1 Max-Planck-Institut für Entwicklungsbiologie, Spemannstr. 35, 72076
Tübingen, Germany
2 Centre for Developmental Genetics, Department of Biomedical Science,
University of Sheffield, Firth Court, Sheffield S10 2TN, UK
3 Oregon Hearing Research Center and Vollum Institute, Oregon Health &
Science University, Portland, OR 97201, USA
* Author for correspondence (e-mail: nicolson{at}ohsu.edu)
Accepted 30 October 2003
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SUMMARY |
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Key words: dfna5, Deafness, Zebrafish, Semicircular canals, Pharyngeal cartilage, UDP-glucose dehydrogenase, Hyaluronic acid
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Introduction |
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DFNA5 is a non-syndromic autosomal dominant form of progressive hearing
loss that was identified in an extended Dutch family (van Camp et al., 1995;
DeLeenheer et al., 2002). High frequency hearing loss starts in the first or
second decade of life. The patients carry a complex intronic
insertion/deletion mutation that leads to truncation of the protein
(Van Laer et al., 1998).
DFNA5 encodes a protein of 496 amino acids that is found in human
cochlea, brain, placenta and kidney cDNA preparations. DFNA5 is a novel
protein, sharing no obvious homology to other proteins, and as there is no
animal model, nothing is known about its molecular function or what role it
plays during vertebrate development. To gain insight into a possible function
for Dfna5, we examined its expression pattern in developing zebrafish embryos
and its function using a morpholino knock-down strategy. Our phenotypic
analysis suggests that Dfna5 function is essential for development of the
semicircular canals and the pharyngeal cartilage, and acts at the level of
extracellular matrix production.
The extracellular matrix plays an important role in cell migration,
differentiation and morphogenesis of organs such as the ear and jaw. The ear
and the pharyngeal cartilage arise from the same region of the embryo. In
zebrafish, the otic placode develops from the cells overlying the second
cranial neural crest (CNC) stream, adjacent to rhombomeres 5 and 6, at about
14 hpf (hours post fertilization). At around 45 hpf, the epithelial
projections of the developing semicircular canal begin to grow out. At 52 hpf,
the opposing projections of the anterior and posterior canals touch and fuse.
This process is completed with the fusion of the ventral column at 64 hours.
The directed outgrowth of the protrusions is dependent on secretion of
extracellular matrix (ECM), including hyaluronic acid (HA), into the extending
lumen between the epithelium and the underlying mesenchyme
(Haddon and Lewis, 1991). The
ECM is later replaced by invading fibroblasts.
(Haddon and Lewis, 1991
;
Haddon and Lewis, 1996
;
Waterman and Bell, 1984
).
The cartilage that gives rise to the jaw and gills is called pharyngeal
cartilage and is of neural-crest origin
(Schilling and Kimmel, 1994).
Pharyngeal cartilage is derived from three streams of cranial neural crest
(CNC) that begin to migrate at 12 hpf. The most anterior two CNC streams give
rise to the cartilage of the upper and lower jaw, and the posterior stream is
subdivided by endodermal pouches to give rise to the five sets of branchial
(gill) cartilage. The pharyngeal cartilage condensations begin to
differentiate starting at 53 hpf as seen by Alcian Blue staining (reviewed by
Kimmel et al., 2001
;
Schilling and Kimmel, 1994
).
The early steps of jaw development are regulated by Sox9, under the control of
bone morphogenetic proteins (BMPs) and sonic hedgehog (Shh)
(de Crombrugghe et al., 2001
).
After condensation, the differentiating chondrocytes begin to express
col2a1, the main collagen of differentiated cartilage, col11a2,
aggrecan and other ECM proteins under the control of Sox5 and Sox6. In
differentiated cartilage, only 5% of the volume is occupied by chondrocytes,
with the remainder consisting of ECM.
Not surprisingly, many genes expressed in the posterior region of the head
regulate the development of both the ear and pharyngeal cartilage. For
example, expression of fgf3 and fgf8 in rhombomere 4 is
required for induction of the ear placode and formation of branchial cartilage
(Maroon et al., 2002;
Leger and Brand, 2002
;
Liu et al., 2003
). A mutation
in another gene, jekyll, leads to interrupted ear columns and a lack
of cranial cartilage (Neuhauss et al.,
1996
). jekyll encodes Udgh, an enzyme required for
synthesis of proteoglycans including HA
(Walsh and Stainier, 2001
). We
show that a knock-down of dfna5 function in zebrafish causes the loss
of ugdh expression and reduction of HA levels, leading to
malformation of the semicircular canals of the ear and pharyngeal
cartilage.
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Materials and methods |
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We analyzed the genomic organization of zebrafish dfna5 partially by blasting the cDNA sequence against genomic zebrafish sequence and partially by PCR on genomic sequence with subsequent cloning and sequencing of the obtained PCR-products.
Whole-mount in situ hybridization
We amplified full-length dfna5 using the forward primer A5-bf
5'-AGCACGAGCTGATCCTCAAA-3' and reverse primer A5-br
5'-AAGCCTCGTCTGTTTCGTGC-3', cloned the 1990 bp fragment and used
it as a template to synthesize a digoxigenin labeled in situ RNA probe. For
col2a1 we used the forward primer
5'-CTGGATCTGATGGTCCACCT-3' and reverse primer
5'-ACATGGCTGGATTTCAGAGG-3' to amplify a 1593 bp fragment and for
ugdh the forward primer 5'-GACGTACGGTATGGGCAAAG-3' and
reverse primer 5'-TTGATTTCCAGCAATGGTCA-3' to amplify a 1304 bp
fragment. Both PCR-product were used as described for the dfna5 in
situ probe. Whole-mount in situ hybridization was performed as described
previously (Thisse et al.,
1993).
Antisense morpholino oligonucleotide and DNA injections
We designed one antisense morpholino oligonucleotide (MO, Gene Tools)
directed against the 5' sequence around the putative start codon to
block dfna5 translation ("ATG-MO") and one MO against the
splice donor site of exon 8 to interfere with splicing ("GT-MO").
The sequences for the ATG-MO and the GT-MO were
5'-TGCAAACATCTTCAATGCTGACAAG-3' and
5'-TGATTTAACTGAACTCACCGTCTAG-3', respectively. An additional
morpholino with four base pair exchanges was designed for control injections.
The sequence for the mismatch MO was
5'-TGCGAACACCTTTAATGCTAACAAG-3'. We injected concentrations
between 10 and 40 ng in single cell embryos in a volume of 5 nl.
For rescue experiments we amplified full-length dfna5 with the primers used for generation of the in situ probe template, cloned it in pCS2+ vector and identified a mutation free clone by sequencing. The plasmid was co-injected in single cell embryos with different concentrations of ATG-MO in a concentration of 50 pg/nl.
Histology and electron microscopy
Whole larvae (day 5) were anesthetized with 0.02% MESAB (3-aminobenzoic
acid ethyl ester) and then fixed in 2.0% glutaraldehyde and 1.0%
paraformaldehyde in PBS overnight to several days at 4°C. Specimens were
fixed with 1.0% OsO4 in H2O for 10 minutes on ice,
followed by fixation and contrast with 1.0% uranyl acetate for 1 hour on ice,
and then dehydrated with several steps in ethanol and embedded in Epon.
Sections (5 µm) were stained with Toluidine Blue. Ultrathin sections were
stained with lead citrate and uranyl acetate.
Alcian-Blue cartilage staining
Cartilage was stained with Alcian Blue as described previously
(Schilling et al., 1996).
Whole mount labeling for hyaluronic acid
Freshly fixed larvae (4% PFA in PBS overnight at 4°C) were
permeabilized for 6 hours at room temperature with 2% Triton, 4% PFA in PBS.
After washing several times in TBS, larvae were incubated with 10 µg/ml
biotinylated hyaluronic acid binding protein (HABP) from Seikagaku corporation
in TBS overnight at 4°C. The HABP was removed and the larvae washed in TBS
three times for 20 minutes. After 4 hours blocking with 2% BSA in TBS at room
temperature, the larvae were incubated with anti-biotin rabbit antibody (Heinz
Schwarz, Tübingen), diluted 1:50 in TBS for 3 hours at room temperature.
The larvae were washed in TBS three times for 20 minutes and incubated
overnight at 4°C with alexa-Fluor-coupled anti-rabbit antibody, diluted
1:500. After rinsing several times, larvae were mounted on a glass slide in
1.5% low melting point agarose and examined.
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Results |
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Knock-down of dfna5 leads to ear and jaw malformation
To determine the function of dfna5 in zebrafish, we analyzed the
phenotype in animals injected with either ATG or GT dfna5
morpholinos. Injection of the ATG-MO (Fig.
4) or the GT-MO caused malformation of the semicircular canals of
the ear. Either anterior, posterior or both columns of the developing
semicircular canals were not fused (Fig.
4E,G,I). At higher doses the lateral canal also did not form
(Fig. 4J). The projections,
which grow out and fuse to form the columns, were present, but short and
thickened. Other structures of the ear, like the cristae or maculae, were
unaffected.
|
As dfna5 mRNA is highly expressed in the intermediate cell mass which gives rise to blood cells, we analyzed this tissue in more detail. We did not see any change in expression of scl, an early blood marker (data not shown). In addition, staining of blood cells with o-dianisidine did not reveal any differences (data not shown).
To exclude the possibility that toxicity of injecting high amounts of morpholino led to the jaw and ear phenotype, we examined cell death in MO-injected animals. Embryos injected with two different doses of ATG-MO (20-40 ng) were stained with Acridine Orange at 35 hpf and 48 hpf. We did not observe elevated levels of cell death in any tissue compared with uninjected embryos (data not shown).
We observed a strong correlation between the amount of MO injected and the resulting phenotype, indicating the specificity of the morpholino knockdown of dfna5 (Fig. 5A). In addition, injection of a 4 bp mismatch control oligonucleotide did not produce any effects on ear or jaw development (Fig. 5B). To gain further evidence of specific interference with dfna5 function, we co-injected dfna5 ATG-MO with an expression construct containing the full-length coding sequence of dfna5 under the control of the cytomegalo virus (CMV) promoter into one cell-stage wild-type embryos. We found a partial rescue of the phenotype (Fig. 5C,D) at day 5 in 20% of the embryos with strongly affected ears and jaw (n=20). In partially rescued larvae, one ear and the lower jaw showed severe malformations typical for injection of high doses of ATG-MO, whereas the other ear was completely normal. A partial rescue was expected because of the highly mosaic expression of injected DNA. Injection of ATG-MO alone never led to the above `rescued' phenotype of abnormal jaw morphology combined with normal ear morphology. Only low doses of ATG-MO or GT-MO yielded defects in one ear accompanied by normal morphology in the other ear. However, this unilateral defect was never present in combination with a jaw defect, as seen in larvae co-injected with the expression construct.
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Hyaluronic acid levels are strongly reduced in dfna5 morphants
The biosynthesis of HA depends upon the activities of a number of enzymes,
including Ugdh. We tested whether the loss of ugdh in the developing
semicircular canals affected the production of HA in these structures. We
examined the levels of HA in vivo using a biotinylated HA-binding protein
(Fig. 9). As shown in
Fig. 9C, HA is highly abundant
in the semicircular projections of 54 hpf wild-type larvae. Morphant embryos,
however, have reduced levels of HA in the protrusions
(Fig. 9D-F). This strongly
suggests that reduced HA levels lead to the failure of semicircular canal
formation in the ear and supports previous findings that HA is important for
projection outgrowth.
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Discussion |
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dfna5 is essential for semicircular canal formation in the inner ear and cartilage formation in the jaw
During development of the inner ear, three projections emerge from the
epithelium lining the otic vesicle (Haddon
and Lewis, 1996; Waterman and Bell, 1994). These projections or
epithelial columns then grow towards the center of the otic vesicle where they
fuse and form the rudiments of the three semicircular canals. In larvae
injected with either ATG or GT-MOs directed against dfna5, the
columns fail to fuse. Instead, they appear as short and thickened masses, with
a loss of an epithelial monolayer. As judged by thick sections, the number of
cells is not reduced, but rather the space in between cells is increased. This
phenotype suggests that these cells were unable to coordinate their movements
to form elongated columns.
An additional phenotype occurs in animals injected with the ATG-MO that is predicted to mimic a loss-of-function allele. The pharyngeal cartilage is reduced in size and distorted. The cells stain weakly with Alcian Blue, suggesting that cartilage differentiation and production of the cartilage-specific extracellular matrix is disrupted. During differentiation, chondrocytes express high levels of collagens. Our results indicate that a collagen highly expressed by chondrocytes, col2a1, is reduced in dfna5 morphants, suggesting that dfna5 is required for differentiation of chondrocytes.
The role of HA in the developing ear
The phenotype seen in dfna5 morphants is similar to the phenotype
observed in zebrafish jekyll mutants
(Neuhauss et al., 1996). These
loss-of-function mutants have short and thickened epithelial columns that fail
to elongate and fuse and cartilage differentiation is also affected. The
jekyll gene encodes Ugdh, an enzyme necessary for the production of
the dissaccharide unit of HA (Walsh and
Stainier, 2001
). Based on the similar phenotypes, we hypothesized
that dfna5 may be involved in the ugdh pathway. In
MO-injected animals, we find that loss of dfna5 function leads to
loss of ugdh expression in the ear and the developing pharyngeal
arches. We also find that HA is reduced in the developing semicircular canals
of the dfna5 morphants. Moreover, loss of HA is associated with
reduced levels of col2a1 expression, affecting differentiation of the
branchial cartilage. By contrast, col2a1 expression in morphant ears
is normal, supporting our hypothesis that loss of ugdh in the
developing ear is not affecting differentiation of the ear cavity, but only
the directed outgrowth of the canal projections.
In the ear, HA has been shown to play a role in the outgrowth of
projections that fuse to form columns
(Haddon and Lewis, 1991). HA
is made of variable numbers of disaccharide units capable of forming filaments
up to several micrometers in length. In the growing columns, epithelial cells
at the tip of each protrusion are thought to secrete these long molecules that
can act as a `propellant'. Secretion of HA by a subset of cells within the
projection may provide a driving force for growth in a particular direction.
When HA is enzymatically removed in the epithelial columns, a similar
phenotype of defective outgrowth is seen
(Haddon and Lewis, 1991
). In
dfna5 morphants, the reduction of HA levels results in an
uncoordinated outgrowth of the epithelial projections. The epithelial cells
are still proliferating, but the projection appears to lack the directional
force of HA secretion by the cells at the leading edge. This causes
disorganization of the epithelium as reflected by the missing basal lamina and
stacking of cells. Controlled breakdown of the basal lamina underlying the
outpocketing epithelium appears to be crucial for proper outgrowth. In
wild-type canal projections, only the cells at the leading edge of the
protrusion lack a basal lamina. In netrin 1 mouse mutants, the opposite
situation is found: the basal lamina does not break down at all. However, this
defect also results in unfused columns
(Salminen et al., 2000
),
suggesting that only spatially and temporally correct remodeling of the basal
lamina allows proper outgrowth and fusion of the epithelial projections.
Members of the protein family that share motifs with DFNA5 that are
described in the literature are cancer-associated
(Saeki et al., 2000;
Thompson and Weigel, 1998
;
Lage et al., 2001
;
Watabe et al., 2001
) (see
http://www.sanger.ac.uk/cgi-bin/Pfam/getacc?PF04598
for DFNA5 family). This is particularly compelling because high levels of HA
are a prognostic indicator for malignancy in clinical cases of human breast,
ovarian and colon carcinomas. HA may promote invasiveness of cancer cells by
providing a highly hydrated microenvironment that facilitates detachment and
migration of cells (Toole, 2002). One possibility is that proteins of this
family are able to regulate HA biosynthesis. All members of this protein
family notably contain one (in the case of DFNA5) or more putative HA-binding
sites. This motif is called BX7B domain because it contains two
basic amino acids (arginine or lysine) separated by seven non-acidic amino
acids (Yang et al., 1994
).
This domain is found in all hyaladherins, proteins that interact with HA, such
as aggrecan or CD44. Because this is a very common motif, it is not clear
whether this domain is sufficient for binding to HA.
If Dfna5 regulates the HA biosynthetic pathway, it may act at the
transcriptional level. Evidence for this notion is supported by a recent
heterologous expression study in which human DFNA5 was expressed in
S. pombe cells (Gregan et al.,
2003). Gregan and coworkers found a genetic interaction with the
S. pombe gene mcm10 (cdc23), which is involved in
DNA replication. Interestingly, both DFNA5 and Mcm10p contain similar
zinc-finger-like motifs that define the Mcm10 family of DNA replication
proteins.
Human DFNA5 hearing loss
How does our study add to the understanding of the human DFNA5
disease? The anatomy of the vestibular inner ear, including the semicircular
canals, is highly conserved among vertebrates
(Riley and Phillips, 2003).
Certain aspects of the development and morphogenesis of semicircular circular
canals are similar. For example, in fish, frogs, birds and mammals, apposing
epithelial walls or protrusions contact and fuse to form the rudiments of the
semicircular canals (Haddon and Lewis,
1991
; Martin and Swanson,
1993
; Haddon and Lewis,
1996
; Fekete et al.,
1997
). Development of the pharyngeal arches is also conserved
among vertebrates (Graham,
2003
). However, individuals with a mutation in DFNA5
display neither obvious vestibular defects nor craniofacial malformations.
Only one family has been found with a single mutation in DFNA5 thus
far. Whether the truncation of DFNA5 in humans results in haplo-insufficiency
remains to be determined. Typically, only severe mutations such as null
mutations cause syndromic hearing loss (Astuto et al., 2002). Syndromic
deafness associated with craniofacial and/or skeletal deformations have been
reported for three genes, COL2A1, COL11A1 and COL11A2
(reviewed by Morton, 2002
).
All three collagens are highly abundant in cartilage extracellular matrix (de
Crombrugghe, 2001). Mutations in COL11A2 cause syndromic as well as
nonsyndromic hearing loss: congenital hearing loss is accompanied by various
skeletal abnormalities in the case of Stickler syndrome (which can also be
caused by mutations in COL2A1 and COL11A1), whereas
individuals with a mutation in DFNA13 suffer from nonsyndromic
progressive hearing loss at high and middle frequencies (Sirko-Osadsa, 1998;
McGuirt et al., 1999
;
Kunst et al., 2000
). The
Marshall syndrome is associated with splicing mutations in COL11A1
and characterized by craniofacial and skeletal abnormalities, cataracts and
progressive sensorineural hearing loss. Computer tomography did not detect any
malformations of ear bones. Therefore, hearing loss is thought to be due to
direct effects on COL11A1 loss in the labyrinth and CNS
(Griffith et al., 2000
). In all
three cases, genes affecting cartilage differentiation can cause progressive
sensorineural hearing loss without causing cartilage malformations in the
human ear.
This leads us to speculate that a complete loss of DFNA5 function in humans
could cause cartilage phenotypes as seen in individuals with Stickler
syndrome. Determination of the expression pattern of Dfna5 in mammals
will shed some light on this paradox. Nevertheless, HA is highly abundant in
many areas of the developing human inner ear. It is suggested to serve there
as a friction-reducing lubricant and molecular filter
(Anniko and Arnold, 1995). If
HA biosynthesis is reduced or lost in the ear, it is possible that this
disruption of extracellular matrix causes increased mechanical stress on hair
cells. This stress may lead to premature aging of hair cells and a progressive
hearing loss, reminiscent of age-related hearing loss that also starts with
high frequency sound.
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