Available online at www.sciencedirect.com

Journal of Genetics and Genomics 39 (2012) 451e462

JGG

ORIGINAL RESEARCH

Ribosome Biogenesis Factor Bms1-like Is Essential for LiverDevelopment in Zebrafish

Yong Wang a, Yue Luo a, Yunhan Hong b,*, Jinrong Peng a,*, Lijan Lo a,b,*

aCollege of Animal Sciences, Zhejiang University, Hangzhou 310058, ChinabDepartment of Biological Sciences, National University of Singapore, Science Drive 4, Singapore 117543, Singapore

Received 6 May 2012; revised 11 July 2012; accepted 12 July 2012

Available online 11 August 2012

ABSTRACT

Ribosome biogenesis in the nucleolus requires numerous nucleolar proteins and small non-coding RNAs. Among them is ribosomebiogenesis factor Bms1, which is highly conserved from yeast to human. In yeast, Bms1 initiates ribosome biogenesis through recruitingRcl1 to pre-ribosomes. However, little is known about the biological function of Bms1 in vertebrates. Here we report that Bms1 plays anessential role in zebrafish liver development. We identified a zebrafish bms1lsq163 mutant which carries a T to A mutation in the genebms1-like (bms1l ). This mutation results in L152 to Q152 substitution in a GTPase motif in Bms1l. Surprisingly, bms1lsq163 mutationconfers hypoplasia specifically in the liver, exocrine pancreas and intestine after 3 days post-fertilization (dpf). Consistent with thebms1lsq163 mutant phenotypes, whole-mount in situ hybridization (WISH) on wild type embryos showed that bms1l transcripts areabundant in the entire digestive tract and its accessory organs. Immunostaining for phospho-Histone 3 (P-H3) and TUNEL assay revealedthat impairment of hepatoblast proliferation rather than cell apoptosis is one of the consequences of bms1lsq163 giving rise to an under-developed liver. Therefore, our findings demonstrate that Bms1l is necessary for zebrafish liver development.

KEYWORDS: Liver development; Digestive organ development; Ribosome biogenesis; Bms1-like; Zebrafish

1. INTRODUCTION

The liver is an essential organ that carries out manyimportant functions. Most studies in liver development arecarried out in mice and chick using reverse genetics and/orexplants culture method (Zaret, 2002; Duncan, 2003).However, there are still a lot of missing gaps in the wholepicture of liver organogenesis due to limitations of suchapproaches and early lethality of liver defects. Zebrafish,

Abbreviations: BSA, bulked-segregant analysis; dpf, days post-fertiliza-

tion; fabp10, liver fatty acid binding protein; fabp2, intestinal fatty acid

binding protein; hpf, hours post-fertilization; SSLP, simple sequence length

polymorphism; WISH, whole-mount in situ hybridization.

* Corresponding authors. Tel/fax: þ86 571 8898 2233.

E-mail addresses: dbshyh@nus.edu.sg (Y. Hong); pengjr@zju.edu.cn

(J. Peng); g0403022@zju.edu.cn (L. Lo).

1673-8527/$ - see front matter Copyright � 2012, Institute of Genetics and Develop

Published by Elsevier Limited and Science Press. All rights reserved.

http://dx.doi.org/10.1016/j.jgg.2012.07.007

a recent model for vertebrate development, is particularlysuitable for studying liver organogenesis through forwardgenetics (Tao and Peng, 2009). In zebrafish, liver organogen-esis begins with the establishment of a population of cellsgaining hepatic competency within the ventral foregut endo-derm, instructed by Foxa and Gata factors; thereafter, meso-dermal signals, including Fgfs, Bmps, Wnt2b and retinoic acid(RA), induce the specification of hepatoblasts; hepatoblaststhen migrate and proliferate to form a discrete liver bud andfinally hepatoblasts in the liver bud undergo rapid proliferationand differentiation, giving rise to bile duct cells and functionalhepatocytes (Allende et al., 1996; Stafford and Prince, 2002;Zaret, 2002; Duncan, 2003; Field et al., 2003; Mayer andFishman, 2003; Chen et al., 2005; Holtzinger and Evans,2005; Ober et al., 2006; Sadler et al., 2007; Shin et al., 2007).

Nucleolus, a sub-cellular organelle in the nucleus, is bestknown as the location for the biosynthesis and processing of

mental Biology, Chinese Academy of Sciences, and Genetics Society of China.

452 Y. Wang et al. / Journal of Genetics and Genomics 39 (2012) 451e462

rRNA precursors and the assembly of two subunits of theribosome. The small subunit (SSU) processosome isa complex responsible for the assembly of the 40S ribosomesmall subunit (Fromont-Racine et al., 2003). Bms1 and Rcl1are two components of the SSU processosome. Bms1 isa G-domain-containing protein characterized first in yeast(Wegierski et al., 2001). By exerting GTPase activity on theconversion of recruited GTP into GDP, the N-terminal domainhas been shown to promote 35S pre-rRNA processing at sitesA0, A1 and A2 during the 40S ribosomal subunit biogenesisevents (Karbstein et al., 2005; Karbstein and Doudna, 2006). Itis believed that Rcl1, a putative endonuclease, activates Bms1by acting as a guanine-nucleotide exchange factor (GEF) topromote GDP/GTP exchange, and that activated (GTP-bound)Bms1 delivers Rcl1 to the pre-ribosomes. Apart from enzy-matic functions, the GTPase domain has also been reported torecruit and to enhance binding affinity of interacting partnerslike Rcl1 via conformational changes. The C-terminal domain,on the other hand, contains a GTPase-activating protein (GAP)motif that functions intra-molecularly (Karbstein et al., 2005;Karbstein and Doudna, 2006). Nevertheless, studies on Bms1thus far are limited to the role of this protein in ribosomebiogenesis, with focus on the GTPase domain, using yeast asthe model system.

Here we report the characterization of a small liver mutant,bms1lsq163 obtained from our genetic screening in zebrafishand the subsequent cloning of the mutant gene through posi-tional cloning. The mutation was identified to be a T to Aconversion in the gene bms1l encoding ribosomal biogenesisprotein Bms1l, which results in L152 to Q152 substitution in theGTPase motif. Genetic evidences from co-segregation anal-ysis, morpholino knockdown and mRNA rescue experimentunequivocally demonstrated that the bms1lsq163 mutation isresponsible for the small liver phenotype we observed. Ourstudy on bms1lsq163 provides the first genetic evidencedemonstrating that Bms1l possibly plays a specific function invertebrate liver development. Since Bms1l is a key componentin the 40S ribosomal biogenesis pathway that recruits manyother ribosomal proteins onto the pre-ribosomeerRNAcomplex, our work provides the first evidence for theinvolvement of a seemingly housekeeping gene in a specificdevelopmental process such as liver formation. This finding isinstrumental in filling up some of the current gaps in ourunderstanding of liver organogenesis.

2. MATERIALS AND METHODS

2.1. Generation of the bms1lsq163 mutant and geneticmapping of the mutant gene

Zebrafish were raised and maintained according to standardprocedures. Ethylnitrosourea (ENU, 3 mmol/L) was used asthe mutagen to mutagenize male fish (AB wild type strain).The mutagenized progenies were screened for small or no livermutants using prox1 as a probe in a high throughput whole-mount in situ hybridization (WISH) approach (Huang et al.,2008). The bms1lsq163 mutant was identified as a small liver

mutant from a total of 524 F2 ENU-mutagenized families.Map-based cloning method was used to clone the bms1lsq163

mutant gene. In order to create mapping families, twoheterozygous pairs (163-5 and 163-10) were used to cross withtwo wild type WIK pairs (WIK-A2 and WIK-C5). The ob-tained homozygous mutant embryos were subjected to bulked-segregant analysis (BSA) (Shimoda et al., 1999) using 226simple sequence length polymorphism (SSLP) markersgenerated by the Fishman (Shimoda et al., 1999) and Zon(http://zfrhmaps.tch.harvard.edu/ZonRHmapper/Maps.htm)groups.

2.2. WISH

For WISH probe labeling, plasmids harboring prox1,fabp10 (liver fatty acid binding protein 10), trypsin, insulin,fabp2 (intestinal fatty acid binding protein 2), hhex, foxA1,foxA3, gata4 and gata6 sequences (Chen et al., 2005; Huanget al., 2008) were used to synthesize their correspondingmRNAs via in vitro transcription using appropriate RNApolymerases. All probes were labeled with digoxigenin (DIG,Roche Diagnostics, USA). WISH was performed as describedpreviously (Chen et al., 2005).

2.3. Mutant phenotype rescue

Full length bms1l cDNA was obtained via RT-PCR(forward primer: 50-ctcgagtctagaacagtggagagagtcacgttg-30;reverse primer: 50-ctcgagtacgtatctcgtgcagcgtcatcat-30) andcloned into the pCS2þ vector. The plasmid harboring bms1lfull length cDNA was used to synthesize bsm1l mRNA. Formutant phenotype rescue, 1 ng of in vitro transcribed bms1lmRNA was injected into fertilized eggs at the one-cell stage.Injected embryos at 3 days post-fertilization (dpf) wereanalyzed by WISH using the liver specific probe fabp10.

2.4. Morpholino (MO) injection

Morpholino (50-ctcttttgcggttacatttattta-30) specifically tar-geting the 50-UTR (50-untranslated region) of bms1l mRNA(Bms1l-50-UTR MO) was designed and synthesized by GeneTools (Philomath, USA). One nanolitre of Bms1l-MO(0.75 nmol/mL) was injected into one-cell stage embryos.

2.5. RNA analysis

Total RNA from different samples was extracted usingTRIzol (Gibco BRL, USA) as instructed by the supplier.Probes were DIG-labeled via polymerase chain reaction (PCR)using plasmids harboring the target probes as templates. RNAgel blot hybridization was performed as previously described(Wen et al., 2005; Cheng et al., 2006).

2.6. Phospho-histone 3 (P-H3) immunostaining

Sectioned samples were fixed in 2% PFA (para-formaldehyde) for 20 min and washed three times of 20 min

453Y. Wang et al. / Journal of Genetics and Genomics 39 (2012) 451e462

each in PBST (140 mmol/L NaCl, 2.7 mmol/L KCl, 12 mmol/L Na2HPO4, 1.4 mmol/L KH2PO4, 0.1% Tween-20, pH 7.5).Samples were blocked in 4% bovine serum albumin for30 min and incubated with primary antibody against P-H3(sc-8656R, Santa Cruz, USA) at a dilution of 1:200 for 1 hat room temperature or overnight at 4�C. Five washes of20 min each were followed to remove excess antibody. Thefluorescence-conjugated second antibody, Alexa Fluor� 568goat anti-rabbit IgG (Hþ L) (SKU# A-11011, MolecularProbes, USA) was applied to sample at a dilution of 1:400 for1 h at room temperature or overnight at 4�C. After the removalof excess antibody by similar washing conditions used to getrid of the first antibody, the samples were mounted forimaging.

2.7. TUNEL assay

Cryo-sections (8 microns) were fixed in 4% PFA for20 min, washed 30 min with PBS, and incubated in per-meabilization solution (0.1% Triton X-100, 0.1% sodiumcitrate) for 2 min on ice. TUNEL assay was carried out with InSitu Cell Death Detection Kit, TMR red (Roche) according tothe instructions provided.

3. RESULTS

3.1. bms1lsq163 confers a small liver phenotype

Fig. 1. bms1lsq163 mutation confers a small liver phenotype.

Wild type (wt) and bms1lsq163 mutant (mt) embryos at 3 dpf (except for Alcian

blue staining) were harvested for WISH or alkaline phosphatase (AP) staining.

A: WISH using a prox1 probe. Red arrow indicates liver bud. B: comparison

of overall appearance of a wt embryo and a bms1lsq163 mutant embryo.

C: WISH using a troponin C probe for somite staining. D and E: AP staining

of the pronephric duct (D) and blood vessels (E). F: Alcian blue staining of the

skeleton cartilage at 5 dpf. GeJ: WISH using the liver-specific marker fabp10

(G), exocrine pancreas marker trypsin (H), endocrine pancreas marker insulin

(I), and intestinal marker fabp2 (J) as probes. Black arrow: liver; red arrow:

exocrine pancreas; black arrowhead: islet; blue arrow: intestine.

Genetic screening for no and/or small liver mutants wascarried out at 3 dpf via WISH when a liver proper has beenwell-defined. Being strongly expressed in the zebrafishembryonic liver from 1.5 dpf to 4 dpf (Ober et al., 2003),prox1 was selected as the probe to assess liver status in the F3progenies. The bms1lsq163 mutant was isolated as a small livermutant with weaker prox1 signals (Fig. 1A), with otherwiseoverall normal morphological development (Fig. 1B). Furthercharacterization showed that mesoderm-derived organs suchas the somite (examined with a troponin C probe) (Fig. 1C),pronephric duct and blood vessels of the mutant embryos(examined with AP staining) (Fig. 1D and E) also appearednormal at 3 dpf. The mutation does not appear to affectskeletal cartilage development up to 5 dpf (Fig. 1F). The smallliver phenotype was confirmed by similar signal reductionwith the liver specific gene fabp10a (Fig. 1G). By checkingtrypsin (exocrine pancreas) and insulin (islet) expression, wefound that the mutant exocrine pancreas was affected signifi-cantly in size (Fig. 1H), while no visible defect in the islet wasobserved (Fig. 1I). The gut of the mutant was also noted toexhibit reduced signal when compared to that observed in thewild type using the intestinal marker fabp2 (Fig. 1G). Thesedefects found on the three major digestive organs in the mutantwere reflected more profoundly at 4 dpf. By 4 dpf, the left lobeof a normal liver should have accumulated in size compre-hensively and extended over forming the right lobe, asobserved in the wild type. However, the liver in bms1lsq163

experienced drastic growth arrest where the size remained thesame as that in 3 dpf, with no sign of the second lobe (data not

shown). The exocrine pancreas in the mutant also sufferedobviously delayed expansion where the organ failed tobroaden and lengthen posteriorly as that seen in wild type(data not shown). Apart from the absence of looping, the

454 Y. Wang et al. / Journal of Genetics and Genomics 39 (2012) 451e462

mutant failed to form a fully expanded intestinal bulb (data notshown). Mutant embryos do not survive beyond 5 dpf.

3.2. bms1lsq163 does not affect liver initiation

Fig. 2. bms1lsq163 affects liver bud growth.

Wild type (wt) and bms1lsq163 mutant (mt) embryos at 2 dpf were analyzed

with early markers to assess liver budding. WISH probes used include pan-

endodermal markers gata4, gata6, foxA1, foxA3, and hepatic markers prox1

and hhex. All markers showed that the liver bud size in bms1lsq163 mutant was

obviously smaller although liver bud initiation occurred normally. Meanwhile,

it appeared that prox1 and hhex might mark different cell populations in the

liver bud. Interestingly, while foxA1, gata4 and gata6 showed similar

expression in the liver bud in both wt and mt, their expression patterns in the

mutant pancreatic bud were different from that in wt. Black arrow: liver; red

arrow: exocrine pancreas; blue arrow: intestine.

The above results indicate that the mutation in bms1lsq163

does not exclusively affect the development of the liver butimpinge on the growth of other digestive organs as well.Meanwhile, despite displaying much reduced expression,positive fabp10a, trypsin and fabp2 signals were detected inbms1lsq163 mutant embryos. This observation suggests that thedifferentiation of hepatocytes, exocrine pancreatic cells andintestinal epithelial cells from their corresponding precursorsdid take place in the mutant although the reduced expressionof these markers could be due to the compromised differen-tiation of these cell types. To determine the onset of observ-able defects, earlier markers were used to assess the status ofthe earlier events of liver formation. One of the earliest eventsof liver development in zebrafish is competency acquisitionwhere factors such as foxA1, foxA3, gata4 and gata6 areknown to confer endodermal cells with competency proper todevelop as hepatic cells and these genes continue to express inthe endodermal lineage during early development of digestiveorgans (Lee et al., 2005; Zhao et al., 2005). Upon comparingthe expression of these four early markers between wild typeand bms1lsq163 at 2 dpf, it was found that though subtle, theliver bud in the mutant is noticeably smaller than that in itswild type siblings. All data at this stage suggests that liver budexpansion but not liver bud initiation is affected in bms1lsq163.Interestingly, the pancreas appeared to be most drasticallyaffected where there was little or no detectable expression offoxA1, gata4 and gata6 in the pancreatic bud whereas the liverwas only slightly smaller (Fig. 2). Intriguingly, unlike thedrastic defects as revealed by foxA1, gata4 and gata6 probes,WISH using a foxA3 probe showed no evidence of anyabnormal phenotype (Fig. 2). One explanation for this obser-vation is that, though present, the mutant pancreatic cellssomehow failed to be stained by foxa1, gata4 and gata6 due tocertain yet to be defined alterations caused by the mutation. Inaddition, we observed that, at as early as 2 dpf, the thickeningand left-looping of the mutant gut tube were also affected(Fig. 2).

3.3. bms1lsq163 does not block hepatoblast specification

The stage following competency acquisition is the specifi-cation of hepatoblast from competent hepatic endoderm cells.prox1 and hhex are the two of the earliest markers of definitivehepatoblasts (Ober et al., 2006). To check the hepatoblasts inbms1lsq163, we examined the expression of these two genes at2 dpf. Consistently, prox1 revealed a smaller liver bud inbms1lsq163 compared to wild type (Fig. 2), implying that themutant suffers deficiency in sustaining definitive hepatoblast.Interestingly, no noticeable difference was detected in the liverbud between bms1lsq163 and wild type when another hepato-blast marker, hhex was used (Fig. 2). Comparison of thestaining patterns of prox1 and hhex suggests that these two

markers appear to mark different sub-type of cells in the liverbud at 2 dpf (Fig. 2).

3.4. Positional cloning of bms1lsq163

After backcrossing four rounds with wild type AB fish topurify the genetic background, bms1lsq163 heterozygousmutants were outcrossed with the WIK line to generatemapping families for positional cloning. To identify markersclosely linked to bms1lsq163 by BSA, DNA from 26 homozy-gous mutant embryos and 26 siblings from the same mappingfamilies were pooled, respectively. DNA from the mutant andsiblings pools was used as PCR templates for each of the266 SSLP markers. Two SSLP markers Z4830 (north marker)and Z4397 (south marker) were found to be linked to each sideof bms1lsq163 on linkage group 12 (Fig. 3A). Further geneticmapping revealed Z35706 as a new north marker closer tobms1lsq163 than Z4830. Screening with Z4397 identified

Fig. 3. bms1lsq163 mutation alters a conserved motif in Bms1l.

A: schematic drawing shows map-based cloning of the bms1lsq163 mutant

gene. bms1lsq163 mutation was finally mapped onto linkage group 12 contig

1189 on BAC clone CH211-214E3. Genetic SSLP markers are shown on the

top with their corresponding number of recombinants shown below. The

mutant gene was mapped between SSLP markers CH211-214E3-11 and

CH211-214E3-1 (in blue) spanning a 155 kb genomic DNA fragment spanned

by two BACs DKEY-288I10 and CH211-214E3. ‘m’ in red: bms1lsq163

mutation locus; N: north; S: south. Diagram is not drawn to scale. B: sequence

trace showing the T to A mutation (underlined in purple) in the bms1l gene in

bms1lsq163 mutant that results in a CTG to CAG codon change (underlined

cyan). C: top panel of a schematic diagram showing the structural organization

of the Bms1l protein. Lower panel shows that the bms1lsq163 mutation leads to

the conversion of the conserved L152 to Q152 (indicated by green asterisk) in

the GTPase domain. NLS: nuclear localization signal.

455Y. Wang et al. / Journal of Genetics and Genomics 39 (2012) 451e462

2 recombinants out of 3500 mutants. At the same time,screening with Z35706 identified 30 recombinants out of 3538mutant embryos (Fig. 3A). Subsequently, fc06f12-9 took overZ35706 to become the closest informative marker that liesjust north of the mutation (Fig. 3A). A search in the Ensembldatabase (http://www.ensembl.org/Danio_rerio/index.html)and Genome Fingerprinting Project database (http://www.sanger.ac.uk/Projects/D_rerio/WebFPC/zebrafish) locked bothfc06f12-9 and Z4397 markers onto one BAC contig 1189,which is composed of 219 overlapping BAC clones, most withfull sequence available. Marker fc06f12-9 was located on BACclone DKEY-145P15 while Z4397 on DKEY-288I10(Fig. 3A). Based on the BAC sequences in these contigs,new SSLP markers were designed with the help of a freesoftware (http://danio.mgh.harvard.edu/markers/ssr.html) andwere tested on recombinants (data not shown). The final2 (south) and 1 (north) recombinants could not be reducedfurther upon assessment with finer north SSLP CH211-214E3-11 and south SSLP CH211-214E3-1 markers, respectively (onCH211-214E3) (Fig. 3A). These two final SSLPs definea critical region with an estimated physical distance of 155 kb(Fig. 3A).

3.5. bms1lsq163 alters a conserved domain in Bms1l

Blast search against the zebrafish genome sequence in theEnsembl database showed that this 155 kb genomic fragmentcontains 5 open reading frames (ORFs) encoding for G-proteincoupled receptor Gpr123 (gi125831439), zinc finger CTCF(gi125831441), Mxtx2 (gi24371281), Bms1l (gi125831426)and SH2-like protein (gi125831443) (data not shown).Systematic exons sequencing was embarked on these genes inhunt for the mutation. Consisting of 23 exons, exon 5 of thebms1l gene was found to harbor a nucleotide change from T toA (Fig. 3B), resulting in an amino acid substitution from Leu toGln on the 152nd amino acid (Fig. 3B) in the conserved GTPasedomain of Bms1l (Figs. 3C and 4). Sequencing the remainderof the bsm1l coding region revealed no other mutations.

3.6. L152 to Q152 substitution in Bms1l causes small liverphenotype in bms1lsq163

To confirm whether the gene identified is indeed themutated gene responsible for the mutant phenotypes observed,segregation analysis was carried out. Male and femalebms1lsq163 heterozygotes were mated and the progenies weresubjected to WISH analysis using fabp10 probe to group theembryos based on liver size. One hundred and five embryosand 26 embryos showing normal and small liver, respectively,were identified after WISH. Genomic DNA from thesesiblings was isolated and DNA fragment containing themutation was amplified via PCR and sequenced. Embryosshowing normal sized liver were expected to show either T/T(wild type) or A/T (heterozygote) whilst embryos showingsmall liver would be A/A (homozygote) at the nucleotide ofquery. Results showed that embryos with normal sized liverexhibited T/T (33/105) and A/T (72/105) at the anticipatedratio of 1:2 while A/A genotype was strictly restricted to themutant homozygotes.

To unequivocally prove that the T to A substitution in bms1lis responsible for the bms1lsq163 phenotype, complementationtest was carried out. The mRNA encoding wild type andmutant bms1l was injected into one-cell stage embryos inde-pendently and the embryos are genotyped later after assessingthe liver status via in situ hybridization. At 5 dpf,w57% of themutants injected with wild type bms1l mRNA (34 out of 60injected mutant embryos examined) had restored the expres-sion of the liver specific gene fabp10 fully or partially(Fig. 5A). In contrast, both the T to A mutant bms1l mRNAand a mRNA harboring a premature stop codon failed torescue the mutant phenotype (0/6 and 0/18 mutant embryosexamined, respectively) (Fig. 5A). These results definitivelyprove that the L152 to Q152 substitution in Bms1l in bms1lsq163

brought about the small liver phenotype we observed.

3.7. Knockdown of bms1l gene phenocopies the smallliver phenotype in bms1lsq163

In zebrafish, morpholino-mediated gene knockdown iscommonly used to study gene function. A 50-UTR morpholino

Fig. 4. Alignment of Bms1l proteins from different species.

Alignment of Bms1l proteins from zebrafish (NM_001111150), human (GI:40788900), mouse (GI:39930555) and baker’s yeast (GI:6325039). The conserved

GTPase domain is underlined. The mutated amino acid Leu152 is marked with a green asterisk.

456 Y. Wang et al. / Journal of Genetics and Genomics 39 (2012) 451e462

was designed to block the 50-UTR region of bms1l preventingefficient translation of the gene. The small liver phenotypeobserved in bms1lsq163 was phenocopied in morphants injected

with bms1l-50-UTR where 100% of the 93 morphants exhibi-ted small liver, a specific phenotype that was not detected inany of the 89 embryos injected with a control morpholino

Fig. 5. bms1lsq163 small liver phenotype can be rescued by bms1l mRNA injection and phenocopied by Bms1l knockdown.

A: the small liver phenotype of the bms1lsq163 mutant (mt) was fully or partially rescued by the wild type bms1l mRNA transcript (bms1lwt) but not the T to A

mutant mRNA (bms1l163) or the mRNA harboring a premature stop codon (bms1lstop). All embryos were sampled at 5 dpf and the liver was visualized by the

fabp10 probe. wt: wild type. Number on the right bottom corner indicates the number of homozygous mutants with liver rescued over total mutants examined.

B: the small liver phenotype of bms1lsq163 can be mimicked by the injection of a bms1l morpholino (bms1l-50-UTR MO). Control morpholino (control MO) did not

elicit similar phenotype indicating the specificity of the bms1l-50-UTR MO. Number on the right bottom corner indicates the number of morphants conferring small

liver phenotype over total morphants examined. C: specificity of the morpholino is further supported by liver recovery in the morphants through co-injection of

wild type bms1lmRNA. Note that the bms1l wild type transcript here begins with ATG start codon, devoid of bms1l-50-UTR MO target region. All embryos were at

5 dpf. The liver was visualized by the fabp10 probe. Number on the right bottom corner indicates the number of morphants conferring full/partial/no rescued liver

over total morphants examined.

457Y. Wang et al. / Journal of Genetics and Genomics 39 (2012) 451e462

(Fig. 5B). The specificity of this observation was furtherverified by the rescue of these morphants by co-injected bms1lmRNA, where more than 80% was fully or partially rescued(Fig. 5B). This provides additional genetic evidence that themutated bms1l is responsible for the small liver phenotypeseen in bms1lsq163.

3.8. Bms1l is highly conserved in different species

Blast search revealed that proteins showing strong sequencesimilarity to Bms1l are encoded in a diverse range of genomes,including that of Homo sapiens, Drosophila melanogaster,Caenorhabditis elegans, Schizosaccharomyces pombe andeven Arabidopsis thaliana, suggesting that it is highly evolu-tionarily conserved. The approximately 400 amino acids longN- and C-terminal domains (domain N and domain C,

respectively) are the most conserved regions of the proteins(Fig. 4), implying functional significance of these two regions.

3.9. Expression patterns of bms1l

To explore the possible roles of Bms1l in the developmentof the liver and other digestive organs, we first analyzed bms1lexpression patterns in zebrafish. WISH using a fragment ofbms1l containing the GTPase domain as a probe on wild typezebrafish embryos revealed strong specific signals in thevarious endoderm-derived organs including liver, pancreas andgut (Fig. 6A). Probes generated from the middle and C-terminal of the gene showed similar enriched signals in theseorgans (data not shown). Northern blot of bms1l in wild typeembryos showed that bms1l transcripts were expressed atsimilar levels from unfertilized eggs (maternal expression) and

Fig. 6. Embryonic expression patterns of bms1l.

A: WISH using a fragment containing the GTPase domain of the bms1l gene as a probe revealed that the transcript is enriched in endodermal organs such as the

liver (L), pancreas (P) and gut (G) at 60 hpf and 84 hpf. Signals are also detected in the retina. B: Northern blot showing bms1l expression at early stages of

embryogenesis. Methylene blue staining of 28S rRNA is used as a loading control. hpf: hours post-fertilization, unf: unfertilized eggs. C: diagram showing the

process of maturation of 28S, 18S and 5.8S rRNAs and intermediate products during processing. ETS: external transcribed spacer. ITS: internal transcribed spacer.

D: Northern blot analysis of 28S, 18S and intermediate products of pre-rRNA processing using corresponding probes as indicated. The ITS1 probe is expected to

pick up products of (a), (b) and (c) corresponding to that shown in C. Asterisk: unidentified rRNA intermediate molecule.

458 Y. Wang et al. / Journal of Genetics and Genomics 39 (2012) 451e462

1e5 dpf, indicating that it is required throughout embryo-genesis (Fig. 6B).

3.10. bms1lsq163 affects the processing of pre-rRNA

All 28S, 18S and 5.8S rRNAs are derived from the samepre-rRNA precursor (Fig. 6C). In yeast, depletion of Bms1caused aberrant processing of 35S pre-rRNA (Karbsteinet al., 2005; Karbstein and Doudna, 2006). To find outwhether Bms1l plays similar function in zebrafish, we per-formed Northern blot to analyze the status of mature 18S and28S rRNA and their intermediate precursors using corre-sponding probes (Azuma et al., 2006). Our result showed thatbms1l-50-UTR morphants accumulated pre-18S precursorsalthough the final amount of 18S rRNA remained stable(Fig. 6D).

3.11. bms1lsq163 small liver phenotype is due to impairedhepatoblast proliferation rather than elevated apoptosis

Growth or otherwise, is an output after balancing prolifer-ation and apoptosis. A direct supposition for the small liverphenotype in bms1lsq163 therefore is reduced proliferation ofthe hepatoblasts or increased apoptosis or both. To find outwhich is the case, P-H3 staining and TUNEL assay wereperformed to compare the proliferation and cell death status,respectively, in wild type and mutants. P-H3 assay uses anantibody against the phospho-histone 3 protein (P-H3) asa marker of proliferating cells. To better distinguish thehepatocytes from the surrounding cells, we performed thisexperiment in the background of Tg(fabp10a:RFP) transgenicfish, in which the reporter RFP is specifically expressed in theliver. By examining sectioned embryos at 3 dpf, we found that

459Y. Wang et al. / Journal of Genetics and Genomics 39 (2012) 451e462

the mutant had approximately 4-fold less P-H3-positive cells(6 positive cells out of 645 cells counted) than the wild type(67 positive cells out of 1744 cells counted) in the liver region,respectively (Fig. 7AeE). Meanwhile, we noticed that the P-H3-positive cells in the mutant intestine were reducedapproximately 1.5 folds at 3 dpf (Fig. 7AeE). The bms1lsq163

mutation does not seem to affect proliferation of the cells inthe neural tube (Fig. 7E). This result suggests that one of thecontributing factors for a reduced size liver observed is theimpairment of hepatoblast proliferation. Concurrently,increased apoptosis could add onto the phenotype observed.Apoptotic activities in both the wild type and mutant embryoswere examined via TUNEL method and no apoptotic cellswere found in wild type or mutant in the liver region examinedat 2 dpf (data not shown) and 4 dpf (Fig. 7F and G).

4. DISCUSSION

Phenotypic characterization of the bms1lsq163 mutant usingspecific digestive organ markers suggests that liver bud growthbut not bud initiation was affected in the mutant. The lesioncontinued to impinge on the expansion of the liver, as well asother digestive organs such as the intestine and pancreas,resulting in their retardation after 3 dpf. The fact that three ofthe major organs were formed suggests that the differentiationof these organs is compromised but not totally abolished.This is in contrast to liverless mutants like mypt1sq181 andprometheus ( prt) (Ober et al., 2006; Huang et al., 2008). Byexploiting the polymorphisms exhibited in 226 pairs of SSLPmarkers and polymorphic mapping families, the BSA protocolmapped bms1lsq163 to linkage group 12. From w7000 meioticevents, subsequent intermediate and fine mapping in combi-nation with candidate gene approach identified a T to Asubstitution in the ribosomal biogenesis protein gene bms1l,which results in L152 to Q152 substitution in a GTPase motif inBms1l. Three lines of evidence from co-segregation analysis,morpholino knockdown and wild type transcript rescue clearlyproved that bms1lsq163 is responsible for the small liverphenotype. Consistent with the bms1lsq163 phenotypes, WISHon wild type embryos showed that bms1l was enriched in theentire digestive tract and its accessory organs. Therefore, themutation in the GTPase domain of Bms1l in bms1lsq163 mightlead to the inability or reduced efficiency of the GTPase ineither the conversion of GTP into GDP, or the recruitment ofproper partners to exert its function in organogenesis. Rcl1 isan interacting partner of Bms1l in ribosomal synthesis andprocessing (Wegierski et al., 2001). One working hypothesis isthat in bms1lsq163, the interaction (either physical or func-tional) between Bms1l and Rcl1 has been compromisedresulting in the small liver phenotype. Future work is requiredto address this proposition.

Ribosome, responsible for protein manufacturing in allliving cells, is a large ribonucleoprotein complex made up of65% rRNA and 35% ribosomal proteins. Eukaryotes have 80Sribosomes, each consisting of a small (40S) and large (60S)subunit. The large subunit is composed of a 5S RNA (120

nucleotides), a 28S RNA (4700 nucleotides), a 5.8S subunit(160 nucleotides) and w49 proteins. The 40S subunit hasa 1900 nucleotide (18S) RNA and w33 proteins (Ben-Shemet al., 2011). Synthesis and processing of rRNAs and theassembly of ribosomes in eukaryotic cells occur in thenucleolus following a complex pathway where rRNA is tran-scribed, processed and assembled with ribosomal proteinsproducing ribosomal subunits. The machinery responsible forrRNA processing is a large ribonucleoprotein complex con-taining multiple ribosomal proteins and accessory nucleolartrans-acting factors that associate with the nascent pre-rRNA(Kressler et al., 1999; Fromont-Racine et al., 2003). rRNAprocessing has been most extensively studied in the yeastSaccharomyces cerevisiae and many trans-acting factors, bothproteins and ribonucleoproteins, required for the process havebeen characterized in the organism as well. Rcl1p is a putativeendonuclease essential for pre-rRNA processing at specificsites of the ribosomes (Billy et al., 2000). Bms1p wasdiscovered via a yeast two-hybrid screen using Rcl1p as thebait (Wegierski et al., 2001). Like Rcl1p, Bms1p is anevolutionarily conserved nucleolar protein required for pre-rRNA processing at specific sites. Bms1lp possesses struc-tural features common to the regulatory GTP/GDP bindingprotein (G-proteins) at the N-termini domain and later wasshown to indeed, function as a GTPase for the biogenesis of40S ribosomal subunits (Karbstein et al., 2005). Almost theentirety of Bms1l functional study to date is focused on itsconserved role of GTPase in ribosomal assembly, wherea large proportion of the investigation is biochemical in nature,exploring the ordering and kinetics of complex formation anddisassociation by components reconstitution assays.

Based on close sequence homology, the structure of theG-domain of Bms1lp has been inferred from the closely relatedeubacterial protein elongation factors EF-Tu (Sanchez and Sali,1998), containing five conserved polypeptide loops designatedas G1 through G5, which form contact sites with guanine ringor coordinate Mg2þ ion. Mutational studies have shown thatcertain residue substitution at the loops of Bms1l affected itsbiological activity (Wegierski et al., 2001), hence definingspecific amino acids crucial for the process. G-domain isknown to be present in many regulatory GTPases, acting asmolecular switches in diverse cellular processes such astranslation, protein trafficking and signal transduction (Bourneet al., 1991). Therefore, it is of no surprise when a mutatedBms1lp resulted in unviable spores that failed to germinate,revealing reduced 40S subunits levels in the yeast mutant asdemonstrated by polysome profiling (Wegierski et al., 2001).Similarly, bms1lsq163 zebrafish could only live up to 6 dpf.However, no significant decrease of mature rRNAwas observedon Northern blots (data not shown). It is possible that thefluctuation of 40S level in the mutant is not sufficient enough tobe detected by Northern blot. Alternatively, the effect of thebms1l mutation was not manifested through the reduction of40S level in zebrafish. A more sensitive detection method willbe needed to address this aspect, such as to monitor the statusof newly synthesized rRNA instead of total rRNA.

Fig. 7. bms1lsq163 impairs hepatoblasts proliferation.

AeD: immunostaining using a P-H3 antibody (green) on 3 dpf wild type (wt) (A,C) and mutant embryo (mt) (B,D) sections. DAPI (blue) is used to mark cell

nucleus. Hepatoblasts are labeled in red by staining RFP which is specifically expressed in the liver under driven by the fabp10a promoter. E: quantitative analysis

of the proliferating cells in the liver, intestinal bulb and neural tube. Sections from three wt and three mt embryos across the liver and the intestinal bulb were used

in counting the PH-3-positive cells and total DAPI positive cells. F and G: TUNEL analysis of apoptotic cells in wild type (wt) and mutant (mt) at 5 dpf. No

apoptotic cells were found in both wt and mu in the liver region. Forty-six sections from three wild type embryos and 51 sections from three mutant embryos were

examined. Ib: intestinal bulb; L: liver; Nt: neural tube; Nc: notochord; Pa: pancreas; Pd: pronephric duct; Ph: pharyngeal.

460 Y. Wang et al. / Journal of Genetics and Genomics 39 (2012) 451e462

461Y. Wang et al. / Journal of Genetics and Genomics 39 (2012) 451e462

One puzzling question raised with the identification ofbms1l as the gene responsible for the specific small liverphenotype in bms1lsq163 is the regional defects instead ofthe more expected general developmental defects with wide-spread phenotypes like those observed in the Minutes flymutants, which were reported to be contributed by quantitativedeficiencies of ribosomal protein (RP) genes (Lambertsson,1998). One likely explanation is due to maternally expressedbms1l that compensates for bms1lsq163 at the early stage ofembryogenesis. Another possible rationalization of thespecific liver phenotype is that ribosomal protein genes couldconceivably display some novel biological function indepen-dent of their role in the ribosome where inhibition of thisfunction leads to specific consequence such as defective liverdevelopment. Such speculation is not groundless since indi-vidual ribosomal proteins have been implicated in a widevariety of biological functions, including cell cycle andprogression, apoptosis and DNA damage responses (Volarevicet al., 2000; Lohrum et al., 2003), and it has also been sug-gested that their roles in these generic processes may ariseindependently of their role in the ribosome itself (Wool, 1996;Soulet et al., 2001). Therefore, a possible novel function ofBms1l in liver organogenesis cannot be ruled out. Alterna-tively, the selective manifestation of endodermal phenotype byan apparently housekeeping gene is due to different geneproduct dosage in different tissue types, which is similar to thereason for explaining why one-third of the mutated rp did notgive rise to tumors (MacInnes et al., 2008). This is probablynot surprising after all since it has been suggested thatdifferent tissues have different requirements for specific rpdosages hence expressing variable amounts of the same tran-script (Bortoluzzi et al., 2001).

While the characterization work on bms1lsq163 reported sofar is still at its infancy and is limited to phenotypic exami-nation via molecular markers, a glimpse into possible reasonsbehind a small liver phenotype revealed impairment of hep-atoblast proliferation. This is in contrast to the previous find-ings in the conditional knockout mouse for S6 (a ribosomalprotein for the 40S subunit) where although the S6-deficientliver cells failed to enter S phase and proliferate, the liversfrom fasted mice were capable of increasing cell size inresponse to the re-addition of food despite a deficiency in 40Sribosomes. These results place a cell proliferation checkpointbut not one on growth as a consequence of deficiency inribosomal biogenesis (Opferman and Zambetti, 2006). Adifferent mechanism might be governed by the other ribo-somal biogenesis proteins such as Bms1l.

ACKNOWLEDGEMENTS

We thank Dr. Jun Chen and Dr. Honghui Huang for theirsuggestions and technical support. This work was supportedby the grants from the National Natural Science Foundation ofChina (NSFC) (No. 31171391) to LJL, an NSFC grant (No.30825025) to JRP and a grant from the National ResearchFoundation of Singapore (R-154-000-529-281) to YHH.

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