Oligodendrocytes (OLs) are the glial cells responsible for the synthesis and maintenance of myelin in the central nervous system. Recently, Hoxa2 was found by our laboratory to be expressed by OLs and down-regulated at the terminal differentiation stage during oligodendrogenesis in mice (Nicolay et al., 2004b). To further investigate the role of Hoxa2 in oligodendroglial development, a tetracycline regulated controllable expression system was utilized to establish two stable cell lines where the expression level of Hoxa2 gene could be up-regulated (CG4-SHoxa2 [sense Hoxa2]) or down-regulated (CG4-ASHoxa2 [Antisense Hoxa2]) in CG4 glial cells. Morphologically, no obvious differences were observed between CG4-SHoxa2 and CG4 wild-type cells, whereas CG4-ASHoxa2 cells exhibited much shorter processes compared with those of CG4 wild-type cells. Data from BrdU uptake assays indicated that an up-regulation of Hoxa2 gene promoted the proliferation of CG4-SHoxa2 cells. PDGF&alphaR (Platelet-derived growth factor [PDGF] receptor alpha), a receptor for the mitogen PDGF that enhances the survival and proliferation of OLs, was assessed at the mRNA level in both CG4 and CG4-SHoxa2 cells, but no significant differences were observed between Hoxa2 up-regulated cells and wild-type CG4 cells with respect to the mRNA level of PDGF&alphaR. In addition, specific investigations of the differentiation of CG4-SHoxa2 cells were carried out by characterizing the composition of stage specific oligodendroglial subpopulations in culture. Our immunocytochemical study did not indicate the differentiation course of the genetically engineered cells was significantly altered compared to CG4 wild-type cells, although results from semi-quantitative RT-PCR of oligodendrocyte-specific ceramide galactosyltransferase (CGT) and myelin basic protein (MBP) indicate that the differentiation of CG4-SHoxa2 cells was delayed when Hoxa2 gene was up-regulated.

Oligodendrocytes (OLs) are the glial cells responsible for the synthesis and maintenance of myelin in the central nervous system. Recently, Hoxa2 was found by our laboratory to be expressed by OLs and down-regulated at the terminal differentiation stage during oligodendrogenesis in mice (Nicolay et al., 2004b). To further investigate the role of Hoxa2 in oligodendroglial development, a tetracycline regulated controllable expression system was utilized to establish two stable cell lines where the expression level of Hoxa2 gene could be up-regulated (CG4-SHoxa2 [sense Hoxa2]) or down-regulated (CG4-ASHoxa2 [Antisense Hoxa2]) in CG4 glial cells. Morphologically, no obvious differences were observed between CG4-SHoxa2 and CG4 wild-type cells, whereas CG4-ASHoxa2 cells exhibited much shorter processes compared with those of CG4 wild-type cells. Data from BrdU uptake assays indicated that an up-regulation of Hoxa2 gene promoted the proliferation of CG4-SHoxa2 cells. PDGF&alphaR (Platelet-derived growth factor [PDGF] receptor alpha), a receptor for the mitogen PDGF that enhances the survival and proliferation of OLs, was assessed at the mRNA level in both CG4 and CG4-SHoxa2 cells, but no significant differences were observed between Hoxa2 up-regulated cells and wild-type CG4 cells with respect to the mRNA level of PDGF&alphaR. In addition, specific investigations of the differentiation of CG4-SHoxa2 cells were carried out by characterizing the composition of stage specific oligodendroglial subpopulations in culture. Our immunocytochemical study did not indicate the differentiation course of the genetically engineered cells was significantly altered compared to CG4 wild-type cells, although results from semi-quantitative RT-PCR of oligodendrocyte-specific ceramide galactosyltransferase (CGT) and myelin basic protein (MBP) indicate that the differentiation of CG4-SHoxa2 cells was delayed when Hoxa2 gene was up-regulated.

Valproic acid (2-propylpentanoic acid, VPA) is a clinically used anti-epileptic drug and an effective mood stabilizer. VPA is also a histone deacetylase inhibitor and can induce embryonic malformations in both humans and mice. The mechanism(s) of VPA-induced teratogenicity are not well characterized. The objectives of my study were three fold, to: (i) investigate the effect of VPA on mouse embryonic development, (ii) characterize the putative mechanism(s) of VPA-induced teratogenicity and, (iii) investigate VPA associated epigenetic regulation of Hoxa2 gene in cell lines and in developing embryos. Whole mouse embryo cultures were treated with VPA at doses of 0, 50 (0.35 mM), 100 (0.70 mM), 200 (1.4 mM), and 400 µg/mL (2.8 mM), encompassing the therapeutic range of 0.35 mM to 0.70 mM. Van Maele-Fabry’s morphologic scoring system was used to quantitatively assess embryonic organ differentiation and development. Hoxa2 gene expression was measured by quantitative real-time RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). To assess epigenetic changes on the Hoxa2 gene promoter, DNA methylation was determined by bisulfite (BSP) sequencing and pyrosequencing. Histone “bivalent domains” H3K4me3 (histone 3 lysine 4 trimethylation) and H3K27me3 (histone 3 lysine 27 trimethylation) associated with gene activation repression, respectively, analyzed qChIP-PCR (quantitative chromatin immunoprecipitation-PCR). Telomere length and telomerase activity were analyzed in mouse embryos and in NIH3T3 cell line treated with VPA. Results indicate significantly increased incidence of dysmorphogenesis in embryos (11.8%, 35.3%, 47.0% and 88.3%) exposed to increasing doses of VPA (0.35 mM, 0.70 mM, 1.4 mM and 2.8 mM respectively). Van Maele-Fabry’s quantitative differentiation assessment of developing embryos demonstrated a significantly lower score for the circulation system, central nervous system, craniofacial development and limb development in VPA treated embryos (0.35 mM to 2.8 mM) compared to the untreated control group. Glutathione homeostasis was altered as indicated by decreased total glutathione content and increased GSSG/GSH ratio in all VPA treatment groups. In addition, a dose-dependent inhibition of Hoxa2 gene expression was observed in embryos and in the NIH3T3 cell line exposed to VPA. Pre-treatment with ascorbic acid [1000 µg/mL (5 mM)] restored glutathione level and normalized Hoxa2 gene expression in embryos exposed to VPA. DNA methylation status was characterized on the Hoxa2 gene promoter at the three CpG islands; CpG island 1 (-277 to -620 bp), CpG island 2 (-919 to -1133 bp), and CpG island 3 (-1176 to -1301 bp) in the two cells lines (NIH3T3 and EG7) and in developing embryos. CpG sites remained unmethylated on the Hoxa2 gene promoter in the NIH3T3 cell line which expresses the Hoxa2 gene, whereas these same CpG sites were methylated in EG7 cells that did not express Hoxa2. CpG island 1 is closest to Hoxa2 transcription start site and its methylation status was most affected. In developing embryos, CpG island 1 was found to be highly methylated at E6.5 when Hoxa2 is not expressed, whereas the methylation status of CpG sites on the CpG island 1 declined between E8.5 and E10.5 when Hoxa2 expression is present. VPA induced methylation of several CpG sites on CpG island 1 in NIH3T3 cell line and in E10.5 embryos when Hoxa2 expression was down regulated following VPA exposure. In addition, embryos and the NIH3T3 cell line treated with VPA impacted the “bivalent domains” resulting in increased H3K27me3 enrichment and decreased H3K4me3 enrichment on Hoxa2 promoter. Pre-treatment with ascorbic acid normalized Hoxa2 expression and histone bivalent domain changes and prevented increased DNA methylation following VPA exposure. Moreover, the telomerase activity and telomere length were both impacted by changes in glutathione redox potential induced by VPA. Oxidative stress following VPA treatment reduced telomerase activity and accelerated telomere shortening. These results are the first to demonstrate: (i) a correlation between VPA dose and total morphologic score in the developing mouse embryos. VPA impacted embryonic tissue differentiation and neural system development in the dose range of 0.35 mM to 2.8 mM; (ii) VPA altered glutathione homeostasis in cultured mouse embryos and inhibited Hoxa2 gene expression; (iii) Histone bivalent domains of H3K27 and H3K4 trimethylation and DNA methylation status at the Hoxa2 gene promoter region were altered following treatment with VPA. This appears to be the epigenetic event in transcriptional silencing of Hoxa2 gene expression after VPA exposure; and (iv) Ascorbic acid normalizes glutathione homeostasis, H3K27 and H3K4 trimethylation and DNA methylation status, restoring Hoxa2 gene expression following VPA exposure. Taken together our results show VPA- induced altered glutathione homeostasis, telomere shortening and telomerase dysfunction, and an inhibition of Hoxa2 gene expression leads to developmental abnormalities. Exposure to ascorbic acid had a protective effect on developing embryos exposed to VPA.

Cleft palate is a common human birth defect caused by any process which interferes with palatogenesis. Studies in Hoxa2 mutant (Hoxa2-/-) mice which exhibit a secondary cleft palate were reported to be due to an abnormal positioning of the tongue which prevents normal palatal shelf fusion to occur. To obtain direct evidence for the importance of Hoxa2 in murine palate development, an in vitro whole organ palatal culture model was developed, eliminating any influences from the tongue. A retroviral gene delivery system was employed, containing either Hoxa2 sense or Hoxa2 antisense cDNA, to respectively enhance or knockdown the expression of Hoxa2 mRNA in the developing palate. <p>Our results show that palatal cultures infected with the lowest titer of Hoxa2 sense virus induce a fusion rate of 72.7%, which is similar to palatal cultures treated with the control virus (81.8%), although fusion rates of 41.2% to 50.0% were observed in palates infected with higher titers. With the antisense virus treated group, a more profound inhibition of the fusion rate was observed (27.7% - 46.1%), which is comparable with the frequency of palatal fusion in Hoxa2-/- mice (44.4%). Additionally, the palatal shelves in both sense and antisense virus treated groups appear to be relatively shorter in length, than those measured in the control group. Interestingly, in the antisense virus treated group, the ratio of the length of the fused portion to the length of palatal shelves appears to be relatively large compared to the control group. Verification and quantification of Hoxa2 mRNA in the developing palate between E12.5 and E15.5 was performed by real-time RT-PCR. Hoxa2 gene expression was observed at all stages studied, with expression being the highest at E12.5 and declining from E13.5. The expression level remained constant from E13.5 through E15.5. These findings demonstrate for the first time that Hoxa2 may play a direct role in murine palate development. Results suggest that both factors (the absence of Hoxa2 gene in the palate causing delayed palatal development, as well as the position of the tongue) appear to act in unison to produce cleft palate in Hoxa2 knockout mice.

Cleft palate is a common human birth defect caused by any process which interferes with palatogenesis. Studies in Hoxa2 mutant (Hoxa2-/-) mice which exhibit a secondary cleft palate were reported to be due to an abnormal positioning of the tongue which prevents normal palatal shelf fusion to occur. To obtain direct evidence for the importance of Hoxa2 in murine palate development, an in vitro whole organ palatal culture model was developed, eliminating any influences from the tongue. A retroviral gene delivery system was employed, containing either Hoxa2 sense or Hoxa2 antisense cDNA, to respectively enhance or knockdown the expression of Hoxa2 mRNA in the developing palate. <p>Our results show that palatal cultures infected with the lowest titer of Hoxa2 sense virus induce a fusion rate of 72.7%, which is similar to palatal cultures treated with the control virus (81.8%), although fusion rates of 41.2% to 50.0% were observed in palates infected with higher titers. With the antisense virus treated group, a more profound inhibition of the fusion rate was observed (27.7% - 46.1%), which is comparable with the frequency of palatal fusion in Hoxa2-/- mice (44.4%). Additionally, the palatal shelves in both sense and antisense virus treated groups appear to be relatively shorter in length, than those measured in the control group. Interestingly, in the antisense virus treated group, the ratio of the length of the fused portion to the length of palatal shelves appears to be relatively large compared to the control group. Verification and quantification of Hoxa2 mRNA in the developing palate between E12.5 and E15.5 was performed by real-time RT-PCR. Hoxa2 gene expression was observed at all stages studied, with expression being the highest at E12.5 and declining from E13.5. The expression level remained constant from E13.5 through E15.5. These findings demonstrate for the first time that Hoxa2 may play a direct role in murine palate development. Results suggest that both factors (the absence of Hoxa2 gene in the palate causing delayed palatal development, as well as the position of the tongue) appear to act in unison to produce cleft palate in Hoxa2 knockout mice.