Fgf8P2A-3×GFP/+: A New Genetic Mouse Model for Specifically Labeling and Sorting Cochlear Inner Hair Cells / 神经科学通报·英文版

The cochlear auditory epithelium contains two types of sound receptors, inner hair cells (IHCs) and outer hair cells (OHCs). Mouse models for labelling juvenile and adult IHCs or OHCs exist; however, labelling for embryonic and perinatal IHCs or OHCs are lacking. Here, we generated a new knock-in Fgf8P2A-3×GFP/+ (Fgf8GFP/+) strain, in which the expression of a series of three GFP fragments is controlled by endogenous Fgf8 cis-regulatory elements. After confirming that GFP expression accurately reflects the expression of Fgf8, we successfully obtained both embryonic and neonatal IHCs with high purity, highlighting the power of Fgf8GFP/+. Furthermore, our fate-mapping analysis revealed, unexpectedly, that IHCs are also derived from inner ear progenitors expressing Insm1, which is currently regarded as an OHC marker. Thus, besides serving as a highly favorable tool for sorting early IHCs, Fgf8GFP/+ will facilitate the isolation of pure early OHCs by excluding IHCs from the entire hair cell pool.

Expression and clinical significance of FGF8b in ovarian cancer tissues / 中华内分泌外科杂志

Objective To investigate the expression of FGF8b in ovarian cancer tissue and its relationship with clinical characteristics of patients.Methods Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the FGF8b mRNA level in 28 cases of human ovarian cancer tissues and the adjacent normal tissues.Western blot method was used to detect the FGF8b protein.The relationship between the clinical characteristics of the patients was analyzed.Results The expression of FGF8b mRNA in ovarian cancer tissue (0.23±0.08) was higher than that of paraplastic tissue (0.71±0.11)(P＜0.05),and FGF8b protein expression in cancerous tissue (0.27±0.03) was higher than that in cancerous tissue (0.44±0.03)(P＜0.05).In ovarian cancer tissues,FGF8b positive rate (76.9 ％) in patients with stage Ⅲ to Ⅳ was higher than in patients with stage Ⅰ to Ⅱ (33.3％) (P＜0.05) The positive rate of lymph node metastasis (60.0％) was higher than that of non-lymphatic metastasis (27.8％)(P＜0.05).Conclusion FGF8b is significantly highly expressed in ovarian cancer tissues,which is correlated with the clinical characteristics of the ovarian cancer patients.

Effect of target silencing FGF8b gene on proliferation and apoptosis of ovarian cancer SKOV-3 cells / 中华内分泌外科杂志

Objective To investigate the effect of targeted silencing FGF8b gene on the proliferation and apoptosis of ovarian cancer SKOV-3 cells and its possible molecular mechanism.Methods The design and synthesis of small interfering RNA targeting FGF8b (siRNA),was transfected into ovarian cancer cell SKOV-3,using four methyl thiazolyl tetrazolium (MTT) method to detect cell proliferation and apoptosis of TUNEL kit cells.The expression of Bcl-2 and Bax protein by Western blot assay in cells.Results FGF8b siRNA could inhibit the expression of FGF8b protein in ovarian cancer SKOV-3 cells.The experimental group showed a high proportion of TUNEL positive cells(22.33±2.517) (P＜0.01).The down-regulation of FGF8b protein significantly inhibited the proliferation of ovarian cancer SKOV-3 cells,reduced the expression of Bcl-2(0.586±0.025) (P＜0.01),and increased the expression of Bax(0.334±0.019) (P＜0.01).Conclusion Down regulation of FGF8b expression can inhibit the proliferation of ovarian cancer SKOV-3 cells and induce cell apoptosis,which may be related to the expression changes of Bcl-2 and Bax protein,suggesting that FGF8b plays an important role in the development of ovarian cancer.

FGF8 is Essential for Functionality of Induced Neural Precursor Cell-derived Dopaminergic Neurons

Induced neural precursor cells (iNPCs) are one source of transplantable dopaminergic neurons used in cell therapy for Parkinson's disease. In the present study, we demonstrate that iNPCs can be generated by transducing Brn2, Ascl1, Myt1L and Bcl-xL in a culture supplemented with several mitogens and subsequently can be differentiated to dopaminergic neurons (DA). However, studies have shown that iDA and/or iNPC-derived DA neurons using various conversion protocols have low efficiency. Here, we show that early exposure of FGF8 to fibroblasts efficiently improves differentiation of DA neurons. So our study demonstrates that FGF8 is a critical factor for generation of iNPC-derived DA neurons.

Delayed Long Bone Formation in Hyperthermia-exposed Mouse Embryos / 체질인류학회지

Maternal hyperthermia, which is currently confirmed as one of major causative factors inducing growth retardation, congenital anomalies and abortion, is known to influence normal development of CNS and various organ system. In addition, maternal hyperthermia could induce severe developmental defects including development of the limb. However, it is not clearly identified how maternal hyperthermia affects the expression of chondrogenesis-related proteins in developing limb of mouse. Thus, this study is aimed to investigate the effects of the maternal hyperthermia on the expression of a various proteins in developing upper limb. To elucidate it, ICR mice were used in this study, and the animals were divided into control and heat shock groups. The heat shock treatment was given to embryonic day (ED) 8. The animals were sacrificed on ED 11, 13, 15 and 17, and the humerus were removed. Chondrogenesis-related factors such as FGF8, SOX9 and collagen II were detected on ED 11, 13 and 15 using western blot and immunohistochemistry. Developing humerus on ED 17 was stained with alizarin red S and alcian blue. The expression of FGF8 of heat shock groups was continued even though the development was succeeded. SOX9 expression in heat shock groups was significantly elevated on ED 13 compared to the control embryos. In addition, collagen II expression of heat groups was significantly higher than that of the control group on ED 13 and 15. The results of this study suggest that hyperthermia causes delayed endochondral ossification in long bone through continuous expression of FGF8, SOX9 and collagen II proteins even though the endochondral ossification is succeeded.

Delayed Long Bone Formation in Hyperthermia-exposed Mouse Embryos / 체질인류학회지

Maternal hyperthermia, which is currently confirmed as one of major causative factors inducing growth retardation, congenital anomalies and abortion, is known to influence normal development of CNS and various organ system. In addition, maternal hyperthermia could induce severe developmental defects including development of the limb. However, it is not clearly identified how maternal hyperthermia affects the expression of chondrogenesis-related proteins in developing limb of mouse. Thus, this study is aimed to investigate the effects of the maternal hyperthermia on the expression of a various proteins in developing upper limb. To elucidate it, ICR mice were used in this study, and the animals were divided into control and heat shock groups. The heat shock treatment was given to embryonic day (ED) 8. The animals were sacrificed on ED 11, 13, 15 and 17, and the humerus were removed. Chondrogenesis-related factors such as FGF8, SOX9 and collagen II were detected on ED 11, 13 and 15 using western blot and immunohistochemistry. Developing humerus on ED 17 was stained with alizarin red S and alcian blue. The expression of FGF8 of heat shock groups was continued even though the development was succeeded. SOX9 expression in heat shock groups was significantly elevated on ED 13 compared to the control embryos. In addition, collagen II expression of heat groups was significantly higher than that of the control group on ED 13 and 15. The results of this study suggest that hyperthermia causes delayed endochondral ossification in long bone through continuous expression of FGF8, SOX9 and collagen II proteins even though the endochondral ossification is succeeded.

The Effects of Hyperthermia on FGF-8 and BMP-4 Expression during Early Odontogenesis in Hsp70 Knock-out Mice Fetuses / 체질인류학회지

During early tooth development, multiple signaling molecules are expressed in the dental lamina and induce the dental mesenchyme. One signal, FGF-8, is expressed in the early dental epithelium, another one, BMP-4, has been shown to induce morphologic changes in dental mesenchyme. Meanwhile, hyperthermic exposure during pregnancy, as one of teratogens, is known to disturbe normal development and induce several congenital anomalies. This study is aimed to investigate the effects of maternal hyperthermia on the expressions of FGF-8 and BMP-4 in early odontogenesis. The pregnant Hsp70 knock-out at gestational day 8 were immersed in 43degrees C water bath until their body core temperature reached at 43degrees C. Thereafter, pregnant mice were given more 5 minutes hyperthermic exposure. Heat-untreated Hsp70 KO mice fetuses were used as the control group. Fetuses were collected at embryonic day (ED) 13, 15 and 17. Developing tooth in the mandible was processed for immunohistochemical study. Tissue sections were immunostained for FGF-8 and BMP-4 and observed with light microscope. The obtained results were as follows: Tooth development in the heat shocked (HS) group is delayed rather than the control group in the given developmental period. FGF-8 immunolocalization in control group at ED 13 was gradually decreased compared to the HS group which showed continuously positive immunoreaction. BMP-4 immunolocalization was detected in dental mesenchyme, however, there was no positive immunoreaction found in HS group. These results suggest that maternal hyperthermia should induce the early odontogenesis, delay the expression of FGF-8 in dental epithelium, and disturbe the expression of BMP-4 in dental mesenchyme. Consequently, hyperthermic exposure during pregnancy affects epithelial-mesenchymal interactions.

Immunohistochemical Study on Early Odontogenesis in Hsp70 Knock-out Mice Fetuses Exposed by Maternal Hyperthermia / 대한해부학회지

To investigate the effects of maternal hyperthermia on early odontogenesis,pregnant Hsp70 knock-out and wild type mice at embryonic day (ED)8.5 were immersed in a 43 degrees C water bath until their core body temperature reached that temperature,and then given a further 5 min of hyperthermia.Untreated Hsp70 WT mice fetuses were used as the control group.Fetuses were collected at EDs 13.5,15.5 and 17.5.Developing teeth in the mandible were processed for histological and immunohistochemical studies.Tissue sections were immunostained for FGF-8 and FGF -4 and observed using light microscopy.In the controls, FGF-8 immunolocalization was observed in cells within the dental lamina and in apically located dental epithelium at ED 13.5.However,a few cells were immunopositive in the heat shocked (HS)group.At EDs 15.5 and 17.5 of the control group,the basal lamina adjacent to the dental pulp showed positive immunostaining.In contrast,most of the dental epithelium was immunopositive at ED 15.5 in the HS group and inner and outer dental epithelial cells were continuously immunopositive by ED 17.5.FGF-4 immunolocalization was found in apical dental epithelium at ED 13.3 in the control group,but no such positive reaction was observed in the HS group.At ED 15.5 in the controls,basal lamina and dental epithelium near the cervical loop were immunopositive.In contrast,early cap-stage teeth had cells near the mouth of the dental bud and cervical loop that were immunopositive to FGF-4 in the HS group.In controls at ED 17.5,cells near the future secondary enamel knot were immunopositive,whereas most of the dental epithelium except for cells in the mouth of the dental lamina was negative in the HS group.Thus,maternal hyperthermia may inhibit normal odontogenesis through sustained production of FGF-8 and downregulation of FGF-4.

Effects of Maternal Hyperthermia on the Palatal Mesenchyme Development of Hsp70 Knock-out Mice Fetuses / 체질인류학회지

To investigate the effects of maternal hyperthermia on the development of the palate, pregnant Hsp70 knock-out mice at gestational day (GD) 8.5 were immersed in 43degrees C water bath until their body core temperature reached at 43degrees C. Thereafter, pregnant mice were given more 5 minutes hyperthermic exposure. Heat-untreated Hsp70 WT mice fetuses were used as the control group. Fetuses were collected at embryonic day 13.5, 14.5 and 15.5 (E13.5, E14, 5 and E15.5). Heads followed by removal of the mandible and the tongue were obtained and photographed for palatal development. Developing palates were processed for histological and immunohistochemical studies. Tissue sections were immunostained for TGF-beta2, FGF-8 and fibronectin, and observed with light microscope. The obtained results were as follows: Cleft palate was formed in heat-treated Hsp70 KO fetuses at E14.5 and E15.5. Immunohistochemical findings indicated that TGF-beta2 expression of the experimental fetuses were more delayed than that of the control fetuses. Mesenchyme under the medial edge epithelium (MEE) and cells of MEE showed continuously strong positive TGF-beta2 reactivity at E15.5. FGF-8 was revealed in both of the mesenchyme and the epithelium at the same time. FGF-8 immunoreactivity in the mesenchyme and the epithelium of the heat-treated fetuses showed strong reactivity at E15.5. In the experimental fetuses fibronectin was revealed the mesenchyma and basal lamina at E15.5. Taken together, it is suggested that maternal hyperthermia induces continuous expression of TGF-beta2 and FGF-8 in the mesenchyme and delayed expression of fibronectin. These should affect the normal palatogenesis and result in cleft palate.