1 MIT, Center for Cancer Research and Biology Department, Building E17 Room 340,
77 Massachusetts Avenue, Cambridge, MA 02139, USA
2 Program in Molecular Medicine, University of Massachusetts Medical School, 373
Plantation Street, Worcester, MA 01605, USA
3 Department of Microbiology, Molecular Biology and Biochemistry (LSS142),
University of Idaho, Moscow, ID 83844, USA
Author for correspondence (e-mail:
nhopkins{at}mit.edu)
Accepted 28 April 2004
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SUMMARY |
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Key words: PKD, Cilium, Zebrafish
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Introduction |
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In addition to ADPKD, kidney cyst formation is also a major component in a
variety of genetic diseases, including autosomal recessive PKD (ARPKD),
nephronophthisis (NPHP) and Bardet-Biedl syndrome
(Gabow and Grantham, 1997).
Multiple genes for these diseases have been cloned, including PKHD1
for ARPKD (Onuchic et al.,
2002
; Ward et al.,
2002
); BBS1, BBS2, BBS4, BBS7 and BBS8 for
Bardet-Biedl syndrome (BBS) (Ansley et al.,
2003
; Badano et al.,
2003
; Mykytyn et al.,
2001
; Mykytyn et al.,
2002
; Nishimura et al.,
2001
) and NPHP1, NPHP2 (INVS Human Gene
Nomenclature Database) NPHP3 and NPHP4 for nephronophthisis
(NPHP) (Hildebrandt et al.,
1997
; Olbrich et al.,
2003
; Otto et al.,
2002
; Otto et al.,
2003
).
Recent evidence suggests that cilia play a central role in the etiology of
PKD (Ong and Wheatley, 2003).
The cilium is a cell surface organelle. It is surrounded by a membrane that is
contiguous with the cell membrane and it has a microtubule axoneme at its
center. Eukaryotic cilia and flagella are assembled by a process called
intraflagellar transport (IFT). This process was first discovered, and remains
best understood, in the green algae Chlamydomonas
(Rosenbaum and Witman, 2002
).
During IFT, large protein particles are carried along the ciliary microtubules
by kinesin and dynein. The IFT particles are thought to be vehicles for
transporting cargo needed for assembly, maintenance and function of flagella
and cilia. These particles are composed of at least 17 polypeptides
(Cole et al., 1998
;
Piperno et al., 1998
) and are
highly conserved. Homologs have been found in all ciliated organisms examined,
including C. elegans, Drosophila and mammals
(Baker et al., 2003
;
Cole et al., 1998
;
Han et al., 2003
;
Haycraft et al., 2003
;
Huangfu et al., 2003
).
Mutations in genes encoding IFT particle proteins block ciliary assembly in
Chlamydomonas (Pazour et al.,
2000
), C. elegans
(Cole et al., 1998
;
Haycraft et al., 2001
;
Qin et al., 2001
),
Drosophila (Han et al.,
2003
) and mouse (Huangfu et
al., 2003
; Murcia et al.,
2000
; Pazour et al.,
2000
). In the mouse, complete null alleles block assembly of cilia
on the embryonic node and result in embryonic lethality during mid-gestation
(Huangfu et al., 2003
;
Murcia et al., 2000
), while a
partial loss of function allele retards cilia formation in the kidney
(Pazour et al., 2000
) and
other organs and causes hydrocephaly, preaxial polydactyly, and cysts in the
kidney, liver and pancreas (Moyer et al.,
1994
), indicating that cilia play important roles in vertebrate
development. In support of the idea that cilia assembly defects cause PKD,
targeted deletions of KIF3A in kidney epithelium block ciliary
assembly and cause cyst formation (Lin et
al., 2003
). KIF3A powers IFT particle movement from the cell body
to the ciliary tip (Rosenbaum and Witman,
2002
). Furthermore, several PKD-associated gene products have been
found on cilia, including the products of the human autosomal dominant
PKD1 and PKD2 genes
(Pazour et al., 2002b
;
Yoder et al., 2002
), the human
autosomal recessive PKHD1 gene
(Ward et al., 2003
), and the
human nephronophthisis NPHP1 and NPHP2 genes
(Otto, 2003
;
Watanabe, 2003
). These
findings lead to a cilia model for PKD
(Rosenbaum and Witman, 2002
;
Pazour and Whitman, 2003
). In
this model, cilia on renal epithelial cells function as antennae that detect
environmental signals. Activation of sensors on the cilium, such as polycystin
2, triggers a Ca2+ influx into the cell and eventually regulates
cell proliferation. However, the link between cilia and PKD is largely based
on `guilt by association'. Additionally, the nature of the signal sensed by
cilia remains controversial and the signaling pathway that couples cilia to
cell proliferation is almost completely unknown.
To further our understanding of PKD, we carried out a forward genetic
screen in zebrafish to identify genes that can cause this disorder. The
zebrafish pronephros is composed of two nephrons with glomeruli fused at the
midline, connected to pronephric ducts that alter the blood filtrate and shunt
the urine outside of the animal (Drummond
et al., 1998). Zebrafish embryos can develop kidney cysts by 2
days post fertilization (Drummond et al.,
1998
; Sun and Hopkins,
2001
). As zebrafish embryos are transparent, cyst formation is
clearly visible under a stereoscope. Moreover, we have demonstrated previously
that mutations in vhnf1 (tcf2 Zebrafish Information
Network) which is associated with human familial GCKD (glomerulocystic kidney
disease) (Bingham et al.,
2001
), can cause kidney cysts in zebrafish
(Sun and Hopkins, 2001
),
suggesting that kidney cyst formation in zebrafish is highly relevant to human
PKD. Taken together, these characteristics of zebrafish make it feasible to
perform large-scale genetic screens to search for genes involved in kidney
cyst formation. From just such a screen, we have identified 12 different genes
that can cause kidney cysts in zebrafish embryos when mutated. We cloned 10 of
these genes, three of which encode IFT particle components. We further show
that a fourth gene is also required for cilia formation through an unknown
mechanism. Thus, through an unbiased genetic screen for cystic kidney mutants,
we not only provide strong support for the cilial model of cystogenesis, but
evidence that this may be the primary cause of human PKD.
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Materials and methods |
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Molecular biology
We cloned genes responsible for the mutant phenotypes, obtained full-length
cDNA and carried out RT-PCR as previously described
(Amsterdam et al., 1999;
Golling et al., 2002
). To
detect maternal expression of tested genes, we extracted RNA from embryos
before the 128-cell stage with trizol reagent (Invitrogen) following the user
manual. To compare gene expression in mutant and wild-type embryos, we
extracted RNA from embryos at day 3-5 post-fertilization. We then reverse
transcribed RNA into cDNA with the superscript II RT-PCR system (Invitrogen)
and subsequently performed PCR with gene-specific primers. The primers used
are as follows: hi409, 5'-GCCTGAAAAGAGAGAGTTTATC-3' and
5'-CGTAATTCTTCAAGATGAGCGA-3'; hi2211,
5'-GATGGAGCTGCTAAAGTCACCTGT-3' and
5'-AAATACTCCGTCGGAGACAGCA-3'; hi3308,
5'-TGCGGTTTTAAGGAGCGTCT-3' and
5'-CACTCTCACATACATTGGCTGAACA-3'; hi3417,
5'CGGGATCCGGGATGGCGGAGGAGGAAGA-3' and
5'-GGAATTCGTGTTTCAATAAGCCTGCGCA-3'; hi4166,
5'-TGGAATTCACGATGAGCTCCAGTCGCGT-3' and
5'-GGGATACGTGCTGTGGTTCTC3'.
Histological analysis
Embryos were fixed in Bouin's fixative overnight at room temperature,
washed three times with PBS, embedded in JB-4 resin from Polysciences and cut
at 4 µm. Slides were then stained with Hematoxylin and Eosin.
Immunohistochemistry
Embryos were fixed in Dent's fixative as described before
(Drummond et al., 1998). A
monoclonal anti-acetylated tubulin antibody from Sigma was used at 1:500.
6F antibody from DSHB was used at 1:10. Alkaline phosphatase-conjugated
(Vector Laboratories) anti-mouse IgG was used at 1:1000. Color was developed
with NBT/BCIP as the chromogenic substrate. Embryos were then cleared with
benzyl benzoate and flattened with coverslips for photography. For confocal
analysis, Alexa fluor 488 conjugate anti-mouse IgG from Molecular Probes was
used at 1:500.
Morpholino injection
We obtained morpholino oligos from Gene Tools and injected them into
embryos at one- to four-cell stages at a concentration of 1 µM. A standard
control oligo (5'-CCT CTT ACC TCA GTT ACA ATT TAT A-3') was
injected as a control. We raised morpholino injected embryos and observed for
phenotypes with stereoscopes. The oligos are designed against the
translational initiation sites of corresponding genes. The sequences for the
morpholino oligos used are as follows: hi459 oligo 1,
5'-TTCGCCATCAGATTGAACATTTCCC-3'; hi459 oligo 2,
5'-TTTCCCCCCTAAATGCTTTCACTGG-3'; hi1055B oligo 1,
5'-TGCTTTTTACTTCTTCGATCTTCAT-3'; hi2211 oligo 1,
5'-CATACTTCACGTTTATAATAAGACT-3'; hi2211 oligo 2,
5'-GACTCAGGGCAGTTATAAGAACGTA-3'; hi4166 oligo 1,
5'-AGGACGAACGCGACTGGAGCTCATC-3'.
Accession numbers
hi409/IFT81, AY618922; hi459/scorpion, AF506213;
hi2211/IFT172, AY618923; hi3417/IFT57, AY618924;
hi3308/seahorse, AY618925; hi3959A/qilin, BC045921;
hi4166/pkd2, AY618926; CrIFT81, AY615519; CrIFT172,
AY615520.
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Results |
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To facilitate the analysis of the mechanisms involved in cytogenesis, we
divided these mutants into two groups based on their phenotypes, e.g. whether
they show body curvature defects in addition to cystic kidney
(Table 1). In Group I are the
vhnf1 and cad1/caudal mutants, neither of which shows body
curvature defects. vhnf1 encodes a homeobox gene. vhnf1
mutant embryos show patterning defects in multiple organs, as we reported
previously (Sun and Hopkins,
2001). cad1 encodes a bHLH transcription factor, and its
fly homolog caudal is involved in the anterior-posterior patterning
of the body plan (Macdonald and Struhl,
1986
). Reminiscent of such a role, the zebrafish cad1
mutant also shows tail truncation in addition to the kidney cyst. Noticeably,
both genes in Group I encode transcription factors.
The rest of the mutants display striking similarities in their phenotypes, including body curvature and kidney cysts (Fig. 1A), suggesting that they may function in the same process or even pathway. Therefore, we put them into a separate group that we designate Group II. Because of the body curvature and kidney cyst phenotype of pkd2 morphants (morpholino knock down animals, Fig. 1D), we include the pkd2 mutant in this group, even though it displays only the body curvature but not the cystic kidney phenotype.
In the entire insertional mutagenesis screen, novel genes account for about
20% of all the genes identified. However, among the genes mutated in the Group
II kidney mutants, six out of eight are novel genes: they have human
homologues, but their predicted encoded proteins had no assigned biochemical
function when we first cloned them. The exceptions are pkd2 and
hi1055B, which encodes pontin, a component of a DNA stimulated ATPase
complex. It is thought that pontin and reptin, a paralog of pontin, antagonize
each other's functions (Bauer et al.,
2000). A zebrafish gain-of-function mutant of reptin displays
curved body and hyperplastic heart
(Rottbauer et al., 2002
). The
function of pontin and reptin in kidney development have not been formally
studied.
Many Group II kidney mutants have defects in ciliary genes
Given the connection between cilia and PKD, we compared the sequences of
the novel genes we identified to a collection of 12 IFT genes whose sequences
were available to us. The IFT machinery was first identified in the green alga
Chlamydomonas and is thought to be needed for assembly, maintenance
and function of flagella and cilia
(Kozminski et al., 1993;
Rosenbaum and Witman, 2002
).
Strikingly, the sequence analyses revealed that three of our group II genes
encode zebrafish homologs of IFT components. hi409 encodes a homolog
of IFT81 (D.G.C. and M.S.M., unpublished), hi2211 encodes a homolog
of IFT172 (Cole et al., 1998
)
and hi3417 encodes a homolog of IFT57
(Haycraft et al., 2003
)
(G.J.P., G. B. Whitman, J. L. Rosenbaum and D.G.C., unpublished)
(Table 1) (see Fig. S1 at
http://dev.biologists.org/supplemental).
We named mutants hi409, hi2211 and hi3417 larry, moe and
curly, respectively. Intriguingly, these three IFT genes are the only
known IFT genes identified in the screen among the 300 different genes cloned
to date. These results provide compelling support for an intimate connection
between cilia and cystogenesis.
hi409/IFT81 is expressed widely in the embryo
Despite the specific phenotype of cyst formation in PKD, a number of PKD
genes have been shown to be widely expressed. To determine if this is also
true for some of the genes we isolated, we performed in situ hybridization to
study hi409/larry gene expression in embryos at the eight-somite
stage, 25 hpf and 34 hpf. At all time points, hi409/larry transcripts
can be detected in all regions of the embryos
(Fig. 3). However, we also
observed enriched expression in specific regions at specific time points. At
the eight-somite stage, the transcript is enriched in the notochord. At 25
hpf, it is concentrated in cells surrounding the brain ventricles. At 34 hpf,
enrichment can be seen in the otic vesicle and to some extent in the
pronephric ducts. Interestingly, cilia are known to be present in the brain
ventricle, the otic vesicle and the kidney.
|
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The functions of the two other novel genes, hi3308/seahorse and hi3959A/qilin, and pontin in our collection of Group II mutants remain unclear. However, as vertebrates might have a wider repertoire of genes that are required for the formation and function of cilia, it is possible that some of them may also be cilia genes. Alternatively, they may be involved in connecting cilia signals to downstream events.
Further analysis of mutant phenotypes provided interesting clues for the
cellular pathogenesis of kidney cysts. All the kidney cysts we observed were
medial to the pectoral fins (Fig.
1A). Histological sections verified that they are located in the
tubular-glomerular region (Fig.
1B,C). Interestingly, the pronephric ducts outside of the cystic
regions were enlarged in all of the mutants, as shown by histological sections
(an example is shown in Fig.
5A,B). We further stained whole embryos with 6F
(Fig. 5C,D), an antibody that
recognizes a subunit of Na+/K+ ATPase enriched in the
basolateral surface of ductal and tubular cells
(Drummond et al., 1998
).
Neither the intensity of the signal, nor the subcellular location, was
significantly affected. However, the appearance of this staining also suggests
that the lumen size of the pronephric ducts is increased in mutants. The size
of the tubules and ducts along their lengths in these mutants also becomes
variable, making it difficult to quantify the size difference.
|
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Discussion |
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In addition to ADPKD, multiple human diseases, including ARPKD,
nephronophthisis and Bardet-Biedl syndrome, show kidney cyst formation. A
growing list of genes involved in these diseases have been shown to encode
proteins involved in cilia formation/function
(Ansley et al., 2003;
Otto et al., 2003
;
Ward et al., 2003
;
Watanabe et al., 2003
). A
model has been proposed in which cilia are directly involved in cystogenesis
(Pazour and Witman, 2003
).
Here, through a nonbiased forward genetic screen for cystic kidney mutants, we
show that a major class of mutants involves defects in cilia formation or
function. This includes three genes (hi409, hi2211 and
hi3417) whose products are part of the IFT particle that is required
for cilia formation, a novel gene (hi459/scorpion) that is also
required for ciliary assembly by an unknown mechanism, and pkd2,
whose products are found in cilia in other organisms. This result suggests
that mutations in ciliary genes are the major cause of PKD, that such genes
are excellent candidate for multiple human diseases that involve kidney cyst
formation, and also that the genes identified in this screen are excellent
candidates for human PKD.
Cilia project from the apical surface of renal epithelial cells into the
lumen of the duct and so are ideally situated to sense the environment and
regulate the proliferation and differentiation of these cells. The mechanism
by which this is accomplished is unknown but may involve detection of liquid
flow through the lumen, by interaction with neighboring cells or by detection
of chemical or ligand signals (Lubarsky
and Krasnow, 2003; Nauli et
al., 2003
; Praetorius et al.,
2003
). Activation of mechanosensory channels (i.e. polycystins) on
the cilium then triggers a signaling cascade that regulates cell proliferation
and/or volume (Fig. 6). Defects
in this pathway can lead to uncontrolled expansion of epithelial tubes and
formation of cysts in local areas.
|
In addition to their role in cystogenesis, cilia also play important roles
in sensory responses. It is thought that nodal cilia are involved in both
generating and sensing nodal flow, thereby breaking the bilateral symmetry of
the body plan (McGrath et al.,
2003; Nonaka et al.,
1998
). The outer segments of photoreceptor cells are specialized
cilia. It has been shown that tg737 mice, in which an IFT gene is
mutated, have abnormal outer segment development and retinal degeneration
(Pazour et al., 2002a
). In
C. elegans, some sensory neurons are ciliated and IFT has been
observed in chemosensory neurons (Orozco
et al., 1999
). In all these systems, cilia function as sensory
organelles. Protruding from the cell surface, they serve as antennae of cells,
albeit adapted to the diverse signals they detect. In the node, they probably
sense nodal flow. In the photoreceptor cells, they sense light. In the worm,
they are involved in sensing chemical signals. Given that cilia can be found
on almost every type of vertebrate cell, it would not be surprising that new
functions will be identified for these important but somewhat ignored
organelles. As IFT genes are thought to be required for the formation or the
normal functioning of cilia, the IFT mutants we identified will undoubtedly
serve as powerful tools in unraveling novel functions of cilia in
vertebrates.
In summary, we have identified 12 cystic kidney genes in a non-biased forward genetic screen that reached about 25% saturation, suggesting that at least 50 genes could be found by this approach. At least five out of the 10 genes we cloned are required either for cilia formation or function. Because of the similarity between cyst formation in zebrafish and human, we predict that a majority of human PKD genes are involved in cilia formation and/or function and that novel genes identified here are excellent candidates for human diseases that involve kidney cyst formation.
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Note added in proof |
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Department of Genetics, Yale University School of
Medicine, 333 Cedar Street, NSB-393, P.O. Box 208005, New Haven, CT 06520,
USA
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