Effects Of P204 Injection On Zebrafish's Embryonic Development And Osteoblastogenesis

Mammalian skeletal development goes through a multi-step process, including pattern formation of early bone, mesenchymal stem cells (MSCs) differentiation into osteoblast precursor, chondro-precursor, hematopoietic stem cells differentiation into osteoclast cell lines as well as their terminal differentiation into three types of cells, namely, osteoblasts, cartilage cells and osteoclasts. Csteogenesis is a very complicated process involving several signaling pathways such as TGF-beta signaling pathway, Wnt signaling pathway, FGF pathway and Hedghog signaling pathway. Among these pathways, core binding factor a-1 (Cbfa 1, also known as Runx2), a member of the runt family of transcription factors, plays a central role in osteoblast differentiation and bone formation.The p204 gene is a member of the interferon-inducible p200 family. Increasing evidences from cultured cells demonstrate that p204 modulates cell proliferation, cell cycling, and the differentiation of various tissues and cells, including skeletal muscle myotubes, beating cardiac type myocytes, osteoblasts, chondrocytes, and macrophages. It has been demonstrated that p204 acts as a coactivator of Cbfa 1 to enhance osteoblast differentiation. In this process, pRb is a necessary linker between p204 and Cbfa 1 during the differentiation of pluripotent C2C12 to osteoblast induced by BMP-2. Together, Cbfa 1, p204 and pRb form a ternary transcriptional complex to enhance cbfa 1's downstream genes'transcription, such as ALP gene, OCL gene.Genetic comparison and analysis between zebrafish and mouse indicates runx2b and pRb gene exsist in zebrafish genome. In zebrafish there are two orthologs of runx2, namely runx2a and runx2b, the latter plays significant role during osteogenesis. It has been demonstrated that runx2b is a materal determinant of ventral zygotic genes in zebrafish and the only known direct regulator of vent at the onset of zygotic transcription. Embryos treated with a runx2b-specific morpholino (MO) are strongly dorsalized. We could not find p204 orthologs in zebrafish genome, thereby we transfer the p204 gene into zebrafish embryos by microinjection to investigate p204's effects on zebrafish's early embryonic development and osteogenesis.Methods:(1) Construction of p204-GFP recombinant vectors. (2) Injection of constructed vectors into 1-cell stage zebrafish embryos. (3) Detection p204's effects on zebrafish's early development and osteogenesis.1) Observation of p204's expression denoted by GFP.2) Detection of some genes involved in dorsoventral patterning and osteogenesis by real-time RT-PCR.3) Observation of runx2b's expression pattern by in situ hybridization.Results:1) p204's injection into zebrafish resulted in striking malformations such as bent spine and expanded belly.2) p204 was widely expressed from 8hpf to 8dpf and disappeared after 11dpf from our GFP observation results.3) The expressions of some genes (vent, runx2b, osn) involved in dorsoventral patterning and osteoblastogenesis were upregulated after p204 injection from real-time RT-PCR.4) The expression of zebrafish runx2b was enhanced after p204 injection though its expression location had little difference by in situ hybridization.Conclusions:Striking malformations such as bent spine and expanded belly were induced after p204's injection and some genes(vent, runx2b, osn) involved in dorsoventral patterning and osteogenesis were significantly upregulated after p204 injection. This study may help to uncover the underlying mechanisms during zebrafish osteoblastogenesis and provide assistance for developing therapeutic means of intervention in skeletal diseases.