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First published online 30 November 2005
doi: 10.1242/dev.02182
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1 Max Delbrück Center (MDC) for Molecular Medicine, Robert-Rössle
Strasse 10, 13125 Berlin, Germany.
2 The Charité, Department of Cardiology, Campus Buch and Campus Virchow
Clinics, Humboldt University, Berlin, Germany.
* Author for correspondence (e-mail: salim{at}mdc-berlin.de)
Accepted 25 October 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Organ morphogenesis, prkci, Myocardium, heart and soul, nagie oko, mpp5, pals1, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Yelon et al., 1999).
Subsequently, the heart cone moves from the dorsoventral into the
anterior-posterior plane, a process referred to as tilting. As a consequence,
the ventricular portion of the heart cone comes to lie more posteriorly.
Finally, the heart cone elongates along the anterior-posterior axis into the
heart tube, which is converted into a looped, two-chambered organ.
The heart and soul (has) mutation causes severe defects
in cardiac morphogenesis, in which tilting of the heart cone is blocked and
heart tube elongation is impaired (Yelon
et al., 1999; Horne-Badovinac
et al., 2001; Peterson et al.,
2001). The has gene encodes PRKCi, which is required for
the establishment of apicobasal polarity of epithelial cells. PRKCs are
components of the apical Par3 protein complex, which has been linked to the
apical Crumbs-Pals1/Mpp5 protein complex
(Hurd et al., 2003;
Nam and Choi, 2003;
Wang et al., 2004). Consistent
with a function in apicobasal cell polarity, has mutants show
defective formation and maintenance of several embryonic epithelia. This
observation led us to investigate whether an epithelial tissue is involved in
cardiac morphogenesis. As heart cone tilting and heart tube elongation occur
in cloche, a zebrafish mutant that lacks endocardial cells,
morphogenesis may largely depend on the myocardium or on tissues external to
the heart (Stainier et al.,
1995). During heart fusion and cone formation, myocardial cells
are organized into two bilateral cell populations that have some
characteristics of polarized epithelia
(Trinh and Stainier, 2004;
Trinh et al., 2005). At these
early stages, PRKCs are localized to the apical junctions of myocardial
cells.
In this study, we expand on the roles of nagie oko (nok)/mpp5 and has/prkci during polarization of the myocardial layer and cardiac morphogenesis. We show that has/prkci and nok/mpp5 function within myocardial cells during heart cone formation and that Has/PRKCi function requires its catalytic activity. Loss of has/prkci disrupts the epithelial organization of myocardial cells and blocks heart cone tilting. Using nok/mpp5 antisense morpholino oligonucleotides (MO), we have identified an early function of this gene in epithelial polarity of myocardial cells before heart cone formation, whereby heart cone fusion is severely impaired. Moreover, zygotic nok/mpp5 mutants display defects in myocardial remodeling that contribute to the subsequent elongation of the heart tube.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Embryos
were staged by hours post-fertilization (hpf) at 28.5°C
(Kimmel et al., 1995) and
according to somite number. The nokm520 allele analyzed in
this study results in a premature stop codon at residue Arg 546 within the GUK
domain (Wei and Malicki,
2002), whereas noks305 causes the loss of the
entire GUK domain, resulting in an even larger truncation (Wei, personal
communication). The nokm520 mutation has been shown to
cause a complete, or almost complete, loss of function
(Wei and Malicki, 2002).
Moreover, the noks305 mutation placed in trans to the
nokm520 mutation produced a phenotype that was
indistinguishable from noks305 homozygotes. Therefore, the
noks305 mutation causes a complete, or almost complete,
loss of function.
The Tg(cmlc2:prkci) and Tg(cmlc2:nok)
transgenic lines were generated by injection of I-SceI
cmlc2:prkci and I-SceI cmlc2:nok transformation
vectors into 1-cell stage embryos (Thermes
et al., 2002). F1 founders were identified by PCR genotyping and
used for the establishment of stable lines. For rescue experiments using the
transgenic lines in has or nok mutant backgrounds,
respectively, embryos were PCR genotyped for the presence of the transgene
insertion using transgene-specific primers (primer sequence information
available upon request).
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DNA constructs and site-directed mutagenesis
The coding region of zebrafish has/prkci was amplified by PCR from
a full-length clone (Horne-Badovinac et
al., 2001), introducing XhoI and XbaI
restriction sites 5' and 3', respectively, and subcloned into the
expression vector pCS2+. Site-directed mutagenesis was performed using the
Quick Change Site Directed Mutagenesis Kit (Stratagene, CA, USA).
Mutagenesis primer sequences used for PRKCi2A
5'-CCCAATTACATTGCAGCAGCGATTCTGAGAGGAGAAG-3'. Further details
are available upon request. pXT7NOK containing full-length nok was a
gift from J. Malicki (Massachusetts General Hospital, Boston, MA). The
cmlc2:mRFP was cloned by introducing the PCR-amplified mRFP
fragment into the I SceI cmlc2 transformation vector.
RNA and morpholino injections
Constructs were transcribed using the SP6 MessageMachine kit (Ambion). In
vitro synthesized capped mRNA was dissolved in water and mixed with the MO
prior to injection. Typically, 60 pg of RNA were injected into WIK or WIK/TL
embryos. For overexpression, 120 pg of prkci2A were
injected into wild-type embryos. MOs (Gene Tools) were injected at a
concentration of 100 µmol/l. MO sequences were: nokMO,
5'-TGAGGTCAGCAGC-GGCTCCAAACAC-3'; hasMO,
5'-TGTCCCGCAGCGTGGGCATTATGGA-3'.
In situ hybridization
Whole-mount in situ hybridization was performed as previously described
(Jowett and Lettice, 1994).
Digoxigenin-UTP-labeled riboprobes were synthesized according to the
manufacturer's instructions (Boehringer Mannheim). The cmlc2 probe
(AF114428) was amplified from cDNA and subcloned into pCS2+. Probes for
amhc and vmhc were a gift from D. Yelon. Embryos were
mounted in Permount (Fisher Scientific) and documented using an Axioplan 2
microscope (Zeiss). Images were processed with Adobe Photoshop software (Adobe
Systems).
Antibody and phalloidin staining
Antibody and phalloidin stainings were performed as previously described
(Horne-Badovinac et al., 2001).
Nuclear stainings were performed using SYTOX green nucleic acid stain
(Molecular Probes) as described (Picker et
al., 1999). The following antibodies were used: rabbit
anti-aPKC
(1:100, Santa Cruz Biotechnology, USA), mouse anti-ZO-1
(1:200, Zymed), rabbit anti-
-catenin (1:200, Sigma), goat anti-rabbit
RRX (1:200), anti-mouse Cy5 (1:100) (Jackson ImmunoResearch). Transverse
sectioning was performed according to Trinh et al. (Trinh et al., 2004).
Hearts were analyzed using a Leica TCS SP2 confocal microscope. For
reconstruction of myocardial cell morphology single sections of recorded
z-stacks were analyzed using Leica software. For quantification of myocardial
cell size, individual scans of cmlc2:mRFP-positive cells were
analyzed with TINA version 2.08e (Raytest Isotpenmeßgeräte GmbH)
and absolute values recalculated as relative values. Standard deviation was
calculated using Excel software (Microsoft).
BrdU labeling
Sixteen-somite stage embryos (noks305 and wild-type
siblings) were yolk injected with 10 mmol/l BrdU in 0.2 mol/l KCl. Embryos
were fixed at 32 hpf in 4% PFA for 2 hours at RT. After rinsing in 1x
PBS, embryos were dehydrated in MeOH and stored overnight at -20°C. After
rehydration, the samples were digested in 10 µg/ml proteinase K for 15
minutes and refixed in 4% PFA for 20 minutes. Incubation in 2N HCl for 1 hour
was performed to relax the chromatin and facilitate the following
immunolabeling using anti-BrdU antibody (Roche 1170376) diluted 1:100 in
blocking solution. BrdU was visualized with Cy3-conjugated goat anti-mouse
(Jackson ImmunoResearch; 1:500).
Apoptosis detection by TUNEL staining
The fragmented DNA of apoptotic cells was detected in 32-hpf-old
noks305 and wild-type siblings using the In Situ Cell
Death Detection Kit TMR Red (Roche) following the protocol from Shepard et al.
(Shepard et al., 2004), with
the exception that the yolk was removed manually after fixation for improved
accessibility to the heart.
Microangiography
Microangiographic images were generated by injection of 70 kD FD into the
dorsal aorta of 36-hpf embryos. Embryos were imaged 5-10 minutes after
injection with a CoolSnap ES camera (Photometrics). Data were collected and
analyzed using Metamorph software version 6.1 (Visitron Systems).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), to
assess heart morphogenesis. Within Tg(cmlc2:prkci)
transgenic embryos, injection of the has/prkci MO produced the
complete range of epithelial defects characteristic of has/prkci but
failed to affect cardiac morphogenesis
(Fig. 1D). has/prkci
morphants that were Tg(cmlc2:prkci) transgenic showed
correct heart cone tilting followed by heart tube elongation. In total, we
examined cardiac morphogenesis in 61 transgenic embryos, 41 of which produced
an elongated heart tube (65.1%). By contrast, almost all of the control
wild-type siblings injected with has/prkci MO produced the
has/prkci cardiac morphogenesis phenotype with an arrested heart cone
(Fig. 1C). The variability of
heart morphogenesis rescue among Tg(cmlc2:prkci) embryos may
be due to the critical period of has/prkci function, which coincides
with the onset of cmlc2 transgene expression. Previously, the
promoter region used for the production of the transgenic line has been shown
to display a variable onset of transgene expression
(Huang et al., 2003).
Therefore, Has/PRKCi acts tissue-autonomously within myocardial cells and is
sufficient for correct heart cone tilting and heart tube elongation. To test whether Has/PRKCi activity within myocardial cells is sufficient for heart function in an otherwise morphant embryo, we injected 70 kD fluorescein dextran into the dorsal aorta of 36-hpf embryos and found that peripheral circulation was indeed present, which is never seen in has/prkci morphants (Fig. 2B,C). A similar result was obtained after outcross of the transgene into the has mutant background (Fig. 2E,F). Therefore, Has/PRKCi activity within myocardial cells is sufficient for heart function.
Heart morphogenesis requires the catalytic activity of Has/PRKCi
To further assess whether Has/PRKCi function during heart morphogenesis
depends on its kinase activity, we generated a mutation of has/prkci
that encodes a kinase-dead version of the protein
(prkci2A; residues Pro 408 and Glu 409 within the
catalytic center exchanged for Ala) (Hanks
and Hunter, 1995). Whereas co-injection of 60 pg of wild-type
prkci mRNA together with has/prkci MO gave a complete rescue
(data not shown), co-injection of prkci2A mRNA with
has/prkci MO resulted in the has/prkci external phenotype.
We refer to these animals as prkci2A mutants. The 32-hpf
wild-type heart is an elongated two-chambered tube with posterior ventricle
and anterior atrium, one-third of prkci2A mutants had the
characteristic dysmorphic cone-like heart of has/prkci mutants. Thus,
normal morphogenetic movements of heart cone tilting and regular elongation
into the two-chambered heart tube had failed. Moreover, 54% of
prkci2A mutants displayed an even more severe phenotype in
which cardiac fusion was not completed, resulting in bilateral wings of cells
(Fig. 1E). Therefore, the
prkci2A mutation affects earlier steps of heart
morphogenesis than the zygotic has/prkci mutation, which we attribute
either to an interference with maternal Has/PRKCi protein or redundant
function of PRKC. Thus, the biological activity of Has/PRKCi during
heart morphogenesis crucially depends on its catalytic kinase activity within
myocardial cells.
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; Horne-Badovinac et al.,
2003). In mammalian cells, Pals1/Mpp5 functions as a scaffolding
protein that links the transmembrane receptor Crumbs with Par6-PRKC
(Hurd et al., 2003;
Wang et al., 2004). We tested
whether Nok is a functional homolog of mammalian Pals1/Mpp5 by injecting
murine Pals1/MPP5 mRNA into nok mutants and morphants.
Murine Pals1/MPP5 mRNA largely prevented ventral curvature of
morphants (n=29/30 morphants rescued;
Fig. 3A-C). To further evaluate
the rescue efficiency of Pals1/MPP5 mRNA, we assayed the organization
of the retinal pigmented epithelium (RPE), which is almost completely lost in
nok mutants but was significantly recovered upon injection of murine
Pals1/MPP5 mRNA (Fig.
3D-F). Finally, we assessed the recovery of heart function by
quantifying the presence of circulation within peripheral blood vessels in
nok morphants at 36 hpf. Injection of Pals1/MPP5 mRNA caused
a significant increase in the number of nok/mpp5 morphants with
functional circulation (Fig.
3G). The failure to completely rescue the nok morphant
phenotype may be attributed to sequence variations between Nok and murine
Pals1. Therefore, Nok is a functional homolog of murine Pals1/Mpp5.
Assessment of heart morphology in nok/mpp5 mutant hearts
(noks305 and nokm520 alleles) revealed
that they largely developed beyond the cone stage. However, expression of
cmlc2 showed that the heart failed to completely elongate by 36 hpf.
The defects observed ranged from severe (the heart tube failed to elongate)
(Fig. 1F) to moderate (the
heart tube was significantly shorter than wild type)
(Fig. 1G) to mild (the heart
tube elongated but was narrow) (not shown). Expression of ventricular
myosin heavy chain (vmhc) and atrial myosin heavy chain
(amhc) revealed that chamber-specific differentiation occurred but
that both the atrium and the ventricle were defective in size and form
(Fig. 1J-M) (Berdougo et al., 2003).
Therefore, we conclude that nok/mpp5 is required for correct heart
tube elongation.
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). Similarly, for has/prkci, we injected an
ATG-directed MO that has been shown to eliminate protein expression
(Horne-Badovinac et al., 2001).
Indeed, myocardial morphogenesis in these morphants resembled the
has/prkci mutant heart phenotype (see below). The co-injection of
nok/mpp5 MO with wild-type nok/mpp5 mRNA completely rescued
myocardial morphogenesis, thereby demonstrating that the MO causes a specific
developmental defect (Fig.
1I).
To assess the effects of nok/mpp5 and has/prkci on the
polarization of the myocardial epithelium, we used a transgenic line of
zebrafish that expresses green fluorescent protein (GFP) under the control of
the cmlc2 promoter region [Tg(cmlc2:GFP)] to
visualize heart morphology (Huang et al.,
2003). We utilized this transgenic line in conjunction with
antibodies against the junctional protein Zonula occludens-1 (ZO-1), against
PRKCi and z (anti-aPKC) at the apical junction of myocardial cells and
-catenin at the adherens junction and lateral membranes, and rhodamine
phalloidin to detect filamentous actin. In wild type, before heart cone
formation (16-somite stage), myocardial cells were positioned in two bilateral
sheets that converged toward the midline
(Fig. 4A). Within each
bilateral sheet, myocardial cells were highly polarized and displayed
junctional belts positive for PRKC and ZO-1
(Fig. 4J,K)
(Trinh and Stainier, 2004;
Trinh et al., 2005). Within
the next 2 hours, myocardial cells converged onto the midline and fused to
form the heart cone (20-somite stage; Fig.
4B). At this stage, the myocardium was a monolayered epithelial
sheet that lay symmetrically near the midline and had the appearance of an
evenly shaped cone structure with PRKC- and ZO-1-positive junctional belts. In
transverse sections, lateral portions of the myocardium were organized as a
monolayered sheet of mostly cuboidal cells. There was asymmetrical
localization of ZO-1 at the apical side and PRKC just basal of ZO-1 and more
diffusely along basolateral membranes (Fig.
5B). The adherens junctional protein, -catenin, largely
co-localized with ZO-1 at the apical membrane
(Fig. 5D).
Before heart cone tilting, myocardial cells further converged and the heart cone elongated along the dorsoventral axis (Fig. 4C). During tilting (Fig. 4D), the wild-type heart cone became highly asymmetrical and revealed the presence of several morphologically elongated myocardial cells that were attached to surrounding tissues (Fig. 4S). These cells were located at the anterior/ventral edge of the heart cone.
In has/prkci and nok/mpp5 morphants, the epithelial
organization of the myocardium was affected already before heart cone
formation (16-somite stage). The localization of ZO-1 was in a spotted
distribution rather than in a contiguous junctional belt
(Fig. 4N,P). Moreover, within
nok/mpp5 morphant myocardial cells, PRKC was mislocalized in a
diffuse pattern (Fig. 4M).
Transverse sections revealed that nok/mpp5 and has/prkci
morphant myocardial layers appeared less coherent and were, in part,
multilayered (Fig. 5C,E). Both
morphants lacked the asymmetric apical distribution of ZO-1, which was,
instead, found to be diffusely distributed. Moreover, PRKC was mislocalized in
a diffuse pattern in nok/mpp5 morphants
(Fig. 5C). In
has/prkci morphants, there was no co-localization of ZO-1 and
-catenin. Instead, -catenin was diffusely distributed along the
membrane (Fig. 5E). Therefore,
Nok/Mpp5 functions to maintain PRKC localization basal of ZO-1. The coherence
of the myocardial sheets was disrupted and heart cone fusion was delayed in
nok/mpp5 morphants (Fig.
4E,G). Indeed, nok/mpp5 morphants failed to undergo heart
cone fusion at the 28-somite stage (Fig.
4G), a stage at which the wild-type heart cone has already tilted
into the anterior-posterior plane of the embryo
(Fig. 4D). In
has/prkci morphants, the coherence of the myocardial layer was
affected as indicated by holes that disrupt the integrity of this layer
(Fig. 4H). Moreover, tilting of
the heart cone did not occur. Myocardial cells that connect the myocardium
with surrounding tissues were not seen in has/prkci mutants. We
conclude that the polarized epithelial organization and coherence of the
myocardium is affected in has/prkci and nok/mpp5 morphants.
Therefore, nok/mpp5 morphants reveal an early gene function that is
not apparent in the two zygotic mutants characterized.
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A smaller cell count may be caused by a reduced rate of cell proliferation or by an increased rate of cell death. We first compared the proliferation rates of wild type and nok/mpp5 mutants that were also Tg(cmlc2:GFP) transgenic using a BrdU assay. The mitotic index of myocardial cells (mitotic cells/100 myocardial cells) that divided between the 16-somite stage and 32 hpf was similar between wild type (10.9%) and nok/mpp5 mutants (10.3%) [wild type, 29.3±8.3 s.d. BrdU-positive cells/heart (n=10 hearts); nok/mpp5, 21.8±15.1 s.d. BrdU-positive cells/heart (n=6 hearts)] (Fig. 6H,I,L). Therefore, cell proliferation contributes only slightly to heart tube elongation between the 16-somite stage and 32 hpf, and the comparable mitotic index does not explain the differences in cell numbers of wild-type and nok/mpp5 mutant myocardia. We next tested whether an increased rate of cell death might cause the reduced myocardial cell count in nok/mpp5 mutants. TUNEL analysis of wild type and nok/mpp5 mutants that were also Tg(cmlc2:GFP) transgenic indicated a low rate of apoptotic cell death that was slightly increased in mutants [wild type, 0.5±1.0 s.d. TUNEL-positive cells/heart (n=10 hearts); nok/mpp5, 2.0±1.2 s.d. TUNEL-positive cells/heart (n=10 hearts)] (Fig. 6J,K,L). Similar results were obtained with Acridine Orange stainings (not shown). Therefore, the smaller cell count in nok/mpp5 mutants is not caused by a reduced cell proliferation rate or increased rates of apoptotic cell death during heart tube elongation. We suggest that a smaller heart is the result of altered proliferation and/or cell survival rates at earlier stages of cardiogenesis, or a smaller pool of progenitor cells.
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Next, we compared the spatial arrangement of myocardial cells and found that nok/mpp5 mutant myocardial cells were more densely arranged than wild-type cells at 36 hpf. The differences were most striking within the atrium, where nok/mpp5 mutant myocardial cells were reminiscent of wild-type heart-cone-stage myocardial cells.
For visualization and measurement of individual myocardial cell shapes in
wild type and nok/mpp5 mutants, we used an expression plasmid
[cmlc2:membrane red fluorescent protein (cmlc2:mRFP)] to
achieve transient and mosaic expression of mRFP within single myocardial
cells. We compared single cells expressing mRFP between wild-type siblings and
mutants within the Tg(cmlc2:GFP)/noks305
and Tg(cmlc2:GFP)/nokm520 transgenic
backgrounds (Fig. 6E-G). The
relative surface extension of mRFP-positive cells was quantified using TINA
2.08 software (Raytest Isotpenmeßgeräte GmbH). In summary, we found
that wild-type myocardial cells were on average about 25-35% more extended
than in nok/mpp5 [wild type, 100±25.4% s.d. (n=53
cells); noks305, 74.1±25.3% s.d. (n=58
cells); nokm520, 64.1±22.6% s.d. (n=42
cells)]. The differences in cell surface extension are highly significant
(P
0.01, according to Student's t-test). Even more
strikingly, wild-type atrial cells had an average surface extension that was
about 40% larger than wild-type ventricle cells [wild-type atrium,
136.01±19.48% s.d. (n=8 cells); wild-type ventricle,
93.61±20.68% s.d. (n=45 cells)]. This demonstrates that the
average wild-type ventricular cell is more extended than the average
nok/mpp5 mutant myocardial cell (the average surface extension of
mutant cells being derived from both atrial and ventricular myocardial cells).
Due to similar cell surface extensions, we did not distinguish between atrial
and ventricular cells in nok/mpp5 mutants. In summary,
nok/mpp5 mutant myocardial cells do not fully expand in size. Both
size reduction of myocardial cells and smaller cell number appear to be
important factors contributing to heart tube elongation defects in
nok/mpp5 mutants. We conclude that nok/mpp5 is required for
the remodeling of myocardial cells during heart tube elongation.
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) (Fig.
6M). Among the homozygous noks305 mutants that
lacked the rescue transgene, most embryos displayed a shortened and malformed
heart tube characteristic of the nok/mpp5 mutant phenotype
(Fig. 6B). Therefore, similar
to Has/PRKCi, Nok/Mpp5 functions tissue-autonomously within the myocardial
layer.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Horne-Badovinac et al., 2003).
Within myocardial cells, Nok/Mpp5 is required for correct junctional
localization of Has/PRKCi. We also show that Nok/Mpp5 and Has/PRKCi function
autonomously within myocardial cells during heart morphogenesis and that
Has/PRKCi function depends on the catalytic kinase activity. Hence, embryonic
heart morphogenesis depends on localized Has/PRKCi catalytic activity at the
apical junction, and Nok/Mpp5 is essential for its stabilization. We have demonstrated that has/prkci and nok/mpp5 regulate heart cone tilting, and that nok/mpp5 also controls the subsequent step of heart tube elongation. The mechanism of heart cone tilting is currently not understood but it is conceivable that a directed motion that displaces the heart cone from the midline toward the left anterior side of the embryo may be involved (compare Fig. 4B,C). This myocardial motion could be caused by an intrinsic motive force and/or by external forces. We have shown that during the process of heart cone tilting myocardial cells are strongly converging to produce a narrow and extended cone that subsequently tilts into the anterior-posterior axis (Fig. 4C). This supports the possibility that myocardial cells are actively moving to cause heart cone tilting and that this movement requires the monolayered, polarized and cuboidal organization of myocardial cells at this stage (Fig. 7). Interestingly, loss of has/prkci or nok/mpp5 does not impede the convergence of myocardial cells toward the midline. Therefore, either heart progenitor cells do not require the integration into an epithelial sheet for migration, or the defective myocardium maintains some epithelial cohesion that is mediated by factors other than the ones tested in our study. We also hypothesize that the morphologically specialized population of myocardial cells that connects the heart cone with surrounding tissues may be necessary to convert external motive forces onto the heart cone or to generate an anchor point that may force the heart cone to always tilt in a stereotypical direction (Fig. 4S).
We have observed that prkci2A mutants frequently
displayed a myocardial phenotype that was more severe than in
has/prkci morphants. In fact, overexpression of high levels of
prkci2A mRNA in wild-type embryos resulted in a phenotype
similar to nok/mpp5 (Fig.
1N,O). Conceivably, PRKCi2A interferes, in a
dominant-negative fashion, with pools of maternal Has/PRKCi or another PRKC
isoform that is within protein complexes associated with Nok/Mpp5.
Immunohistochemistry performed on has/prkci mutants using an antibody
against the 20 C-terminal amino acids of murine PRKCz demonstrated the
presence of another PRKC isoform different from zygotic PRKCi
(Horne-Badovinac et al.,
2001).
As mammalian Pals1 physically interacts via an evolutionarily conserved
region with Par6, and as physical association of the two apical protein
complexes, Crumbs-Stardust/Mpp5 and Bazooka-DaPKC-DmPar6, has also been mapped
in Drosophila, we propose that a similar protein complex is likewise
present in zebrafish and that it plays an essential role in zebrafish
epithelial maintenance (Wang et al.,
2004; Nam and Choi,
2003). The results presented in this study indicate a previously
unknown function for these proteins in myocardial polarization and remodeling.
The mechanisms by which heart tube elongation is regulated and the
contribution of nok/mpp5 to the remodeling of atrial myocardial cells
from a cuboidal shape (at heart cone stages;
Fig. 7) to a squamous and
elongated shape during heart tube elongation stages currently remain elusive.
Moreover, it is possible that cellular rearrangements of myocardial cells
during heart tube elongation that are reminiscent of convergence/extension
type movements are essential for this process and that they could be regulated
by nok/mpp5. Further studies into the roles of the apical
Crumbs-Nok/Mpp5 and Par6-PRKC protein complexes in this process are
warranted.
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ACKNOWLEDGMENTS |
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