Znf179 is a member of the RING finger protein family. The expression of Znf179 is abundant in the brain and is regulated during brain development. In our previous study, we first demonstrated that Znf179 is a novel cell-intrinsic factor that is critical for neuronal differentiation. In this proposal, we will further investigate the physiological functions of Znf179 in animal models. First, we generated conventional Znf179 knockout mice and found that more than 75% of Znf179-/- mice from Znf179+/- intercrosses died in utero. Further investigation revealed that Znf179-/- embryos exhibited blood vascular defects and died between E14.5 and birth. This phenotype was observed in both 129/sv and C57BL/6 mice. To further evaluate the blood vascular structure in Znf179-/- embryos, India ink will be used for visualization of the possible vascular rupture. Immunostaining of platelet endothelial cell adhesion molecule and collagen IV, which are important components of basement membranes of vasculature, will be also performed to evaluate the blood vessel structure in Znf179-/- embryos. To clarify the underlying mechanisms contributing to blood vascular defects in Znf179 knockout embryos, the expression of vascular endothelial growth factors (VEGFs), vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor (PDGF) and other important factors involved in blood vessel maturation will be examined by quantitative RT-PCR, Western blotting, immunostaining and microarray analysis. In addition, we found that the survival rate of homozygous Znf179-knockout mice in 129/sv and C57BL/6 mixed genetic background is increased. The homozygous mice were smaller than control littermates and displayed impairment of brain functions including motor balance, and spatial learning and memory. The differences in hematology, serum biochemistry and histopathology among wild type, Znf179+/- and Znf179-/- mice will be examined. More behavioral tests including open field activity and novel object recognition will be also used to assess locomotion, exploration, anxiety, and cognitive ability in the Znf179 knockout mice. Before and after training in the Morris water maze, the neuronal density and the number of dendritic spines in the hippocampus will be measured by Golgi staining and immunohistochemistry. Microarray analysis will be used to determine gene expression changes before and after training in wild type and Znf179-/- mice and to identify the molecular mechanism of spatial learning and memory impairment in Znf179-/- mice. Furthermore, the Znf179 conditional knockout and Znf179 overexpressed transgenic mice will be generated and serve as more powerful tools to study the physiological and pathological roles of Znf179 in vivo.
|Effective start/end date||8/1/13 → 7/31/14|