The Expression Of HIF-1α In The Developing Nervous System Of Mouse And The Experimental Studies Related To RNAi

The development of neural tube is an important embryologic event involved in the primordium establishment of the central nervous system. During this process, nervous system can not develop normally until the neural plate closes on time accurately, or the neural tube defect (NTD) would occur, such as spina bifida, encephalocele, anencephaly, etc. Patients suffered from spina bifida, especially spina bifida occulta, brought large burdens to the whole society. Nowadays, in the field of developmental neurobiology, the studies of molecular mechanisms of the development of nervous system have been increasingly focused on. Scientists on embryology and developmental neurobiology continuously used various animal models and experimental technics trying to find the concrete mechanisms from the emergence of neural plate to the closure of neural tube in order to avoid and prevent the occurrence of NTD. Following the closure, neuroepithelium continues to proliferate, differentiate and migrate, which plays an important role in the establishment of normal morphology and function of nervous system. Subsequently, prosencephalon (forebrain), mesencephalon (midbrain) and rhombencephalon (hindbrain) appear in the anterior region of neural tube. When the neural tube is completely closed, these regions are termed the primary brain vesicles. Then the prosencephalon subdivides into the two lateral telencephalon vesicles (presumptive cerebrum), and midline diencephalon (presumptive thalamus). The rhombencephalon divides into a cranial portion, the metencephalon (which gives rise to the pons and cerebellum), and a cauda region, the myelencephalon (the eventual medulla). The proliferation, differentiation and migration of the neuroepithelium are essential for the structural formation and functional establishment of the nervous system. In terms of gene expression, such a process results from the expression and interaction of an array of genes that work in a highly specific, spatio-temporal manner. As known, oxygen is the most fundamental and important factor to guarantee various vital activities. Hypoxia regions have been found in the development of human embryos and murine embryos in recent studies. So, could it be possible the hypoxia-inducible factor-1 (HIF-1), as an important factor of regulating hypoxia, may be involved? HIF-1α-/-mice have been shown to exhibit defects in neural tube development,including neural tube patency and cerebral vascular malformation, whereas the mechanisms by which HIF-1αexpression defects influences the closure of neural tube remains unknown. Also what's the exact function of HIF-1αand molecular mechanisms of this? And among the genes involved in the NTD, could it be an organizer? Recent studies have revealed that a number of genes participated in the regulation and control of development of the central nervous system and some environmental factors exert their functions via these genes as well. As an important hypoxia regulating factor, could it be possible that through promoting different target genes, HIF-1 participates in the proliferation, differentiation and migration of the neuroepithelium? Up to date, however, little is known about gene expression and regulation of this complicated process. In addition, no report is available on the spatio-temporal pattern of HIF-1αexpression during embryonic development. Up to date, however, little is known about gene expression and regulation during neural tube genesis. RNA interference (RNAi) is an innate cellular process, which is activated by a double stranded RNA molecule with 19-23 nucleotide duplexes in cells from Caenorhabditis elegans to mammals. The RNAi is triggered by degradation of single-stranded RNAs of identical sequences. Therefore, RNAi technology can be used to silence gene expression by directly targeting its specific sequence of mRNA. Besides the widely used strategies for knocking down gene expression in academic research, RNAi technology, generated by small interfering RNAi (siRNA), has been used in therapeutic studies of human diseases including cancer, neurodegenerative diseases, viral infectious diseases, etc. Nowadays increasingly more researchers have begun to use siRNA to inhibit the definite genes expression of mammals. After designing, some vectors may express short hairpin RNA (shRNA) and it may be processed to be a siRNA molecule with 21nt in vivo. Subsequently, RNAi processes begin and give rise to post-transcriptional gene silencing (PTGS). By this means, the expression of siRNA can be continuous in the transfected mammals'cells and results in the effect of continuously, steadily inhibiting target genes. Therefore, as an idealtool instead of gene knockout, RNAi technology can be used to study the function of a certain gene in the developmental biology. In the present study, the spatio-temporal expression of HIF-1αmRNA and protein were investigated during the nervous system of mouse embryonic development using the whole-mount embryo in situ hybridization, immunohistochemistry, etc in order to provide morphological evidence for revealing the possible mechanisms of HIF-1αin the development of central nervous system. Subsequently, HIF-1αgene is selected to be the target gene to interfere by using RNAi technology. Using molecule clone technologies, mouse HIF-1αplasmid of eukaryotic expression and the specific siRNA for HIF-1αprotein are successfully constructed. And in the level of cell lines, both Western blot and immunostaining experiments consistently suggested that the DNA vectors carrying the siRNA hairpin of targeting the overtransfected HIF-1αgene in cells are effective and specific. We also showed that the mRNA level was decreased when siRNAs were transfected into the cells. Meanwhile, the feasibility on neural cell lines of siRNA has also been tested. All above is to decrease or block the expression of HIF-1αgene. This has established a theory basis and experimental evidence for later using it on whole mount embryo culture in vitro to examine the functions of genes, observe the changes of embryo morphology and related downstream genes after transfection, and reveal its exact molecular mechanisms during the neural tube closure, the development of neuroepithelium and the genesis of NTD. The results are as follows: 1. The expression of HIF-1αin the developing nervous system of mouse 1.1 The expression of HIF-1αmRNA during developing nervous system was examined by using whole-mount embryo in situ hybridization. Results show: At E8.5d, expression of HIF-1αmRNA was weakly detected at the cranial portion and caudal end of the neural tube in mouse embryo. With closure of the neural tube the expression of HIF-1αmRNA was increased dramatically in the prosencephalon, branchial arches 1, branchial arches 2, and metencephalon at E9.5d, especially it was showed that there was the higher level of HIF-1αmRNA in the developing eyes and the lower level in the mesencephalon and primary heart. At E10.5d, besides the regions where positive signals were detected on E9.5d, HIF-1αmRNA was clearly observed in the telencephalon, diencephalons, developing limbs, and caudal regions. At E11.5d HIF-1αmRNA was strongly detected in the commissural plate,rostral region, branchial arches 1 and 2, metencephalon, myelencephalon, limbs, and terminal caudal region of the neural tube. The present results suggest that HIF-1 might be involved in the development of the mouse neurulation. 1.2 The expression of HIF-1αprotein in the nerve tissue was detected from E12.5d to E17.5d by immunohistochemistry section staining. At E12.5d, the positive signal of HIF-1αprotein expression was detected in the telencephalon, mesencephalon, the surrounding regions of the fourth cerebral ventricle, myelencephalon, developing eyes and retina, spinal cord and the surroundings of the vertebral canal, heart and liver, etc. At E13.5d, HIF-1αprotein could be detected in the cupular part of neocortex (namely the future cerebral cortex), the posterior of mesencephalon, diencephalon (the surroundings of the third cerebral ventricle), myelencephalon, the juncture regions of myelencephalon and spinal cord, developing eyes and retina, heart and liver, etc. It is the time of E12.0～E13.0d that neuroepithelium migrates from the mantle layer to the marginal layer. The expression of HIF-1αprotein in many regions suggests that HIF-1αmay be involved in the differentiation and migration of the neural progenitor cells. At E14.5d, the positive signals was clearly observed in the telencephalon, mesencephalon, diencephalon (hypothalamus), the fourth cerebral ventricle choroid plexus, the dorsal and ventral part of myelencephalon, developing eyes and retina, spinal cord, heart and liver, etc. At E15.5d, besides the regions where positive signals were detected on E14.5d, HIF-1αprotein was also found in the surroundings of aqueduct of mesencephalon. During this period, primary cortex notablely thickens, cell layers dramatically increase and cells of individual encephalic regions continue to differentiate and migrate, therefore, the positive signals can be detected in most encephalic regions such as telencephalon, mesencephalon, diencephalon, myelencephalon, etc. Till E17.5d, positive cells mainly localized in the central and posterior parts of telencephalon, the third cerebral ventricle choroid plexus and its surroundings. During this period, the expression regions of HIF-1αprotein diminish, suggesting that the expression of HIF-1αprotein decreased after the elementary completion of the differentiation and migration of cells. 2. The construction of HIF-1αsubclone and its eukaryotic expression In order to construct the eukaryotic expression plasmid of HIF-1αgene, cDNA of HIF-1αgene was reversely transcribed from the total RNA of HIF-1αgene. Then the cDNAwas subcloned into the eukaryotic expression vector pcDNA3.1-HA. This construct was transfected into 293T cells and the expression of HIF-1αprotein was measured by Western blot. As known, HIF-1αprotein is not stable with a very low expression level under normoxia conditions; cells were treated under hypoxia conditions for an hour after transfection and harvest. Thus, the significantly increasing expression of HIF-1αprotein could be detected. It may be analyzed that in these HIF-1αoverexpressing cells, HIF-1αwas detectable in a small cell population which was treated with an hour hypoxia even in normoxia, indicating that the normal normoxic proteasomal degradation capacity for HIF-1αis overridden under these conditions. On the whole, successfully constructing the eukaryotic expression plasmid of mouse HIF-1αgene has established the base of the studies which focus on testing the siRNA of HIF-1αgene. 3. The construction of interfering plasmid of HIF-1αand identification of its effectiveness The U6 promoter vector was adopted and a 22bp siRNA was constructed through the hairpin, which could be produced by the DNA vector. The hairpin cDNAs were generated through annealing of the complementary oligos synthesized, where ApaⅠand EcoRⅠsites were constructed. The hairpin cDNA as an insert was subcloned into pBS/U6 vector through ApaⅠand EcoRⅠsites. The correct clones were verified by KpnⅠ/EcoRⅠand KpnⅠ/XhoⅠdigestion. To determine whether the siRNA we generated could effectively reduce the expression of HIF-1αprotein in cultured cells, we first transfected the siRNA vectors pBS/U6/HIF1αi-Ⅰ～Ⅲinto 293T cells where HIF-1αprotein was overexpressed. To show the specificity of the siRNA targeting, we used EGFP as a control. The data of Western blot, immunostaining and RT-PCR demonstrated that pBS/U6/HIF1αi-Ⅰ～Ⅲcould specifically silence the expression of HIF-1αprotein to some extent in cells., The effectiveness and specificity of siRNA has been further verified on the neuroblastoma cell lines (SH-SY5Y) to ensure the feasibility on neural cells. We also proved that some dose-effect relationship does exist between the effects of HIF-1αgene silencing and the dose of plasmid pBS/U6/HIF1αi. This has established a theory basis and experimental evidence for later using it on whole mount embryo culture in vitro to examine the functions of genes, observe the changes of embryo morphology and related downstream genes after transfection, andreveal its exact molecular mechanisms during the neural tube closure, the development of neuroepithelium and the genesis of NTD.