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Nucleosome regulator Xhmgb3 is required for cell proliferation of the eye and brain as a downstream target of Xenopus rax/Rx1.
Abstract Rax/Rx is a paired-type homeodomain-containing transcription factor that is essential for cell proliferation in the developing eye and brain. The molecular mechanisms that regulate cell proliferation by rax, however, are largely unknown. Here, we identify the high mobility group B3 gene (hmgb3) as a downstream target of Xenopus rax (Xrax/XRx1). Overexpression of Xhmgb3 results in an increase in eye and brain sizes due to promoted cell proliferation, while morpholino-oligo-mediated knock down of Xhmgb3 reduces eye and brain sizes. In addition, ChIP assays showed that Xhmgb3 is recruited around the promoter region of c-myc to enhance c-myc transcription. We also found that XOptx2 requires rax for its initial expression. Furthermore, we show that Xhmgb3 and XOptx2 are required for retinal development mainly at different developmental stages. Our findings reveal a novel aspect of progenitor cell proliferation during embryonic central nervous system (CNS) development.
Fig. 1. Expression of Xhmgb3 mRNA and regulation of Xhmgb3 by rax. (A–E) Regulation of Xhmgb3 mRNA (A–D) or Xhmgb3a mRNA (E) by rax. Induction of Xhmgb3 (A and B) by rax overexpression. Reduction of Xhmgb3 (C and D) or Xhmgb3a (E) by rax MO injection. Northern blot analysis of total RNA isolated from animal caps injected with water, rax or rax + smad10 RNAs. The cDNA fragment encoding for entire protein of Xhmgb3 was used as a labeled probe (A). The lower panel shows Ethidium Bromide (EtBr) staining of ribosomal RNA (A). In situ hybridization using a probe of Xhmgb3 for embryos injected with rax and β-gal RNAs (stages 18/19) (B) or rax MO (1 pmol) and β-gal RNA (stages 17/18) (C) into one dorsal blastomere at 4- or 8-cell stage on the animal side. Anterior view with dorsal to the top (B) or anterior–dorsal view (C). Section in situ hybridization using a probe of Xhmgb3 (D) or Xhmgb3a (E) followed by immunostaining against β-gal for embryos injected with rax MO (0.5 pmol) and β-gal RNA. The injected side or areas are indicated by β-gal activity in blue (B and C) or by β-gal immunostaining in red (D and E) and also indicated by green arrows (B, D and E). White brackets at both sides of midline indicate the anterior expression of Xhmgb3 in rax MO-injected and control side (C). (F–U) The expression of Xhmgb3 (F–R) or Xhmgb3a (S–U). Animal view at stage 10.5 (F). Dorsal views with anterior to the bottom at stage 12.5 (G) and stage 15 (H). Lateral views with the anterior to the left (I–N) at stages 18/19 (I), stage 25 (J), stage 28 (K), stages 33/34 (L), higher magnifications of panels K and L (M), and stage 39 (N). Yellow arrowheads indicate otic vesicles (M). (O) A cryosection of Xenopus optic vesicle at stage 25 following whole mount in situ hybridization using a probe of Xhmgb3. Black dotted lines delineate an optic vesicle. (P–R) Section in situ hybridization using a probe of Xhmgb3 for Xenopus forebrain (P) or retina at stages 40–41 (Q and R). (S–U) Expression of Xhmgb3a in embryos (S and T) or in the retina (U). Yellow arrows indicate the expression of Xhmgb3 or Xhmgb3a around CMZ (Q, R and U).
Fig. 4. Rax is required for the initial expression of XOptx2 mRNA in Xenopus. (A–I) Embryos were injected at 4- or 8-cell stage into one dorsal blastomere on the animal side and injected side is indicated by β-gal activity in blue. Embryos (stages 17/18) injected with rax MO or rax-EnR RNA with β-gal RNA (A–H). Expression of XOptx2 (A, B and F), Pax6 (C, D and G) or Six3 (E, H). Anterior views with dorsal to the top (A–H). (I) Embryos (stages 37/38) injected with rax MO (0.5 pmol). Dorsal views with anterior to the top (I). Yellow arrows indicate reduced eyes (I).
Fig. 6. Functional activity of Xhmgb3 in the anterior neural plate. An embryo (stage 13) (A), embryos (stages 17/18) (B–L). Embryos were injected with synthetic RNAs and/or Xhmgb3 MO as indicated at the top of each panel (A–L). Expression of rax (A, B, C, F, G and H), Pax6 (D), Six3 (E), XOptx2 (I and J) or XEn2 (K and L). The injected side is indicated by β-gal activity in blue (B–L) or by a yellow arrow (A).
Fig. 7. Xhmgb3 directly activates the transcription of Xenopus c-myc mRNA. (A) Northern blot analysis of total RNA from animal caps injected with water or Xhmgb3 RNA. A cDNA fragment of Xenopus c-myc was used as a labeled probe. Similar results were obtained in at least three independent experiments. The lower panel shows EtBr staining of ribosomal RNA. (B–F) Expression of Xenopus c-myc (B–D) or N-myc (E and F) or Xhmgb3 MO and β-gal RNA (C, E) or Xhmgb3 MO, Xhmgb3 RNA and β-gal RNA (D, F) into one dorsal blastomere at 4- or 8-cell stage on the animal side. Expression was examined at stages 13/14 (B–D) or stages 17/18 (E and F). The injected side is indicated by β-gal activity in blue (C–F). (G–K) ChIP analysis of Xhmgb3 for the c-myc promoter region. (G) Promoter region of Xenopus c-myc gene and designs of primers for PCR. (H–K) ChIP assay with primers for c-myc promoter region (H and I), for distal region with respect to promoter (J) or for c-myc protein-coding region (K). Immunocomplex was obtained from the extract of animal caps expressing 6x myc tag (H) or 6x myc-Xhmgb3 (I–K). ChIP assay was performed at least 4 times independently and showed same results. (L) Model of rax function in CNS development. Each molecule regulated by rax acts at different stages through distinct mechanisms, leading to normal development of the eye and brain.