| The birth prevalence of anophthalmia and microphthalmia has been generally estimated to be up to 30 per 100,000 population, Both anophthalmia and microphthalmia are more commonly bilateral; the exception appears to be isolated microphthalmia, which is usually unilateral. Microphthalmia is reported in 3.2–11.2% of blind children. The precise pathogenesis of anophthalmia and microphthalmia remains unknown.The zebrafish has become a powerful model system to study genetic mechanisms of vertebrate development and disease. Much of the current success can be traced back to the pioneering work of George Streisinger and colleagues. The similarity between zebrafish and human genome is about 87%. Streisinger first recognized many of the oft-cited advantages for the use of zebrafish as a genetic model. Zebrafish, small freshwater teleosts, are easily adapted to the laboratory setting and can be maintained in a relatively small space. The fish typically reach sexual maturity in 3 to 4 months, and a breeding pair of fish can produce >200 fertilized eggs per mating. Fertilization is external, and the egg and embryo are transparent, facilitating visual identification ofmorphogenetic movements and organogenesis with a standard dissecting microscope.Development is rapid; by 24 hours post-fertilization (hpf) all of the major organ systems have formed and spontaneous muscle flexures soon begin. Many of the methods in use today, making zebrafish as one of the major animal models for organ development, gene function, chemical screen and so forth. The anatomy, histology, circuitry and biochemistry of the eye are strikingly conserved among most classes of vertebrate. Zebrafish is not an exception, The first visually evoked startle responses are observed 68-79hpf. By 73-80hpf, many larvae demonstrate an optokinetic reflex.(OKR) in response to moving objects, and by 4dpf, >95% of zebrafish larvae display smooth pursuit and saccade eye movements in response to illuminated rotating stripes. Unlike rodent models, the zebrafish is diurnal and its retina contains a large number of diverse cone subtypes in addition to rods. In recent years, scientists develop a series of methods to test the zebrafish optic function. Such as optokinetic response, electroretinography. The highly conserved mechanisms of development and function have contributed significantly to our overall understanding of visual processes and led to rapid advances in understanding the basic mechanisms of diseases.Our research is mainly focus on the gdf6 (growth differentiation factors), gdf6 is a member of the bone morphogenetic proteins (BMP) sub-family of transforming growth factor-beta (TGF-b) signaling ligands, The BMPs were originally identified through their capacity to induce bone formation and are now recognized to have critical roles patterning a diverse range of tissues including bone, heart, lungs and kidney.aslo bmp expresses in neuro system. Any direct evidence of protein processing, structure or physical characteristics specific to gdf6 are as yet unavailable or unpublished.Annek detected the base point mutation at the beginning of the open reading frame in gdf6 gene, c.1 A→G. p. Met l Val, our research will focus on the mechanism of gdf6 function in the eye regarding to the following aspects: the phenotype of the gdf6 mutants, the expression pattern in the optic tissue, the cell death detection, the function between the retina and the lens, the signal pathway detection.Purpose:To study the mechanism of gdf6 functions in the retina and lens by using zebrafish as a model.Method: Using chemical ENU to induce gene mutation,identified gdf6 mutants via traditional genetic methods. Compared the differences of lens and retina development between the mutant and widetype zebrafish. Performed in situ hybridization by using gdf6 RNA as a probe, confirmed the expression pattern of gdf6 in the zebrafish retina. Detected the cell death in the zebrafish eye by acridine orange staining in different development stages. Transplanted the mutant lens into widetype eye, in order to figure out whether the widetype retina can rescue the mutant lens. Using cyclopamine to block shh function, detect if the gdf6 is involved in the shh pathway.Result1.The phenotype of the gdf6 mutantsDuring zebrafish development, from embryo to adult, the eye structure and lamination seems normal comparing mutant to widetype. Mutant lens starts to grow smaller than widetype lens at about 30hpf, it may be half sized when at 5 dpf. At 30hpf,3dpf,5dpf,the retina and lens architecture remains intact.Some mutants can survive until adulthood, although the eye size varies among the mutants, several mutants began to lose the entire eye ball at 3 months, while the other eye remains intact appearance, but the overall eye size is much smaller than the widetype, about 20% sized. The swimming pattern is obviously abnormal among the smaller eye ball fish, they can't avoid the objectives in the surrounding. Some zebrafish developed irregular stripes in their pectoral fin and tail fin.Mutants can be reproductive, the female mutants and male mutants can't mate because of the poor eye sight, but the female mutants can mate with the male heterozygotes, and their offspring don't exist the maternal effect.2.The expression pattern of gdf6 in the zebrafish retina.In situ hybridization shows gdf6 expressed in the dorsal retina robustly around 24hpf,then the expression level decreased gradually, the signal is nearly absent until 72hpf.3.Cell death detectAcridine orange staining shows that at 24hpf and 30hpf a lot of dead cells in the lens and retina. At 48hpf, the cell death level decreased, we can just detect cell death at the surface of lens, excluding the retina. At 72hpf, there are barely cell death in the retina or lens.4.The function between the retina and the lensWe performed the lens transplantation at 30hpf, after incubation until 48hpf, the transplant lens can be observed under UV microscope, the embryos were fixed at 72hpf, mutant lens size increased after transplanted into the widetype retina, but remains smaller than the wildtype control eye.5.The signal pathway detectionCompared with the control group, the cyclopamine treated mutant embryos developed heart edema, the eye balls growed nasal toward. But there are not statistically significance between the two groups.Conclusion:1.gdf6 mutant zebrafish has smaller eye size, but the archetacture remains normal.2.gdf6 expresses in the dorsal retina of zebrafish, it can express strongly during 24-30hpf, decreases obviously after48hpf, when at 72hpf, it is almost absent.3.At 24-30hpf, there are a lot of cell death in the retina and lens. gdf6 can inhibit cell death in the retina and lens.4.The mechanism of gdf6 functions in lens can be partially mediated by retina.5.Direct interations between dgf6 and shh appear very weak,gdf6 function may be conveyed either by genes downstream of shh or by gdf6-independent bmp signaling.Disscusion:The development of the eye is highly complex. It is determined by sequential and coordinated expression of eye development genes within the developing tissues. Although some individuals with anophthalmia or microphtha- lmia have relatives with other eye malformations, the frequent lack of clear Mendelian inheritance in these conditions has made identifying the genes for eye development very challenging. However, using a variety of techniques, some genes involved in anophthalmia or microphthalmia have now been identified. These include genes principally involved in ocular development, such as CHX10, many of which are involved in the development of substructures within the eye and genes that are involved in eye and brain development including SOX2, OTX2, and PAX6.In this study, we mainly focus on gdf6's function, like other bmps, gdf6 at a minimum possessed an appreciable skeletal developmental role. gdf6 characterized by defective cervical, thoracic or lumbar vertebral segmentation, is frequently associated with other skeletal (scoliosis, rib abnormalities, and non-skeletal (deafness, cardiovascular, ocular and renal) anomalies. Some of these (e.g. deafness and rib anomalies) correlate with the extensive expression pattern of gdf6. In light of these factors supporting gdf6 involvement in human ocular and skeletal disorders, Some researchers screened patients with ocular and skeletal developmental anomalies for gdf6 mutations. gdf6 also has been found for the essential role in keeping the vascular intacticy. In ophthalmologial study, gdf6 expresses in the dorsal part of retina in different models, such as mouse, newts and zebrafish. When knowdown the gdf6, the microphthalmia and abnormal retina will occur, the mechanism is still unknown. Now there are not effective way to cure the microphthalmia and anophthalmia. In our study, gdf6 mutant zebrafish has smaller eye size, but the archetacture remains normal. Some mutants can survive until adulthood, mutants can be reproductive.gdf6 expresses in the dorsal retina of zebrafish, it can express strongly during 24-30hpf, decreases obviously after48hpf, when at 72hpf, it is almost absent. At 24-30hpf, there are a lot of cell death in the retina and lens. gdf6 can inhibit cell death in the retina and lens. The mechanism of gdf6 functions in lens can be partially mediated by retina. Direct interations between dgf6 and shh appear very weak,gdf6 function may be conveyed either by genes downstream of shh or by gdf6-independent bmp signaling. The mechanism of gdf6 plays an essential role, we will try to find the specific pathway of gdf6 in the next step, it will be helpful in the gene therapy for the microphthalmia and anophthalmia. |
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