LBH is a highly conserved protein whose role during vertebrate development is relatively under-studied. In collaboration with the Albertson lab, our lab has previously shown that it is necessary for cranial neural crest cell migration in the zebrafish and in Xenopus laevis. The molecular mechanisms through which it acts are not well understood. In Xenopus, LBH is a maternally deposited protein. As such, studying its role in early development has not been feasible through the morpholino-mediated knockdown techniques that prevent translation of target genes. Recently, a technique for degrading endogenous proteins was developed, called Trim-Away. This was developed in mammalian systems and utilizes the E3 ubiquitin ligase Trim21 in conjunction with an antibody against a protein of interest in order to degrade the protein. In order to observe the effects of a knockdown of LBH during early embryonic development, we sought to modify the technique for use in Xenopus. We injected embryos with mRNA encoding the human form of trim21 along with a monoclonal antibody against LBH that our lab developed (2B8) and tracked degradation of the protein over time, monitoring embryos for any phenotypes arising during early development. Our results demonstrate that Trim-Away can be utilized in Xenopus. LBH depleted embryos display a variety of defects during gastrulation, the process by which the three germ layers are properly organized. These appear to be mainly due to defects in fibronectin fibrillogenesis and mesodermal migration.

Retinoblastoma-binding protein 4 (RBBP4) is a subunit of chromatin remodeling factor 1 (CAF-1) and is essential for mammalian oocyte maturation, embryo survival, and embryo implantation. RBBP4 also localizes to the chromatin and is a ubiquitously expressed nuclear protein. Previous methods used to study this protein include short interfacing RNAs (siRNAs) and CRISPR/Cas9. These techniques have limitations such as determining an indirect depletion of proteins, may trigger compensatory mechanisms, and may not be useful in non-dividing primary cells. A new, acute, and rapid endogenous protein depletion technique called Trim-Away, can overcome these limitations. Trim-Away is also widely applicable since it can be used with many off-the-shelf reagents. Trim-Away utilizes the TRIM21-antibody interaction within the cytosol and the ubiquitin-proteasome pathway (UPP) to target and degrade a protein of interest. Studying RBBP4 using Trim-Away can offer insight into possible new functions of RBBP4 and its maternal effect, and increase the knowledge on a new, acute, and endogenous protein depletion technique. Here we report that, RBBP4 is required for proper blastocyst development and RBBP4 is more abundant in MII oocytes than GVBD oocytes. We also report that the loss of RBBP4 hinders RNA synthesis and causes cell death in later stages of embryo development. While our Trim-Away methodology can deplete RBBP4 as early as the 2-cell stage in embryos, our oocyte Trim-Away protocol needs to be optimized.

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