Generation of a human SOX10 knock-in reporter iPSC line for visualization of neural crest cell differentiation.

SOX10 (SRY-box transcription factor 10) is not only a definitive molecular marker of neural crest cells (NCCs) but also an essential transcription factor for the differentiation of NCCs in vertebrate embryogenesis. Here, we report the establishment of a human SOX10 knock-in reporter iPSC line (SOX10-tdT) by CRISPR/Cas9-mediated homologous recombination, in which the expression of SOX10 can be monitored as tdTomato fluorescence. This iPSC line can provide a useful tool to model the differentiation process of human NCCs in vitro.

Generation of human GAPDH knock-in reporter iPSC lines for stable expression of tdTomato in pluripotent and differentiated culture conditions.

Human induced pluripotent stem cells (iPSCs) can differentiate into multiple cell types and are utilized for research on human development and regenerative medicine. Here, we report the establishment of human GAPDH knock-in reporter iPSC lines (GAPDH-tdT1 and 2), via CRISPR/Cas9-mediated homologous recombination, that stably express tdTomato as a constitutive cell label in both iPSCs and their differentiated derivatives. These cell lines will provide useful tools to trace cell locations and fates in 2D cultures and 3D organoids and will facilitate in vivo experiments.

Reduction of Kiss1 expression in the anteroventral periventricular nucleus is associated with atrazine-induced attenuation of the luteinizing hormone surge in female rats.

Atrazine, a commonly used herbicide, suppresses the luteinizing hormone (LH) surge in female rats, although the underlying mechanism remains unclear. Kisspeptin, encoded by the Kiss1 gene, is a hypothalamic peptide that controls gonadotropin-releasing hormone (GnRH) release from the GnRH neurons. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) are involved in regulating pre-ovulatory GnRH and LH surge. To clarify the effect of atrazine on the LH surge in female rats, we investigated its effects on hypothalamic GnRH and kisspeptin. Ovariectomized female rats in a high-dose estradiol supplementation model were orally administered vehicle or 100 mg/kg of atrazine once daily for 5 days. This attenuated the LH surge but did not affect baseline LH levels, with no difference in hypothalamic GnRH levels between the vehicle-treated and atrazine-treated animals. After the fifth treatment, subcutaneous administration of kisspeptin (at 0, 0.1, 1, and 10 nmol/kg) induced a dose-dependent LH release almost equivalent in the vehicle- and atrazine-treated animals, suggesting that GnRH neurons maintain normal responsiveness to kisspeptin. However, Kiss1 mRNA expression levels in the AVPV were significantly reduced in the atrazine-treated animals. Given the normal response of GnRH neurons to exogenously administered kisspeptin, the suppressive effect of atrazine may be explained by suppression of Kiss1 expression in the AVPV leading to the attenuation of kisspeptin release from kisspeptin neurons in the AVPV. Further studies are warranted to elucidate more precisely the mechanism of atrazine's involvement in the suppression of Kiss1 mRNA expression in the AVPV.

Effects of thalidomide on Fgf8, Bmp4 and Hoxa11 expression in the limb bud in Kbl:JW rabbit embryos.

Thalidomide (TM) induces limb defects in humans and some animal species including rabbits. Although the mechanism of TM-induced limb defects has been investigated for a long period, the limb development-related genes expressions have not been vigorously characterized in rabbits. In this study, we investigated the Fgf8, Bmp4 and Hoxa11 expressions in TM-treated JW rabbit embryos on gestation days (GDs) 10, 11 and 12 by whole mount in situ hybridization. On GDs 10 and 11, growth retardation of the embryo was induced by TM treatment. The Fgf8 expression lengths on GDs 10 and 11 in the forelimb bud were significantly or tended to be decreased in the TM-treated embryos, which was correlated to the growth retardation and was not considered to be directly relevant to the teratogenic effect of TM in the forelimb. The TM-induced characteristic changes in the expression pattern of Hoxa11 and Bmp4 on GDs 10 and/or 11 were not noted. On GD 12, TM-induced growth retardation was not noted and the Fgf8 and Bmp4 expressions were not changed. On the contrary, Hoxa11 expression was narrowed at the anterior region, which was located on the radial side, and was not changed at the middle and posterior regions in the forelimb bud and in all regions in the hindlimb bud. Because the radius malformations were induced by TM treatment, we concluded the decrease in the Hoxa11 expression was related to the TM-induced limb defects and can be a good marker for early prediction of the teratogenic effect of TM.

Reversible membrane permeabilization of mammalian cells treated with digitonin and its use for inducing nuclear reprogramming by Xenopus egg extracts.

Plasma membranes can be reversibly permeabilized by Streptolysin O. The permeabilized cells can be reprogrammed and partially dedifferentiated in the cell-free system from egg extracts. However, the permeabilizing activity of Streptolysin O is not stable, and therefore it is difficult to control its activity. An alternative method for reversible permeabilization is useful for establishing a cell-free system. Here, we used a nonionic detergent, digitonin, for permeabilization. A low concentration of digitonin induced reversible permeabilization of the plasma membrane in bovine, mouse, and porcine somatic cells. The permeabilized cells were treated with Xenopus laevis egg extracts. The treated cells showed exchange of nuclear proteins from extracts such as incorporation of Xenopus-specific histone B4 and Lamin LIII into nuclei. After resealing of the membrane, the cells showed upregulation of OCT4, SOX2, and NANOG expression. Our results suggest that reversible permeabilization with digitonin can be used to induce nuclear reprogramming and to activate pluripotent genes by a cell-free system.