The Role Of Sox19b On The Early Development Of Nervous System In Zebrafish

ObjectiveResearch on the sox gene family began with the seminal discovery of the mammalian testis-determining factor, Sry. Sry carries a characteristic high-mobility-group (HMG) domain that binds DNA in a sequence-specific manner. During development, the sox family is a good candidate as coordinators of some embryonic processes, including sex determination, chondrogenesis, nervous system development and lens development.In zebrafish, soxl9b, as an important member of Sox B1, is expressed in the blastula, whereas the corresponding early expression in mice is covered by Sox2. Following this stage, the sox19b gene is thought to be important for specification of the embryonic ectoderm into the neuroectoderm lineage. During this process, their expression becomes confined to the neuroectoderm. As development proceeds to the neurula stage, expression of soxl9b continues in the developing central nervous system (CNS) including forebrain, midbrain, hindbrain and spinal cord. Together, these studies have demonstrated the importance of soxl9b in the development of nervous system.In our research, we performed knockdown of sox19b in zebrafish embryos to analysis the function and mechanism during the early development of nervous system. However the pathogenic mechanism and the therapeutic methods of the diseases related to neural system are still unknown. So the mechanism of sox19b during the development of neural system can provide new clues and theoretical foundations for clinic treatment of the diseases related to neural system.Methods We use zebrafish as the model organism to explore the mechanisms of soxl9b during the development of neural system in zebrafish via microinjection of Morpholinos to inhibit the expression of soxl9b by the whole-mount in situ hybridization, RT-PCR, tissue section, H&E staining, BrdU uptake and acridine orange staining.Results:1、Spatial and temporal expression pattern of soxl9b in zebrafish during embryogenesisTo explore the function of soxl9b during embryogenesis, we first examine the spatial and temporal expression of sox19b by the whole-mount in situ hybridization (WISH) and RT-PCR. In zebrafish, soxl9b is expressed in the blastula. Following this stage, the soxl9b gene is thought to be important for specification of the embryonic ectoderm into the neuroectoderm lineage. During this process, their expression becomes confined to the neuroectoderm. As development proceeds to the neurula stage, expression of soxl9b continues in the developing CNS including forebrain, midbrain, hindbrain and spinal cord.2、The effect of sox19b on the development of neural system in zebrafishTo identify neural cells affected by soxl9b MO, we performed WISH to detect sox3(neroectoderm), otx2(anterior neroectoderm), and dlx3b (the edge of anterior neural plate). The analyses revealed that all these neural genes are abolished. Next, to assess cell death as a possible mechanism, we analyzed apoptotic cells in embryos by acridine orange staining. Acridine orange staining of living embryos revealed increased staining of brain cells especially the CNS region of sox19b MO treated embryos at24hpf. At the same time, we analyzed BrdU uptake at24hpf, to determine whether knockdown of soxl9b reduces cell proliferation in brain. BrdU uptake was significantly reduced in morphants compared with controls.3-. The effect of sox19b on the development of eyes in zebrafishInjection of soxl9b morpholino resulted in microphthalmia or anophthalmia in67%of injected embryos. The retina is a well-suited model for understanding a series of important events of neural system development that include neural induction, cell proliferation, apoptosis, differentiation and migration. Thus, we used this retina model to evaluate the effect and molecular mechanism of soxl9b during neural system development. The WISH analyses revealed that expression of many specification factors during retinogenesis is abolished in the morphant embryos, including rx3, pax2a as well as vsx2.4、The mechanisms of sox19b on the development of neural system in zebrafishComponents of BMP、FGF、Wnt signaling are major regulators of neural system development, including neural induction and neurogenesis. Since these movements are severely impaired in the morphant embryos, we investigated expression profiles of genes related to these processes by RT-PCR. The results showed that the expression of some ligands and targent genes of BMP and FGF signal pathway decreased apparently by RT-PCR.Conclusions1、Regulation of sox19b on neural induction and neurogensis.2、Regulation of Sox19b on proliferation and apoptosis of the neural stem cells and neural progenitor cells.3、Regulation of Soxl9b on the development of eyes.4、Regulation of Soxl9b on BMP and FGF signal pathway.