The Function Of Tmp21 In The Liver Development Of The Zebrafish

In the early 21st century, Vertebrate development processes has caused wide concern over the recent years. In vertebrate development process, endoderm, mesoderm and ectoderm differentiation is one of the initial cell fate and differentiation events. The understanding of development of the mesoderm and ectoderm is more systematic and in-depth, but the development of mesoderm cell fate decisions is still poorly understood.The Tmp21 (transmembrane emp24-like trafficking protein 10) also termed p23, p24c, or p248, this gene is a member of the p24 endoplasmic reticulum/Golgi cargo family and plays critical roles in protein transport, organization of membrane structure, and the secretory pathway. The p24 proteins family are a phylogenetically-conserved family of type I transmembrane proteins that are highly enriched in the ER, Golgi, and coat protein (COP) Ⅰ and Ⅱ transport vesicles, whose main function is to regulate anterograde and retrograde transport in the early secretory pathway between the endoplasmic reticulum (ER) and Golgi apparatus.The p24 family proteins, including Tmp21, are ubiquitously expressed in mouse brain tissues. Embryos with targeted deletion of Tmp21 die at E 4.5 prior to implantation, demonstrating that p23 function is essential for mouse embryonic development.The protein is also a member of a heteromeric secretase complex and regulates the complex’s gamma-secretase activity without affecting its epsilon-secretase activity. Mutations in this gene have been associated with early-onset familial Alzheimer’s disease. Knockdown of Tmp21 selectively regulated pathogenic gamma-secretase activity, without affecting the epsilon-cleavage of Notch. siRNA knockdown of Tmp21 expression in cultured neuronal and non-neuronal cells enhances secretory trafficking of APP as well increased secretion of soluble APP derivatives and AP, suggesting that Tmp21 is a negative modulator of Aβ production. Previously we reported that Tmp21 is widely expressed in major brain areas, and co-localizes in neurons with γ-secretase core subunits presenilinl and nicastrin. Interestingly, the steady-state Tmp21 levels decline during postnatal development in rat and mouse brain, and are also reduced in the brains of individuals with AD. In this present study, we will use the zebrafish as a model organism try to have a comprehensive understanding the Tmp21 in embryos development.According to the previous studies, firstly, we cloned Tmp21 of zebrafish. Then knocked down the translation of endogenous Tmp21 by injecting antisense morpholino oligonucleotide into the 1-2 cell stage embryos, and observed internal mesoderm in zebrafish, we found that at 4dpf the liver was significantly smaller compare with the control. Endoderm development process is relatively conservative between the vertebrates and mammals.So Tmp21 gene plays an important role in the early liver morphological development of the zebrafish, then we use transgenic zebrafish system Tg (Lfabp: GFP), Tg (Gut:GFP) and Tg (sox17:GFP) for further study. In the former transgenic line, green fluorescence marks liver cells, in the latter one, green fluorescence marks the early endoderm cells. We knocked down Tmp21 gene, tracked cell in vivo by confocal microscopy, and found that the number of the early liver cells is significantly reduced. And by injecting Tmp21 mRNA, we can partially rescue the defects caused by this genes’knocking down.Subsequently, we use the whole embryo in situ hybridization to detect the Tmp21 gene expression pattern in zebrafish embryos at different developmental stages. We found that at 3dpf, this gene specificly expressed in the liver area, In order to gain a better understanding of the specific role of Tmp21 in the liver during development, we used specific marker genes hhex, proxl, sepb and cp. When knocking down the endogenous Tmp21, the sepb and cp expression is significantly reduced, indicating that Tmp21 gene has an impact on hepatic specification and the differentiation process of the liver.Then we want to detect whether the Tmp21 specifically express in endoderm, so by flow cytometry sorting techniques isolate endoderm cells of early developmental stages and non-endoderm cells of the transgenic fish Tg (sox l7:GFP),acquired the result Tmp21 gene expressed ubiquitously in early zebrafish embryos, not only expressed in the endoderm cells.In order to detect whether the injection influenced the phenotype of the zebrafish embryos, we did double knockdown of p53 and Tmp21. It showed that p53 deficiency didn’t rescued Tmp21 deficient embryos completely, so excluded the possibility of the injection causing apoptosis in zebrafish embryos which may lead to affected liver development. PKCδ, as a kinase widely implicated in apoptosis, inhibition of cell cycle progression, associates with p23 and determined the potential functional implications of this interaction. And the PKCδ Clb domain associates with p23 and identified two key residues (Asp245 and Met266) implicated in this interaction. In the present study, we will make use of the TUNEL assay in the Tmp21 knocking down embryos to detect hepatic apoptosis., in the Tmp21 morpholinos injected embryos, apoptotic cells significantly increase.In zebrafish, endoderm organ development experienced cell differentiation and organ morphogenesis, the processes are very complex. Normal growth and development of these organs are very important, any abnormalities in embryos growth may affect the body’s normal development, leading to disease and even causes organism malformation. In summary, this study was carried out to find in-depth understanding of Tmp21 gene function in liver development process.