3232 RESEARCH ARTICLE
Development 139, 3232-3241 (2012) doi:10.1242/dev.077107
© 2012. Published by The Company of Biologists Ltd
Ribosomal biogenesis genes play an essential and p53independent role in zebrafish pancreas development
Elayne Provost1, Karen A. Wehner3,*, Xiangang Zhong1,*, Foram Ashar2, Elizabeth Nguyen4, Rachel Green3,
Michael J. Parsons2 and Steven D. Leach1,2,‡
SUMMARY
Mutations in the human Shwachman-Bodian-Diamond syndrome (SBDS) gene cause defective ribosome assembly and are associated
with exocrine pancreatic insufficiency, chronic neutropenia and skeletal defects. However, the mechanism underlying these
phenotypes remains unclear. Here we show that knockdown of the zebrafish sbds ortholog fully recapitulates the spectrum of
developmental abnormalities observed in the human syndrome, and further implicate impaired proliferation of ptf1a-expressing
pancreatic progenitor cells as the basis for the observed pancreatic phenotype. It is thought that diseases of ribosome assembly
share a p53-dependent mechanism. However, loss of p53 did not rescue the developmental defects associated with loss of zebrafish
sbds. To clarify the molecular mechanisms underlying the observed organogenesis defects, we performed transcriptional profiling
to identify candidate downstream mediators of the sbds phenotype. Among transcripts displaying differential expression, functional
group analysis revealed marked enrichment of genes related to ribosome biogenesis, rRNA processing and translational initiation.
Among these, ribosomal protein L3 (rpl3) and pescadillo (pes) were selected for additional analysis. Similar to knockdown of sbds,
knockdown or mutation of either rpl3 or pes resulted in impaired expansion of pancreatic progenitor cells. The pancreatic
phenotypes observed in rpl3- and pes-deficient embryos were also independent of p53. Together, these data suggest novel p53independent roles for ribosomal biogenesis genes in zebrafish pancreas development.
INTRODUCTION
Shwachman-Diamond syndrome (SDS) is an autosomal recessive,
multi-system pediatric disorder characterized by pancreatic
insufficiency, skeletal abnormalities and chronic neutropenia.
Infants present early with failure to thrive and require treatment for
chronic infection and pancreatic enzyme deficiency. In 2002, the
causal gene for SDS, SBDS, was identified (Boocock et al., 2003).
SBDS is a highly conserved, essential gene that functions in late
maturation of the large 60S ribosomal subunit. Studies in a variety
of organisms and SDS patient lymphoblasts suggest that mutations
in SBDS result in ribosomal subunit joining defects associated with
altered subunit ratios (Zhang et al., 2006; Menne et al., 2007; Finch
et al., 2011; Wong et al., 2011).
Ribosome biogenesis is normally accomplished in a tightly
controlled, multi-step process. In addition to the 80 known
ribosomal proteins (RPs), Sbds is one of ~150 non-ribosomal
proteins required to assemble a functional 80S ribosome
(Deisenroth and Zhang, 2010). In addition to SDS, other diseases
with causal mutations in genes related to ribosome assembly have
been described and are collectively referred to as ribosomopathies
(Luft, 2010; Narla and Ebert, 2010). One well-characterized
ribosomopathy, Diamond-Blackfan anemia (DBA), is commonly
associated with mutations in the RP gene RPS19 (Draptchinskaia
et al., 1999). Models of DBA in mice and zebrafish have suggested
a role for p53 (Tp53/Trp53) in mediating the disease phenotype
1
Department of Surgery and 2Institute for Genetic Medicine, Johns Hopkins
University, Baltimore, MD 21205, USA. 3The Howard Hughes Medical Institute and
Department of Molecular Biology and Genetics, Johns Hopkins University, Baltimore,
MD 21205, USA. 4Department of Biology, Barry University, Miami, FL 33176, USA.
*These authors contributed equally to this work
Author for correspondence ([email protected])
‡
Accepted 7 June 2012
(Danilova et al., 2008; McGowan et al., 2008; Uechi et al., 2008).
Mice with mutations in Rps19 are phenotypically rescued by
homozygous loss of p53 (McGowan et al., 2008), as are other
mouse models of human ribosomopathies, including 5q-syndrome
and Treacher Collins syndrome (Jones et al., 2008; Barlow et al.,
2010). Thus, it is generally appreciated that ribosomopathies
mediate their phenotype through p53-dependent mechanisms.
In order to determine whether the SDS phenotype exhibits similar
p53 dependence, we used antisense morpholinos to recapitulate the
composite SDS phenotype in zebrafish. Extending a previous report
of the zebrafish sbds morphant phenotype (Venkatasubramani and
Mayer, 2008), we found that sbds knockdown resulted in loss of
neutrophils, skeletal defects and pancreatic hypoplasia, as well as
changes in ribosomal subunit ratios. In addition, we have identified
impaired proliferation but normal differentiation of ptf1a-expressing
pancreatic progenitor cells as the primary defect underlying the
pancreatic hypoplasia phenotype. Moreover, the observed
organogenesis defects were unaffected by simultaneous inactivation
of either p53 or its 113p53 variant, suggesting that SDS is a largely
p53-independent ribosomopathy.
As a means to more fully understand the consequences of sbds
loss, we used a discovery-based microarray platform to evaluate
global transcriptional changes in sbds morphants. Functional group
analysis suggested that loss of sbds results in widespread changes
in the expression of genes related to ribosome biogenesis, rRNA
processing and translational initiation. Among the genes displaying
altered expression in sbds morphants, we performed additional
functional analysis of ribosomal protein L3 (rpl3) and pescadillo
(pes). Using antisense morpholinos or available mutants, we found
that inactivation of either rpl3 or pes phenocopied the sbds
pancreatic progenitor phenotype, also in a p53-independent
manner. Together, these data identify novel developmental roles for
genes related to ribosome biogenesis, independent of p53.
DEVELOPMENT
KEY WORDS: Shwachman-Diamond syndrome, Ribosome, Zebrafish, p53 (tp53), rpl3, pescadillo, sbds
MATERIALS AND METHODS
Fish strains, genotyping and morpholinos
Fish were raised using standard husbandry procedures. The following
strains were used: Tg BAC ptf1a:eGFPjh1 (herein ptf1a:eGFP) (Godinho
et al., 2007), Tg Tol2 ins:mCherryjh2 (herein ins:mCherry) (Pisharath et al.,
2007), Tg (gcga:GFP)ia1 (herein gcga:GFP) (Pauls et al., 2007), Tg
(T2KTp1hglob:hmgb1-mCherry)jh11 (herein Tp1:hmgb1-mCherry)
(Parsons et al., 2009), Tg (kdrl:GRCFP)zn1 (Cross et al., 2003), tp53zdf1
[M214K (Berghmans et al., 2005), obtained from the Zebrafish
International Resource Center (ZIRC), Eugene, OR, USA], peshi2Tg
(Allende et al., 1996) (ZIRC) and rpl3hi2347 (ZIRC). Tail fins of adult fish
were digested in low TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) plus
proteinase K. PCR genotyping was performed to confirm the genotype of
tp53zdf1 homozygous embryos. peshi2Tg fish were functionally genotyped
for clutches with 25% phenotypic embryos. Morpholinos (MOs) included:
SbdsATG MO, SbdsSpl MO, p53ATG MO, p53Spl MO, Rpl3ATG MO and
PesATG MO, all obtained from GeneTools (Philomath, OR, USA). MOs and
genotyping primers are listed in supplementary material Table S1.
Polyribosome analysis
Zebrafish embryos were either mock injected or injected with SbdsATG MO
48 hours prior to polyribosome analysis. Approximately 150 embryos were
washed and de-yolked in ice-cold PBS containing 150 g/ml
cycloheximide. Embryos were mechanically dissociated in ice-cold lysis
buffer (300 mM NaCl, 15 mM Tris-HCl pH 7.5, 15 mM MgCl2, 0.75%
Triton X-100, 100 g/ml cycloheximide, 1 mg/ml heparin) and incubated
on ice for 10 minutes. Lysates were cleared at 16,300 g at 4°C for 5
minutes. Cleared lysates were layered onto 10-50% sucrose gradients (in
lysis buffer without the Triton X-100) with a 60% sucrose cushion.
Gradients were spun in an SW 41 rotor (Beckman Coulter) at 40,000 rpm
at 4°C for 3 hours and fractionated with an Isco Foxy R1/UA-6 detector
system. Polyribosome profiles were analyzed using a custom macro in
IGOR Pro (WaveMetrics).
Immunofluorescence
Whole-mount immunofluorescence was performed as previously described
(Lin et al., 2004).
In situ hybridization
Whole-mount in situ hybridization was conducted as previously described
(Lin et al., 2004). To perform in situ hybridization on paraffin sections, 12m sections were deparaffinized in Histoclear (Fisher Scientific) and
rehydrated. Sections were prehybridized (50% formamide, 5⫻ SSC pH 4.5
with citric acid, 50 g/ml yeast tRNA, 1% SDS, 50 g/ml heparin) at 55°C
for 2 hours. Primers used to generate in situ probes are listed in
supplementary material Table S1. Antisense in situ probes were boiled in
prehybridization buffer (10 l probe/ml), added to tissue sections, covered
with a coverslip and hybridized at 68°C in a humidity chamber overnight.
Tissue sections were washed in 5⫻ SSC at 68°C, followed by 0.2⫻ SSC
at 68°C and maelic acid buffer (MAB) at room temperature. Sections were
blocked in 10% sheep serum diluted in MAB for 1 hour and incubated with
anti-digoxigenin alkaline phosphatase antibody at 1:5000 overnight at 4°C.
Slides were washed with MAB containing 0.1% Tween 20 and signal
detected using BM Purple (Roche) at room temperature. Reactions were
stopped with 1 mM EDTA pH 8.0, dehydrated, mounted and imaged.
Alcian Blue and Alizarin Red staining
Embryos were stained as described previously (Walker and Kimmel, 2007).
cDNA microarray analysis
Embryos were carefully staged at 24 hpf and RNA was extracted from ~50
embryos per condition in Trizol (Invitrogen), followed by purification
using RNA Miniprep columns (Qiagen). Four experimental replicates were
analyzed. cDNA microarrays were performed at the Sidney Kimmel
Comprehensive Cancer Center at Johns Hopkins Microarray Core Facility
using 4⫻44K zebrafish gene expression microarray slides (Agilent, Santa
Clara, CA, USA). Differential expression was calculated using a moderated
eBayes t-test as implemented in the Limma Bioconductor package (Smyth,
2004). The resulting P-values were false discovery rate (FDR)-adjusted
RESEARCH ARTICLE 3233
across the whole microarray dataset to correct for multiple testing.
Statistically significant changes in gene expression were determined at
three different levels of stringency (P<0.001, P<0.01, P<0.05). Gene set
enrichment analysis (GSEA) was performed with the preranked moderated
t statistics obtained from Limma analysis (Smyth, 2004), according to the
GO functional categories for the indicated microarray comparison.
Additional analysis was carried out using Partek Genomics Suite (St Louis,
MO, USA). Microarray data are available at Gene Expression Omnibus
under accession number GSE39399.
RESULTS
Knockdown of zebrafish Sbds recapitulates the
developmental defects observed in SDS
The sbds ortholog in zebrafish is highly homologous to the mouse
and human Sbds/SBDS genes. Previously, it was shown that
zebrafish sbds is expressed in the area of the pancreas, with highest
expression
in
peri-pancreatic
mesenchymal
tissues
(Venkatasubramani and Mayer, 2008). We confirmed this
expression pattern at 5 days postfertilization (dpf) (Fig. 1A), and
further identified expression in the acinar epithelia of adult
zebrafish (Fig. 1B). In addition, analysis of publicly available
mouse ChIP-Seq datasets (http://www.betacell.org/resources/data/
studies/view.php?study_id3994) revealed overlapping binding of
Ptf1a and Rbpj1 (two members of the larger pancreas-specific
PTF1 transcriptional complex) in the 5⬘ promoter region of the
murine Sbds locus (supplementary material Fig. S1A).
To evaluate the role of Sbds during development we designed
two morpholinos (MOs), one targeting the start codon (SbdsATG
MO) and one targeting the splice junction between exon 2 and
exon 3 (SbdsSpl MO) of zebrafish sbds. Injection of the SbdsSpl MO
resulted in a loss of sbds transcript, as assessed by RT-PCR, that
persisted beyond 72 hours postfertilization (hpf) (Fig. 1C).
Although gene inactivation following MO injection is typically
transient, embryos injected with either MO did not recover from
the early loss of sbds. Embryos injected with the SbdsATG MO
developed significant edema at later stages of development and did
not survive past 7 dpf (Fig. 1D).
Work in yeast (Menne et al., 2007), mouse liver (Finch et al.,
2011), Dictyostelium and SDS patient lymphoblasts (Wong et al.,
2011) has suggested a role for Sbds in late ribosome assembly, with
loss of Sbds function resulting in a subunit joining defect. To
identify whether our SbdsATG MO-injected embryos exhibited
similar ribosome assembly defects, we generated ribosome profiles
at 48 hpf under native polysome assembly conditions. We
identified altered ratios of the 40S subunit, 60S subunit and 80S
monosome, as well as a decrease in the number of heavy
polysomes in the SbdsATG MO-injected embryos (Fig. 1E,F,
supplementary material Fig. S1B,C). This reduction in the number
of heavy polysomes suggests an overall loss of fully functional
ribosomes in SbdsATG MO-injected embryos.
Using the SbdsATG MO we evaluated three components of the
developmental phenotype observed in SDS: the number of
neutrophils, the size of the exocrine and endocrine pancreas, and the
structure of the developing cartilage and bone. As in other
vertebrates, myeloperoxidase (mpo; mpx – Zebrafish Information
Network) is an early marker of neutrophils in zebrafish (Chen and
Zon, 2009). We therefore evaluated neutrophil numbers in control
and SbdsATG MO-injected embryos using an antisense mpo in situ
probe. In mock injected embryos at 24 hpf, mpo-positive neutrophils
were observed in the anterior lateral mesoderm (Fig. 2A, top arrow)
and the intermediate cell mass (Fig. 2A, bottom arrow). By contrast,
we observed a profound loss of mpo-positive neutrophils in SbdsATG
DEVELOPMENT
Ribosome biogenesis genes in pancreas development
3234 RESEARCH ARTICLE
Development 139 (17)
Fig. 1. Zebrafish sbds is an essential developmental
gene with high-level expression in pancreas and
surrounding tissues. (A)Expression of sbds examined by
in situ hybridization in wild-type zebrafish embryos at 120
hpf. The boxed pancreatic region is shown at higher
magnification to illustrate pancreatic and peri-pancreatic
expression (arrowheads) of sbds. (B)Adult zebrafish
pancreas (boxed) expresses sbds. (C)SbdsSpl MO results in
durable loss of sbds transcript as assessed by RT-PCR.
(D)Survival curve for recipients of SbdsATG MO illustrates
near complete embryonic lethality by 7 dpf, as compared
with mock injected embryos. (E)Ribosome profile of lysate
prepared from 48-hpf control and SbdsATG MO-injected
embryos. (F)Quantification of ribosomal subunit ratios,
monosomes and polysomes in control and SbdsATG MOinjected embryos. Error bars indicate mean ± s.e.m. i,
intestine; L, liver; P, pancreas.
2D). Despite their dramatic reduction in number, eGFP-positive
pancreatic progenitor cells were able to appropriately migrate away
from the gut tube and associate with the preserved principal islet at
48 hpf (supplementary material Fig. S3A). To further document the
pancreatic phenotype in the SbdsATG MO-injected embryos, we
observed a dose-dependent reduction in pancreatic volume in
ptf1a:eGFP larvae over multiple doses of both the SbdsATG MO
(supplementary material Fig. S3B) and an additional spliceblocking MO, SbdsSpl (supplementary material Fig. S3C,D).
Loss of Sbds results in defective proliferation of
pancreatic progenitors
Although the human SDS pancreatic phenotype is well characterized,
its developmental basis has not been elucidated. To further explore
the mechanism underlying the hypoplastic pancreas phenotype of
SbdsATG MO-injected embryos, we analyzed phospho-histone H3
(pHH3) labeling of ptf1a-expressing pancreatic progenitor cells at 48
hpf. Similar to previous reports suggesting that Sbds might influence
cellular proliferation (Zhang et al., 2006; Orelio et al., 2009), we
observed a decrease in the number of pHH3-positive mitotic cells
within the ptf1a:eGFP-defined pancreatic progenitor population at
48 hpf (Fig. 2E), with several sbds morphant embryos showing a
complete absence of pHH3 labeling within the ptf1a:eGFP
expression domain. When the total volume of pHH3-positive nuclei
was normalized to the total volume of eGFP-expressing pancreatic
progenitor cells, sbds morphants displayed a 39% reduction in pHH3
labeling. In contrast to this defect in the proliferation of early
pancreatic progenitor cells, we observed no defects in the ability of
progenitor cells to undergo normal exocrine differentiation. At 72
hpf, the digestive enzymes carboxypeptidase and trypsin were
detected even in the reduced pancreatic mass of SbdsATG MOinjected embryos (supplementary material Fig. S3E,F).
In contrast to the abnormal expansion of ptf1a-expressing
pancreatic progenitor cells, ptf1a-independent endocrine progenitors
were able to appropriately expand and form the principal islet in
DEVELOPMENT
MO-injected embryos (Fig. 2A). Other aspects of hematopoietic
development were unaltered in SbdsATG MO-injected embryos,
including normal numbers of L-plastin (Lcp1)-positive macrophages,
normal vasculature, normal expression of gata1 in erythroid
progenitors and normal expression of the neutrophil/macrophage
progenitor marker spi1 (pu.1) (supplementary material Fig. S2AC,E,F). As in other species, zebrafish neutrophils are dynamically
recruited to sites of injury (Mathias et al., 2006; Renshaw et al.,
2006). Even when SbdsATG MO-injected embryos were subjected to
epithelial injury, no recruitment of mpo-positive neutrophils was
observed (supplementary material Fig. S2D). Thus, we conclude that
loss of Sbds results in a specific defect in the neutrophil lineage at a
point beyond the formation of common neutrophil/macrophage
progenitors.
When we evaluated the skeletal phenotype of SbdsATG MOinjected embryos versus controls at 5 dpf by staining with Alcian
Blue (cartilage) and Alizarin Red (bone), we detected defects in both
the cartilage and bone. Both the cerotohyal cartilage of the lower jaw
and the ceratobranchial cartilage of the gill arches (Kimmel et al.,
2001) were malformed (Fig. 2B). At 5 dpf, the opercal, a bone of the
inner ear, was misshapen and thinner in SbdsATG MO-injected
embryos than in control embryos (Fig. 2B, arrowheads).
Finally, we evaluated the effect of SbdsATG MO injection on the
developing pancreas using a ptf1a:eGFPjh1 transgenic line to
visualize early pancreatic progenitor cells and later differentiating
acinar cells (Park et al., 2008), in combination with an
ins:mCherryjh2 transgenic line (Pisharath et al., 2007) to visualize
beta cells in the principal islet. At 72 hpf, embryos injected with
the SbdsATG MO exhibited dramatic pancreatic hypoplasia, initially
manifested by a marked decrease in the number of undifferentiated
ptf1a-expressing pancreatic progenitor cells (Fig. 2C).
Quantification of the volume of eGFP-positive cells in ptf1a:eGFP
larvae at 72 hpf revealed that the SbdsATG MO-injected embryos
had an almost 75% reduction in the volume of ptf1a-expressing
pancreatic progenitor cells compared with control embryos (Fig.
Ribosome biogenesis genes in pancreas development
RESEARCH ARTICLE 3235
Fig. 2. sbds knockdown in zebrafish embryos recapitulates
human SDS organogenesis phenotypes. (A)At 24 hpf, loss of
neutrophils in SbdsATG MO-injected embryos is revealed by in situ
hybridization for the neutrophil marker mpo. mpo-positive cells were
absent from both the anterior lateral mesoderm and the intermediate
cell mass (top and bottom arrows, respectively). (B)At 120 hpf,
abnormal cartilage of the gill arches and cerotohyal (ch) cartilage and
bone (arrowheads) were observed in SbdsATG MO-injected embryos.
(C)Visualization of ptf1-expressing pancreatic progenitor cells and
differentiated beta cells in 72-hpf ptf1a:eGFP;ins:mCherry embryos
(arrows). SbdsATG MO-injected embryos showed defects in ptf1expressing pancreatic progenitors (green), whereas beta cells in the
principal islet appeared normal (red). (D)Quantification of the volume
of ptf1a:eGFP-expressing cells reveals an almost 75% reduction of
pancreatic volume in SbdsATG MO-injected embryos. Error bars indicate
s.e.m. (E) Immunofluorescence of phospho-histone H3 (pHH3, blue)
marks proliferation in the exocrine pancreatic mass. Proliferation was
decreased in the SbdsATG MO-injected embryos at 72 hpf. (F)Highresolution images of the pancreatic islet using gcga:eGFPia1;ins:mCherry
double-transgenic zebrafish reveal normal islet architecture with
peripheral alpha cells (green) and central beta cells (red) in SbdsATG MOinjected embryos at 72 hpf.
SbdsATG MO-injected embryos (Fig. 2F). Using the transgenic
zebrafish line gcga:GFPia1, which expresses GFP from the glucagon
a promoter, we were able to visualize the alpha cells of the endocrine
pancreas in conjunction with ins:mCherryjh2-expressing beta cells.
In both control and SbdsATG MO-injected embryos, alpha cells
appropriately localized to the periphery of the islet surrounding a
central core of beta cells (Fig. 2F). Despite normal islet architecture,
we observed a tendency toward diminished islet size in SbdsATG
Loss of p53 does not rescue the sbds
organogenesis phenotypes
When normal ribosome biogenesis is disrupted, a resulting p53
response is commonly accepted to play a role in the associated
disease phenotypes (Lohrum et al., 2003; Dai and Lu, 2004; Jin et
al., 2004; Danilova et al., 2008; Jones et al., 2008; Barlow et al.,
2010; Duan et al., 2011; Sasaki et al., 2011). To determine whether
SDS disease phenotypes are similarly p53 dependent, we evaluated
the influence of p53 inactivation on the organogenesis defects
induced by SbdsATG MO injection, using both MO-mediated p53
knockdown and available p53 mutant alleles.
As previously reported (Robu et al., 2007), injection of 1 ng of
a p53ATG MO had no adverse effect on developing zebrafish
embryos. Overall, co-injection of the p53ATG MO in combination
with the SbdsATG MO resulted in healthier-appearing embryos than
following injection of SbdsATG MO alone. At 48 hpf we observed
a twisted tail phenotype in SbdsATG MO-injected embryos, and coinjection of p53ATG MO relieved this axis defect (Fig. 3A). To
further validate the efficacy of the p53 knockdown, we performed
semi-quantitative PCR for the p53 target genes 113p53 and p21
(cdkn1a – Zebrafish Information Network). Injection of the
SbdsATG MO increased both 113p53 and p21 expression, but this
expression was attenuated when the SbdsATG MO and p53ATG MO
were co-injected (Fig. 3B).
Next we evaluated whether knockdown of p53 could rescue
SbdsATG MO-induced organogenesis phenotypes. Despite the
improved gross appearance of the embryos, co-injection of SbdsATG
MO and p53ATG MO did not rescue the pancreatic progenitor defect
(Fig. 3C, supplementary material Fig. S4A). Likewise, mpopositive neutrophils were not rescued by concomitant p53
knockdown (Fig. 3D, supplementary material Fig. S4B). Finally,
the cartilage phenotype was not rescued in embryos co-injected
with SbdsATG and p53ATG MOs, as assessed by Alcian Blue staining
at 72 hpf (supplementary material Fig. S4C,D). We conclude that
even though p53 knockdown effectively minimizes the pleiotropic
effects associated with Sbds knockdown, it is unable to rescue
SbdsATG MO-induced organogenesis phenotypes.
DEVELOPMENT
MO-injected embryos, although not at a level of statistical
significance (P0.072) (supplementary material Fig. S3G). This
result mirrors the recently reported conditional knockout of sbds,
where the adult islet structure is normal but islet volume is modestly
reduced (Tourlakis et al., 2012).
Islet development in zebrafish involves early specification (prior
to 24 hpf) of the principal islet, followed by the formation of
secondary islets (after 5 dpf). These secondary islets arise from
Notch-responsive progenitor cells that reside in the main pancreatic
duct (Parsons et al., 2009; Wang et al., 2011). Although our SbdsATG
MO-injected embryos die before secondary islet formation occurs,
we were able to determine the influence of Sbds knockdown on this
Notch-responsive islet progenitor population using Tp1:hmgb1mCherry transgenic Notch reporter fish. Following Sbds knockdown,
Tp1:hmgb1-mCherry-expressing cells failed to form a main
pancreatic duct, thus corresponding to the failure of ptf1a-expressing
progenitor cells to undergo normal morphogenesis. However, there
was no change in the ratio of eGFP to mCherry volume in SbdsATG
MO-treated ptf1a:eGFP;Tp1;hmgb1-mCherry double-transgenic
embryos (supplementary material Fig. S3H,I). Although the early
lethality of Sbds knockdown precludes direct observation of lateroccurring secondary islet formation, these observations suggest that
the relative number of secondary islet progenitor cells is retained,
even in the absence of Sbds.
3236 RESEARCH ARTICLE
Development 139 (17)
Fig. 3. Loss of p53 does not rescue sbds organogenesis defects. (A)The body curvature observed in SbdsATG MO-injected zebrafish embryos is
attenuated in SbdsATG/p53ATG MO-injected embryos at 48 hpf. (B)The p53 transcriptional targets 113p53 and p21 are induced upon injection of
SbdsATG MO, as assessed by semi-quantitative PCR; co-injection of p53ATG MO abrogates this response. Error bars indicate s.e.m. (C) The 72-hpf
pancreatic progenitor phenotype induced by the SbdsATG MO is not rescued by co-injection of p53ATG MO (arrows). (D)The absence of mpoexpressing neutrophils in the intermediate cell mass of SbdsATG/p53ATG MO-injected embryos was detected at 24 hpf (arrowheads). (E)Co-injection
of p53Spl MO and SbdsATG MO into ptf1a:eGFP;ins:mCherry embryos does not rescue the defective expansion of pancreatic progenitor cells
(arrows). (F)Genetic loss of p53 in p53zdf1/zdf1 embryos does not rescue the SbdsATG MO-induced neutrophil phenotype, as assessed by in situ
hybridization for mpo at 24 hpf (arrowheads). (G)The SbdsATG MO-induced pancreatic progenitor defect is not rescued in
p53zdf1/zdf1;ptf1a:eGFP;ins:mCherry embryos at 72 hpf.
was generated by ENU mutagenesis, harbors an M216K missense
mutation in the p53 DNA-binding domain, preventing activation of
p53 target genes (Berghmans et al., 2005). Because the 113p53
variant is dependent upon full-length p53 for its expression,
113p53 is also lost in the tp53zdf1 (p53zdf1) mutant (Chen et al.,
2009). Consistent with our previous results, mpo-positive
neutrophils were absent at 24 hpf in SbdsATG MO-injected
p53zdf1/zdf1 embryos (Fig. 3F). Likewise, there was no rescue of the
pancreatic phenotype in SbdsATG MO-injected p53zdf1/zdf1 embryos
(Fig. 3G). In conclusion, neither p53 knockdown using two
different MOs nor genetic loss of p53 is able to rescue defective
organogenesis in SbdsATG MO-injected embryos. Thus, we
conclude that the organogenesis defects observed following Sbds
knockdown are p53 independent.
Loss of Sbds is associated with a broad change in
ribosome-related gene expression
To begin to understand additional cellular consequences of Sbds
knockdown in developing zebrafish embryos, we analyzed global
transcriptional profiles in SbdsATG MO-injected embryos with and
without co-injection of p53ATG MO. Clutches produced from
ptf1a:eGFP outcrosses were injected with the SbdsATG and/or the
p53ATG MO, and embryos were carefully staged to 24 hpf. Nontransgenic embryos were then harvested for isolation of RNA. We
chose 24 hpf as our earliest phenotypic time point because embryos
can be more precisely staged early in development, allowing
correction for any developmental delay. The remaining ptf1a:eGFP
transgenic embryos were evaluated at 72 hpf to confirm defective
pancreas organogenesis as a measure of successful SbdsATG MO
injection. Four experimental replicates were run on the Agilent
DEVELOPMENT
113p53 (human) is both a shortened isoform and target gene of
full-length p53 (Chen et al., 2005; Chen et al., 2009). Although
full-length p53 is dispensable for normal development, work in the
zebrafish mutant def (digestive organ expansion factor, or diexf)
has demonstrated that 113p53 plays an important role in
endoderm development. def mutants have properly differentiated
but severely hypoplastic digestive organs, which are partially
rescued by knockdown of 113p53 (Chen et al., 2005).
To evaluate the role of 113p53 in the zebrafish sbds phenotype,
we used a splice-blocking p53 MO targeting the exon 5-intron 5
boundary (Chen et al., 2005). This p53Spl MO effectively depletes
both full-length p53 and 113p53. As we observed no rescue
associated with loss of full-length p53, any ability of the p53Spl MO
to rescue normal pancreas development was expected to be the
result of 113p53 knockdown. Co-injection of the SbdsATG and
p53Spl MOs into ptf1a:eGFP;ins:mCherry embryos did not rescue
defective pancreatic organogenesis (Fig. 3E, supplementary
material Fig. S4E). RT-PCR of p53Spl MO-injected embryos
confirmed the near complete loss of the 113p53 transcript, its
absence persisting through 72 hpf (supplementary material Fig.
S4F). Thus, we conclude that knockdown of p53 and/or 113p53
is unable to rescue the pancreas progenitor defect observed in
SbdsATG MO-injected embryos.
In these p53 knockdown experiments, we noted that co-injection
of SbdsATG and p53ATG MOs reduced, but did not eliminate,
transcription of the p53 target genes 113p53 and p21 (Fig. 3B).
To be certain that the persistent SbdsATG MO-induced phenotype
observed following p53ATG MO injection was not due to residual
p53 activity, we extended our p53 loss-of-function analysis using
an available p53 null mutation. The zebrafish tp53zdf1 allele, which
Ribosome biogenesis genes in pancreas development
RESEARCH ARTICLE 3237
zebrafish oligo microarray 4⫻44k platform. Using FDR-adjusted Pvalue cut-offs of 0.05, 0.01 and 0.001 to identify differentially
expressed genes at increasing levels of statistical stringency, we
identified genes differentially expressed in SbdsATG MO-injected
versus control embryos, as well as in SbdsATG plus p53ATG
(SbdsATG/p53ATG) MO-injected versus p53ATG MO-injected embryos.
The numbers of differentially expressed genes at each level of
statistical significance are presented in supplementary material Table
S2, and the list of individual differentially expressed genes including
fold changes is presented in supplementary material Table S3.
Among 24,278 annotated zebrafish genes in the platform, we
identified 4251 to be differentially expressed (P<0.01) in SbdsATG
MO-injected versus control embryos. By comparison, 931 genes
were found to be differentially expressed in SbdsATG/p53ATG MOinjected versus p53ATG MO-injected embryos, with 556 genes
differentially expressed in both comparisons (Fig. 4A).
Gene set enrichment analysis using Gene Ontology (GO)
functional group categories demonstrated that transcripts displaying
altered expression following sbds knockdown were markedly
enriched for genes assigned to the ‘ribosome biogenesis’, ‘rRNA
processing’, ‘nucleolus’ and ‘translational initiation’ categories.
These trends were especially robust among genes that were
upregulated in the absence of Sbds, where we observed enrichment
for ribosome-related GO functional groups in both the presence and
absence of p53 (Fig. 4B,C, supplementary material Fig. S5A).
Loss of rpl3 phenocopies the p53-independent
sbds pancreatic phenotype
Significant changes in the expression of ribosome-related genes were
previously noted in the analysis of human SDS patient bone marrow
samples (Rujkijyanont et al., 2009). Similar to that report, we
identified ribosomal protein L3 (rpl3) as a gene downregulated in the
absence of Sbds. Rpl3 is an RP that is associated with the large 60S
subunit. In 24-hpf zebrafish embryos, expression of rpl3 is initially
broad, but becomes increasingly restricted to the endoderm as
development proceeds (Fig. 5A). Our microarray studies indicated
that embryos injected with SbdsATG MO or SbdsATG/p53ATG MOs
had a significant reduction in rpl3 expression (Fig. 5B). We
confirmed this reduction in rpl3 expression in Sbds knockdown
embryos by in situ hybridization for rpl3 in SbdsATG MO-injected
p53zdf1/zdf1 embryos. At 24 hpf, expression of rpl3 was reduced in the
somites and nervous system of SbdsATG MO-injected embryos (Fig.
5C). This reduction in rpl3 expression became more pronounced at
72 hpf, particularly in the pharyngeal arches and intestine (Fig. 5C).
In order to determine whether loss of Rpl3 phenocopies loss of
Sbds, we designed an ATG MO to rpl3 and evaluated its role
during development. We identified an MO dose-dependent defect
in pancreatic volume in Rpl3ATG MO-injected p53zdf1/zdf1 embryos
(Fig. 5D, supplementary material Fig. S5A). Similar to SbdsATG
MO-injected embryos, the rpl3 pancreatic phenotype originated in
a failure of progenitor expansion, rather than differentiation, as
cells in the hypoplastic pancreas showed normal activation of
trypsin expression (Fig. 5E). As with our SbdsATG MO, Rpl3ATG
MO-injected embryos appeared healthier on a p53zdf1/zdf1
background. In addition to the pancreatic organogenesis defect, we
also observed swelling in the brain and reduced eye size at a
phenotypic dose of Rpl3ATG MO (Fig. 5F).
A retroviral mutagenesis screen in zebrafish had identified a
mutagenic insertion in the rpl3 locus, rpl3hi2347 (Gaiano et al.,
1996; Lai et al., 2009). We therefore examined pancreatic
development at 72 hpf in progeny of an incross of
rpl3hi2347/+;ptf1a:eGFP;ins:mCherry adults. Similar to the effects
of the Rpl3ATG MO, embryos homozygous for the rpl3hi2347 allele
developed a defect in pancreatic progenitor expansion, while the
primary islet appeared unaffected. This effect was not rescued by
injection of the p53ATG MO (Fig. 5G).
We conclude that Rpl3 is essential for the normal expansion of
pancreatic progenitors. Furthermore, loss of Rpl3 phenocopies the
hypoplastic pancreas phenotype observed in SbdsATG MO-injected
embryos and does so in a p53-independent manner.
Loss of pescadillo also phenocopies the p53independent sbds pancreatic phenotype
In yeast, Rpl3 is an early-assembling RP that is essential for
nucleolar ribosome biogenesis. Additionally, Rpl3 is a
component of the multi-protein Pescadillo complex involved in
DEVELOPMENT
Fig. 4. Gene set enrichment analysis of genes differentially expressed in SbdsATG MO-injected versus control and in SbdsATG/p53ATG
MO-injected versus p53ATG MO-injected embryos. (A)Venn diagram depicting differentially expressed genes with a P<0.01 statistical cut-off.
The red circle indicates genes differentially expressed in SbdsATG MO-injected versus control embryos, whereas the blue circle represents genes
differentially expressed in SbdsATG/p53ATG MO-injected versus p53ATG MO-injected embryos. (B)The most highly enriched GO categories for genes
upregulated in SbdsATG/p53ATG MO-injected versus p53ATG MO-injected embryos. (C)The most highly enriched GO categories for genes upregulated
in SbdsATG MO-injected versus control embryos. For B and C, enriched GO categories are ranked based on the negative logarithm of their P-value.
Note the high-level enrichment of genes related to ribosome biogenesis and rRNA processing in both comparisons.
3238 RESEARCH ARTICLE
Development 139 (17)
both ribosome biogenesis and cell cycle control (Iouk et al.,
2001; Schaper et al., 2001; Du and Stillman, 2002; Killian et al.,
2004). A gene encoding another member of this complex,
pescadillo (pes), was among those displaying altered expression
in sbds morphants. The pes gene was originally identified in a
zebrafish mutant characterized by major axis defects, small eyes
and early embryonic lethality (Allende et al., 1996). Both in vitro
and in vivo, disruption of pes has been reported to induce a p53
response (Gessert et al., 2007; Rohrmoser et al., 2007). We
therefore undertook an analysis of pancreatic development in pes
mutant and morphant embryos.
At 24 hpf, zebrafish pes is highly expressed in the embryonic
eye and midbrain. At 72 hpf, pes expression is detected broadly in
the endoderm, including the developing pancreas (Fig. 6A). An
incross of peshi2Tg/wt fish results in 25% of the clutch exhibiting a
profoundly curved axis, a small degenerating head and small eyes.
Heterozygous and wild-type siblings develop normally (Fig. 6B).
Based on previous work with pes and our observation that
peshi2Tg/hi2Tg embryos display head degeneration characteristic of a
p53 apoptotic response (Fig. 6B), we analyzed 113p53 and p21
expression by quantitative PCR. Both were strongly upregulated in
peshi2Tg/hi2Tg embryos compared with their phenotypically normal
siblings (24-fold increase for 113p53, P<0.0001; 2.4-fold increase
for p21, P<0.01) (Fig. 6C). We next tested whether knockdown of
p53 could rescue the peshi2Tg/hi2Tg phenotype. When incrossed
peshi2Tg/wt embryos were injected with p53ATG MO or p53Spl MO,
neither was able to rescue the peshi2Tg/hi2Tg phenotype (Fig. 6D,
supplementary material Fig. S5B). We confirmed these results
genetically by incrossing peshi2Tg/wt;p53zdf1/zdf1 fish. Again, we were
unable to rescue the peshi2Tg/hi2Tg phenotype, even on a p53 null
background (Fig. 6D, supplementary material Fig. S5C).
It has been suggested that peshi2Tg/hi2Tg mutants have disrupted
endoderm development (Allende et al., 1996). Imaging of
peshi2Tg/hi2Tg embryos in combination with ptf1a:eGFP and
ins:mCherry reporters revealed a profoundly hypoplastic pancreas
(Fig. 6E). As with the gross phenotype of peshi2Tg/hi2Tg fish, neither
p53 knockdown nor genetic inactivation of p53 in p53zdf1/zdf1
embryos was able to rescue the observed defect in pancreatic
organogenesis (Fig. 6E). Thus, we conclude that loss of p53 is
insufficient to rescue multiple components of the peshi2Tg/hi2Tg
phenotype, including the axis defects, small eyes and abnormal
pancreas development.
Because the peshi2Tg/hi2Tg phenotype is severe, we reasoned that
abnormal pancreas development might occur due to a more general
disruption of endodermal patterning. To address this, we titrated
MO
in
increasing
doses
of
PesATG
zdf1/zdf1
;ptf1a:eGFP;ins:mCherry embryos. Embryos injected
p53
with the PesATG MO showed an MO dose-dependent pancreatic
development defect (Fig. 7A,B). PesATG MO-injected embryos also
exhibited swelling in the brain and an underdeveloped eye (Fig.
7C), but overall appeared much healthier than peshi2Tg/hi2Tg
embryos. Because we detected a persistent pancreatic progenitor
defect even in embryos with otherwise grossly normal
development, we suggest that pes is specifically required for
normal pancreas development.
In our microarray studies, pes transcripts were significantly
increased in SbdsATG MO-injected and SbdsATG/p53ATG MO-injected
embryos (Fig. 7D). We confirmed this by in situ hybridization. At 24
hpf, pes expression is expanded in the somites of p53zdf1/zdf1 SbdsATG
MO-injected embryos. This difference becomes more striking at 48
hpf, when expression of pes is stronger and more widespread in the
SbdsATG MO-injected embryos (Fig. 7E). Based on the convergent
DEVELOPMENT
Fig. 5. Knockdown of rpl3 phenocopies SbdsATG MO-induced defects in pancreas development. (A)Expression of rpl3 is widespread at 24
hpf and becomes progressively restricted to the endoderm, with high-level pancreatic expression at 120 hpf. The principal islet (I) is labeled by in
situ hybridization for insulin (red). (B)rpl3 expression is decreased in SbdsATG MO-injected embryos, as quantified by microarray analysis of transcript
abundance. Error bars indicate s.e.m. (C) In situ hybridization for rpl3 in SbdsATG MO-injected p53zdf1/zdf1 embryos confirms loss of rpl3 expression in
somites (black arrows), central nervous system (white arrows), pharyngeal arches (red arrows) and intestine (yellow arrows) of developing embryos.
(D)An MO dose-dependent defect in pancreas progenitor expansion was observed in Rpl3ATG MO-injected ptf1a:eGFP;ins:mCherry;p53zdf1/zdf1
embryos. (E)The small pancreas (arrow) of p53zdf1/zdf1 embryos injected with 0.5 ng Rpl3ATG MO is positive for trypsin expression by in situ
hybridization. (F)p53zdf1/zdf1 embryos injected with 0.5 ng Rpl3ATG MO at 72 hpf exhibit small heads and hydroencephaly (yellow arrowhead).
(G)rpl3hi2437/+;ptf1a:eGFP;ins:mCherry heterozygotes bearing a mutagenic rpl3 transgenic insertion were incrossed. rpl3hi2437/hi2437 homozygous
embryos displayed disrupted pancreas progenitor expansion at 72 hpf. Injection of rpl3hi2437;ptf1a:eGFP;ins:mCherry with the p53ATG MO did not
rescue the pancreatic progenitor phenotype of homozygous rpl3hi2437hi/2437 embryos. L, liver; P, pancreas; I, islet; Ex, exocrine pancreas.
Ribosome biogenesis genes in pancreas development
RESEARCH ARTICLE 3239
phenotypes observed following knockdown of Sbds, Rpl3 and Pes,
these results suggest that disruptions in pathways crucial for
ribosome biogenesis result in the failure of normal pancreas
development, mediated by defective expansion of ptf1a-expressing
pancreatic progenitors.
DISCUSSION
Our work demonstrates that loss of sbds function in zebrafish
effectively recapitulates the organogenesis defects observed in
human SDS: loss of neutrophils, abnormal skeletal architecture and
pancreatic hypoplasia. These results confirm and extend a previous
analysis of the zebrafish sbds morphant phenotype
(Venkatasubramani and Mayer, 2008). They also complement a
recent analysis of pancreas-specific deletion of murine Sbds
(Tourlakis et al., 2012), and implicate defective proliferation of ptf1aexpressing pancreatic progenitor cells as a basis for the observed
pancreatic phenotype. The zebrafish Sbds knockdown phenotype
was not rescued by loss of p53, suggesting that, in contrast to
Treacher Collins syndrome and 5q-syndrome (Jones et al., 2008;
Barlow et al., 2010), SDS is a largely p53-independent
ribosomopathy. Furthermore, by unbiased screening of genes
exhibiting altered expression following Sbds knockdown, we have
identified a previously unappreciated requirement for additional
genes related to ribosome assembly in early pancreas development.
Importantly, the exquisite sensitivity of pancreatic progenitor cells to
the altered expression of ribosome assembly genes is
developmentally apparent well before the elaboration of extensive
rough endoplasmic reticulum associated with subsequent exocrine
differentiation. This suggests that a later requirement for high-level
synthesis of digestive zymogens is not the sole basis for the resulting
pancreatic hypoplasia.
Although loss of p53 failed to rescue the organogenesis
phenotypes and the changes in expression of multiple ribosomerelated genes observed following Sbds knockdown, it did improve
the overall health of the embryos. The twisted tail phenotype and
CNS degeneration we observed in SbdsATG MO-injected embryos
were relieved by loss of p53 (Fig. 3A; data not shown). Similarly,
the majority of genes displaying altered expression following Sbds
knockdown were normalized by concomitant loss of p53. Using
our present MO-based model, we cannot rule out the possibility
that the p53 dependence of these events reflects non-specific MO
toxicity (Robu et al., 2007). However, it remains a formal
possibility that the composite SDS phenotype includes both p53dependent and p53-independent aspects. Support for p53dependent and p53-independent aspects of ribosome biogenesis
disorders is also provided by studies of rps19, the gene responsible
for DBA. Initial analyses of the zebrafish rps19 morphant
phenotype described a loss of red blood cells (RBCs) and other
gross developmental defects, and further reported that concomitant
knockdown of p53 improved the health and survival of the
embryos (Danilova et al., 2008; Uechi et al., 2008). However, more
recent work found that only gross developmental defects were
rescued by p53 knockdown, whereas loss of RBCs persisted in a
p53-independent manner (Torihara et al., 2011).
It is fascinating to contemplate how mutations in genes
responsible for an essential cellular process, such as ribosome
assembly, result in largely tissue-restricted phenotypes. One
possibility is that individual assembly factors are expressed in a cell
type-specific or tissue-restricted manner. A recent report using an
Rpl38 mutant mouse described a previously unappreciated
enrichment of Rpl38 expression during development in tissues that
are affected by its loss (Kondrashov et al., 2011). This also appears
to be the case for pes, where tissues with high-level pes expression,
including brain, eye and pancreas, are those most severely affected
in the pes mutant phenotype (Fig. 6A,B). However, unlike pes, sbds
appears to be expressed in all tissues except muscle (Boocock et al.,
2003). Despite this, the sbds loss-of-function phenotype
disproportionately affects the pancreas, the neutrophil lineage and the
developing skeleton. It is still unclear why different tissues display
differential sensitivity to sbds loss. However, the convergence of the
DEVELOPMENT
Fig. 6. The pes mutant phenocopies SbdsATG MOinduced p53-independent defects in pancreas
development. (A)In situ hybridization at 24 hpf
demonstrates pes expression in the eye and central
nervous system. At 72 hpf, pes expression is detected in
endoderm, including the developing pancreas. (B)The
peshi2Tg/hi2Tg phenotype includes axis defects (tail
curvature), a small dark head and a small eye. (C)Semiquantitative RT-PCR of peshi2Tg/hi2Tg embryos reveals
activation of the p53 target genes 113p53 (24-fold,
P<0.0001) and p21 (2.4-fold, P<0.01). Error bars
indicate s.e.m. (D) Loss of p53 does not rescue axis
defects in peshi2Tg/hi2Tg embryos. Representative images
of siblings (sib) generated either from a peshi2Tg/WT
incross and injected with or without (Mock) p53ATG MO
or p53Spl MO, or from a peshi2Tg/WT;p53zdf1/zdf1 incross.
Asterisks indicate siblings displaying the pes phenotype.
See supplementary material Fig. S6 for quantification.
(E)peshi2Tg/hi2Tg embryos exhibit a p53-independent
defect in expansion of ptf1-expressing pancreatic
progenitor cells. The pancreatic progenitor defect in 72hpf peshi2Tg/hi2Tg;ptf1a:eGFP;ins:mCherry embryos is not
rescued by p53 MO-mediated knockdown, nor by
genetic inactivation of p53 as assessed by incross of
peshi2Tg/WT;p53zdf1/zdf1;ptf1a:eGFP;ins:mCherry fish.
3240 RESEARCH ARTICLE
Development 139 (17)
Fig. 7. MO knockdown of pes phenocopies
SbdsATG MO-induced p53-independent defects in
pancreas development. (A)Injection of PesATG MO
into p53zdf1/zdf1 embryos results in a dose-dependent
defect in pancreatic progenitor expansion, as assessed
in 72-hpf ptf1a:eGFP;ins:mCherry embryos.
(B)Quantification of three independent experiments
scored for the percentage of embryos with wild-type
pancreas following injection of PesATG MO into a
p53zdf1/zdf1;ptf1a:eGFPjh1;ins:mCherryjh2 background.
(C)The gross appearance of PesATG MO-injected
embryos at 72 hpf includes a small head and eye but
normal body axis. (D)Microarray quantification of pes
transcript levels indicates an increase in pes expression
in SbdsATG MO-injected p53zdf1/zdf1 embryos.
(E)Increased abundance of pes transcripts in SbdsATG
MO-injected p53zdf1/zdf1 embryos is confirmed by in situ
hybridization. Error bars indicate s.e.m.
unaffected. Likewise, loss of Rrb1p expression results in increased
expression of other RP genes, but not rpl3 (Iouk et al., 2001). In
addition to nuanced transcriptional control of RPs, posttranslational modification of RPs and non-ribosome-related roles
for RPs have been described (Bhavsar et al., 2010). This suggests
that our previous understanding of the role of ribosome assembly
genes during normal development and in disease states is
incomplete, with additional tissue- and cell type-specific roles
likely to emerge.
Acknowledgements
We thank Danielle Blake and Seneca Bessling for expert technical support and
Dr Raymond MacDonald and the Beta Cell Biology Consortium for publicly
available ChIP-Seq datasets.
Funding
This work was supported by grants from the National Institutes of Health (NIH)
[DK077480 to E.P., DK080730 and DK082060 to M.J.P., DK61215 and
DK56211 to S.D.L.]; an NIH National Institute of General Medical Sciences
(NIGMS) RISE Grant [5R25GM059244-11 to Barry University]; and the Johns
Hopkins Brain Science Institute (to K.A.W.). R.G. is an HHMI Investigator. S.D.L.
is further supported by the Paul K. Neumann Professorship in Pancreatic
Cancer at Johns Hopkins University. Deposited in PMC for release after 6
months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.077107/-/DC1
References
Allende, M. L., Amsterdam, A., Becker, T., Kawakami, K., Gaiano, N. and
Hopkins, N. (1996). Insertional mutagenesis in zebrafish identifies two novel
genes, pescadillo and dead eye, essential for embryonic development. Genes
Dev. 10, 3141-3155.
Barlow, J. L., Drynan, L. F., Hewett, D. R., Holmes, L. R., Lorenzo-Abalde, S.,
Lane, A. L., Jolin, H. E., Pannell, R., Middleton, A. J., Wong, S. H. et al.
(2010). A p53-dependent mechanism underlies macrocytic anemia in a mouse
model of human 5q– syndrome. Nat. Med. 16, 59-66.
Berghmans, S., Murphey, R. D., Wienholds, E., Neuberg, D., Kutok, J. L.,
Fletcher, C. D., Morris, J. P., Liu, T. X., Schulte-Merker, S., Kanki, J. P. et al.
(2005). tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors.
Proc. Natl. Acad. Sci. USA 102, 407-412.
Bhavsar, R. B., Makley, L. N. and Tsonis, P. A. (2010). The other lives of ribosomal
proteins. Hum. Genomics 4, 327-344.
DEVELOPMENT
sbds, rpl3 and pes phenotypes, together with the observed changes
in ribosome profiles and the p53 independence of the sbds
organogenesis phenotypes, all argue that these developmental defects
represent the direct consequence of altered ribosome biogenesis.
As an additional component of the sbds loss-of-function
phenotype, we identified widespread changes in the expression of
genes related to ribosome biogenesis, rRNA processing and
translational initiation. We functionally characterized two additional
genes related to ribosome biogenesis, rpl3 and pes, with loss-offunction phenotypes that converge upon a similar p53-independent
defect in pancreatic progenitor expansion. The fact that the rpl3 and
pes loss-of-function phenotypes are similar even though rpl3 is
downregulated and pes is upregulated in sbds morphants emphasizes
that directional changes in gene expression do not necessarily
correlate with gain- or loss-of-function phenotypes. Indeed, we
observed increased expression of many ribosome-related genes in
sbds MO-injected embryos, even though ribosome biogenesis is
clearly disrupted in these embryos. These data suggest that the
observed increased expression of pes and other ribosome-related
genes is likely to represent a compensatory mechanism by which
cells attempt to overcome disruptions in ribosome assembly. Rather
than using our microarray analysis as a means of inferring function
based on the directionality of observed changes in gene expression,
these data serve primarily as a discovery tool to identify additional
genes and pathways altered by sbds knockdown. Using this
approach, we were able to identify a previously unappreciated role
for multiple genes involved in ribosome biogenesis as early essential
genes in pancreas development.
Because of the general understanding that genes encoding RPs
are transcribed in excess (Lam et al., 2007), it was surprising to
find that loss of Sbds was associated with profound changes in the
expression of multiple RP genes. However, this view of RP
transcription is likely to be an oversimplification, and evidence
exists for uncoupling of individual RPs from otherwise coordinated
expression. For example, both Rpl3 and Pes interact with another
member of the yeast Pescadillo complex, Rrb1p (GRWD1 in
human) (Iouk et al., 2001; Killian et al., 2004). The function of
Rrb1p in ribosome biogenesis is linked to its physical interaction
with Rpl3. Overexpression of Rrb1p results in an increase in the
expression of rpl3, whereas the expression of other RP genes is
Boocock, G. R., Morrison, J. A., Popovic, M., Richards, N., Ellis, L., Durie, P. R.
and Rommens, J. M. (2003). Mutations in SBDS are associated with ShwachmanDiamond syndrome. Nat. Genet. 33, 97-101.
Chen, A. T. and Zon, L. I. (2009). Zebrafish blood stem cells. J. Cell. Biochem. 108,
35-42.
Chen, J., Ruan, H., Ng, S. M., Gao, C., Soo, H. M., Wu, W., Zhang, Z., Wen, Z.,
Lane, D. P. and Peng, J. (2005). Loss of function of def selectively up-regulates
Delta113p53 expression to arrest expansion growth of digestive organs in
zebrafish. Genes Dev. 19, 2900-2911.
Chen, J., Ng, S. M., Chang, C., Zhang, Z., Bourdon, J. C., Lane, D. P. and Peng,
J. (2009). p53 isoform delta113p53 is a p53 target gene that antagonizes p53
apoptotic activity via BclxL activation in zebrafish. Genes Dev. 23, 278-290.
Cross, L. M., Cook, M. A., Lin, S., Chen, J. N. and Rubinstein, A. L. (2003). Rapid
analysis of angiogenesis drugs in a live fluorescent zebrafish assay. Arterioscler.
Thromb. Vasc. Biol. 23, 911-912.
Dai, M. S. and Lu, H. (2004). Inhibition of MDM2-mediated p53 ubiquitination and
degradation by ribosomal protein L5. J. Biol. Chem. 279, 44475-44482.
Danilova, N., Sakamoto, K. M. and Lin, S. (2008). Ribosomal protein S19
deficiency in zebrafish leads to developmental abnormalities and defective
erythropoiesis through activation of p53 protein family. Blood 112, 5228-5237.
Deisenroth, C. and Zhang, Y. (2010). Ribosome biogenesis surveillance: probing
the ribosomal protein-Mdm2-p53 pathway. Oncogene 29, 4253-4260.
Draptchinskaia, N., Gustavsson, P., Andersson, B., Pettersson, M., Willig, T.
N., Dianzani, I., Ball, S., Tchernia, G., Klar, J., Matsson, H. et al. (1999). The
gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia.
Nat. Genet. 21, 169-175.
Du, Y. C. and Stillman, B. (2002). Yph1p, an ORC-interacting protein: potential
links between cell proliferation control, DNA replication, and ribosome biogenesis.
Cell 109, 835-848.
Duan, J., Ba, Q., Wang, Z., Hao, M., Li, X., Hu, P., Zhang, D., Zhang, R. and
Wang, H. (2011). Knockdown of ribosomal protein S7 causes developmental
abnormalities via p53 dependent and independent pathways in zebrafish. Int. J.
Biochem. Cell Biol. 43, 1218-1227.
Finch, A. J., Hilcenko, C., Basse, N., Drynan, L. F., Goyenechea, B., Menne, T.
F., González Fernández, A., Simpson, P., D’Santos, C. S., Arends, M. J. et al.
(2011). Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes
Shwachman-Diamond syndrome. Genes Dev. 25, 917-929.
Gaiano, N., Amsterdam, A., Kawakami, K., Allende, M., Becker, T. and
Hopkins, N. (1996). Insertional mutagenesis and rapid cloning of essential genes
in zebrafish. Nature 383, 829-832.
Gessert, S., Maurus, D., Rössner, A. and Kühl, M. (2007). Pescadillo is required
for Xenopus laevis eye development and neural crest migration. Dev. Biol. 310,
99-112.
Godinho, L., Williams, P. R., Claassen, Y., Provost, E., Leach, S. D.,
Kamermans, M. and Wong, R. O. (2007). Nonapical symmetric divisions
underlie horizontal cell layer formation in the developing retina in vivo. Neuron 56,
597-603.
Iouk, T. L., Aitchison, J. D., Maguire, S. and Wozniak, R. W. (2001). Rrb1p, a
yeast nuclear WD-repeat protein involved in the regulation of ribosome
biosynthesis. Mol. Cell. Biol. 21, 1260-1271.
Jin, A., Itahana, K., O’Keefe, K. and Zhang, Y. (2004). Inhibition of HDM2 and
activation of p53 by ribosomal protein L23. Mol. Cell. Biol. 24, 7669-7680.
Jones, N. C., Lynn, M. L., Gaudenz, K., Sakai, D., Aoto, K., Rey, J. P., Glynn, E.
F., Ellington, L., Du, C., Dixon, J. et al. (2008). Prevention of the
neurocristopathy Treacher Collins syndrome through inhibition of p53 function.
Nat. Med. 14, 125-133.
Killian, A., Le Meur, N., Sesboüé, R., Bourguignon, J., Bougeard, G.,
Gautherot, J., Bastard, C., Frébourg, T. and Flaman, J.-M. (2004). Inactivation
of the RRB1-Pescadillo pathway involved in ribosome biogenesis induces
chromosomal instability. Oncogene 23, 8597-8602.
Kimmel, C. B., Miller, C. T. and Moens, C. B. (2001). Specification and
morphogenesis of the zebrafish larval head skeleton. Dev. Biol. 233, 239-257.
Kondrashov, N., Pusic, A., Stumpf, C. R., Shimizu, K., Hsieh, A. C., Xue, S.,
Ishijima, J., Shiroishi, T. and Barna, M. (2011). Ribosome-mediated specificity in
Hox mRNA translation and vertebrate tissue patterning. Cell 145, 383-397.
Lai, K., Amsterdam, A., Farrington, S., Bronson, R. T., Hopkins, N. and Lees, J.
A. (2009). Many ribosomal protein mutations are associated with growth
impairment and tumor predisposition in zebrafish. Dev. Dyn. 238, 76-85.
Lam, Y. W., Lamond, A. I., Mann, M. and Andersen, J. S. (2007). Analysis of
nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins.
Curr. Biol. 17, 749-760.
Lin, J. W., Biankin, A. V., Horb, M. E., Ghosh, B., Prasad, N. B., Yee, N. S., Pack,
M. A. and Leach, S. D. (2004). Differential requirement for ptf1a in endocrine
and exocrine lineages of developing zebrafish pancreas. Dev. Biol. 270, 474-486.
Lohrum, M. A., Ludwig, R. L., Kubbutat, M. H., Hanlon, M. and Vousden, K. H.
(2003). Regulation of HDM2 activity by the ribosomal protein L11. Cancer Cell 3,
577-587.
Luft, F. (2010). The rise of a ribosomopathy and increased cancer risk. J. Mol. Med.
88, 1-3.
RESEARCH ARTICLE 3241
Mathias, J. R., Perrin, B. J., Liu, T. X., Kanki, J., Look, A. T. and Huttenlocher,
A. (2006). Resolution of inflammation by retrograde chemotaxis of neutrophils in
transgenic zebrafish. J. Leukoc. Biol. 80, 1281-1288.
McGowan, K. A., Li, J. Z., Park, C. Y., Beaudry, V., Tabor, H. K., Sabnis, A. J.,
Zhang, W., Fuchs, H., de Angelis, M. H., Myers, R. M. et al. (2008). Ribosomal
mutations cause p53-mediated dark skin and pleiotropic effects. Nat. Genet. 40,
963-970.
Menne, T. F., Goyenechea, B., Sánchez-Puig, N., Wong, C. C., Tonkin, L. M.,
Ancliff, P. J., Brost, R. L., Costanzo, M., Boone, C. and Warren, A. J. (2007).
The Shwachman-Bodian-Diamond syndrome protein mediates translational
activation of ribosomes in yeast. Nat. Genet. 39, 486-495.
Narla, A. and Ebert, B. L. (2010). Ribosomopathies: human disorders of ribosome
dysfunction. Blood 115, 3196-3205.
Orelio, C., Verkuijlen, P., Geissler, J., van den Berg, T. K. and Kuijpers, T. W.
(2009). SBDS expression and localization at the mitotic spindle in human myeloid
progenitors. PLoS ONE 4, e7084.
Park, S. W., Davison, J. M., Rhee, J., Hruban, R. H., Maitra, A. and Leach, S. D.
(2008). Oncogenic KRAS induces progenitor cell expansion and malignant
transformation in zebrafish exocrine pancreas. Gastroenterology 134, 2080-2090.
Parsons, M. J., Pisharath, H., Yusuff, S., Moore, J. C., Siekmann, A. F., Lawson,
N. and Leach, S. D. (2009). Notch-responsive cells initiate the secondary transition
in larval zebrafish pancreas. Mech. Dev. 126, 898-912.
Pauls, S., Zecchin, E., Tiso, N., Bortolussi, M. and Argenton, F. (2007). Function
and regulation of zebrafish nkx2.2a during development of pancreatic islet and
ducts. Dev. Biol. 304, 875-890.
Pisharath, H., Rhee, J. M., Swanson, M. A., Leach, S. D. and Parsons, M. J.
(2007). Targeted ablation of beta cells in the embryonic zebrafish pancreas using
E. coli nitroreductase. Mech. Dev. 124, 218-229.
Renshaw, S. A., Loynes, C. A., Trushell, D. M., Elworthy, S., Ingham, P. W. and
Whyte, M. K. (2006). A transgenic zebrafish model of neutrophilic inflammation.
Blood 108, 3976-3978.
Robu, M. E., Larson, J. D., Nasevicius, A., Beiraghi, S., Brenner, C., Farber, S. A.
and Ekker, S. C. (2007). p53 activation by knockdown technologies. PLoS Genet.
3, e78.
Rohrmoser, M., Hölzel, M., Grimm, T., Malamoussi, A., Harasim, T., Orban,
M., Pfisterer, I., Gruber-Eber, A., Kremmer, E. and Eick, D. (2007).
Interdependence of Pes1, Bop1, and WDR12 controls nucleolar localization and
assembly of the PeBoW complex required for maturation of the 60S ribosomal
subunit. Mol. Cell. Biol. 27, 3682-3694.
Rujkijyanont, P., Adams, S. L., Beyene, J. and Dror, Y. (2009). Bone marrow cells
from patients with Shwachman-Diamond syndrome abnormally express genes
involved in ribosome biogenesis and RNA processing. Br. J. Haematol. 145, 806815.
Sasaki, M., Kawahara, K., Nishio, M., Mimori, K., Kogo, R., Hamada, K., Itoh,
B., Wang, J., Komatsu, Y., Yang, Y. R. et al. (2011). Regulation of the MDM2P53 pathway and tumor growth by PICT1 via nucleolar RPL11. Nat. Med. 17, 944951.
Schaper, S., Fromont-Racine, M., Linder, P., de la Cruz, J., Namane, A. and
Yaniv, M. (2001). A yeast homolog of chromatin assembly factor 1 is involved in
early ribosome assembly. Curr. Biol. 11, 1885-1890.
Smyth, G. K. (2004). Linear models and empirical Bayes methods for assessing
differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3,
Article3.
Torihara, H., Uechi, T., Chakraborty, A., Shinya, M., Sakai, N. and Kenmochi,
N. (2011). Erythropoiesis failure due to RPS19 deficiency is independent of an
activated Tp53 response in a zebrafish model of Diamond-Blackfan anaemia. Br. J.
Haematol. 152, 648-654.
Tourlakis, M. E., Zhong, J., Gandhi, R., Zhang, S., Chen, L., Durie, P. R. and
Rommens, J. M. (2012). Deficiency of SBDS in the mouse pancreas leads to
features of Shwachman-Diamond syndrome, with loss of zymogen granules.
Gastroenterology (in press).
Uechi, T., Nakajima, Y., Chakraborty, A., Torihara, H., Higa, S. and Kenmochi,
N. (2008). Deficiency of ribosomal protein S19 during early embryogenesis leads
to reduction of erythrocytes in a zebrafish model of Diamond-Blackfan anemia.
Hum. Mol. Genet. 17, 3204-3211.
Venkatasubramani, N. and Mayer, A. N. (2008). A zebrafish model for the
Shwachman-Diamond syndrome (SDS). Pediatr. Res. 63, 348-352.
Walker, M. B. and Kimmel, C. B. (2007). A two-color acid-free cartilage and bone
stain for zebrafish larvae. Biotech. Histochem. 82, 23-28.
Wang, Y., Rovira, M., Yusuff, S. and Parsons, M. J. (2011). Genetic inducible fate
mapping in larval zebrafish reveals origins of adult insulin-producing -cells.
Development 138, 609-617.
Wong, C. C., Traynor, D., Basse, N., Kay, R. R. and Warren, A. J. (2011).
Defective ribosome assembly in Shwachman-Diamond syndrome. Blood 118,
4305-4312.
Zhang, S., Shi, M., Hui, C. C. and Rommens, J. M. (2006). Loss of the mouse
ortholog of the shwachman-diamond syndrome gene (Sbds) results in early
embryonic lethality. Mol. Cell. Biol. 26, 6656-6663.
DEVELOPMENT
Ribosome biogenesis genes in pancreas development