Ribosome Biogenesis Protein Urb1 Acts Downstream Of MTOR Complex 1 To Modulate Digestive Organ Development In Zebrafish

Ribosomes are essential as the machinery of protein synthesis.The process of ribosome biogenesis is highly conserved and organized.Ribosome synthesis requires enough nutrient and friendly environments.Under conditions of nutrient deprivation,the ribosome activity will be disrupted,which leads to reduction of protein synthesis and growth arrest.Mammalian/mechanistic target of rapamycin(m TOR)plays key roles in the modulation of cell proliferation.During embryonic development,deletion of mtor would cause embryonic lethal in mice.si RNA-mediated knockdown of m TOR in human ESCs reduced the levels of Sox2 and Oct4,suggesting roles of m TORC1 in the maintenance of stem cell pluripotency.Urb1,containing a conserved Npa1 domain,predominantly localizes in the nucleolus and plays a vital role in the formation of 60 S ribosomal particles in yeast.Here we describe a zebrafish mutant urb1cq31,which exhibits hypoplastic digestive organs and impaired ribosomal subunits synthesis.This study demonstrates that digestive organ development requires hyperactive protein synthesis,which is regulated by Urb1 downstream of m TORC1.From an ENU forward genetic screen for liver development,we identified a cq31 mutant.Under the Tg(lfabp:Ds Red)transgenic background,the homozygous cq31 mutant exhibited reduced liver size at 72 hours post fertilization(hpf)in contrast to the wild type,without causing obvious body phenotype.Under the Tg(p48:GFP)and Tg(ifabp:Ds Red)cq32 transgenic backgrounds,the cq31 mutant showed a smaller exocrine pancreas at 72 hpf and an underexpanded anterior intestine at 120 hpf,respectively.However,the endocrine pancreas remained unaffected in the mutant.These results suggest the hypoplastic digestive organs in the cq31 mutant.Positional cloning for the cq31 mutant identified a point mutation in the exon 2 of the urb1 genomic loci located at linkage group 15.The T to C replacement in the exon 2 resulted in a Phe to Leu conversion.This Phe is conserved among vertebrates.The urb1 c DNA comprised 4815 base pairs according to the Zv9 database,which was confirmed by the Northern blot.An antisense morpholino oligonucleotide(MO)against the urb1 m RNA led to notably reduced expressions of the liver marker ceruloplasmin(cp),the exocrine pancreas marker trypsin,and the intestine marker ifabp,displaying the same phenotypes as in the cq31 mutant.These data further validate urb1 as the causative gene of the cq31 phenotypes.To confirm whether the Phe to Leu conversion caused degradations of urb1 m RNA or protein,we generated two constructs,a heat shock promoter-driven wild type urb1-Flag-P2A-GFP and a mutated urb1P80L-Flag-P2A-GFP,which obtained the same mutation as in the cq31 mutant.The plasmids were injected and the GFP-positive cells at 54 hpf were sorted for western blot.The expression levels of Urb1-Flag and Urb1P80L-Flag were comparable using GFP as the internal control.These data suggest that the P80 L mutation affect the protein function rather than stability.Expression of urb1 was absent at 12 hpf and weakly detectable at 24 hpf.Enrichment of urb1 transcripts in the head and foregut endoderm became visible at 48 hpf.At 72 hpf and 96 hpf,urb1 expression was detected in head,liver bud,intestine and exocrine pancreas.These expression patterns were consistent with the digestive organs defects in the cq31 mutant.To examine whether the hypoplastic digestive organs in the urb1cq31 mutant was due to a differentiation failure of endodermal progenitors,we detected liver differentiation with cp at different developmental stages.The liver buds of the wild type and mutant embryos were of similar sizes at 48 hpf.However,while the liver size kept increasing in the wild type embryos at 54 hpf and 60 hpf,the liver bud of the mutant stopped growing.These data suggest that hepatoblast specification occurs in the mutant,but the liver bud fails to grow.At 5 dpf,the expressions of mature hepatocyte markers fatty acid-binding protein 10(fabp10)and betaine-homocysteine methyltransferase(bhmt)were both downregulated in the mutant,indicating that the differentiation of hepatocytes also occurs in the urb1cq31 mutant.To explore the reasons underlying the arrest of liver growth,phospho-Histone H3(p H3),a proliferating cell marker,and TUNEL assays were performed.Under the Tg(gut:GFP)s854 transgenic background,the numbers of gut GFP+p H3+ cells in the wild type and mutant embryos were comparable at 42 hpf,whereas the numbers of double positive cells in the wild type were significantly more than those in the mutant at 48 hpf,54 hpf,and 60 hpf.These results indicate that the proliferative capability of endodermal cells was gradually impaired from 42 hpf to 60 hpf in the urb1cq31 mutant.At these stages,cell apoptosis was rarely detectable in the digestive organs of both wild type and mutant embryos.Injection of p53 MO could not rescue the digestive organs deficiency of the urb1cq31 mutant,indicating urb1 is dispensable for the p53-dependent cell apoptosis.All these results demonstrate that ubr1 regulates cell proliferation rather than progenitor differentiation or cell apoptosis during digestive organ development.Mammalian/mechanistic target of rapamycin(m TOR)plays key roles in the modulation of cell proliferation(Wullschleger,2006).To explore the functional correlation between Urb1 and m TOR signaling in the digestive organ development,expression patterns of mtor,raptor and rictor were first analyzed.At 96 hpf,expression of raptor,but not rictor,was enriched in the liver,pancreas,and gut,similar to the urb1 expression pattern.mtor was ubiquitously expressed in the embryo at this stage.Double fluorescent in situ hybridizations(FISH)validated the enrichments of urb1 and raptor in the digestive organs at 54 hpf.Furthermore,treatment of rapamycin,an inhibitor of m TORC1,led to defects in the digestive organ development,similar to the urb1cq31 phenotypes.On the contrary,leucine activates m TOR pathway to regulate cell growth and m RNA translation.Embryos incubated with 500 ?M L-leucine exhibited enlarged liver and exocrine pancreas,similar to phenotypes of the Tg(hsp70l:urb1-Flag)cq33 transgenic embryos overexpressing Urb1.These data suggest a potential functional correlation between Urb1 and m TORC1.To characterize the functional relationship of Urb1 and m TORC1 in the digestive organ development,FISHs were carried out to analyze the expression of urb1 in the mtor and raptor morphants.In contrast to the wild type,injection of mtor MO or raptor MO led to decreases in the urb1 expression in the digestive organs.To perform rescue experiments,we continued using the Tg(hsp70l:urb1-Flag)cq33 transgenic line with urb1-Flag driven by a heat shock promoter.Beware that application of heat shock at 36 hpf led to expanded sizes of liver and exocrine pancreas in the control morphants at 72 hpf,substantiating the positive role of Urb1 in the cell proliferation of digestive organs.Overexpression of Urb1 efficiently rescued the hypoplastic liver and exocrine pancreas phenotypes in the urb1,mtor,and raptor morphants.On the contrary,overexpression of Rheb,an upstream factor of m TORC1,could not rescue the phenotypes of urb1 morphant.All the data above demonstrate that Urb1 acts downstream of m TORC to regulate digestive organ development.Urb1 plays a vital role in the formation of 27 S pre-r RNA.We performed sucrose density gradients to study the functions of Urb1 and m TORC1 as well as their functional correlation in the ribosomal subunit assembly in zebrafish embryos.In contrast to the wild type control,all the free 40 S,60S,and 80 S r-particles as well as polysomes were reduced in the urb1cq31 mutant and raptor morphant,in particular the 60 S r-particles in the urb1cq31 mutant.Overexpression of Urb1-Flag on one hand increased the free 60 S,80S,and polysomes,on the other hand rescued the reduction of ribosomal subunits in the raptor morphant.These data indicate that Urb1 controls ribosomal subunits syntheses downstream of m TORC1.Functions of Urb1 in the assembly of ribosomal subunits prompt its roles in protein synthesis.Dual-luciferase reporter assay exhibited that the luciferase activity was dramatically repressed in urb1cq31 mutant,whereas RT-PCRs showed the unaffected levels of m RNAs.These results indicate that Urb1 is required for the translation rather than transcription.According to the sucrose density gradients using the sorted gut:GFP+ cells and gut:GFP-cells,we ensured that the gut:GFP+ cells at 60 hpf contained more free r-particles and polysomes.That means,the developing digestive organs maintain relatively high levels of protein synthesis.The active protein synthesis in the developing digestive organs and the effects of m TORC1 and Urb1 were further confirmed by the injection of O-propargyl-puromycin,an indicator of protein synthesis activity.In contrast to control embryos,raptor morphant showed weak OPP staining,which could be rescued by the overexpression of Urb1-Flag.Overexpression of Urb1-Flag in the control morphant led to strong OPP staining.All these results demonstrate that Urb1 controls protein synthesis downstream of m TORC1,thus modulating digestive organ development.In this study,loss of a ribosome biogenesis protein Urb1 results in hypoplastic digestive organs,revealing key roles of Urb1 in the formation of digestive organs.Our data indicate that cells of developing digestive organs contain more ribosomes to maintain hyperactive protein synthesis for both proliferation and metabolism.This further highlights the requirement of hyperactive protein synthesis for the digestive organ development.Our study firstly presents that a ribosome biogenesis protein plays downstream roles of m TORC1 to regulate the development of digestive organs.The mechanisms underlying the regulation of urb1 expression by m TORC1 remain unclear and need further investigations.