kdm5c is expressed in neural tissues including the neural crest and eyes
To investigate the specific roles of KDM5C during embryogenesis, we first analyzed its gene expression pattern in Xenopus. For this purpose, we conducted RT-PCR and WISH analyses. RT-PCR revealed that kdm5c is a maternal gene, as it was found expressed throughout embryonic development from the single-cell stage to the tadpole stage (Fig. 1a). The temporal expression pattern of kdm5c indicated that this gene possesses essential functions during Xenopus development.
WISH analysis was performed to determine the spatial expression patterns of kdm5c during Xenopus embryonic development at different developmental stages (st. 6, 9, 13, 16, 22, 32, and 36; Fig. 1b–k′). The expression pattern of kdm5c indicated that this gene is expressed in the animal hemisphere of developing embryos at developmental stage 6 (Fig. 1b). Additionally, kdm5c expression was observed in late blastula-stage embryos showing enhanced expression in the animal pole (st. 9; Fig. 1c). Tissue-specific expression of kdm5c was observed during the neurula stage of embryonic development and was found expressed in the early eye field region at stage 13 (Fig. 1d). We also observed kdm5c expression in the anterior neural tissues of neurula stage embryos (st. 16; Fig. 1e) with dorsal expression in the neural plate border region (Fig. 1e′). Vibratome section analysis confirmed kdm5c expression in neural plate and neural plate border regions (Fig. 1f). We also examined the expression patterns of kdm5c during early and late tailbud stages by focusing on the lateral views of developing embryos and by transverse sectioning these embryos for detailed analysis (st. 22, 32, and 36). Our data demonstrated kdm5c expression in the branchial arches and eyes of early tailbud stage (Fig. 1g) as well as in the whole brain of late tailbud stage Xenopus embryos (Fig. 1h, h′). Although kdm5c expression was observed in the whole brain, elevated expression levels of kdm5c were detected in the forebrain and hindbrain regions of developing embryos (Fig. 1h′) as well as in the midbrain regions as revealed by transverse embryo sections (Fig. 1i, k). In addition to the whole brain, enhanced kdm5c expression was also observed in the retina and lens of Xenopus embryos (Fig. 1h′, j′). A detailed view of the embryos through vibratome transverse sections indicated that kdm5c is predominantly expressed in the lens and retina (Fig. 1i, k); furthermore, kdm5c was found strongly expressed in the ganglion cell layer of the eye (Fig. 1k′). Based on these findings, it is evident that KDM5C is significant during embryogenesis.
Knockdown of kdm5c leads to small-sized head and reduced cartilage size
To gain insights into the physiological functions of KDM5C during Xenopus embryogenesis, we conducted knockdown studies using kdm5c MOs by microinjecting kdm5c MO (48 ng) into one-cell stage embryos. To investigate the specificity of kdm5c MOs in the knockdown of kdm5c, analyzing endogenous KDM5C levels by using anti-KDM5C antibodies is the most suitable; however, due to the lack in availability of anti-KDM5C antibodies for Xenopus, we synthesized kdm5c mutant RNA using wobble base pairing (kdm5c**) and carried out western blot analysis of control embryos, embryos injected with MO-bound kdm5c mRNA, kdm5c*, and kdm5c* together with the MO. Our results revealed that kdm5c translation was blocked in MO-bound kdm5c mRNA (Additional file 1: Fig. S1). Moreover, KDM5C protein expression of embryos injected with mutated kdm5c or coinjected with mutated kdm5c and MO verified the specificity of the kdm5c MO (Additional file 1: Fig. S1). Microinjection of kdm5c MO resulted in phenotypic abnormalities, such as small-sized heads and reduced cartilage size (Fig. 2a–d). Compared with control embryos, over 80% of kdm5c MO-injected embryos exhibited smaller-sized heads (Fig. 2b). Moreover, we investigated head size by measuring the head area of kdm5c morphants relative to the head area of control MO-injected embryos and observed significantly smaller head areas of approximately 70% upon kdm5c depletion (Fig. 2c). To further investigate these cartilage defects, we carried out alcian blue staining of kdm5c MO-injected embryos (st. 46). The results indicated that kdm5c morphants exhibited a marked reduction in cartilage size compared with that of control MO-injected embryos, whereas cartilage structure was not affected (Fig. 2d).
To rule out the unspecific side effects of MOs using kdm5c RNA, we carried out rescue experiments by microinjecting Xenopus embryos with kdm5c mutant RNA along with kdm5c MO. Injection of mutant kdm5c* RNA (1.6 ng) rescued all phenotypic malformations induced by the kdm5c MO (Fig. 2a–d); embryos injected with mutant kdm5c* recovered approximately 88% of head area reduction (Fig. 2c). Taken together, these findings indicate that KDM5C is specifically involved in head and cartilage development during embryogenesis.
KDM5C regulates apoptosis and cell proliferation
Cell number plays a significant role in determining organ as well as whole organism size. To maintain constant size, cell number is tightly controlled by different mechanisms including apoptosis and cell proliferation, which are indispensable for regulating cell number and consequently organ size [28]. To elucidate whether the reduced head and cartilage sizes induced by kdm5c knockdown were due to perturbation of apoptosis and cell proliferation, we coinjected kdm5c MO and β-galactosidase mRNA unilaterally into one blastomere of two-cell stage embryos and performed terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and pH3 (phospho-histone H3) staining at stage 32 (Fig. 3). The uninjected side of the embryos served as an internal control, while β-galactosidase mRNA was used as a lineage tracer.
TUNEL staining revealed a significant increase in TUNEL-positive cells after kdm5c depletion in the kdm5c MO-injected side compared with the uninjected side of the embryos (Fig. 3a, b), indicating involvement of KDM5C in apoptosis regulation. Moreover, pH3 staining indicated significant reduction in cell proliferation in the kdm5c MO-injected side of the embryos compared with that of the uninjected side (Fig. 3c, d). Therefore, the mechanism underlying reduced head and cartilage sizes may be the result of a significant increase in apoptosis and marked decrease in cell proliferation due to kdm5c depletion.
Loss of kdm5c affects migration and differentiation of neural crest cells
Neural crest development is regulated by the dynamic expression of a number of genes, including sox3 [29], pax3 [30], twist [31], slug [32], and members of the soxE family, i.e., sox8, sox9, and sox10 [29]. To evaluate the functions of kdm5c in neural crest development, we performed a loss-of-function analysis using the kdm5c MO and examined its effects on expression of neural crest specifiers. Embryos at the two-cell stage were unilaterally coinjected with kdm5c MO and β-galactosidase mRNA into one blastomere of two-cell stage embryos, after which WISH was performed using these neural crest specifiers. Our results showed sox3 and pax3 expressions in the expanded neural plate regions of the kdm5c MO-injected side of embryos (Fig. 4a, b), while RT-PCR analysis indicated similar expression levels of sox3 and pax3 between control and kdm5c MO-injected embryos (Fig. 4c). In contrast to sox3 and pax3, downregulated expression was observed for twist, slug, sox8, and sox10 in the kdm5c MO-injected side; however, sox9 expression remained unaffected (Fig. 5a, b). sox8, sox9, and sox10 belong to the SoxE protein family and play a significant role along with other neural crest specifiers (i.e., twist and snail). sox9 is expressed in cranial and cardiac neural crest cells and precedes the expression of sox8 and sox10 [33]. Thus, we speculated that the unaffected expression levels of sox9 in the kdm5c MO-injected side are why the cranial cartilage did not exhibit deformations, only a reduction in size (Figs. 2d, 5a, b). To further clarify the expression of these neural crest-specific genes, RT-PCR analysis indicated that expression levels of twist, slug, sox8, and sox10 were considerably reduced, while sox9 remained unaltered in kdm5c MO-injected embryos compared with control MO (Fig. 5c).
We further examined the twist expression pattern during the late tailbud stage (st. 32) to analyze the effect of kdm5c knockdown on neural crest migration. Perturbed twist expression was observed during later stages of embryonic development after kdm5c knockdown, indicating abnormal migration of neural crest cells (Fig. 5d, e). Moreover, the abnormal expression patterns of neural crest specifiers were significantly rescued by injecting kdm5c mutant RNA, ruling out any unspecific side effects of the kdm5c MOs (Figs. 4a–c, 5a–e). Altogether, these results indicate that KDM5C is required for the expression of neural crest specifiers; thus, perturbation of kdm5c expression altered expression patterns and influenced neural crest migration.
KDM5C is involved in eye development
As our spatial expression analysis of kdm5c in Xenopus embryos indicated enriched expression of kdm5c in the eye regions (Fig. 1g–k′) and on the basis of the well-established Xenopus fate maps, we sought to investigate the involvement of kdm5c in eye development during Xenopus embryogenesis. Thus, we conducted unilateral microinjection of kdm5c into eight-cell stage Xenopus embryos and found that kdm5c morphants exhibited significantly smaller and deformed eyes, i.e., coloboma/optic fissures, compared with that of control embryos (Fig. 6a, d). Statistical analysis revealed that compared with control embryos, more than 80% of kdm5c MO-injected embryos exhibited eye defects (Fig. 6b) and among the kdm5c morphants, approximately 20% exhibited small-sized eyes and 60% possessed deformed eyes (Fig. 6c). Additionally, histological analysis of the eye structure through vibratome sections indicated that kdm5c morphants exhibited abnormal retinal pigment epithelium (Fig. 6a). For validating the specificity of kdm5c MO-induced eye defects, we performed rescue experiments that confirmed the eye malformations observed in kdm5c morphants were specifically caused by a depletion of kdm5c and not through unspecific side effects of kdm5c MOs (Fig. 6a–c). In short, our results implicate kdm5c in eye development during Xenopus embryogenesis.
Loss of kdm5c induced phenotypic eye defects of coloboma/optic fissures in the morphant embryos (Fig. 6a–d), which may have resulted from non-closure of the choroid fissure, leading to coloboma. Dorsoventral (DV) patterning of the retina is important for the choroid fissure, and impairment of DV patterning can result in colobomas. Retina DV patterning is controlled by asymmetric expression of transcription factors, such as vax1 (optical stalk-specific), vax2 (optical stalk and ventral retina-specific), pax6 (ventral and dorsal retina-specific), and tbx5 (dorsal retina-specific), that regionalize optic vesicle into three compartments, i.e., optic stalk, dorsal retina, and ventral retina [34]. Thus, we examined the effects of kdm5c knockdown on DV patterning by analyzing the expression of DV-patterning markers (vax1, vax2, pax6, and tbx5) through WISH. We found that depletion of kdm5c significantly downregulated vax1, vax2, pax6 expressions, while tbx5 expression was slightly reduced (Fig. 6e); thus, the reduced expression of DV-patterning markers may be responsible for the colobomas observed in kdm5c morphants.
In addition to colobomas, vibratome sections of kdm5c morphants indicated retinal lamination defects (Fig. 6a). Therefore, we performed WISH with the well-known eye-specific markers arr3 (photoreceptor cell-specific), prox1 (horizontal cell-specific), vsx1 (bipolar cell-specific), and pax6 (ganglion and amacrine cell-specific) to further analyze kdm5c morphant eyes (st. 40). We obtained both mild and severe phenotypes through WISH analysis as well as severe disorganization of retinal cell layers (Fig. 6f). Overall, our findings indicate that kdm5c knockdown induced severe eye defects, including colobomas and perturbed retinal lamination.
KDM5C is significant for early eye field induction and differentiation
We further investigated the roles of KDM5C at the molecular level during eye development by coinjecting kdm5c MO and β-galactosidase mRNA unilaterally into one dorsal blastomere of eight-cell stage embryos. WISH analysis of these kdm5c MO/β-galactosidase mRNA-coinjected embryos was performed to evaluate the effect of kdm5c knockdown on eye field induction and differentiation by examining the expression patterns of otx2 [35], rax [36], and pax6 [37] at stage 16 of embryogenesis. Compared with control, otx2, rax, and pax6 expressions were downregulated in the kdm5c MO-injected side of the embryos (Fig. 7a, b). Furthermore, we examined the effect of kdm5c knockdown on eye differentiation at stage 32 (Fig. 7c, d) and found that all tested eye-specific markers exhibited reduced expression levels in the kdm5c MO-injected side, whereas normal expression was observed on the uninjected side of the embryos. WISH analysis with cryba1 specific for the vertebrate eye lens [38] was also conducted at stage 32; however, kdm5c knockdown did not affect lens development during Xenopus embryogenesis (Fig. 7e, f). RT-PCR analysis further confirmed that the presence of KDM5C is significant during eye field induction and differentiation but is not required during eye lens development (Fig. 7g). Moreover, rescue experiments effectively recovered the reduced expression levels of eye-specific markers induced by kdm5c knockdown (Fig. 7a–f), verifying the specificity of KDM5C in eye development during Xenopus embryogenesis. Altogether, our results demonstrate that KDM5C plays an important role during eye field induction and differentiation and that loss of kdm5c results in anomalies of retina formation during Xenopus embryogenesis.
KDM5C is required for organogenesis and morphogenesis
To pinpoint target genes specifically affected by kdm5c knockdown, we performed a transcriptome analysis of kdm5c morphants. Total RNA of kdm5c morphants was extracted and processed for transcriptome and RNA sequence analysis. RNA sequence analysis identified important gene groups (Additional file 2: Fig. S2); genes were classified into 19 groups using PANTHER gene ontology; and a bar chart was plotted based on the downregulated expression of these gene groups in the kdm5c morphants (Additional file 2: Fig. S2). These analyses indicated that kdm5c plays significant roles in morphogenesis.
To validate RNA sequence analysis, genes with high fold-change values were selected and RT-PCR was performed to analyze the expression of these genes in kdm5c morphants. epha4, epha2, efnb2, sox8, sox10, aldh1a2, and wnt8a are all genes involved in the regulation of eye and neural crest development during embryogenesis [39,40,41,42,43]. We found that epha4, epha2, efnb2, sox8, sox10, aldh1a2, and wnt8a were downregulated among several other genes (Fig. 8). RT-PCR showed reduced gene expression of sox8, sox10, and wnt8a, confirming that kdm5c is essential for the regulation of neural crest development (Figs. 5c, 8). Moreover, the downregulated expression patterns of epha4, epha2, efnb2, and aldh1a2 validated the involvement of KDM5C in eye development during embryogenesis (Fig. 8). Overall, our results demonstrate that KDM5C is critical for morphogenesis and specifically influences neural crest development and eye formation during embryonic development.