Ubiquitin-dependent protein modification process is critical not only for the target proteins destruction but also for the cellular signaling, protein trafficking, endocytosis, DNA damage tolerance and kinase activation. The protein ubiquitylation is mediated by the consecutive action of E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase, which determines substrate specificity. In human, there are at least 600 different E3 ubiquitin ligases estimated to be expressed. Several types of human diseases are implicated in the defective protein ubiquitylation, reflecting the importance of the protein ubiquitylation in the cellular physiology. To data, the ubiquitin-dependent protein modification mechanism has been extensively studied in in vitro and homogenous cell culture systems. However, the in vivo functions of individual E3 ubiquitin ligases during vertebrate embryonic development still remain as an enigma. The early vertebrate embryonic development is largely dependent upon the crosstalk and collaboration of Wnt, TGFβ and FGF signaling. The combined action of the signaling pathways initially determines dorso-ventral body axis followed by anterior-posterior axis and left-right body asymmetry. The aforementioned signaling pathways are also important at the post-embryonic pathogenesis. For example, over the 85% of human colon cancer patient harbors mutated adenomatous polyposis coli (APC) gene that encodes a scaffold protein negatively regulating the Wnt signaling. Although TGFβ has been known as a cancer suppressor, it is also involved in cancer metastasis. Therefore, it is important to fully understand the intricate signaling cascades of the TGFβ signaling for the diagnosis and treatment of TGFβ related human diseases. In addition, aberrant regulation of RAS-RAF-MAPK signaling pathway, which can be activated by FGF/EGF/VEGF stimuli, is the one of the main causative factors of the cardio-facio-cutaneous (CFC) syndrome and malignant melanoma. Since these signaling pathways play important roles for the embryonic axis formation as well as diverse post- embryonic pathogenesis, it is valuable to study the molecular mechanisms using early developing embryos with suitable model system to gain insights into genetic disorders elicited by aberrant modulation of the signaling pathways.
The zebrafish model organism is suitable for the study of vertebrate embryonic development and cellular signaling mechanism. The attributes of zebrafish include its small size, fecundity, relatively small genome size and production of optically transparent embryos that undergo exceptionally rapid development ex utero. In addition, the relatively large size of zebrafish embryos, which is approximately 20 times bigger than mouse embryo makes the system amenable to knockdown or gain-of-function experiments using morpholino or mRNA injection respectively, and generate transgenic animal through micro-manipulation with relatively simple equipment (Fig. A). This experimental advantage renders the zebrafish an excellent animal model for the development of human disease models and for high-throughput chemical modifier screening. Importantly the recent advent of TALEN, CRISPR/Cas9-based genome editing technology has overcome the weakness in reverse genetics of zebrafish.
Fig. cdx4-EGFP transgenic zebrafish embryo. (A) EGFP reporter gene is under the control of cdx4 promoter. (B) The equivalent stage of embryos stained with anti-cdx4 riboprobe to detect endogenously expressed cdx4 transcripts. Note that EGFP expression mimics endogenous expression 37 pattern of cdx4. 12-somites stage embryos. Lateral view. Dorsal is up, anterior is left.
To date, few E3 ubiquitin ligases have been reported to play an important role for the early vertebrate embryonic development. Given that there are over 600 E3 ubiquitin ligases in human genome and that approximately 30% of genes are estimated being critical for the early embryonic development, around 200 different E3 ubiquitin ligases are predicted to be involved in tissue specification and/or differentiation during prenatal stage. So far the molecular mechanisms underlying the ubiquitin dependent protein modification processes have been extensively studied in the fields of cell biology, molecular biology and biochemistry. However, how specific modification of proteins with ubiqutin/ubiquitin-like molecules affects embryonic development remains largely unknown. As mentioned before, understanding of early embryonic development is important to gain insights into the in vivo function of genes, which are relevant to postnatal human diseases. Taken together, it opens up promising analytical prospects in the field of biology to study the molecular functions of E3 ubiquitin ligases during early embryonic development.