Martini D et al. (2024), Kdm7a expression is spatiotemporally regulated ...

XB-ART-60379

Dev Dyn 2024 May 01;2535:508-518. doi: 10.1002/dvdy.670.

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Kdm7a expression is spatiotemporally regulated in developing Xenopus laevis embryos, and its overexpression influences late retinal development.

Martini D , Digregorio M , Voto IAP , Morabito G , Degl'Innocenti A , Giudetti G , Giannaccini M , Andreazzoli M .

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BACKGROUND: Post-translational histone modifications are among the most common epigenetic modifications that orchestrate gene expression, playing a pivotal role during embryonic development and in various pathological conditions. Among histone lysine demethylases, KDM7A, also known as KIAA1718 or JHDM1D, catalyzes the demethylation of H3K9me1/2 and H3K27me1/2, leading to transcriptional regulation. Previous data suggest that KDM7A plays a central role in several biological processes, including cell proliferation, commitment, differentiation, apoptosis, and maintenance. However, information on the expression pattern of KDM7A in whole organisms is limited, and its functional role is still unclear. RESULTS: In Xenopus development, kdm7a is expressed early, undergoing spatiotemporal regulation in various organs and tissues, including the central nervous system and the eye. Focusing on retinal development, we found that kdm7a overexpression does not affect the expression of genes critically involved in early neural development and eye-field specification, whereas unbalances the distribution of neural cell subtypes in the mature retina by disfavoring the development of ganglion cells while promoting that of horizontal cells. CONCLUSIONS: Kdm7a is dynamically expressed during embryonic development, and its overexpression influences late retinal development, suggesting a potential involvement in the molecular machinery regulating the spatiotemporally ordered generation of retinal neuronal subtypes.

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Species referenced: Xenopus laevis
Genes referenced: kdm7a sub1

Phenotypes: Xla Wt + kdm7a mRNA + GFP (Fig. 7 D)

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	Figure 1. Real-time reverse transcription-polymerase chain reaction (RT-qPCR) analysis of kdm7a expression in developing embryos. cDNAs were synthesized from embryos and larvae at the indicated developmental stages (St.: stage), and amplified by RT-qPCR using primers specific for kdm7a, as well as for sub1.S, slc35b1.L, and ppp1ca.L, used for normalization. n: number of samples. Asterisks () indicate statistical comparisons between each stage and stage 4 (St. 4); hash marks (#) indicate statistical comparisons between each stage and stage 7 (St. 7). Data are expressed as meanstandard error of the mean. ANOVA followed by Tukey post-hoc test: p<.05; ***p<.001; ##p<.01.
	Figure 2. In situ hybridization analysis of kdm7a during segmentation and gastrulation. Embryo developmental stages are indicated at the bottom-left corner of each panel (St.: stage). Lateral view of St. 4, 7, 10.5, 11 embryos (A, B/B', C, D/D, respectively). Whole-mount embryos (A, B, D), emi-gastrulae (C), or sagittal sections from whole-mount-stained embryos (B, D) are represented in the panel. Animal pole to the top, vegetal pole to the bottom. Black arrowheads point to the dorsal lip of the blastopore; brackets indicate the dorsal involuting mesoderm. An average of 25 embryos was used for each of the analyzed stages. Scale bar 150m.
	Figure 3. In situ hybridization analysis of kdm7a during neurulation. Embryo developmental stages are indicated at the top-right corner of each panel (St.: stage). Whole-mount embryos (A, B, C; frontal view, dorsal to the top) and respective sagittal (A'; frontal side to the left and dorsal side to the top) and transverse (B, C'; dorsal side to the top) sections from whole-mount-stained embryos are represented in the panel. Black arrow indicates the anterior neural plate. Yellow arrow indicates neural crests. Red arrowheads indicate the neural tube. Black arrowhead indicates the optic vesicles. Yellow arrowheads indicate migrating neural crest cells. An average of 25 embryos was used for each of the analyzed stages. Scale bar: 300m. cg, cement gland; cp, cardiac progenitors
	Figure 4. In situ hybridization analysis of kdm7a in tail bud embryos. Embryo developmental stages are indicated at the top-right corner of each panel (St.: stage). Lateral views of whole-mount hybridized embryos, anterior to the left (A, B). (A', B) magnified views of (A, B), respectively. White lines in (B) indicate the section planes shown in (CG). Transverse sections from whole-mount St. 32 embryos, dorsal to the top (CG). Black arrowheads indicate lateral line placode, red arrowhead indicates epibranchial placode, and green arrowhead indicates olfactory placode. An average of 25 embryos was used for each of the analyzed stages. Scale bar in (AG, A', B): 500m. ba, branchial arches; e, eye; le, lens; me, mesencephalon; n, notochord; ov, otic vesicle; p, proctodeum; pn, pronephros; pr, prosencephalon; pw, posterior wall; re, retina; rh, rhombencephalon; s, somites; tt, tail tip.
	Figure 5. In situ hybridization analysis of kdm7a on stage 42 larvae cryosections. Transverse sections, dorsal side to the top (A, B, B, C, D). Lateral views of the larvae, anterior side to the left (E). White lines in (E) indicate the section planes shown in (A, B, B, C, D). Black arrows indicate the Jacobson's organ in (A) and the external ocular muscles in (B). (B) high magnification of the eye represented in (B). Asterisk in (B) and (C) indicate the absence of hybridization signal at the level of the gray matter. For this analysis 10 embryos were used. Scale bars: 200m in (A, B, C, D); 500m in (E); 100m in (B). cm, cranial muscles; CMZ, ciliary marginal zone; gc, gill chamber; GCL, ganglion cell layer; in, intestine; INL, inner nuclear layer; ipl, inner plexiform layer; irc, infrarostral cartilage; le, lens; me, mesencephalon; mm, mandibular muscles; ONL, outer nuclear layer; op, olfactory pit; opl, outer plexiform layer; ov, otic vesicle; ph, pharynx; pr, prosencephalon; re, retina; rh, rhombencephalon; sc, spinal cord; vp, velar plate.
	FIGURE 6. Whole-mount in situ hybridization analysis of neural plate markers. Frontal view (dorsal side to the top) of St. 13–16 embryos. The side of the embryos microinjected with kdm7a mRNA, together with nuclear β-galactosidase mRNA as a tracer (red spots), is on the right of each panel. The markers analyzed are indicated at the top-right corner of each panel. Images are representative of the phenotypes observed in 57/62 (A), 48/51 (B), 61/67 (C), 56/62 (D), 50/56 (E), and 48/52 (F) embryos analyzed, the remaining embryos displayed minor variations of the expression pattern. Scale bar: 300 μm.
	FIGURE 7. Evaluation of the relative area distribution of neuronal cells subtypes in mature retina. Representative images from cryosections of St. 42 tadpoles, showing GFP+ retinal clones (A, green), immunofluorescent horizontal and amacrine/displaced amacrine cells (B, red), and merge (C). White and blue arrowheads indicate examples of horizontal cells, notoriously localized in the outermost part of the INL (white: GFP+; blue: GFP). White and blue arrows in the INL indicate examples of amacrine cells (white: GFP+; blue: GFP). Orange arrows in the GCL indicate some displaced amacrine cells (all GFP- in this section). GFP+ cells of the clone that did not show immunofluorescence were unequivocally considered photoreceptors, ganglion cells, or bipolar cells, based on their location in the ONL, GCL, and INL, respectively, and based on their morphology. Scale bar (A, B, and C): 50m. (D) Quantitative analysis of relative area index obtained from n=9 (gfp) and n=12 (gfp+kdm7a) retinas. Data are expressed as meanstandard error of the mean. Unpaired Student's t-test: p<.05 (ganglion cells); MannWhitney U-test: p<.05 (horizontal cells). CMZ, ciliary marginal zone; GCL, ganglion cell layer; INL, inner nuclear layer; le, lens; ONL, outer nuclear layer.
	kdm7a ( lysine (K)-specific demethylase 7A) gene expression in X. laevis embryo, NF stage 4, assayed via in situ hybridization, view.
	kdm7a ( lysine (K)-specific demethylase 7A) gene expression in X. laevis embryo, NF stage 7, assayed via in situ hybridization, view.
	kdm7a ( lysine (K)-specific demethylase 7A) gene expression in X. laevis embryo, NF stage 7, assayed via in situ hybridization, viewed in sagittal section, animal pole to the top, vegetal pole to the bottom.
	kdm7a ( lysine (K)-specific demethylase 7A) gene expression in X. laevis embryo, NF stage 16, assayed via in situ hybridization, view. Panel B in anterior view, dorsal up; panel B' in sagittal section, dorsal up.
	kdm7a ( lysine (K)-specific demethylase 7A) gene expression in X. laevis embryo, NF stage 19, assayed via in situ hybridization, panel C, anterior view, dorsal up; panel C' in sagittal section, dorsal up. Yellow arrow indicates neural crests. Red arrowheads indicate the neural tube.
	kdm7a ( lysine (K)-specific demethylase 7A) gene expression in X. laevis embryo, NF stage 25, assayed via in situ hybridization, lateral view, dorsal up.
	kdm7a ( lysine (K)-specific demethylase 7A) gene expression in X. laevis embryo, NF stage 32, assayed via in situ hybridization, later view of head region, anterior left, dorsal up. Scale bar in 500m. Key: ba, branchial [pharyngeal] arches; e, eye; le, lens; me, mesencephalon; ov, otic vesiclepn, pronephros; pr, prosencephalon; rhombencephalon.
	kdm7a ( lysine (K)-specific demethylase 7A) gene expression in X. laevis embryo, transverse section through eye, retina and lens, at NF stage 36, assayed via in situ hybridization. Scale = 100m. Key:.CMZ, ciliary marginal zone; GCL, ganglion cell layer; INL, inner nuclear layer; ipl, inner plexiform layer; le, lens; ONL, outer nuclear layer; opl, outer plexiform layer.
	Cryosections of mature retina from a NF stage 42 tadpole showing normal distribution of neuronal cells types and cell subtypes, imaged via immunohistochemistry with GFP (green) . White arrowheads indicate examples of horizontal cells, notoriously localized in the outermost part of the INL (white: GFP+; blue: GFP). White arrows in the INL indicate examples of amacrine cells (white: GFP+; blue: GFP). Key: CMZ, ciliary marginal zone; GCL, ganglion cell layer; INL, inner nuclear layer; le, lens; ONL, outer nuclear layer. Scale bar = 50m.
	Cryosections of mature retina from a NF stage 42 tadpole showing normal distribution of neuronal cells types and cell subtypes, imaged via immunohistochemistry with amacrine cells in red. White and blue arrowheads indicate examples of horizontal cells, notoriously localized in the outermost part of the INL (white: GFP+; blue: GFP). White and blue arrows in the INL indicate examples of amacrine cells (white: GFP+; blue: GFP). Orange arrows in the GCL indicate some displaced amacrine cells (all GFP- in this section). GFP+ cells of the clone that did not show immunofluorescence were unequivocally considered photoreceptors, ganglion cells, or bipolar cells, based on their location in the ONL, GCL, and INL, respectively, and based on their morphology. Key: GCL, ganglion cell layer; INL, inner nuclear layer; le, lens; ONL, outer nuclear layer. Scale bar = 50m.
	Cryosections of mature retina from a NF stage 42 tadpole showing normal distribution of neuronal cells types and cell subtypes, imaged via immunohistochemistry with GFP (green) and amacrine cells (red) . White and blue arrowheads indicate examples of horizontal cells, notoriously localized in the outermost part of the INL (white: GFP+; blue: GFP). White and blue arrows in the INL indicate examples of amacrine cells (white: GFP+; blue: GFP). Orange arrows in the GCL indicate some displaced amacrine cells (all GFP- in this section). GFP+ cells of the clone that did not show immunofluorescence were unequivocally considered photoreceptors, ganglion cells, or bipolar cells, based on their location in the ONL, GCL, and INL, respectively, and based on their morphology. Key: GCL, ganglion cell layer; INL, inner nuclear layer; le, lens; ONL, outer nuclear layer. Scale bar = 50m.