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First published online 7 February 2007
doi: 10.1242/dev.02806
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1 Nephrology Division, Massachusetts General Hospital, 149 13th Street,
Charlestown, MA 02129, USA.
2 Renal Division, University Hospital Freiburg, Zentrale Klinische Forschung
(ZKF), Breisacher Str. 66, 79106 Freiburg, Germany.
* Author for correspondence (e-mail: idrummon{at}receptor.mgh.harvard.edu)
Accepted 28 December 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Pronephros, Jagged 2, Multiciliated cell, Notch3, double bubble, mind bomb, Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Vize et al.,
2002). Functionally similar cell types in the nephron are
typically arranged in discrete tubule segments that process filtered blood
sequentially as it passes through the nephron. In other parts of the nephron,
functionally distinct cells are intermingled or `intercalated' between
non-like cells. For instance, in the collecting system, intercalated cells
that regulate pH homeostasis are morphologically and functionally distinct
from adjacent principal cells, whose primary function is water transport
(Schuster, 1993;
Seldin and Giebisch, 1992).
Similar intermingling of functionally distinct cell types is found in other
epithelial organs; for example, in the lung, intestine and stomach. In the
lung, neuroendocrine cells, which have specialized sensory and secretory
functions, are dispersed among airway epithelial cells
(Ito et al., 2000). In the
intestinal epithelium, mucous-secreting goblet cells are interspersed among
villus epithelial cells (Zecchini et al.,
2005). In the stomach of the chick, glandular epithelial cells
differentiate from an initially homogeneous layer of epithelium and lie
interspersed with lumenal epithelial cells
(Matsuda et al., 2005). The
existence of adjacent non-like cells in these organs suggests that a lateral
inhibition mechanism, similar to well-defined mechanisms underlying the
development of proneural clusters in Drosophila
(Artavanis-Tsakonas et al.,
1995), may play a role in vertebrate organogenesis. In peripheral
neural development in Drosophila, clusters of cells destined to
become nerves and supporting cells are partitioned by the expression of the
Notch ligand Delta in future nerve cells. Delta, acting on Notch receptors in
adjacent cells, prevents them from adopting a neural fate
(Artavanis-Tsakonas et al.,
1995). Using loss- and gain-of-function approaches targeting
various elements of vertebrate Notch signaling pathways, lateral inhibition
mechanisms have in fact been demonstrated to control the differentiation of
neuroendocrine cells in the lung (Ito et
al., 2000), goblet cells in the intestine
(Crosnier et al., 2005;
Zecchini et al., 2005) and
gland cells in the stomach (Matsuda et
al., 2005).
In Notch-mediated lateral inhibition, a specialized cell type expresses a
transmembrane DSL (Delta/Serrate/Lag-2) ligand of Notch which binds a Notch
receptor in neighboring cells (Kadesch,
2004). The Notch receptor undergoes regulated intramembrane
cleavage to generate an active intracellular domain peptide that transits to
the nucleus to participate in transcriptional regulation
(Mumm and Kopan, 2000). By
binding to and activating the CSL [CBF-1, Suppressor of Hairless (Rbpsuh),
Lag-1] transcriptional complex, the Notch intracellular domain (NICD)
initiates a transcriptional cascade involving the expression of Hes and
HRT/HER/Hey transcriptional repressors
(Kadesch, 2004). These, in
turn, repress genes that are expressed in Notch-ligand-expressing cells, often
including the ligand gene itself, and thereby direct the neighboring cell
along a different developmental pathway
(Kadesch, 2004). In kidney
organogenesis, Notch signaling has been implicated in the development of
glomerular vasculature and in segmentation of the nephron
(Cheng et al., 2003;
McCright, 2003). Expression of
the Notch ligand Jagged 1 in endothelial cells and Notch2 in glomerular
epithelial cells is required for proper development of the glomerular
capillary tuft (McCright et al.,
2001). Broadly activating or inhibiting Notch signaling by the
expression of activated or dominant-negative Suppressor of hairless proteins
have been shown to perturb proximal versus distal nephron fate in
Xenopus embryos (McLaughlin et
al., 2000). Mouse kidney explant cultures treated with the
gamma-secretase inhibitor DAPT to inhibit Notch cleavage also show a loss of
proximal versus distal nephron segments
(Cheng et al., 2003). These
experiments demonstrate a role for Notch signaling in the specification of
relatively large nephron domains. However, there is currently no evidence
implicating Notch signaling in the generation of `salt-and-pepper' patterns of
interdigitating cell types within the kidney nephron.
The zebrafish pronephros is an accessible model for nephron cell
specification and patterning because it consists of typical vertebrate kidney
cell types, and is amenable to both forward and reverse genetic manipulation
(Drummond, 2003). Expression
of jagged and notch genes in the pronephros suggests that they could play an
important role in patterning the pronephric nephron
(Zecchin et al., 2005).
Evidence for nephron patterning in the pronephros is seen in the
segment-specific expression of ion transporters
(Nichane et al., 2006;
Shmukler et al., 2005), in the
expression of receptors and transcription factors in discrete segments
(Bisgrove et al., 1997;
Marcos-Gutierrez et al., 1997;
Van Campenhout et al., 2006),
and in the disruption of segment-boundary formation in the pax2.1
(also known as pax2a - Zebrafish Information Network) mutant no
isthmus (Majumdar et al.,
2000). In our examination of cilia function in the kidney
(Kramer-Zucker et al., 2005)
we have found that two types of ciliated cells exist in the pronephros: single
ciliated cells that have the morphology of typical transporting epithelial
cells, and multiciliated cells that, by several criteria, appear specialized
for fluid propulsion in the pronephros. We show here that multiciliated cells
are isolated, specialized cells surrounded by non-like transporting epithelia
in the distal segment of the pronephros. We present evidence that Notch
signaling via Jagged 2-Notch interactions, as well as the downstream
transcriptional regulation of ciliogenic genes, is essential in the generation
of the `salt-and-pepper' pattern of multiciliated and transporting epithelial
cells in the pronephros. We also demonstrate that transfating cells to the MCC
lineage is sufficient to overcome defects in ciliogenesis in a zebrafish cyst
mutant and prevent the formation of pronephric cysts.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Dechorionated embryos were kept at 28.5°C in E3 solution with or without
0.003% PTU (1-phenyl-2-thiourea, Sigma) to suppress pigmentation and were
staged according to somite number (som) or hours post-fertilization (hpf)
(Westerfield, 1995). The Na,
K-ATPase alpha1A4:GFP transgenic line was created by amplifying an 8112
base-pair (bp) fragment of the Na,K-ATPase alpha1A4-subunit promoter (cDNA
accession: NM_131689) from genomic DNA using nested PCR with the following
primers: LNaK-F1: 5'-GGTCGCTGGGCTAAGTCACAGAC-3'
LNaK-F2: 5'-TGGGCTAAGTCACAGACCGCCTA-3'
LNaK-R: 5'-TCCACTCACTCCAAGCCCCATTT-3'
NaKB-R2: 5'-CGCGGATCCCTCACTCCAAGCCCCATTTTCCT-3'.
The 8112 bp fragment was cloned in pGI upstream of EGFP and injected as a
circular plasmid into TuAB wild-type embryos at the 1-cell stage. Founders
were screened by GFP fluorescence in the pronephros and were out crossed to
create a stable transgenic line. For notch1a NICD induction, the
Tg(uas:notch1a-intra) zebrafish line was crossed to the
Tg(hsp70:gal4) line (Scheer and
Campos-Ortega, 1999; Scheer et
al., 2002) and heat shocked for 60 minutes at 37°C at either
8, 14 and 28 hpf or at 9, 15 and 29 hpf.
Plasmid clones
Selection of in situ probes was based on information derived from the
high-throughput in situ screen carried out by Thisse et al. (ZFIN gene
expression data,
http://zfin.org).
Plasmid probes were ordered from either Open Biosystems or from the Zebrafish
International Resource Center (ZIRC): shippo1 (BC054587);
rfx2 (B1325076); trpM7 (NM_001030061); slc13a1
(NM_199281); her9 (AF301264). jagged 2, NM_131862 and
jagged 1b, NM_131863 (previously jagged 3) were gifts from
A. Chitnis (Laboratory of Molecular Genetics, NIH/NICHD, USA), and the
zebrafish notch3, NM_131549 was a gift from B. Weinstein (Laboratory
of Molecular Genetics, NIH/NICHD, USA). The fleer (flr) cDNA
encodes a TPR protein (N.P. and I.A.D., unpublished).
In situ hybridization
Whole-mount in situ hybridization was performed as previously described
(Thisse and Thisse, 1998). For
two-color in situ hybridization, probes were synthesized using DIG-UTP or
FITC-UTP. Stained embryos were cleared in Benzylbenzoate:Benzyl alcohol and
photographed on a Lietz MZ12 or Nikon E800 microscope equipped with a Spot
Image digital camera. Two-color stained embryos were cleared in glycerol and
photographed after removal of the yolk extension. Two-color fluorescent in
situ hybridization (S. Holley, Yale University, New Haven, CT, personal
communication) was performed using DIG-UTP and FITC-UTP-labeled RNA probes
followed by sequential detection using HRP labeled anti-DIG and HRP-labeled
anti-FITC antibodies (Boehringer Mannheim) and fluorescent tyramide
amplification. Following RNA-probe hybridization, embryos were blocked in 150
mM maleic acid, 100 mM NaCl pH 7.5 with 2% Boehringer blocking agent, and were
then incubated with HRP anti-FITC at 1:500 dilution for 4 hours in blocking
solution. After washing, anti-FITC antibody was visualized with TSA Plus
Fluorescein Solution (Perkin Elmer) for 45 minutes, dehydrated in methanol and
quenched with 1% H2O2 in methanol for 30 minutes. After
rehydrating, embryos were blocked again and incubated with HRP anti-DIG at
1:1000 dilution for 4 hours, then washed and reacted with TSA Plus Cy3
solution (Perkin Elmer) for 45 minutes. For combined fluorescent
shippo1 DIG in situ/anti-acetylated tubulin staining, embryos were
incubated with both anti-acetylated tubulin (clone 6-11B-1; Sigma) and HRP
anti-DIG (Boehringer Mannheim) after hybridization. After complete fluorescent
in situ hybridization, embryos were refixed with 4% PFA, and were then washed
and blocked in 150 mM maleic acid, 100 mM NaCl pH 7.5 with 10% NGS for 2 hours
at room temperature. Acetylated tubulin was visualized with Alexa Fluor 546
goat anti-mouse IgG (Molecular Probes) in blocking solution. Embryos were
cleared in glycerol, mounted and viewed on a Zeiss LSM5 PASCAL confocal
fluorescence microscope.
Morpholino antisense oligonucleotides
Morpholino antisense oligonucleotides (Gene Tools, Philomath, OR) were
designed to target either the translation start or an exon splice-donor site
causing splicing defects of the mRNA. The following morpholinos were used:
j2exon 20: 5'-TCTTTGAGATACTCACTGATACGGC-3'
j2exon 20 control: 5'-CGGCATAGTCACTCATAGAGTTTCT-3'
rfx2exon 6: 5'-GGGTGTAGTCTGACCTGGTAC-3'
rfx2exon 6 control: 5'-CATGGTCCAGTCTGATGTGGG-3'
notch3exon 27: 5'-TGACCAACTCACTTCATGCCCAGTG-3'
jagged 2 ATG: 5'-TCCTGATACAATTCCACATGCCGCC-3'
jagged 1b(3) ATG: 5'-CTGAACTCCGTCGCAGAATCATGCC-3'.
Morpholino oligos were diluted in 100 mM KCl, 10 mM HEPES, 0.1% phenol red (Sigma) and injections were performed using a nanoliter2000 microinjector (World Precision Instruments). Injection concentrations were 0.2-0.5 mM and injection volume was 4.6 nl/embryo (7-16 ng morpholino/embryo). The molecular defect caused by splice-donor morpholinos was verified by reverse-transcriptase PCR from total RNA from a single morphant embryo with nested primers in flanking exons.
Histochemistry and immunohistochemistry
For histology, embryos were fixed with 4% formaldehyde in PBS at 4°C
overnight followed by dehydration and embedding in JB-4 (Polysciences), and
were then sectioned at 5-7 µm. Slides were stained in methylene blue/azure
II (Humphrey and Pittman,
1974) and examined using a Nikon E800 microscope. For acetylated
tubulin staining, the embryos were fixed in Dent's Fix (80% methanol/20% DMSO)
at 4°C overnight. After rehydration they were washed in PBS plus 0.5%
Tween20 and blocked in PBS-DBT (PBS plus 1% DMSO, 1% BSA and 0.5% Tween20)
with 10% normal goat serum (NGS) (Sigma) at room temperature for 2 hours.
Primary-antibody incubation in PBS-DBT 10% NGS [1:500 monoclonal
anti-acetylated tubulin 6-11B-1 (Piperno
and Fuller, 1985), Sigma] was at 4°C overnight. The embryos
were washed in PBS/0.5% Tween20 and blocked in PBS-DBT 10% NGS at room
temperature for 1 hour, and were then incubated in 1:1000 goat anti-mouse
Alexa Fluor 546 (Molecular Probes) in PBS-DBT 10% NGS at 4°C overnight.
After rinsing in PBS, the embryos were washed with methanol, equilibrated in
clearing solution (1:2 benzoyl-alcohol:benzoyl-benzoate) and examined using a
Zeiss LSM5 Pascal confocal microscope. For combined jagged 2 DIG in
situ/anti-acetylated-tubulin staining, embryos were incubated with both
anti-acetylated tubulin 6-11B-1 and anti-DIG after hybridization. Acetylated
tubulin was visualized with diaminobenzidine histochemistry using a HRP ABC
kit (Vector Laboratories) before being embedded and sectioned. For analysis of
GFP expression in tissue sections, GFP-transgenic embryos were stained with
polyclonal anti-rabbit GFP antibody (G1544, Sigma) in whole mount at 1:100
followed by an Alexa Fluor 488 (Molecular Probes) goat anti-rabbit secondary
antibody at 1:500.
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).
High-speed video microscopy
PTU-treated embryos were put in E3 egg water containing 40 mM
2,3-butanedione monoxime (BDM, Sigma) for 5 minutes to stop the heart beat and
were then changed to 20 mM BDM, which contained egg water, for observation.
The embryos were then analyzed using a 40x/0.55 water-immersion lens on
a Zeiss Axioplan microscope (Zeiss, Germany) equipped with a high-speed
Photron FastCAM-PCI 500 video camera (Photron). Images of beating cilia were
acquired at 250 frames per second using Photron FastCAM version 1.2.0.7
(Photron Ltd). Image processing was done using Photoshop 7.0 (Adobe) and
movies compiled in Graphic Converter version 4.5.2 (Lemke Software, Germany)
and Quicktime (Apple).
DAPT treatments
DAPT was used at a final concentration of 100 µM diluted in embryo
medium from a 10 mM stock in DMSO (Geling
et al., 2002). Zebrafish embryos treated with DAPT were kept at
28°C until analysis at 34 hpf. Control embryos were mock treated with
embryo medium containing the same concentration of DMSO carrier only.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). More-detailed examination of ciliated cells in the
pronephros showed that the majority of cells possessed a single cilium, while
a subset of cells were multiciliated and extended up to 20 cilia from their
apical surface into the pronephric lumen
(Fig. 1). Acetylated tubulin
immunofluorescence of whole-mount zebrafish larvae at 52 hpf revealed bright
bundles of cilia in the pronephric lumen, which were interspersed among cells
with a single cilia (Fig.
1A,B). Ultrastructurally, these bundles of cilia originated from
individual cells characterized by multiple apical basal bodies and apically
oriented mitochondria (Fig.
1C-F). By contrast, the surrounding single-ciliated cells
possessed a microvillar brush border and basolaterally oriented mitochondria,
a morphology typical of transporting epithelia. The distribution of
multiciliated cells (MCCs) as isolated individual cells along the pronephros
was also apparent in high-speed micro video images of the pronephros (see
Movie 1 in the supplementary material). Bundles of cilia emanating from MCCs
beated in a coordinated wave pattern, in contrast to the single cilia on
neighboring transporting epithelial cells, which appeared to beat autonomously
(see Movie 1 in the supplementary material).
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) (Fig. 2A) and
rfx2 belongs to the family of X-box-binding transcription factors,
which act as master regulators of the ciliogenesis program
(Bonnafe et al., 2004;
Efimenko et al., 2005;
Swoboda et al., 2000).
rfx2 was initially expressed throughout the pronephros, implying a
role for this transcription factor in the development of both single- and
multiciliated cells (data not shown). By 34 hpf, however, rfx2 was
concentrated in a spotted pattern similar to the distribution of multiciliated
cells (Fig. 2B).
shippo1- and rfx2-positive cells are present throughout the
distal segment of the pronephric nephron
(Nichane et al., 2006) but
were not present in the most-caudal nephron segment
(Fig. 2A,B). Because cells
directly adjacent to MCCs appear by electron microscopy to be more typical of
transporting epithelia (Fig.
1D-F), we examined ion channel gene expression in similar regions
of the pronephros. Cells in the `early distal' segment of the pronephric
nephron expressed the cation transporter trpM7
(Fig. 2C) and the
sodium-sulfate co-transporter slc13a1
(Fig. 2D). Whereas epithelial
cells expressing these two channel genes appeared to coexist with
shippo1- and rfx2-positive cells in the early distal
segment, the distribution of shippo1- and rfx2-positive
cells further extends distally into the `late distal' segment of the
pronephros (Nichane et al.,
2006).
To confirm that shippo1/rfx2 and trpM7/slc13a1 marked
nonequivalent, but adjacent, cell types in the pronephros, we performed
two-color whole-mount in situ hybridization and double fluorescent in situ
hybridization with these probes. Two-color whole-mount in situ hybridization
verified that trpM7/slc13a1-expressing transporting epithelial cells
and shippo1-expressing cells are indeed two distinct cell types that
exist side by side in the pronephros (Fig.
2E,F). To further distinguish MCCs from transporting epithelia, we
created a transgenic line of zebrafish that expresses GFP from the Na,K-ATPase
alpha1A4 gene promoter. This Na,K-ATPase subunit is highly expressed in the
pronephros, where, by analogy to mammalian kidney epithelia, it is likely to
power a variety of ion-transport activities
(Seldin and Giebisch, 1992).
In the distal segment of the pronephros, the Na,K-ATPase:GFP transgene was
expressed in a subset of cells (Fig.
2G) and, based on co-staining for cilia bundles with
anti-acetylated tubulin, was excluded from MCCs
(Fig. 2H,I). The complementary
expression pattern of axonemal genes/cilia bundles with genes for ion
transporters suggests that MCCs and transporting epithelia are distinctly
different cell types, intermixed in the distal segment of the pronephros.
Using a combination of shippo1 fluorescent in situ hybridization and anti-acetylated tubulin immunofluorescence, we found that shippo1-positive cells were multiciliated (Fig. 3A,B). Neighboring cells with a single cilia were shippo1-negative (Fig. 3A,B). Using shippo1 as a reference probe for MCCs in double fluorescent in situ hybridization with ciliogenic or ion channel gene probes, we confirmed that other ciliogenic genes - including fleer (flr), a tetratricopeptide-repeat protein required for ciliogenesis (N.P. and I.A.D., unpublished) (Fig. 3C-E), and rfx2 (Fig. 3F-H) - were co-expressed with shippo1 in MCCs. Conversely, the ion channel genes for slc13a1 (Fig. 3I-K) and trpM7 (Fig. 3L-N) were expressed in a non-overlapping, adjacent set of cells in the pronephros.
The Notch ligand Jagged 2 is expressed in multiciliated cells
The apposition of different epithelial cell types in the pronephros
suggested that a lateral inhibition mechanism, possibly involving Notch
signaling, might play a role in pronephric cell patterning. Because the Notch
receptor notch3 and the Notch ligands jagged 2 and
jagged 1b (previously jagged 3) have recently been reported
to be expressed in the pronephros (Lawson
et al., 2001; Zecchin et al.,
2005), we examined the expression of Notch signaling genes in
relation to MCCs and transporting epithelial cells. Interestingly, jagged
2 was expressed in the pronephric mesoderm during early somitogenesis
(Fig. 4A) and was later
expressed in a subset of cells in the pronephros in a pattern similar to that
of the MCC-marker gene expression (Fig.
4B). Double staining by jagged 2 in situ hybridization
and anti-acetylated tubulin immunohistochemistry demonstrated that jagged
2-positive cells were indeed multiciliated
(Fig. 4C). A bundle of cilia
could be seen originating from a single jagged 2-expressing cell in a
cross section of the pronephros (Fig.
4C). To confirm further the identity of jagged
2-expressing cells, we performed two-color and double fluorescent in situ
hybridization with jagged 2 and shippo1 probes. By both
methods, we found that shippo1 was co-expressed with jagged
2 in a distinct subset of cells (Fig.
4D,E-G), confirming that jagged 2 is specifically
expressed in MCCs.
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) and the J2ex20MO was found to delete exon 20 and generate an
in-frame stop codon immediately following the coding sequence of exon 19, with
the resultant mRNA predicted to encode a truncated jagged 2
extracellular domain peptide (see Fig. S1 in the supplementary material).
Injection of either morpholino oligo gave rise to the same phenotype: the
number of MCCs was dramatically expanded in the pronephros
(Fig. 5A-I). Injection of a
control invert sense morpholino had no effect on the expression pattern of the
MCC markers shippo1, flr or rfx2
(Fig. 5). Sections of
whole-mount stained embryos confirmed that the single-cell pattern of
MCC-marker expression (Fig. 5J)
expanded to include all tubule cells in the section in jagged 2
morphants (Fig. 5K,L).
Interestingly, expression of jagged 2 itself was also dramatically
expanded in jagged 2 morphants
(Fig. 5R,U). One interpretation of the enhanced expression of MCC markers in jagged 2 morphants would be that MCC cell number was increased by the transfating of transporting epithelial cells to MCCs. Alternatively, it is possible that MCC and transporting-cell phenotypes could co-exist in the same cells and that jagged 2 loss of function may simply be derepressing axonemal gene expression. We therefore examined jagged 2 morphants for expression of the transporters trpM7, slc13a1 and Na,K-ATPase alpha1A4:GFP. Strikingly, jagged 2 morphants showed a complete loss of all midsegment ion-transporter gene expression (Fig. 5M-Q) as well as a segment-specific reduction in Na,K-ATPase alpha1A4:GFP expression (Fig. 5S,T). As a further test for specificity, we also knocked down jagged 1b expression and assessed MCC and transporter gene expression. jagged 1b knockdown had no effect on marker gene expression, indicating that the observed effects were specific to jagged 2 (data not shown). The results suggest that jagged 2 acts as a repressor of a genetic pathway leading to MCC differentiation and that part of this patterning mechanism involves jagged 2 repression of its own mRNA synthesis in a subset of pronephric cells. Additionally, the fates of MCCs and transporting epithelial cells appear to be mutually exclusive, with jagged 2 repression of MCC fate being necessary to allow neighboring cells to acquire a secondary, transporting-epithelial-cell fate.
|
) and was
a reasonable candidate for mediating the effects of Jagged 2. We confirmed
that notch3 mRNA was expressed strongly in the proximal and distal
pronephros, and is diffusely expressed in the pronephric midsegment
(Fig. 6A,B). Knockdown of
notch3 with a morpholino oligo targeting notch3 exon 27
produced a morphant `allele' truncated after exon 26 (stop codon following 20
mis-sense amino acids; see Fig. S1 in the supplementary material) and lacking
both the transmembrane domain and the notch3 intracellular domain
required for nuclear signaling. Similar to jagged 2 morphants, both
shippo1-positive (Fig.
6C,D) and flr-positive
(Fig. 6E,F) cells were
significantly increased in number in notch3 morphants, whereas the
expression of slc13a1 (Fig.
5G,H) and trpM7 (Fig.
5I,J) was nearly completely abolished in the early-distal
pronephros.
Regulation of pronephric cell patterning requires mind bomb activity
mind bomb mutants lack an E3 ubiquitin ligase that is required to
process Notch ligands and to facilitate Notch signaling
(Itoh et al., 2003). To test
whether Jagged 2 signaling required mind bomb activity, we examined
MCC and transporting-cell markers in mind bomb homozygotes.
Consistent with the jagged 2- and notch3-morphant
phenotypes, expression of the MCC markers shippo1 and jagged
2 was dramatically expanded in mind bomb homozygotes
(Fig. 7A-D). shippo1
expression in single cells of the distal segment
(Fig. 7A) was expanded to
include nearly all cells of this segment
(Fig. 7B, ds). In
addition, the number of shippo1-positive cells was significantly
increased in the proximal segment of the pronephros
(Fig. 7B, ps).
Expression of the transporting-cell markers trpM7 and
slc13a1 was nearly completely abolished
(Fig. 7E-H). These results
indicate that mind bomb-processing activity is required for
jagged 2/notch3 patterning of the pronephros.
Timing of Jagged 2/Notch3 signaling during pronephric development
jagged 2 mRNA expression is initiated early in the intermediate
mesoderm and is maintained in fully differentiated MCCs
(Fig. 4A,B). To determine when
Jagged 2/Notch3 signaling acts to restrict the MCC fate and to assess whether
continued Jagged 2/Notch3 signaling is required to generate the mature pattern
of cell types in the pronephros, we used the gamma-secretase inhibitor DAPT to
block notch cleavage (Geling et al.,
2002) at different time points during pronephric development.
Expansion of MCC fate (increased shippo1 expression;
Fig. 8A-C) and the loss of
trpM7-expressing transporter cells
(Fig. 8G-I) occurred when DAPT
was added to egg water at 9.5 hpf, but not if it was added later, at 24 hpf
(Fig. 8). Interestingly,
slc13a1 expression was sensitive to inhibition of Notch signaling at
later developmental stages (24+ hpf; Fig.
8J-L), indicating that, although expressed in the same cells,
trpM7 and slc13a1 may respond to Notch signaling in
different ways. The effect of DAPT on MCC differentiation suggested that cell
fate choice is decided during late gastrulation and early somitogenesis, and
that continued Notch3 signaling after 24 hpf may not be required to limit the
number of MCCs. To further refine the time window in which Jagged 2/Notch
signaling acts, we treated embryos with DAPT starting at progressively later
time points and assayed the number of MCCs at 34 hpf by counting the number of
shippo1-positive cells in each pronephric nephron. As shown in
Fig. 9A, control pronephric
tubules contained, on average, 26±4 (±s.d., n=17) MCCs,
and treatment with 100 µM DAPT at 9.5 hpf increased this number to
67±14 (n=8; although this is possibly an underestimate of MCC
number given the difficulty in counting overlapping shippo1-positive
cells). MCC differentiation was sensitive to DAPT only during a narrow time
window (around 9-10 hpf), and later treatment at 12 or 15 hpf did not
significantly affect the number of MCCs
(Fig. 9A).
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Enhanced ciliogenesis and rescue of the double bubble kidney cyst mutant by jagged 2 knockdown
The strong expression of ciliogenic genes and the absence of transporter
gene expression suggested that loss of Jagged 2/Notch signaling could cause
cells to fully transfate to the MCC cell type. To assess whether jagged
2-morphant pronephric cells acquired the functional character of MCCs, we
examined the abundance and motility of cilia in morphant embryos.
Immunostaining with anti-acetylated tubulin revealed that the loss of
jagged 2 function increased the abundance of lumenal cilia in the
pronephros (Fig. 10A,B).
Moreover, the newly formed cilia in jagged 2-morphant pronephroi all
beated rapidly (see Movie 2 in the supplementary material), similar to the
behavior of wild-type pronephric cilia (see Movie 1 in the supplementary
material). To better visualize beating cilia, the pronephric opening of these
embryos was mechanically obstructed to expand the pronephric lumen. Notch3
morphants also showed an increase in cilia bundles in the pronephros (data not
shown). These results indicate that loss of Jagged 2/Notch signaling
not only enhanced axonemal gene expression, but was sufficient to transfate
pronephric cells to fully functional MCCs.
Motile cilia in the pronephros are required to maintain high rates of fluid
output and prevent backpressure, which can lead to pronephric cyst formation
(Kramer-Zucker et al., 2005).
The transfating of epithelial cells to MCCs in jagged 2 morphants
raised the possibility that the enhancement of the ciliogenic program might be
sufficient to compensate for deficiencies in ciliogenesis that are observed in
a class of pronephric cyst mutants. We therefore tested whether jagged
2 knockdown in pronephric cyst mutant embryos would have any effect on
cilia structure, cilia motility and/or pronephric cyst formation. We screened
five different pronephric cyst mutants [flr, oval (ift88),
tg238a, schmalhans and double bubble] for potentially
beneficial effects of jagged 2-morpholino injection on ciliogenesis.
Based on an apparent improvement in phenotype (absence of cysts and edema),
one of these mutants, double bubble (m468)
(Drummond et al., 1998), was
chosen for further analysis. The double bubble (dbb) mutant is
characterized by a loss of cilia structure
(Fig. 10E,G) with consequent
lumen dilatation and pronephric cyst formation
(Drummond et al., 1998).
dbb homozygotes can also be identified by a characteristic ventral
axis curvature (Drummond et al.,
1998). High-speed micro videos showed that dbb
homozygotes lack detectable motile cilia (see Movie 4 in the supplementary
material) in comparison to similar pronephric segments in wild-type embryos
(see Movie 3 in the supplementary material). Significantly, jagged 2
knockdown rescued normal cilia formation in double bubble-mutant
embryos (Fig. 10C,D). Rescued
cilia bundles in dbb homozygotes were actively motile (see Movie 5 in
the supplementary material), similar to wild-type cilia (see Movie 3 in the
supplementary material). Remarkably, jagged 2 knockdown completely
prevented pronephric cyst formation in dbb homozygotes and restored
wild-type pronephric structure to mutant embryos
(Fig. 10E-H). In three
separate experiments, injection of jagged 2 exon 20 donor-morpholino
into embryos from pair matings of dbb heterozygotes prevented cyst
formation (embryos with cysts/total embryos in clutch: 0/42, 0/46 and 0/41).
Control morpholino-injected embryos developed cysts at standard Mendelian
frequencies (25%) and injection of the same concentration of jagged
1b morpholino or control morpholinos had no effect on the
dbb-mutant phenotype (data not shown). We also tested whether DAPT
treatments that significantly increased MCC marker gene expression
(Fig. 8B,
Fig. 9A) would affect cyst
formation in double bubble mutants. Treating dbb-mutant
embryos with 100 µM DAPT at 9.5 hpf significantly reduced cyst formation
(Fig. 10I,J). These results
strengthen our previous findings that motile cilia are key organelles in fluid
transport in the pronephros (Kramer-Zucker
et al., 2005) and further suggest that the capacity of the kidney
for fluid transport can be modulated by Notch-mediated control of the number
of multiciliated versus transporting cells during a crucial stage of
kidney-nephron patterning.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In
the pronephros, multiciliated cells form the biological equivalent of a `screw
pump' by beating in a helical wave pattern in a tight-fitting tubule lumen to
propel fluid out of the pronephric opening
(Kramer-Zucker et al., 2005),
while transporting epithelial cells recover specific ions from filtered blood
depending on the spectrum of ion transporters that they express. Very little
is known about how kidney-tubule cells achieve their different identities.
What we have shown is that a patterning mechanism underlying cell-type
specification in the pronephros involves Jagged 2/Notch signaling. Our
findings point to a lateral inhibition mechanism in generating a
`salt-and-pepper' pattern of multiciliated cells intercalated among
transporting cells.
Notch signaling and kidney development
Previous studies on mouse kidney development have demonstrated roles for
Notch signaling in glomerulus formation and in specifying the fates of
proximal versus distal nephron tubule segments. Notch2 expression in
glomerular epithelial cells and Jagged 1 expression in endothelial cells is
required for development of the glomerular vasculature
(McCright, 2003). Defects in
glomerular vasculature structure have also been reported in combined
jagged 2/jagged 1b morphants in zebrafish, suggesting that similar
developmental mechanisms may underlie zebrafish pronephric development
(Lorent et al., 2004). General
inhibition of Notch signaling by gamma-secretase inhibition or by mutations in
mouse presenilin genes results in a loss of both glomerular and proximal
tubule cells, and in the preservation of the distal nephron fate
(Cheng et al., 2003). Jagged 1
signaling has also been shown to regulate branching morphogenesis of the renal
collecting system (Kuure et al.,
2005). In pronephric development in Xenopus, Notch
signaling also regulates the patterning of proximal versus distal tubule types
(McLaughlin et al., 2000).
Together, these studies point to a role for Notch signaling in nephron
precursor structures (S-shaped bodies or their equivalents) in broadly
defining major nephron segments and structures.
|
). Although mechanisms regulating intercalated cell
differentiation are currently unknown, the expression of multiple notch and
jagged genes in the nephron suggests that notch signaling could play a role
(Challen et al., 2006). In the
zebrafish larval intestine, secretory and adsorptive cells exist intermixed,
and this balance of cell types is controlled by a Notch-mediated lateral
inhibition mechanism involving deltaD expression in secretory cells
(Crosnier et al., 2005). In the
zebrafish liver, the hepatocyte/cholangiocyte cell-fate choice also appears to
be under the control of Notch signaling
(Lorent et al., 2004). In the
pancreas, disruption of Notch signaling by mutation in mind bomb or
by expression of a dominant-negative Suppressor of Hairless construct
accelerates differentiation of exocrine acinar cells
(Esni et al., 2004). The
picture that emerges from these studies is that Notch signaling may act at
multiple stages of organogenesis to first regulate the timing and overall
pattern of organ progenitor cell differentiation, and may later serve to
modify cell-type specification by superimposing a repression mechanism that
regulates the proportion of different cell types in the mature organ.
Notch signaling pathways and the emergence of multiciliated cells
The work presented here suggests that, early in pronephric cell
differentiation (8-10 hpf), cells expressing high levels of Jagged 2 interact
with neighboring Notch-expressing cells (summarized in
Fig. 11). Jagged 2/Notch
signaling requires the E3 ligase mind bomb, and initiates proteolysis
of Notch3 and the liberation of the transcriptionally active NICD. The NICD
activates expression of unknown repressors that downregulate rfx2
(and downstream cilia genes) and jagged 2. Later in development, a
subset of cells becomes multiciliated while neighboring cells acquire a
transporting-epithelial-cell fate and express ion channel genes.
In other Notch signaling contexts, the hairy/enhancer of split family of
genes exert the transcriptional-repressor function of Notch signaling
(Iso et al., 2003). We have
found that her9 is expressed in the pronephros (see Fig. S2 in the
supplementary material); however, it is clear that other factors must act to
repress ciliogenic gene expression, because the expression of rfx2
and shippo1 was unaffected by a her9-translation-blocking
morpholino (Y.L. and I.A.D., unpublished). Whatever the identity of
Notch-induced transcriptional repressors may be, it is likely that the
transcription factor rfx2 may be a primary target of repression,
because it belongs to a gene family known to serve as master regulators of
ciliogenic gene expression in organisms ranging from C. elegans to
mammals (Bonnafe et al., 2004;
Efimenko et al., 2005;
Swoboda et al., 2000).
Repression of rfx2 in neighbors of MCC would be expected to restrict
abundant ciliogenic gene expression to the MCCs and limit MCC number. This
idea is supported by our finding that rfx2 morpholino injection
results in pronephric cyst formation and in an absence of motile pronephric
cilia (Y.L. and I.A.D., unpublished).
|
; Heitzler and Simpson,
1991), this cell interaction would be predicted to exaggerate
differences between neighboring cells in the intermediate mesoderm, where
jagged 2 is first expressed in kidney cells, and to drive the initial
emergence of MCC progenitors from a common pool of cells. Although we have characterized several elements of Notch signaling in pronephric patterning, aspects of our data indicate that the framework is incomplete. In embryos in which Notch signaling has been generally inhibited (i.e. mind bomb mutants and DAPT-treated embryos), the transfating of cells to MCCs is observed not only in the pronephric distal segment but also in the proximal segment, where MCC cells are not as frequently seen, suggesting that additional notch genes or Notch ligands may regulate MCC differentiation. These factors are unlikely to include jagged 1b, because single knockdown of jagged 1b or double knockdown of jagged 1b and jagged 2 did not replicate the mind bomb phenotype (data not shown).
Development and functions of cilia
The importance of cilia in normal organ function is highlighted by the
pathology observed in organisms with non-functional cilia: kidney cystic
disease, retinal degeneration, organ laterality defects and hydrocephalus
(Pazour, 2004). Mutations in a
variety of ciliaassociated proteins cause kidney tubules to become cystic
(Barr et al., 2001;
Blacque et al., 2004;
Fan et al., 2004;
Haycraft et al., 2001;
Kim et al., 2004;
Kramer-Zucker et al., 2005;
Morgan et al., 2002;
Murcia et al., 2000;
Mykytyn et al., 2004;
Otto et al., 2003;
Pazour et al., 2000;
Pazour and Rosenbaum, 2002;
Qin et al., 2001;
Sun et al., 2004;
Yoder et al., 2002). In
cultured mammalian epithelial cells, cilia are proposed to function as
non-motile fluid-flow sensors that regulate epithelial responses to flow via
calcium signaling (Nauli et al.,
2003; Praetorius and Spring,
2001). We have demonstrated a role for motile cilia in cystic
kidney pathology that affects the zebrafish pronephros
(Kramer-Zucker et al., 2005).
Multiciliated cells similar to what we describe here in the zebrafish have
also been shown to exist intercalated between proximal tubule cells in the
human kidney (Duffy and Suzuki,
1968; Hassan and Subramanyan,
1995; Katz and Morgan,
1984; Ong and Wagner,
2005). The function of these cells is currently unknown. Although
there is no evidence for Notch signaling in the development of these cells,
studies of epidermal development in Xenopus have highlighted a role
for Notch signaling in the context of ciliated-cell development. The regularly
spaced pattern of MCC differentiation in the Xenopus epidermis has
been shown to be regulated by X-Delta-1 expression in a subset of epidermal
cells, which later abundantly express alpha-tubulin and become multiciliated
(Deblandre et al., 1999). Taken
together with our work, the results suggest that Notch signaling might be a
general feature of specialized ciliated cell development.
Our finding that jagged 2 knockdown can rescue cilia assembly in the double bubble mutant and prevent cyst formation strengthens our previous results that cilia-driven fluid flow in the pronephros is a key factor in cystic pathology. This result also raises questions concerning the process of ciliogenesis and the nature of the dbb mutation. It is not clear at present why only dbb and not other ciliogenesis mutants are rescued by jagged 2 knockdown. The dbb allele we are studying (m468) could be a hypomorphic allele or, alternatively, the dbb protein may not be absolutely essential for ciliogenesis. Other intraflagellar transport (IFT) proteins, if expressed in high enough amounts, may be sufficient to compensate for the loss of dbb. Further characterization of the dbb mutation and its interactions with other proteins involved in IFT will be necessary to resolve these issues.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/6/1111/DC1
|
ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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