Loss-of-function of zebrafish cdt1 causes retarded body growth and underdeveloped gonads resembling human Meier-Gorlin syndrome

The cell cycle consists of four distinct phases:GO/Gl,S(DNA synthesis),G2,and M(mitosis).The Gl to S transition is typified by an accumulation of 4',6-diamidino-2-phenylindole(DAPI)signal that indicates the rapid DNA synthesis initiated at special sites on DNA,generally called the Ori(origin of replication)(Alfa et al.,1989).

Stability and function of RCL1 are dependent on the interaction with BMS1

During ribosome biogenesis,the small subunit(SSU)processome is responsible for 40S assembly.The BMS1/RCL1 complex is a core component of the SSU processome that plays an important role in 18S rRNA processing and maturation.Genetic studies using zebrafish mutants indicate that both Bms1-like(Bms1l)and Rcl1 are essential for digestive organ development.In spite of vital functions of this complex,the mutual dependence of these two nucleolar proteins for the stability and function remains elusive.In this study,we identified an RCL1-interacting domain in BMS1,which is conserved in zebrafish and humans.Moreover,both the protein stability and nucleolar entry of RCL1 depend on its interaction with BMS1,otherwise RCL1 degraded through the ubiquitination-proteasome pathway.Functional studies revealed that overexpression of RCL1 in BMS1-knockdown cells can partially rescue the defects in 18S rRNA processing and cell proliferation,and hepatocyte-specific overexpression of Rcl1 can resume zebrafish liver development in the bms1l substitution mutant bms1l^(sq163/sq163)but not in the knockout mutant bms1l^(zju1/zju1),which is attributed to the nucleolar entry of Rcl1 in the former mutant.Our data demonstrate that BMS1 and RCL1 interaction is essential for not only pre-rRNA processing but also the communication between ribosome biogenesis and cell cycle regulation.

Zebrafish hhex-null mutant develops an intrahepatic intestinal tube due to de-repression of cdx1b and pdx1

The hepatopancreatic duct (HPD) system links the liver and pancreas to the intestinal tube and is composed of the extrahepatic biliary duct, gallbladder, and pancreatic duct. Haematopoietically expressed-homeobox (Hhex) protein plays an essential role in the establishment of HPD;however, the molecular mechanism remains elusive. Here, we show that zebrafish hhex-null mutants fail to develop the HPD system characterized by lacking the biliary marker Annexin A4 and the HPD marker sox9b. The hepatobiliary duct part of the mutant HPD system is replaced by an intrahepatic intestinal tube characterized by expressing the intestinal marker fatty acid-binding protein 2a (fabp2a). Cell lineage analysis showed that this intrahepatic intestinal tube is not originated from hepatocytes or cholangiocytes. Further analysis revealed that cdx1b and pdx1 are expressed ectopically in the intrahepatic intestinal tube and knockdown of cdx1b and pdx1 could restore the expression of sox9b in the mutant. Chromatin-immunoprecipitation analysis showed that Hhex binds to the promoters of pdx1 and cdx1b genes to repress their expression. We therefore propose that Hhex, Cdx1b, Pdx1, and Sox9b form a genetic network governing the patterning and morphogenesis of the HPD and digestive tract systems in zebrafish.

Hepatocytes in a normal adult liver are derived solely from the embryonic hepatocytes

In vertebrates,body weight increases many folds as a consequence of body growth from the childhood to adulthood（e.g.,～20 folds for a mouse）.Considering the fact that the liver-to-body weight ratio（LBR）stays relatively constant within species（Weglarz and Sandgren,2000;Kan et al.,2009）,the cell number in a liver must therefore keep increasing along with the growth of an individual organism. For example, during the growth of the zebrafish from 5 days post-fertilization （dpf） to 1.5 years old, the number of liver cells increased -900 folds （Figs. 1A and S1 ）.

Nucleolar GTPase Bms1 displaces Ttf1 from RFB-sites to balance progression of rDNA transcription and replication

18S,5.8S,and 28S ribosomal RNAs(rRNAs)are cotranscribed as a pre-ribosomal RNA(pre-rRNA)from the rDNA by RNA polymerase I whose activity is vigorous during the S-phase,leading to a conflict with rDNA replication.This conflict is resolved partly by replication-fork-barrier(RFB)-sites sequences located downstream of the rDNA and RFB-binding proteins such as Ttf1.However,how Ttf1 is displaced from RFB-sites to allow replication fork progression remains elusive.Here,we reported that loss-of-function of Bms1l,a nucleolar GTPase,upregulates rDNA transcription,causes replication-fork stall,and arrests cell cycle at the S-to-G2 transition;however,the G1-to-S transition is constitutively active characterized by persisting DNA synthesis.Concomitantly,ubf,tif-IA,and taf1b marking rDNA transcription,Chk2,Rad51,and p53 marking DNA-damage response,and Rpa2,PCNA,Fen1,and Ttf1 marking replication fork stall are all highly elevated in bms1l mutants.We found that Bms1 interacts with Ttf1 in addition to Rc1l.Finally,we identified RFB-sites for zebrafish Ttf1 through chromatin immunoprecipitation sequencing and showed that Bms1 disassociates the Ttf1–RFB complex with its GTPase activity.We propose that Bms1 functions to balance rDNA transcription and replication at the S-phase through interaction with Rcl1 and Ttf1,respectively.TTF1 and Bms1 together might impose an S-phase checkpoint at the rDNA loci.

Interaction between Bms1 and Rcl1,two ribosome biogenesis factors,is evolutionally conserved in zebrafish and human

Ribosomes are large RNA and protein complexes that function as the machinery for translation protein synthesis （Boisvert et al., 2007; Ben-Shem et al., 2011： Henras et al., 2015： Kbatter et al., 2015; McCann et al., 2015）. The eukaryotic ribosome is composed of two subunits, the 60S large subunit （LSU） and the 40S small sub- unit （SSU）, which collectively comprise of four different ribosomal RNA （rRNA） species and more than 70 proteins （Ben-Sbem et al., 2011： Henras et al., 2015; Khatter et al., 2015）. The LSU contains the 28S, 5.8S and 5S rRNAs and the SSU contains the 18S rRNA. The assembly of each subunit is initiated in the nucleolus using the respective rRNAs as backbones （Ben-Shem et al., 2011; Henras et al., 2015; Khatter et al., 2015）.