dermal tissues depending on the level and type of specific inducers (34,51). dnbr induces the formation of neural tissue from animal cap explants by inhibiting bone morphogenetic protein (BMP) signaling during early development of Xenopus and increasing expression of neurogenesis-specific genes (34).

Gene expression levels in animal caps dissected from kdm3a MO and dnbr mRNA co-injected embryos were analyzed by qPCR to determine the neural marker gene expression. Depletion of kdm3a led to the inhibition of expression of cement gland marker, anterior gradient 1 (ag1) (52), anterior markers, orthodenticle homeobox 2 (otx2), HESX hmeobox1 (hesx1) and forkhead box G1 (foxg1) (53), pan-neural marker, neural cell adhesion molecule 1 (n-cam) (48), and retina marker, SIX homeobox 3 (six3) (54), consistent with the kdm3a morphant phenotype (Fig. 4). By contrast, the expression levels of neural crest marker, ZIC family member 3 and posterior neural marker, engrailed homeobox 2 (55), were not suppressed by the knockdown of kdm3a (Fig. 4). Our qPCR data for neural marker gene expression are consistent with the previously published data (45). Furthermore, the present study indicated that kdm3a depletion inhibited the expression of six3. It may be hypothesized that kdm3a serves a role in the expression of retina specific markers at the early stage of embryonic development.

The present study further conducted a transcriptomics analysis to elucidate the role of Kdm3a during Xenopus embryogenesis. Transcriptomics analysis demonstrated that kdm3a is important for metabolism, cell-cell adhesion and mesoderm development during Xenopus embryogen-esis (Fig. 5). Knockdown of kdm3a led to downregulation of a number of genes involved in mesoderm patterning and development, including fibroblast growth factor 13 (fgf13), signal transducer and activator of transcription 3 gene 1 (stat3) and T-box 5 (tbx5) (56). Furthermore, the depletion of kdm3a induced the downregulation of multiple genes involved in mesoderm induction and patterning (Fig. 5). Knockdown of kdm3a suppressed the expression of genes involved in cell-cell adhesion, including angiopoietin 2 (angpt2), cadherin 5 (cdh5) and fibrinogen β chain (fgb) (Fig. 5).

qPCR analysis using animal caps and the transcriptomics data indicated that kdm3a depletion altered the expression of various genes involved in fundamental developmental processes. Therefore, Kdm3a may serve an important role in normal embryonic development of Xenopus laevis.