A role for N-cadherin in mesodermal morphogenesis during gastrulation
Rachel M. Warga, Donald A. Kane⁎
Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008, USA
Received for publication 14 March 2007; revised 26 June 2007; accepted 28 June 2007
Available online 6 July 2007
Cell adhesion molecules mediate numerous developmental processes necessary for the segregation and organization of tissues. Here we show
that the zebrafish biber (bib) mutant encodes a dominant allele at the N-cadherin locus. When knocked down with antisense oligonucleotides, bib
mutants phenocopy parachute (pac) null alleles, demonstrating that bib is a gain-of-function mutation. The mutant phenotype disrupts normal
cell–cell contacts throughout the mesoderm as well as the ectoderm. During gastrulation stages, cells of the mesodermal germ layer converge
slowly; during segmentation stages, the borders between paraxial and axial tissues are irregular and somite borders do not form; later, myotomes
are fused. During neurulation, the neural tube is disorganized. Although weaker, all traits present in bib mutants were found in pac mutants. When
the distribution of N-cadherin mRNA was analyzed to distinguish mesodermal from neuroectodermal expression, we found that N-cadherin is
strongly expressed in the yolk cell and hypoblast in the early gastrula, just preceding the appearance of the bib mesodermal defects. Only later is
N-cadherin expressed in the anlage of the CNS, where it is found as a radial gradient in the forming neural plate. Hence, besides a well-established
role in neural and somite morphogenesis, N-cadherin is essential for morphogenesis of the mesodermal germ layer during gastrulation.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Mesoderm; Muscle; Notochord; Cell adhesion; N-Cadherin; Gain-of-function; Gastrulation
the biber (bib) mutant identified in the Tübingen large-scale
screen for mutations that affect morphogenesis in the zebrafish,
carried out at the Max Planck Institut für Entwicklungsbiologie
it was placed into the gastrulation and tail class, where it was
described briefly as a simple homozygous recessive lethal,
having indistinct somites and a disorganized tail (Hammersch-
midt et al., 1996). Phenotypically unique amongst the mutants
found in the screen, bib was never successfully placed in a
In the first portion of this work, we demonstrate that the bib
mutation is in a single gene that encodes the zebrafish homolog
of N-cadherin. One of the earliest calcium-dependent cell
adhesion molecules identified, N-cadherin was first described in
the CNS of the mouse and chicken (Hatta and Takeichi, 1986),
of cadherins known as the classic cadherins (Takeichi et al.,
1988). In its most well-known role, N-cadherin is strongly
et al., 1997; Radice et al., 1997; Redies et al., 1993; Redies and
Takeichi, 1993), where its expression pattern is complementary
to the expression of E-cadherin in the epidermal ectoderm
(Takeichi, 1995). In general, the pattern of expression during
motility, suggesting a potential role in the epithelial to mesen-
chymal transition (Duband et al., 1988; Hatta et al., 1987; Oda
et al., 1998). N-Cadherin tends to be expressed in mesenchymal
cell types whereas E-cadherin is expressed in epithelial cell
often E-cadherin expression is lost in metastastic cells, while the
expression of N-cadherin is elevated (Derycke and Fracke,
The N-cadherin protein is characterized by five extracellular
cadherin (EC) repeats, a single pass transmembrane domain and
a cytoplasmic domain. The EC repeats are extremely conserved
among theirhomologs inother species, and arenecessary forthe
specific adhesion properties of the cadherins (Blaschuk et al.,
Developmental Biology 310 (2007) 211–225
E-mail address: firstname.lastname@example.org (D.A. Kane).
0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
1990; Yagi and Takeichi, 2000). The cytoplasmic domain of the
protein binds alpha- and beta-catenin, and indirectly actin. This
anchors the protein to the cytoskeleton of the cell and connects
the molecule to the WNT and other signaling pathways
(Christofori, 2003; Derycke and Fracke, 2004; Gumbiner and
McCrea, 1993; Herrenknecht et al., 1991; Kintner, 1992;
McCrea et al., 1991; Sanson et al., 1996). The protein is thought
to function as a dimer, and the EC repeats are necessary for this
dimerization (Brieher et al., 1996; Nagar et al., 1996; Shapiro et
al., 1995). Based on EC domain switching studies, the adhesive
specificity of cadherins is primarily conferred by the first EC
domain (Blaschuk et al., 1990; Nose et al., 1990), a specificity
that is mediated through a twofold symmetric interaction
between EC1 domains on opposing cells (Boggon et al., 2002;
Harrison et al., 2005).
In the second portion of this work, we examine the role of
N-cadherin in the development of the mesoderm during
gastrulation and early segmentation. Although N-cadherin is
expressed in the mesoderm, targeted knockout studies in the
mouse suggests that it is not essential for mesodermal
morphogenesis during gastrulation; defects are seen only later
during somitogenesis where they are limited to malformations
of somitic and cardiac structures (Radice et al., 1997; Horikawa
et al., 1999). When N-cadherin function is rescued in the heart,
allowing the mutant embryos to develop further, dramatic
defects occur in brain development (Luo et al., 2001). Even
more subtle are targeted N-cadherin mutations in Drosophila.
Null mutant embryos are partly viable with no defects in
mesodermal structures and, when examined more closely, the
mutants show axonal pathfinding errors in the central nervous
system (Iwai et al., 1997).
Recently N-cadherin mutations have been identified in the
al., 2002; Masai et al., 2003; Wiellette et al., 2004). AlthoughN-
cadherin expression is reported as fairly ubiquitous throughout
the zebrafish embryo during early development (Bitzer et al.,
1994; Lele et al., 2002), the phenotypes observed in pac alleles
where it is necessary for the convergence of the ectoderm during
neurulation, and later, the cellular organization of the brain and
retina (Lele et al., 2002; Malicki et al., 2003; Masai et al., 2003).
Outside the nervous system, N-cadherin seems only required
during late somitogenesis for the radial migration of a select
population of slow muscle fibers to the surface of the myotome
(Cortes et al., 2003). Hence, to date, the studies in zebrafish and
other animals support the idea that N-cadherin has no role in
mesodermal morphogenesis during gastrulation.
In this report, we demonstrate that the zebrafish mutant bib is
a dominant gain-of-function mutation in the N-cadherin gene.
We focus primarily on cellular defects that are observed in the
bib mutant mesoderm, especially during gastrulation, and
show that pac mutants exhibit similar albeit more subtle defects
in the same cell types. Analysis of RNA expression during the
blastula and gastrula stages correlates the presence of N-
cadherin RNA in the hypoblast with the time of appearance and
location of the bib mutant phenotypes, showing a surprisingly
The gain-of-function nature of the bib allele reveals that
N-cadherin is playing a far greater role in morphogenesis of
the mesoderm during gastrulation then previously thought.
The bibtb8allele was isolated in a large-scale mutagenesis screen (Haffter
type fish for mapping. Subsequent generations were out-crossed to various wild-
type strains of fish that were polymorphic for the appropriate microsatellite
markerssurroundingthe biblocus.Thepactm101alleleof N-cadherin(Leleet al.,
2002)was usedfor complementationstudies.The trim209alleleof Van Goghlike
were used for double mutant studies.
Mapping of the bib locus
bib was initially mapped to one arm of Linkage Group 20 by half tetrad
analysis (Johnson et al., 1995). Fine-resolution mapping was obtained using a
larger panel of 353 EP-diploid embryos to identify two closely linked
microsatellite markers (Knapik et al., 1998). An independent panel of 657
related diploid embryos was used to further characterize recombination
frequencies between closely linked microsatellite markers and the bib locus.
Microsatellite markers were mapped as simple sequence length polymorphisms
and genes were mapped as single-stranded conformation polymorphisms as in
McFarland et al. (2005).
Isolation of the bib mutation and genotypic characterization
To locate the site of the bib mutation we used the RT–PCR approach
the published cDNA. We confirmed the mutation by isolating genomic DNA
from individual mutant and wild-type embryos to generate sequence for the site
CCT CAT C 3′).
Characterization of individual embryos to determine genotype in subsequent
experiments was performed using the above reverse primer and one of the two
following forward primers (bib: F-5′ CCC ATT GAC ATC ATC AA 3′, 55 °C
annealing; wild type: F-5′ CCC ATC GAC ATC ATC AT 3′, 60 °C annealing)
which took advantage of a polymorphism based on a silent nucleotide change in
the mutant background as well as the mutant lesion, see examples in Fig. 2C.
Alternatively, embryos were genotyped by the closely linked markers z6425 or
z78809 (distal) and z3964, z20582, z61014, z59566 or z9343 (proximal).
Time-lapse and data analysis
For in vivo observations, embryos derived from bib heterozygotes were
mounted and recorded in multi-plane as previously described (Warga and Kane,
between 30° and 60° lateral from the shield. Afterwards, the recordings were
analyzed using Cytos Software, looping half hour segments from the time lapse
recording. In the case of Fig. 6, black and white images from one plane of the
time-lapse video recording were imported into Adobe Photoshop and pseudo-
colored to aid in presentation.
Embryo manipulation, immunohistochemistry and RNA in situ
Morpholino antisense oligonucleotides (5′-TCT GTA TAA AGA AAC
CGA TAG AGT T 3′) corresponding to −40 to −16 of the N-cadherin cDNA
(Lele et al., 2002) were injected into 1-cell stage embryos at a final con-
centration of 50 μM. At the 18-somite stage embryos were photographed and
212R.M. Warga, D.A. Kane / Developmental Biology 310 (2007) 211–225
Transplantation was carried out between doming and 30% epiboly stage as
described elsewhere (Ho and Kane, 1990) with the following modification:
donor embryos were injected with either fixable FITC–dextran or a combination
of neutral rhodamine–dextran and fixable biotinylated–dextran (Molecular
Probes). Hosts were examined at 30 h and recorded live as described (Warga and
Kane, 2003), or fixed in 4% paraformaldehyde and processed for the fixable
To detect the FITC–tracer we used a peroxidase conjugated anti-FITC
Detection of the biotin tracer was performed using the Vectastain Elite ABC
(peroxidase) kit and Vector SG enzyme substrate (Vector laboratories), giving a
blue reaction product.
RNA in situ hybridization was carriedout as describedin Thisse et al. (1994)
with modifications from a more up to date protocol by the same authors at http://
zfin.org/zf_info/monitor/vol5.1/vol5.1.html. Embryos were cleared in 70%
glycerol,photographed ona ZeissAxioskopequippedwitha SonyF-707Digital
Camera, and in most instances, genotyped.
Cell contact is perturbed in the bib mesoderm
The bib phenotype first becomes apparent near the end of
gastrulation, when the anlage of the notochord begins to
characteristically kink in the mutant (Figs. 1A, A′) and cells in
the paraxial mesoderm appear to loose cell contact, phenotypes
easily observed on thestereomicroscope.By the3-somite stage,
the paraxial mesoderm of the mutant has disaggregated into a
loose association of spherical cells (Figs. 1B, B′). Curiously,
where paraxial cells contacted the notochord they appeared to
adhere to it, accentuating its knobby and uneven appearance. In
the notochord region itself, cell adhesion defects did not appear
as severe, although cells were not as closely packed as usual and
did not organize into the uniform rows as seen in normal
embryos (Figs. 1B, B′). All these presumptive adhesion defects
were particularly severe in the tailbud of the mutant (which can
mesodermal tissues remained disorganized, somite boundaries
were fused (Figs. 1C–E′), and the tail appeared vacuolated,
flatter, and often curved up, the basis for the moniker “biber”,
German for “beaver”.
Although cellular defects were first detected within meso-
the mutant became disorganized and it was difficult to define
Eventually, by 30 h, both the brain and spinal cord were filled
with rounded cells and both structures acquired an uneven
appearance (Figs. 1E, E′). Despite these morphological abnorm-
alities, the basic body plan was normal at the gross level, and, at
the cellular level, mutant tissues differentiated into histotypes
muscle formed in the myotomal regions (Figs. 1G, G′) and
differentiated neurons formed in the CNS (data not shown).
bib is a mutation in N-cadherin
Using microsatellite markers and half-tetrad analysis, we
mapped bib to one arm of Linkage Group 20 (Fig. 2A) close to
several centromeric markers (Fig. 2B; Table 1). This placed bib
in the vicinity of the gene N-cadherin (also termed cdh2).
Ironically, this gene was initially rejected as a candidate because
the bib phenotype was distinct from the phenotype of alleles of
parachute (pac) (compare Figs. 1E′ to 1F), mutations in the
zebrafish N-cadherin locus (Birely et al., 2005; Lele et al., 2002;
Malicki et al., 2003; Masai et al., 2003; Wiellette et al., 2004).
Nevertheless, fine mapping placed bib very close to N-cadherin
(0 recombinants in 192 meioses), and subsequently, matings
between bib/+ and pactm101/+ adults revealed that bib and pac
failed to complement. Interestingly, bib/pac transheterozygotes
resembled bib homozygous mutants, the more extreme pheno-
type. Normally a transheterozygote phenotype resembles an
intermediate phenotype close to the weaker of the two
The bib allele was sequenced from cDNA generated by RT–
PCR. The mutation is a transversion in a nucleotide within the
coding region of the first EC domain of the N-cadherin protein,
changing the well-conserved isoleucine242to an asparagine
(Figs. 2D–F). To confirm that the bib mutation was in N-
Fig. 1. Morphology of the bib phenotype. (A) Dorsal view; arrows indicate
kinked mutant notochord, inset shows high magnification of rounded cells in
paraxial mesoderm, dotted line outlines ventral portion of anterior neural plate.
(B) High magnification of notochord (nc) and paraxial mesoderm (pm). (C) Side
view: arrows indicate position of somite furrows; arrowhead indicates flatter
mutant tailbud. (D) Side view: arrowhead indicates defects in brain subdivi-
sions; arrow indicates defects in muscle segmentation. Side views of (E) wild-
type and bib siblings and (F) pac mutant; arrowheads indicate uneven surface of
neural tube, arrows indicate vacuoles in disorganized tail. (G) High magni-
fication of wild type and bib striated muscle.
213R.M. Warga, D.A. Kane / Developmental Biology 310 (2007) 211–225
pression pattern of N-cadherin in the early gastrula coincides
with the bib and pac mutant phenotypes. Understanding the
detailed role that N-cadherin plays in balancing the subtle forces
that underlie adhesion and de-adhesion will be essential for a
complete understanding of vertebrate morphogenesis. Mutants
such as bib, that change that balance, will be valuable tools in
such an investigation.
Solnica-Krezel for comments and discusions on earlier versions
of this paper, and to Dr. Matthias Hammerschmidt for his kind
gift of the antisense oligonucleotide for the knockdown
experiment. The research was supported by grants from the
National Institutes of Health and startup funds from Western
Michigan University to D.A.K.
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