Establishment Of The Zebrafish Model Of Karsch-Neugebauer Syndrome And Behavior Observation

Objective:To investigate the role of FGF8 and LBX1 gene in the pathological mechanism of the hereditary diseases Karsch-Neugebauer syndrome.Materials and Methods:We worked with zebraf ish as a model animal. By means of the Tol2 and Gal4:UAS system, we successfully constructed the expression plasmids T2K-UAS-lbxla-clmc2-mCherry and T2K-Gal4VP16-gata2-FGF8. HS34. The two expression plasmids were injected separately into wild-type zebrafish TAB-WT in single-cell stage fertilized zygote, in order to establish the transgenic zebrafish Tg (FGF8.HS346:Gal4VP16) and Tg (UAS:lbxla;clmc2:mCherry). By crossing transgenic zebrafish Tg (FGF8.HS346:Gal4VP16) and Tg (UAS:lbxla; clmc: mCherry), we manufactured Tg (FGF8. HS34, Gal4VP16; UAS:lbxla) transgenic line, in which FGF8. HS34 enhancer could drive ectopic expression of lbxla gene. Then we observed the phenotypic differences in the jaw and apical ectodermal ridge, and functional changes in the optic nerve of transgenic zebrafish Tg(lbxla UAS FGF8.HS34, Gal4VP16).Results:The sequencing results confirmed the expression plasmid T2K-UAS-lbxla-clmc2-mCherry and T2K-Gal4VP16-gata2-FGF8. HS34 was constructed correctly. By crossing transgenic zebrafish Tg (FGF8. HS346: Gal4VP16) and Tg (UAS:lbxla; clmc2:mCherry), we manufactured Tg (FGF8.HS34, Gal4VP16; UAS:lbxla) transgenic line. Whole mount in situ hybridization confirmed the lbxla gene ectopic expression under the control of FGF8.HS34 enhancer. Observe the Tg (FGF8.HS34:Gal4VP16; UAS: lbxla) zebrafish phenotype under the microscope. On 3dpf, the apical ectodermal ridge of bilateral pectoral fin buds were thickened, and no longer stretched out, and the fin buds was bent. While on 6dpf, mandibular arch was widened like a half oval, and the distal mandibular arch was significantly shorter than the ethmoid bone; hyoid arch lost radians, and the remaining pairs of pharyngeal arches which should have a 60° angle widened to about 120°. The optic nerve function of 5dpf Tg (FGF8.HS34: Gal4VP16; UAS:lbxla) line was significantly decreased compared to TAB-WT when tested by the Optokinetic Response (P<0.001).Conclusion:The transgenic zebrafish Tg (FGF.HS34, Gal4VP16; UAS:lbxla) had similar pharyngeal arch and apical ectodermal ridge deformities, as well as abnormal vision function with Karsch-Neugebauer syndrome. Indicating the FGF8. HS34 enhancer induced ectopic lbxla gene expression could result in similar performance with Karsch-Neugebauer syndrome. Then we concluded the pathological hypothesis of hereditary disease with tandem duplications:the tandem duplication of chromosome fragments may lead enhancers to locate just before the gene near the recombination site, which could caused ectopic expression of the gene near the recombination site. Especially when the tandem duplication mutations were close to a developmental relevant gene, the ectopic expression of that gene may lead to significant abnormalities.