Temporal And Spatial Expression Of Fibroblast Growth Factor-8 In Chicken Embryo Lip Tissue Effect Of Citral

Objective Citral beads were implanted into the right nasal pit of chicken embryos at stage 20, to establish the model of chicken embryos cleft lip. Probe for FGF8 of chicken were labeled with digoxingenin-UTP, to detect the temporal and spatial expression of FGF8 in the chicken embryo cleft lip by the method of Whole-mount in situ hybridization. Then to investigate the mechanism of FGF8 in the development of chicken embryo’s lip, and provide the theretical evidence for preventive action.Methods1、Preparation of probe for FGF8 of chicken labeled with digoxingenin-UTPRecombinant plasmid were presentation by Marion Wassef PhD, then plasmids were linearized with the restriction enzymes of EcoRI and XhoI respectively. The sense and anti-sense DIG labeled RNA probes were synthetized in vitro by T3 and T7 RNA polymerase respectively, and RNA probes for Whole-mount in situ hybridization were prepared.2、Establishment the model of chicken embryo cleft lipAt the stage 20, normal development of chicken embryos were randomly divided into 3 groups:CT group, DMSO group and normal group. CT group:beads which were soaking in citral with concentration of 0.6 g/ml which were dissolved in the DMSO were implantation on the right side of the nasal pit under the ectoderm. DMSO group:beads which were soaking in DMSO were implantation on the right side of the nasal pit under the ectoderm. Normal group without any operation. Then the chicken embryos continued incubating until stage20+1h,22,24,26 and 28, and selected 5 embryos in different groups of each stage.3、Temporal and spatial expression pattern of the FGF8 genesThe temporal and spatial expression of FGF8 during the development of chicken embryo’s lip were detected by the the method of whole-mount in situ hybridization.Results1、The probes for FGF8 of chicken labeled with DIG were synthetized in vitro and positive signals were viewed in chick embryos.2、There was no significant difference of morphology between the Normal group, DMSO group and normal side of CT group. At the stage 20+1h of normal group and DMSO group and normal side of CT group, nasal placodes were became fossa. The nasal pit were formed with frontonasal prominences, lateral nasal prominences and maxillary prominences at stage 22. By the development of chicken embryos the facial process continued proliferation, growth and fusion with each other. While on the right side of CT group, it was not obviously abnormal at the stage20+1h, but was observed that the maxillary prominences and lateral nasal prominences defected lightly after stage22, and the right nasal pit was fusional impairment.3、The expression of FGF8 in the craniofacial of chicken embryos were dynamics, and there was no significant difference of the expression between the Normal group, DMSO group and normal side of CT group. At the stage 20+1h of normal group and DMSO group, FGF8 was high expression in the frontonasal prominences and the margin of median nasal prominences, lower edge and bottom of maxillary prominences, lateral superior border of mandibular prominences. With development the expression of FGF8 limitations in the middle of frontonasal prominences and median nasal prominences, lower edge of maxillary prominences, lateral superior border of mandibular prominences. At stage 24, the expression were disappear in the frontonasal prominences and maxillary prominences, and the median lateral nasal prominences extended to the lateral nasal prominences. After stage 24, the expression of FGF8 began to decline,and significantly reduced or even disappear after fusion contact of the prominences. While on the right side of CT group, the expression of FGF8 was not obviously abnormal with the normal group and the normal side of CT group at the stage20+1h, but was down obviously at stage 22 to 24. While after stage 24, it was high expression than the normal group.Conclusions1.The antisense probe of chicken has high specificity, and be effectively to detect the expression of FGF8 gene during chicken development.2. Citral can regulate the expression of FGF8, and cause the lip tissue defect of chicken embryos.