Functional Study Of Nucleolar Factor Bms1l In Zebrafish Liver Development

The liver initiation and development are complex processes which is precisely regulated. By studies in the model system, people have gained certain knowledge about the origin, morphogenesis and outgrowth of the liver, and identified several key regulatory factors involving in the process. As an ideal model system of studying early development of embryo, zebrafish has been paid more attentions by investigators, especially when combined with the newly CRISPR-Cas9 technology, which provides a more convenient approach for the investigation of gene functions.In this study, we combined the approaches of genetics, developmental biology, molecular biology and cell biology to investigate the biological functions of bmsll gene during zebrafish early embryogenesis. Through ENU-mediated mutagenesis, we obtained a zebrafish bmsll mutant, bmsllsq163. Our previous study showed that there was a T to A mutation in the 5th exon of bmsll gene, which lead to the amino acid substitution from leucine (CTG) to glutamine (CAG) of the 154th codon. Several phenotypic analysis showed that the digestive organs, including liver, exocrine pancreas and gut, suffered from growth arrest specifically. Further study showed that the initiation and differentiation of liver were not affected. In order to verify the essential role of bmsll in liver development, one of my colleagues in our group generated another mutant line, bmsllzju1, by CRISPR-Cas9 technology. In this mutant, there was a 13-base insertion in the 2th exon of bmsll gene, which disrupted the bmsll coding region. As a result of total loss of Bmsll protein, the bmsllzju1 mutant displayed much more severe growth deficiency of the digestive organs. Based on these studies, we took further investigation of the function of Bmsll in zebrafish liver development.Previous study in yeast showed that Bms 1 was a nucleolar protein and could form a complex with Rcll, which is involved in the pre-rRNA processing. Thus, Bmsl play an important role in maturation of 18S rRNA and ribosome biogenesis. This prompted us to investigate whether the zebrafish homolog gene bmsll is also essential for 18S rRNA maturation and ribosome biogenesis. Firstly, we found that both of Bmsll and Rcll were nucleolar proteins and could interact with each other as verified by immunofluorescence co-localization staining and co-immunoprecipitation experiments. This conserved function was also confirmed for human Bmsl and Rcll. Secondly, both of the pre-rRNA processing and biogenesis of 18S rRNA were affected in bms1lsq163 and bmsjlju1 mutants, indicating that Bmsll indeed play a role in the pre-rRNA processing pathway. Besides, the nucleoli morphology of hepatocytes changed greatly in the mutant, likely due to the abnormal pre-rRNA transcription and processing.Meanwhile, we found the rDNA transcription level was significantly increased in the bmsll mutant, which made the DNA replication fork, moving in the opposite direction, stalled. The prolonged stalling of replication fork induced the DNA damage response. We analyzed the P53 pathway in detail and found that the expression level of P53 and its isoforms were increased, as well as the downstream genes regulated by P53. As P53 activation would arrest the cell cycle progression, we examined the rescue level of digestive organs of bms1lsq163 mutant in p53 mutation background. The result revealed that the small liver and pancreas phenotype can be partially rescued in the bmsllsq163/p53M214K double mutant.Next, we went to determine the exact stage when the cell cycle is arrested in the bms1lsq163 mutant hepatocytes. Combined with the immunofluorescence and Edu labeling experiments, we found the ratio of hepatocytes in S phase was significantly increased in the mutant and the ratio of G2 to M phase was decreased, accordingly. We further verified the exuberant DNA replication phenomenon in bmsll mutant hepatocytes by Edu and Brdu sequential double labeling. All results suggest that the high ratio of aneuploidy and polyploidy in the bmsll mutant liver is most likely caused by combination of exuberant DNA replication and cell cycle arrest.Based on the above study in the bmsllsq163 and bmsllzju1 mutants, we not only verified the conserved biological function of Bmsll, but also found that Bmsll play an essential role in zebrafish liver development. In addition, we also established a role for Bmsll in the regulation of cell cycle and rDNA transcription/replication, which lays the foundation of exploring the novel function and mechanism of Bms 11 in the future.