Xirp proteins mark injured skeletal muscle in zebrafish.
ABSTRACT Myocellular regeneration in vertebrates involves the proliferation of activated progenitor or dedifferentiated myogenic cells that have the potential to replenish lost tissue. In comparison little is known about cellular repair mechanisms within myocellular tissue in response to small injuries caused by biomechanical or cellular stress. Using a microarray analysis for genes upregulated upon myocellular injury, we identified zebrafish Xin-actin-binding repeat-containing protein1 (Xirp1) as a marker for wounded skeletal muscle cells. By combining laser-induced micro-injury with proliferation analyses, we found that Xirp1 and Xirp2a localize to nascent myofibrils within wounded skeletal muscle cells and that the repair of injuries does not involve cell proliferation or Pax7(+) cells. Through the use of Xirp1 and Xirp2a as markers, myocellular injury can now be detected, even though functional studies indicate that these proteins are not essential in this process. Previous work in chicken has implicated Xirps in cardiac looping morphogenesis. However, we found that zebrafish cardiac morphogenesis is normal in the absence of Xirp expression, and animals deficient for cardiac Xirp expression are adult viable. Although the functional involvement of Xirps in developmental and repair processes currently remains enigmatic, our findings demonstrate that skeletal muscle harbours a rapid, cell-proliferation-independent response to injury which has now become accessible to detailed molecular and cellular characterizations.
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ABSTRACT: Regenerative medicine has the promise to alleviate morbidity and mortality caused by organ dysfunction, longstanding injury and trauma. Although regenerative approaches for a few diseases have been highly successful, some organs either do not regenerate well or have no current treatment approach to harness their intrinsic regenerative potential. In this Review, we describe the modeling of human disease and tissue repair in zebrafish, through the discovery of disease-causing genes using classical forward-genetic screens and by modulating clinically relevant phenotypes through chemical genetic screening approaches. Furthermore, we present an overview of those organ systems that regenerate well in zebrafish in contrast to mammalian tissue, as well as those organs in which the regenerative potential is conserved from fish to mammals, enabling drug discovery in preclinical disease-relevant models. We provide two examples from our own work in which the clinical translation of zebrafish findings is either imminent or has already proven successful. The promising results in multiple organs suggest that further insight into regenerative mechanisms and novel clinically relevant therapeutic approaches will emerge from zebrafish research in the future.Disease Models and Mechanisms 07/2014; 7(7):769-776. · 5.54 Impact Factor
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ABSTRACT: The Smyth line (SL) chicken is the only animal model for autoimmune vitiligo that spontaneously displays all clinical and biological manifestations of the human disorder. To understand the genetic components underlying the susceptibility to develop SL vitiligo (SLV), whole genome resequencing analysis was performed in SLV chickens compared with non-vitiliginous parental Brown line (BL) chickens, which maintain a very low incidence rate of vitiligo.BMC genomics. 08/2014; 15(1):707.
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ABSTRACT: Scoliosis is a complex genetic disorder of the musculoskeletal system, characterized by three-dimensional rotation of the spine. Curvatures caused by malformed vertebrae (congenital scoliosis (CS)) are apparent at birth. Spinal curvatures with no underlying vertebral abnormality (idiopathic scoliosis (IS)) most commonly manifest during adolescence. The genetic and biological mechanisms responsible for IS remain poorly understood due largely to limited experimental models. Here we describe zygotic ptk7 (Zptk7) mutant zebrafish, deficient in a critical regulator of Wnt signalling, as the first genetically defined developmental model of IS. We identify a novel sequence variant within a single IS patient that disrupts PTK7 function, consistent with a role for dysregulated Wnt activity in disease pathogenesis. Furthermore, we demonstrate that embryonic loss-of-gene function in maternal-zygotic ptk7 mutants (MZptk7) leads to vertebral anomalies associated with CS. Our data suggest novel molecular origins of, and genetic links between, congenital and idiopathic forms of disease.Nature Communications 09/2014; 5:4777. · 10.74 Impact Factor
Xirp Proteins Mark Injured Skeletal Muscle in Zebrafish
Ce ´cile Otten1, Peter F. van der Ven2, Ilka Lewrenz2, Sandeep Paul3,4, Almut Steinhagen2, Elisabeth
Busch-Nentwich5, Jenny Eichhorst6, Burkhard Wiesner6, Derek Stemple5, Uwe Stra ¨hle3, Dieter O. Fu ¨rst2,
1Max Delbru ¨ck Center (MDC) for Molecular Medicine, Berlin, Germany, 2Department of Molecular Cell Biology, Institute of Cell Biology, University of Bonn, Bonn,
Germany, 3Institute for Toxicology and Genetics, Karlsruhe, Germany, 4University of Southern California Keck School of Medicine, Los Angeles, California, United States of
America, 5Vertebrate Development and Genetics, The Wellcome Trust Sanger Institute, Cambridge, United Kingdom, 6Leibniz Institute for Molecular Pharmacology,
Myocellular regeneration in vertebrates involves the proliferation of activated progenitor or dedifferentiated myogenic cells
that have the potential to replenish lost tissue. In comparison little is known about cellular repair mechanisms within
myocellular tissue in response to small injuries caused by biomechanical or cellular stress. Using a microarray analysis for
genes upregulated upon myocellular injury, we identified zebrafish Xin-actin-binding repeat-containing protein1 (Xirp1) as a
marker for wounded skeletal muscle cells. By combining laser-induced micro-injury with proliferation analyses, we found
that Xirp1 and Xirp2a localize to nascent myofibrils within wounded skeletal muscle cells and that the repair of injuries does
not involve cell proliferation or Pax7+cells. Through the use of Xirp1 and Xirp2a as markers, myocellular injury can now be
detected, even though functional studies indicate that these proteins are not essential in this process. Previous work in
chicken has implicated Xirps in cardiac looping morphogenesis. However, we found that zebrafish cardiac morphogenesis is
normal in the absence of Xirp expression, and animals deficient for cardiac Xirp expression are adult viable. Although the
functional involvement of Xirps in developmental and repair processes currently remains enigmatic, our findings
demonstrate that skeletal muscle harbours a rapid, cell-proliferation-independent response to injury which has now
become accessible to detailed molecular and cellular characterizations.
Citation: Otten C, van der Ven PF, Lewrenz I, Paul S, Steinhagen A, et al. (2012) Xirp Proteins Mark Injured Skeletal Muscle in Zebrafish. PLoS ONE 7(2): e31041.
Editor: Henry H. Roehl, University of Sheffield, United Kingdom
Received July 18, 2011; Accepted December 30, 2011; Published February 15, 2012
Copyright: ? 2012 Otten et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Among lower vertebrates, many organs have a remarkable
regenerative potential. This is particularly evident in the case of
the zebrafish heart which can regenerate large injuries by
stimulating the proliferation of dedifferentiated cardiomyocytes
[1,2,3]. Similarly, loss of skeletal muscle can be compensated by
the activation and proliferation of muscle stem cells (for a recent
review, see ). Whereas such regenerative processes require
extensive proliferation and occur over days to months, smaller
injuries that constantly occur due to excessive exercise and
biophysical or cellular stress require more rapid repair mecha-
nisms. A better molecular characterization of such repair processes
may provide alternative routes for tissue healing with beneficial
implications for humans.
Xin-repeat proteins are striated muscle-specific actin-binding
multi-adaptor proteins that interact with sarcomeric proteins or F-
actin associated proteins and localize to the intercalated discs
(ICD) of cardiomyocytes or to the myotendinous junction (MTJ) of
skeletal muscle cells [5,6,7,8,9,10,11]. Xirp1 was found to be
upregulated in mouse models of hypertension [12,13], in other
mouse models based on eccentric exercise [14,15], in a
spontaneous mouse mutant with regenerating muscle tissue
[15,16], and in the dystrophic zebrafish mutant runzel .
Currently, a coherent functional understanding of Xirp family
proteins is lacking. Whereas cXin, the only Xirp family member
present in chicken, has been proposed to regulate cardiac
morphogenesis , both mammalian Xirps, Xirp1 and Xirp2,
have been implicated in hypertrophic cardiomyopathy in humans
and mice [18,19,20,21]. Here, we show that, although not being
functionally essential during development, Xirp1 is strongly
induced within injured skeletal muscle in the zebrafish embryo.
Because Xirp1+injured muscle can recover in the course of hours
and does not involve cell proliferation, our study may have
potential implications for understanding the molecular and cellular
biology of myocellular repair in humans.
xirp1 is upregulated upon myocellular injury
In a search for proteins involved in zebrafish embryonic muscle
repair, we performed a transcriptome analysis in a pharmacolog-
ically induced model of muscle injury. Zebrafish embryos treated
with the acetylcholinesterase (AChE) inhibitor Galanthamine (Gal)
develop a severe disarray of somitic muscle organization after
48 hours post fertilization (hpf) due to an over-activation of muscle
cells by the accumulation of the neurotransmitter acetylcholine
. We assessed the efficacy of the treatment based on the
impaired motility of embryos which strongly correlated with a
disarray of myofibrils within skeletal muscle cells (Fig. 1A,B,C).
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The effects of Gal-treatment were reversible within several hours
of recovery although the cellular and molecular mechanisms
involved in this repair process are not known (Fig. 1B). One of the
most strongly affected genes upon Gal-treatment was the three-
fold upregulated xirp1 gene. In comparison, no other muscle-
specific gene was significantly upregulated except Desmin
(Table 1). This finding was verified by immunohistochemistry
using a zebrafish-specific anti-Xirp1 antibody in Gal-treated
embryos. Immediately after Gal-treatment, skeletal muscle cells
displayed disordered arrays of myofibrils that contained high levels
of Xirp1 protein (Fig. 1B,C). Within 8 hours of recovery, Gal-
induced lesions had recovered and ectopic Xirp1 was not further
detectable (Fig. 1B).
Zebrafish Xirp proteins have overlapping but distinct
The zebrafish xirp gene family comprises three members which
are orthologous to their two mammalian counterparts Xirp1 and
Xirp2 (Fig. 2A) . As in mammals, zebrafish xirp1 is encoded by
a large exon and can yield three isoforms as a result of intraexonic
splicing (Fig. 2B). The gene structure of both xirp2a and xirp2b
consists of several short exons (xirp2a: exon 1–8; xirp2b: exon 1–7)
preceding a major large exon encoding the Xin-repeats (xirp2a:
exon 9; xirp2b: exon 8), and followed by two short exons (xirp2a:
exon 10–11; xirp2b: exon 9–10). Evolutionarily conserved splicing
mechanisms result in large isoforms with Xin-repeats and short
isoforms lacking the Xin-repeats, but encoding a LIM domain
created by splicing together the short exons. Our sequence
analyses have revealed the expression of both large and short
isoforms. However, the short isoforms lack the characteristic Xin-
We performed whole-mount in situ hybridizations and raised
antibodies against Xirp1, Xirp2a and Xirp2b to determine their
expression and subcellular localization patterns. The expression of
xirp1, xirp2a and xirp2b starts in a precise temporal order and
mirrors the progression of skeletal muscle cell differentiation
(Fig. 2C; Fig. 3A). xirp1 has the earliest and broadest expression
and is present in all striated muscles (Fig. 2C). On the protein level,
Xirp1 localizes to the MTJ and the Z-discs and is continuously
Figure 1. Expression and localization of Xirps within Galanthamine- and laser-induced myocellular wounds. (A) Schematic diagram
summarizing the temporal order of Xirp expression within somitic muscle and in myocellular wounding assays. (B) Galanthamine (GAL) treatment
between 80% epiboly and 2 dpf causes severe disruptions of somitic muscle organization and myofibrillar disarray (red: Actin) in 2 dpf zebrafish
embryos. Notably, Xirp1 (green) is strongly expressed and localizes within cells most strongly disrupted by the treatment. These effects are
completely reversible within several hours of recovery. Scale bar: 50 mm. (C) Details from inserts indicated in B (green: Xirp1; red: Actin). Scale bar:
10 mm. (D, E) Similarly, laser-induced muscle injury induces ectopic Xirp1 and Xirp2a localization to damaged myofibrils. In comparison, Xirp2b is not
yet expressed at 33 hpf. Green: Xirp1, Xirp2a or Xirp2b; red: Actin. Arrows indicate the position of laser-induced injury within somitic tissue. Scale
bars: 10 mm. Embryos were injured at 27 hpf (D) or 29 hpf (E) and fixed at 33 hpf (D) or 31.5 hpf (E), respectively.
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expressed in all striated muscle from earliest developmental stages
until adulthood (Fig. 3). Importantly, Xirp1 is the only zebrafish
Xirp protein containing Xin-repeats that is expressed in the heart
(Fig. 2C; Fig. 3A,C). In comparison, xirp2a and xirp2b are
expressed later, their expression is restricted to skeletal muscle or
somitic muscle, respectively, and their mRNAs are localized to the
MTJ (Fig. 2C). Xirp2a and Xirp2b proteins localize exclusively to
the MTJ (Fig. 3A).
Xirp1 and Xirp2a localize along injured myofibrils
In contrast to xirp1, neither xirp2a nor xirp2b were represented in
our initial transcriptome analysis of myocellular injury. To
characterize the mode of myocellular repair in more detail, as
well as to assess expression levels and localization of all three Xirps
within myocellular lesions, we used a micropoint laser to induce
(Fig. 1A,D,E; movie S1). We first assayed by immunohistochem-
istry the expression of Xirps in response to laser-induced muscle
injury. Indeed, the localization of Xirp1 and Xirp2a was altered in
response to myocellular injury at 32 hpf when Xirp2b was not yet
expressed (Fig. 1A,D). Most strikingly, Xirp1 and Xirp2a were
enriched along non-striated portions of injured myofibrils
(Fig. 1D,E). After laser-induced micro-injury, there was a complete
recovery from tissue damage within 2–8 hours. This observation
was indicative of myocellular repair that is considerably more
rapid than expected for regenerative processes that involve cell
Xirp1 marks wounded skeletal muscle cells prior to de
novo cell proliferation
Next, we aimed at analyzing whether upregulation of Xirp1
within damaged tissue correlated with contributions of proliferat-
ing myogenic progenitor cells. To this end, embryos were laser-
injured and subsequently analyzed either with 5-bromodeoxyur-
idine (BrdU)-labelling to detect proliferating cells or with an
antibody against Pax7 which detects external cells, a type of
muscle stem cells found in zebrafish embryos [24,25]. As expected
for such a rapid repair mechanism, within 2 hours of recovery,
laser-injured regions contained Xirp1+cells all of which were
BrdU2(Fig. 4A). Thus, repair of the injured tissue occurred in the
absence of cells that had recently undergone DNA synthesis. In
comparison, laser injuries induced some proliferation within
damaged superficial cell layers (data not shown).
To further substantiate this finding, we next used Aphidicolin to
inhibit cell proliferation . Continuous Aphidicolin-treatment
during recovery after laser-induced micro-injury neither prevented
localization of Xirp1 to injured myofibrils nor tissue repair
(Fig. 4A). To assess the efficiency of wound healing, we performed
histochemistry using rhodamine phalloidin to visualize disrupted
myofibrils and found that less than half of the wounds were still
detectable 6 hours after injury, as in wild-type (WT) [number of
open, visible wounds/number of injuries: WT, (n=8/32; 25%);
Aphidicolin-treated WT, (n=17/40; 42.5%); no significant
difference according to t-test: p-value 0.2149]. Moreover, Xirp1+
cells did not overlap with Pax7+cells upon Gal treatment (Fig. 4B).
After wound healing, Xirp1 was not further detectable with
ectopic localization. Together, these findings suggested that Xirp1
marks injured skeletal muscle which can recover in the absence of
significant cell proliferation.
Another possibility for skeletal muscle recovery could be that
cellular rearrangements of cells neighboring localized lesions may
be involved. To investigate this possibility, we genetically labeled
single muscle cells with a Tg[bactin:a-actinin-GFP] expression
construct and performed laser-assisted micro-injuries only on
those cells expressing the fusion protein (Fig. 5). This analysis
revealed that Xirp1 was expressed within those cells that had
directly been targeted by the laser and within their direct
neighbors (Fig. 5A,B). Immunohistochemical analysis of injury
sites using an antibody against Tropomyosin revealed that strong
Xirp1 expression correlates with a striation pattern of Tropomoy-
sin that is different from unaffected regions of the somite and
which indicates that myofibrillar remodeling is occurring within
injured cells (Fig. 5C) . Since Xirp1 was detected on non-
striated portions of myofibrils, correlated with a unique pattern of
Tropomyosin, and was expressed directly within injured cells or
Table 1. Transcriptome analysis for genes regulated by Galanthamine treatment in zebrafish.
Compugen Serial NumberFC at 56 hpfFC at 72 hpfGene name
CGENZEB_456004831_03,323,22 cardiomyopathy associated 1 (xirp1)
CGENZEB_456003428_0 2,142,43activating transcription factor 3
CGENZEB_456009990_02,063,67complement component 6
CGENZEB_456006757_02,051,88 Q6PBK2_BRARE (GenBank: AAH59678.1)
CGENZEB_456002498_02,00 2,19coagulation factor XIII, A1 polypeptide
CGENZEB_456003229_01,622,07 phosphoenolpyruvate carboxykinase 1
CGENZEB_456001821_01,442,23 uncoupling protein 4
Summary table listing those genes that are most strongly upregulated in the acetylcholinesterase inhibitor Galanthamine-induced model of muscle injury. For
treatment, zebrafish embryos were continuously incubated with Galanthamine between the end of gastrulation and several days of development. Comparisons of fold
changes (FC) at 56 hpf and 72 hpf are shown for those genes showing the strongest expression changes. xirp1 is more than three-fold upregulated at both time-points
under conditions of myocellular injury.
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Figure 2. Structure of the zebrafish Xirp family and gene expression patterns. (A) Phylogenetic analysis of Xirp family members. The zebrafish Xirp
family comprises three members which are orthologous to their two mammalian counterparts. Phylogenetical distances calculated according to the ClustalW
slow/accurate method are indicated. Danio rerio (D.r.); Gallus gallus (G.g.); Mus musculus (M.m.). (B) As for mammalian Xirp1, intraexonic splicing of zebrafish
xirp1 results in three alternative isoforms: xirp1a (6894 bp), xirp1b (4452 bp), xirp1c(2006 bp). Xirp1b is identicalto theN-terminus ofXirp1a andharbours Xin-
repeats; Xirp1c is identical to the C-terminus of Xirp1a and harbours a Filamin C binding domain (FilC BD). Antibodies specific against isoforms Xirp1a/b (P43)
and Xirp1a/c (P47) were generated. Black asterisks indicate the position of protein truncation in the xirp1sa0046mutant. Concerning Xirp2a and Xirp2b,
differential splicing leads to various isoforms classified as long (containing Xin-repeats) or short (lacking a large exon encoding Xin-repeats and resulting in the
encoding of a LIM domain). Here, we represented schematically the long isoforms and their respective antibody. (C) Summary panel of xirp gene family
expression with whole-mount in situ hybridization probes designed to exclusively cover the long isoforms of these genes. xirp1 has the earliest and widest
strongly expressed within somitic muscle after 24 hpf where they localize to the somite boundaries. Both xirp1 and xirp2a are expressed within other types of
skeletal muscle including those of the fin buds, whereas xirp2b expression remains restricted to somitic muscle. Asterisks indicate the heart; arrowheads point
at fin buds. Black scale bars: 250 mm, red scale bars: 200 mm.
Figure 3. Expression and localization of Xirp proteins during skeletal and cardiac muscle development in WT. (A) Xirp expression
patterns overlap within skeletal muscle between 20–48 hpf. In comparison, only Xirp1 is expressed within the cardiac tissue at 5 dpf. Green: Xirp1,
Xirp2a or Xirp2b; red: Actin. Red scale bar: 25 mm, black scale bars: 50 mm. (B) Top panel shows cross section planes of confocal z-scan projections
through somitic tissue. Note that Xirp1 (green) is most strongly expressed within the outer layer of slow muscle cells, as marked by Alcam (blue)
expression (red: Actin). Whereas most of Xirp1 localizes in a staircase-like pattern to the MTJ, some protein is also detectable in a repetitive pattern at
the z-discs of myofibrils and at the lateral plasma membrane, depending on the tissue. Different skeletal muscle types are shown at 5 dpf, whereas
somitic muscle is shown at 32 hpf. Green: Xirp1; red: Actin; blue:a- Actinin. Black scale bars: 10 mm, yellow scale bars: 5 mm. (C) Expression and
localization of Xirp1 during cardiogenesis and in adult cardiac tissue. Bottom row represents higher magnification images (from inserts, if there is a
white box on the top picture) with details on Xirp1 subcellular localization at the ICD of cardiomyocytes (arrowheads). Green: Xirp1; red: myl7:GFP, b-
Catenin or Actin. Scale bars: 50 mm.
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within cells neighboring the injury site, Xirp1 appears to be part of
a myocellular injury response that involves myofibrillar remodel-
Myocellular repair occurs in the absence of Xirp1 and
To elucidate the functional role of Xirp1 in myocellular repair,
we obtained the xirp1sa0046mutant allele which causes a premature
stop codon replacing Lys271. Expression of xirp1 mRNA was not
detectable in cardiac or somitic tissue of xirp1sa0046mutants,
suggesting that the mutation is a null allele causing a complete loss
of Xirp1 protein (Fig. 6A). Consistent with the absence of mRNA
expression, immunohistochemical analyses with isoform-specific
antibodies confirmed the complete lack of all three expected
Xirp1a/b/c protein isoforms in cardiac and skeletal muscles
(Fig. 6B,C; Fig. 7B,C; Fig. 8). Unexpectedly, xirp1sa0046mutants
did not exhibit any obvious developmental defects and were adult
viable and fertile (see below). Similarly, MO-mediated knock-
down of xirp2a  in WT as well as in xirp1sa0046mutant embryos
did not visibly affect embryonic development (see below;
Fig. 7C,D; Fig. 9).
Next we analyzed recovery of skeletal muscle upon laser-injury
in xirp1sa0046/xirp2aMO mutant/morphant embryos. Myocellular
injuries efficiently recovered within 6 hours with normal temporal
dynamics after laser-injury [number of wounds recovered/number
of wounds: WT (n=11/20; 55%); xirp2a morphant (n=12/20;
60%); xirp1sa0046mutant (n=14/20; 70%); xirp1sa0046/xirp2aMO
mutant/morphant (n=12/20; 60%)]. These observations did not
reveal any indispensable involvement of Xirp1 or Xirp2a in rapid
skeletal muscle recovery.
Figure 4. Xirp1 marks wounded skeletal muscle cells prior to de novo cell proliferation. (A) Laser-induced myocellular injuries in 33 hpf old
zebrafish embryos after BrdU pulse labeling (between 24–33 hpf). Embryos were wounded at 31 hpf and left to recover for 2 hours. Xirp1+tissue is
devoid of BrdU+proliferative cells. Treatment of laser-induced myocellular injuries with the proliferation inhibitor Aphidicolin (together with BrdU
between 24–33 hpf) does not affect expression and localization of Xirp1 within damaged tissue. The efficacy of the Aphidicolin treatment is evident
from strongly reduced BrdU labelling. Asterisks indicate the position of laser-induced injury within damaged tissue. Red inserts show Xirp1
localization within damaged tissue. Green: BrDU; red: Actin; blue: Xirp1. (B) Consistent with the lack of proliferating cells within Xirp1+damaged
tissue, the distribution of Pax7+external cells is not changed compared to control conditions. Arrowheads mark ectopic Xirp1. Red inserts show Xirp1
localization within damaged tissue. Green: Xirp1; red: Actin; blue: Pax7. All scale bars: 50 mm.
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Figure 5. Clonal analysis of laser-induced myocellular injuries. (A–B) Laser-injury and recovery within the same embryo. (A) Left: DIC image
superimposed with image of individual muscle cells marked by Tg[bactin:a-actinin-gfp] expression (false colored in yellow). Right: laser-injury was
performed by targeting one of the a-Actinin-GFP positive muscle cells. Immediately upon laser-injury, a-Actinin-GFP expression within the damaged
cell is diminished (arrowheads). Scale bar: 50 mm. (B) Confocal images of two z-scan planes of an immunohistochemical staining show strong Xirp1
expression within 2.5 hours after laser injury in muscle cells directly adjacent to the targeted cell that was most severely affected by the laser-injury
(arrowheads). Pictures on the right are details from the insert (white box on the bottom left picture). Green: bactin:a-Actinin-GFP; red: Actin; blue:
Xirp1. Scale bar: 50 mm. (C) Confocal z-scan projection of an immunohistochemical staining 5 hours after laser-induced injury reveals that strong
Xirp1 expression correlates with a pattern of Tropomyosin distribution that is different from unaffected regions of the somite. Green: Tropomyosin;
red: Actin; blue: Xirp1. Scale bar: 20 mm.
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To determine the temporal dynamics of xirp1 transcriptional
response to micro-injury, we performed whole-mount in situ
hybridizations after laser-injury. Whereas in WT xirp1 expression
was detectable already one hour after injury in damaged somitic
tissue and persisted for not more than 3.5 hours, expression of the
mutant mRNA was completely absent in xirp1sa0046mutants
(Fig. 10). These experiments suggested that either Xirp1 is
involved in its own rapid transcriptional regulation upon injury
or that nonsense mediated mRNA decay prevents gene expression
in xirp1sa0046mutants. Together, the above experiments showed
that Xirp1 expression is strongly responsive to myocellular injury,
albeit with unknown function.
Xirp1 is dispensable for cardiac development and
Functional analysis of chicken cXin had been suggestive of a
crucial role in cardiac looping . Because in mouse disruption
of either one of two murine Xirp family members, Xirp1 or Xirp2,
expressed in the heart did not cause early cardiac defects, it had
been suggested that these two Xirps function redundantly [18,21].
Since Xirp1 is the only Xirp protein containing Xin-repeats
expressed in embryonic zebrafish cardiac tissue and since
redundancies with other Xirps could therefore be excluded, we
assessed its developmental role during cardiac morphogenesis.
However, neither early steps of cardiac morphogenesis nor
myofibrillogenesis were affected in xirp1sa0046mutants (Fig. 6B;
Fig. 7A). Furthermore, homozygous xirp1sa0046mutants raised to
adulthood were fully viable [survival by 17 months of age: WT,
(n=62/96; 64.6%); xirp1sa0046, (n=11/17; 64.7%)] and undistin-
guishable from WT. Immunohistochemical analyses confirmed
that neither Xirp2a nor Xirp2b were upregulated to compen-
sate for lack of Xirp1 within hearts of embryonic or adult
xirp1sa0046mutants (Fig. 6A,C; Fig. 7B). Therefore, unlike reported
for chicken, Xirps are not essential for zebrafish cardiac
This unexpected result prompted us to further investigate
potential roles of Xirps during the development of other
myocellular subtypes. To elucidate potentially redundant roles of
Figure 6. Normal cardiogenesis and skeletal muscle development in the xirp1sa0046mutant. (A) Whole-mount in situ hybridization of xirp1,
xirp2a and xirp2b on 24 hpf WT and xirp1sa0046mutants reveal a loss of xirp1 expression in heart and muscle of xirp1sa0046mutants. In contrast, neither
xirp2a nor xirp2b expression are affected by lack of xirp1. In particular, neither xirp2a nor xirp2b are upregulated in the hearts of xirp1sa0046mutants.
Arrowheads indicate the position of the heart tube. Asterisks indicate the tip of the tail. Red scale bar: 200 mm, black scale bar: 250 mm. (B) Lack of
xirp1 mRNA expression corresponds with the complete absence of Xirp1 (green) within the 20 hpf heart cone, 32 hpf heart tube, and 21 hpf skeletal
muscle. The Actin counterstaining (red) demonstrates that cardiogenesis and skeletal muscle organization at these stages is comparable between WT
and xirp1sa0046mutants. Scale bars: 50 mm. (C) Immunohistochemical stainings for Xirp1, Xirp2a and Xirp2b on 24 hpf WT and xirp1sa0046mutant
hearts show complete absence of Xirp2a and Xirp2b. Top inserts depict overlay images of Actin (red) and Xirp1, Xirp2a or Xirp2b (green). Within the
WT somitic muscle, both Xirp1 and Xirp2a are expressed whereas Xirp1 is absent in xirp1sa0046mutant somitic tissue (bottom inserts). Scale bars:
PLoS ONE | www.plosone.org8February 2012 | Volume 7 | Issue 2 | e31041
all zebrafish Xin-repeats containing proteins in this process, we
analyzed skeletal muscle development in xirp1sa0046/xirp2aMO
mutant/morphants prior to 24 hpf since Xirp2b is not yet
expressed at this stage. Indeed, xirp1sa0046/xirp2aMO mutant/
morphant embryos lacked expression of all three Xirps prior to
24 hpf while displaying normal somitic muscle development and
correctly striated myofibrils (Fig. 7C,D). Together, these findings
suggest that zebrafish Xirps, irrespective of their early develop-
mental expression and their evolutionarily conserved interactions
with the sarcomeric and actin-regulatory proteins Filamin C, Enah
and Vasp (Fig. 11), are not essential myocellular developmental or
In summary, we have outlined evidence for rapid recovery from
myocellular injury within the zebrafish embryo which occurs
within hours of injury and which does not involve progenitor cell
proliferation. Precise laser-induced injuries, as well as pharmaco-
logically induced muscle damage, caused a strong upregulation of
Xirp1 expression within injured tissue. We could also show that
the ectopic localization of Xirp1 and Xirp2a within damaged cells
is a cell-autonomous process that is different from stimulating a
progenitor cell proliferation program. However, since many
wounds are not entirely closed after 6 hours, additional cell
proliferation may also be necessary for complete wound healing.
Laser- and pharmacologically-induced injuries can provoke
varying degrees of damage, ranging from disrupted myofibrils to
complete destruction of the cell. This in turn means that injured
cells may be removed, replaced by neighboring cells, or cell-
autonomously fully recover from damage. Up to date, the
mechanisms underlying the healing of injured muscles are not
understood in detail and we provide here first evidence for a rapid
repair mechanism which may or may not suffice for complete
recovery, depending on the severity of the injury. We propose that
Xirp1 is a conserved marker for myocellular injury, not just during
embryonic development, since it is upregulated within murine
tissue upon excessive exercise or injury as well [12,14,15,16].
However, it is not known whether Xirp1 expression within adult
murine myocellular tissues marks a repair process. One difference
with our observations is that expression of murine Xirp1 has also
been shown in Pax7+muscle progenitor cells . The fact that
Xirp1 is strongly upregulated in such various models of muscle
damage, hints at a function of Xirp1 during muscle repair. In our
assays of injured zebrafish embryos, we found that Xirp1 is
dispensable for rapid repair; however, we cannot exclude that
Xirp1 has an active function in other contexts of muscle
regeneration (e.g. in adult tissues, or when stem cell proliferation
is involved). Further functional long-term assessments of the role of
Xirp1 in different disease or injury conditions such as muscular
dystrophy models are warranted.
Our work in a lower vertebrate has clearly demonstrated that
cardiac morphogenesis is normal in embryos that completely lack
all Xirps within cardiac tissue. Notably, our findings are
unexpected given that functional studies in chicken had implicated
Xirps in cardiac morphogenesis and in the evolution of the
vertebrate multi-chambered heart [11,23]. In contrast, our
findings and studies on the role of murine Xirps [18,21] now
raise serious doubts about the relevance of Xirps for cardiac
morphogenesis in any vertebrate species. Murine Xirp knock-out
Figure 7. Normal cardiogenesis and myofibrillogenesis in the absence of all Xirps. (A) Neither cardiac nor skeletal muscle sarcomeric
organization of myofibrils is affected in 48 hpf xirp1sa0046mutants. Green: a-Actinin or Myosin; red: Actin. Red scale bars: 10 mm, white scale bars:
5 mm. (B) Sectioned adult cardiac tissue reveals that neither Xirp2a nor Xirp2b are expressed to compensate for the loss of Xirp1. Also, the xirp1sa0046
mutant lacks all three Xirp1 isoforms. Therefore, Xirps are not required for development or maintenance of cardiac tissue. Green: Xirp1; red: Actin.
Scale bar: 10 mm. (C) Complete absence of all Xirps within skeletal muscle prior to 24 hpf in xirp1sa0046/xirp2aMO mutant/morphants. Localization of
Xirp1 is not affected in xirp2a morphants and, conversely, Xirp2a localization is normal in xirp1sa0046mutants. Scale bar: 50 mm. (D) Complete loss of
Xirps in xirp1sa0046/xirp2aMO mutant/morphants does not impair early cardiogenesis (at 21 hpf) or myofibrillogenesis (at 32 hpf). Green: Xirp1 or
Myosin; red: Actin. Red scale bar: 50 mm, white scale bar: 5 mm, yellow scale bar: 20 mm.
PLoS ONE | www.plosone.org9February 2012 | Volume 7 | Issue 2 | e31041
models lack obvious developmental defects and develop mild
cardiac phenotypes as adults [18,19,20,21] which is consistent with
our finding that zebrafish Xirp proteins are not required for early
cardiac morphogenesis. It has been suggested that mice lacking all
Xirp proteins in the heart would have more severe cardiac defects
than single Xirp knock-outs. However, we could not detect any
cardiac defects in zebrafish adult xirp1sa0046mutants that entirely
lack cardiac Xirps.
Under normal conditions, Xirp proteins localize to the MTJ, a
site of myofibrillar growth which involves actin remodeling. Since
Xirp1 and Xirp2a are also ectopically present at sites of muscle
injury, Xirp proteins may have in common a high affinity for non-
striated actin-rich myofibrils or their binding proteins. This
subcellular distribution and the direct association of Xirps with
other actin-regulatory proteins suggest that Xirp proteins may
contribute to the organization of actin filaments, together with
other unidentified proteins. We could verify the evolutionary
conservation of interactions with several conserved sarcomeric or
actin-regulatory binding partners (Fig. 11). These analyses suggest
that zebrafish Xirp1 may be involved in conserved protein-protein
interactions at the ICD or the MTJ. Even though a functional
involvement of Xirp1 in cardiac and skeletal muscle development
or in myocellular repair is speculative, we could show that Xirp1 is
rapidly responsive to myocellular injury. What determines this
transcriptional response also awaits further clarification. Further
characterization of the molecular signatures during normal
myocellular repair will be informative for human cardiac or
skeletal muscle repair.
Figure 8. Characterization of the P47 antibody. (A) Myotendinous
junction labeling by the P47 antibody which recognizes Xirp1a/c is
absent in xirp1sa0046mutant embryos at 24 hpf. This staining was
performed upon mild fixation. (B) Clonally expressed Xirp1c is
sensitively detected upon standard fixation conditions by the P47
antibody within somitic muscle tissue. Truncated Xirp1cDFilCBD-GFP
which lacks the Filamin C binding domain including the P47 epitope is
not detected by the antibody. Under standard fixation conditions,
Xirp1a/c is not detected at the myotendinous junctions. Green: GFP or
Xirp1c-GFP fusion protein; red: Actin; blue: Xirp1a/c. All scale bars:
Figure 9. Efficient Morpholino antisense oligonucleotide-
mediated knock-down of xirp2a does not affect myofibrillogen-
esis. (A) Phenotypically, xirp2a morphants are indistinguishable from
WT embryos at 24 hpf. Scale bar: 250 mm. (B) Efficient gene knock-down
of xirp2a is assessed by immunohistochemistry at 24 hpf. Green: Xirp2a;
red: Actin. Scale bar: 20 mm. (C) Complete loss of Xirp2a does not affect
myofibrillogenesis and correct sarcomeric organization of skeletal
muscle as determined by immunohistochemistry using an antibody
against the A/I junction epitope of Titin and rhodamine phalloidin to
label sarcomeric Actin at 24 hpf. Green: Titin; red: Actin. Scale bar: 5 mm.
Figure 10. Rapid xirp1 transcriptional response to myocellular
injury. Whole-mount in situ hybridizations on 33 hpf embryos which
were laser-injured either at 29 hpf (bottom row) or at 32 hpf (middle
row) at the level of three different somites (dotted lines). xirp1 mRNA
transcriptional response occurs within one hour after injury and is no
longer detectable at 3.5 hours after injury in WT. There is a lack of xirp1
mRNA expression upon laser-induced myocellular injury in xirp1sa0046
mutants. Scale bar: 100 mm.
PLoS ONE | www.plosone.org10 February 2012 | Volume 7 | Issue 2 | e31041
Materials and Methods
Fish stocks and maintenance
General zebrafish maintenance and embryo collection was
carried out according to standard conditions. The characterization
of adult xirp1 mutant fish was performed with permission by the
local authorities (Senatsverwaltung fu ¨r Gesundheit, Umwelt und
Verbraucherschutz, Berlin [Lageso Berlin]) under animal exper-
imentation project G0069/10. Embryos were staged at 28.5uC.
The following fish strains were used: AB/WIK, Tu ¨LF/WIK,
Tg[myl7:gfp] . The xirp1sa0046allele was obtained from the
Wellcome Trust Sanger Center (Cambridge, UK). Mutant fish
were genotyped and sequenced using the following primer pair:
Pharmacological treatment of zebrafish embryos
Cell proliferation was determined by BrdU labeling according
to established conditions using 5 mg/ml BrdU in E3 medium
(B5002, Sigma) . We used 150 mM Aphidicolin in E3 medium
(Sigma, A0781) to block cell proliferation . Both treatments
started at 24 hpf followed by wounding of embryos at 31 hpf and
recovery in BrdU or BrdU+Aphidicolin solutions for 2 hours at
For scoring wound healing depending on cell proliferation, wild-
type embryos were incubated with or without 150 mM Aphidicolin
from 24 hpf onwards, laser-injured at 28 hpf and left to recover in
with or without Aphidicolin until 34 hpf (6 h recovery), or
wounded at 33.5 hpf and fixed immediately (no recovery) for
further analysis. Each embryo was tracked individually: after
wounding and imaging of the wounds, each embryo was kept
separately to be imaged again at a later timepoint. Actin staining
of the embryos with rhodamine phalloidine was used to visualize
myofibrils and detect open wounds. Large laser injuries were
inflicted in two distinct somite blocks in each fish and scored
separately. Numbers of open, visible wounds/number of injuries
after 6 hours recovery were counted and an unpaired t-test was
performed to analyze the statistical significance of Aphidicolin
treatment on wound closure. There is no significant difference
whether with or without recovery: p-value 0.2149 (with 6 hours
recovery), p-value 0.6985 (without recovery period).
To induce myocellular injury, zebrafish embryos were incubat-
ed in 1 mM Gal (Enzo life sciences, ALX-550-336-M050) in E3
medium between 80% epiboly and 2 or 3 dpf .
Figure 11. Characterization of interactions between Xirp1 and Filamin C or Enah/Vasp family members. (A) Table summarizing the
results of direct yeast two hybrid experiments with the C-terminus of zebrafish Xirp1 (residues 2097–2297) and the GST-tagged domains 19–21 of
zebrafish Filamin Ca (FlnCa) and b (FlnCb). The Filamin CbD (FlnCbD) isoform represents a splice variant lacking the unique insertion within domain
20 that is present in all Filamin C proteins. (B) Western blot overlay experiments show specific binding of the GST-tagged domains 19–21 of FlnCa and
FlnCb to the blotted C-terminus of Xirp1. GST and FlnCbD do not bind, confirming that the unique insertion within domain 20 of Filamin Cb is
necessary for the interaction. (C) Co-immunoprecipitation experiments confirm these interactions: FlnCa and FlnCb are co-precipitated, whereas GST
alone is not. (D) Western blot overlay experiments show specific binding of T7-tagged N-terminus of zebrafish Xirp1 (residues 1–58) containing
proline-rich repeat1 (PR1) to the blotted EEF-tagged EVH1 domains of zebrafish Enah, VaspI and VaspII. (E) Summary of binding assays shows the
isoforms of Xirp1 identified in our study, and the respective binding sites for EVH1 domains of Enah and Vasp (PR1) and Filamin C (carboxy-terminus).
Binding to F-actin is presumably mediated by Xin-repeats (red boxes). PRs are depicted by blue boxes.
PLoS ONE | www.plosone.org11 February 2012 | Volume 7 | Issue 2 | e31041
Microarray analysis, data preprocessing, quality control,
transformation, and normalization were performed as previously
described . All data is MIAME compliant; the raw data with
the accession number GSE30729 has been deposited in the public
repository GEO at NCBI.
Laser-induced myocellular injuries
Tricaine anesthetized embryos were wounded and then left to
recover in E3 medium at 28.5uC for different times and finally
fixed and immunostained for analysis. A micropoint laser
connected to a Zeiss Axioplan was used for injuring according
to manufacturers’ instructions using a 206 objective and a laser
pulse at a wavelength of 435 nm for cell ablation (Photonics, Inc.).
Timelapse recordings were generated using a camera mounted on
a Zeiss Axioplan; wounding and recording was performed using a
206/dry and a 406/oil objective, respectively.
Zebrafish Morpholino antisense-oligonucleotide
The xirp2a splice-blocking MO (Gene-Tools) was injected at a
concentration of 200 mmol/L:
Generation of antibodies and immunohistochemistry
For the immunization of rabbits, parts of the large exon of all
zebrafish xirp genes encoding different fragments [Xirp1: (P43;
Xirp1a/b) aa1117–1422, (P47; Xirp1a/c) aa2097–2297; Xirp2a:
aa2974–3276; Xirp2b: aa1697–2049] were amplified from adult
zebrafish cDNA using Pfu polymerase . Proteins were expressed
and purified as described . Rabbits were immunized four times
with the purified recombinant proteins according to a standard
protocol (Biogenes, Berlin, Germany). Antibodies were purified as
described . Immunohistochemistry was performed as described,
with an Aceton permeabilization step of 5–10 minutes at 220uC
prior to the blocking step . For tissue sectioning of Xirp
immunostainings, transverse sectioning was performed according to
. Unless stated otherwise, Xirp1 immunostainings were
performed with the P43 antibody which detects Xirp1a/b, as they
are the dominantly expressed isoforms. To detect Xirp1a/c, P47
staining was performed upon very mild fixation (4% PFA for
30 min). The following antibodies were used: rabbit anti-Xirp1a/b
(1:500; P43), rabbit anti-Xirp1a/c (1:500; P47), rabbit anti-Xirp2a
(1:500), rabbit anti-Xirp2b (1:500), mouse anti-b-catenin (1:500,
Sigma), mouse anti-Alcam (1:500, zn-8, DSHB), mouse anti-Titin
A/I (1:10; D.O. Fu ¨rst), mouse anti-a-Actinin (1:500; Sigma), mouse
anti-Myosin (1:10; S46 and F59, both DSHB), mouse anti-
Tropomyosin (1:10; DSHB), mouse anti-Pax7 (1:10; DSHB), mouse
anti-BrdU (1:100; Roche Diagnostics), goat anti-mouse FITC
(1:200; Jackson), goat anti-mouse Cy5 (1:200; Jackson), goat anti-
rabbit FITC (1:200; Jackson), goat anti-rabbit Cy5 (1:200; Jackson),
rhodamine phalloidin (1:100; Invitrogen). Recordings of immuno-
stained tissues were performed at the LSM Meta510 FSC or LSM
Meta510 NLO (Zeiss) with 406/oil or 1006/oil objectives. Images
were analyzed with the LSM image browser (Zeiss) and processed
using Image J (Rasband, W.S., ImageJ, U.S. National Institutes of
Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/,
1997–2008) or Adobe Photoshop (Adobe).
Analysis of the gene structure of the xirp1, xirp2a and
Databases (http://genome.ucsc.edu; www.ensembl.org, http://
zfin.org) were searched for sequences homologous to the consensus
sequence encoding Xin-repeats as they are found in mammalian
and chicken Xirps. To identify splice variants of xirp1, EST
databases were examined for the presence of sequences corre-
sponding to the genes. EST sequences were aligned with the
genome using BioEdit software in order to identify intron and
RT-PCR and 59 RACE
Gene predictions were verified by RT-PCR and 59RACE
experiments. For both types of experiments, total RNA was
purified from 24 hpf zebrafish embryos using the RNeasy mini kit
(Qiagen, Hilden, Germany). The Superscript One-Step RT-PCR
Kit with Platinum Taq was used according to the manufacturers’
suggestions (Invitrogen) for products of approximately 1000 bp or
less. For the analysis of longer fragments, cDNA was synthesized
using either a gene-specific primer or random nonamers, and M-
MLV Reverse Transcriptase RNase H Minus, according to the
instructions of the manufacturer (Promega, Mannheim, Germany).
59 RACE was performed using the 59 RACE System
(Invitrogen). cDNA was synthesized using primers corresponding
to a site close to the 59 end of the large exon of the xirp genes. A
further primer corresponding to a site more downstream within
this exon was used to perform nested PCR. All amplicons were
cloned into the pGEM-T vector (Promega, Mannheim, Germany)
and sequenced (LGC, Berlin, Germany).
Whole-mount in situ hybridization
Probes for whole mount in situ hybridizations were amplified
from genomic zebrafish DNA using the following primers:
All amplicons were cloned in pGEMT and sequenced. Whole
mount in situ hybridizations were performed as previously
described . For documentation, stained embryos were cleared
in benzyl:benzoate (2:1) and embedded in Permount. Images were
recorded on a Zeiss Axioplan microscope with 106 objective by
using a SPOT digital camera (Diagnostic Instruments) and Meta
Morph software (Visitron). Images were processed with Photoshop
Cloning of Expression Constructs
To generate the plasmids bactin:a-actinin-gfp, bactin:xirp1c-gfp and
bactin:xirp1c-ires-gfp, human ACTININ2 and zebrafish xirp1c were
subcloned into the Gateway pDONOR 221 vector (Invitrogen,
USA) using the following primers containing flanking attB1 and
For generation of the bactin:xirp1cDFilCBD-gfp construct, the
attB1-xirp1c-attB2 intermediate was PCR amplified using the
following primer pair, digested with AvrII and religated upon
itself, thereby creating a deletion within Xirp1c:
PLoS ONE | www.plosone.org 12February 2012 | Volume 7 | Issue 2 | e31041
In subsequent steps, these plasmids were recombined with
destination vector pDEST Tol2pA (gift of N. Lawson), vector p5E-
b-actin2 (gift of C.-B. Chien) containing the b-actin promoter,
vector p3E-EGFPpA (gift of C.-B. Chien) encoding a C-terminal
EGFP tag, or vector p3E-IRES-EGFPpA encoding an IRES GFP
cassette (gift of C.-B. Chien).
Expression Constructs and Purification of Recombinant
cDNA fragments covering domains 19-21 of Filamin Ca and
Filamin Cb, were cloned into pGEX6P3 (Amersham Biosciences),
and the carboxy-terminus of Xirp1 into pET23-T7. Constructs
were transformed to the E. coli strain BL21-CodonPlus(DE3)-RP
(Stratagene). Expression and purification were carried out
essentially as described . Protein concentrations were deter-
mined as described .
Western Blot Overlay and Co-Immunoprecipitation
Extracts of bacterial cells expressing the recombinant polypep-
tide Xirp1 aa2097–2297 were used for SDS-PAGE using 12%
polyacrylamide gels, and separated proteins were transferred to a
nitrocellulose membrane. Nitrocellulose strips were either incu-
bated with a tag-specific antibody and a HRP-conjugated
secondary antibody to detect the expressed polypeptide, or
overlaid with recombinant polypeptide (GST-tagged Filamin C
fragments). Bound protein was immunodetected using an anti-
GST antibody (Santa Cruz), HRP-conjugated secondary antibod-
ies (Jackson Immuno Research Laboratories, Soham, UK), and
ECL using ‘‘SuperSignal West Pico Chemiluminescent Substrate’’
(Pierce, Rockford, IL, USA) and Kodak XAR-351 film. For co-
immunoprecipitation experiments the purified bacterially ex-
pressed T7 and His6-tagged carboxy-terminus of Xirp1 was
mixed with purified recombinant GST-tagged Filamin C frag-
ments. T7 antibody was added to the mixture and complexes were
immunoprecipitated using ProteinG-coupled Dynabeads. Immu-
noprecipitated proteins were separated by PAA gel ectrophoresis
and blotted to nitrocellulose. The presence of the recombinant
proteins on the blots was analyzed using anti T7-tag or anti-GST
antibodies using standard procedures.
Yeast two hybrid assays
To investigate whether the carboxy-terminus of Xirp1 interacts
with Filamin C, the cDNA fragment of Xirp1 encoding residues
aa2097–2297 was cloned into the pLexA vector. Fragments
homologous to human Filamin C domains 19–21 (the Filamin C
fragment that interacts with human Xirp) from both zebrafish
Filamin Ca/b cDNAs, were amplified by RT-PCR using total
RNA isolated from 24 hpf zebrafish embryos as a template. All
cDNA fragments were cloned into the pAct2 vector, sequenced
and cotransformed with the pLexA vector with the cDNA
fragment derived from Xirp1. Growth on SD-LWH agar plates
and activity of ß-galactosidase were assayed as described .
tion of the laser wounding assay within 28 hpf zebrafish skeletal
muscle. The two laser pulses are marked (yellow circle) and the
retraction of myofibrils at both ends is indicated by red arrows.
Changes also occur within neighboring cells.
Live laser wounding assay. Live stream acquisi-
We are indebted to Robby Fechner for expert technical assistance with the
fish facility. We would like to thank Nicole Cornitius and Jana Richter for
help with the molecular lab work. We would like to thank Robert Fischer,
Thomas Willnow, Michael Bader and members of the Abdelilah-Seyfried
lab for technical help, discussions and comments on the manuscript.
Thanks to Nathan Lawson and Chi-Bin Chien for providing us with
Conceived and designed the experiments: CO PvdV SP US DF SAS.
Performed the experiments: CO IL SP PvdV AS EBN. Analyzed the data:
CO SP PvdV IL US DF SAS. Contributed reagents/materials/analysis
tools: EBN DS JE BW SP US. Wrote the paper: CO SAS.
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