Gene expression pattern analysis of the tight junction protein, Claudin, in the early morphogenesis of Xenopus embryos.
ABSTRACT To study how epithelial layers are formed during early development in Xenopus embryos, we have focused on Claudin, the major component of the tight junction. So far, 19 claudin genes have been found in the mouse, expressed in different epithelial tissues. However, though a number of cytological studies have been done for the roles of Claudins, their expression patterns and functions during early embryogenesis are largely unknown. We found three novel Xenopus claudin genes, which are referred to as claudin-4L1, -4L2, and -7L1. At the early gastrula stage, claudin-4L1, -4L2, and -7L1 mRNAs were detected in the ectoderm and in the mesoderm. At the late gastrula stage, claudin mRNAs were detected in the ectoderm through the involuting archenteron roof. At the neurula stage, claudin-4L1/4L2 and -7L1 mRNAs were differentially expressed in the neural groove and the epidermal ectoderm. At the tailbud stage, the claudin mRNAs were found in the branchial arches, the otic vesicles, the sensorial layer of the epidermis, and along the dorsal midline of the neural tube. In addition, claudin-4L1/4L2 mRNAs were detected in the pronephros and the endoderm, whereas claudin-7L1 mRNA was observed in the epithelial layer of the epidermis.
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ABSTRACT: Der Großteil der in eukaryontischen Zellen ablaufenden chemischen Reaktionen finden in Zellkompartimenten statt, die durch Membranen eingeschlossen werden. SNARE-Proteine sind essentielle Komponenten für die Fusion von gleichartigen (homotypischen) als auch verschiedenartigen (heterotypischen) Membranen. SNAREs sind meist über eine C-terminale Transmembran-Domäne in der „Geber“-Membran verankert. N-terminale 60-70 Aminosäuren lange Hepta-Peptide (SNARE-Motiv) können über Coiled-Coil Formierung den Kontakt mit einem SNARE an der „Empfänger“-Membran herstellen (trans-SNARE Komplex). In der vorliegenden Arbeit wurden die Plasmamembran SNAREs SNC1 und SNC2 in Saccharomyces cerevisiae auf posttranslationale Modifikation durch Ubiquitin untersucht. Dabei stellte sich heraus, dass die Ubiquitylierung an mindestens zwei Lysinen im Substrat erfolgt. Die kovalente Verknüpfung erfolgt durch die Ubiquitin-Ligase RSP5. In Mutanten, die die Sekretion von SNC1/SNC2 zur Plasmamembran inhibieren (sec17-1, sec18-1, sed5-1 und sec1-1), liegen die SNAREs in nicht-modifizierter Form vor. In den Endozytose-Mutanten end3 und end4, die für die Invagination von endozytotischen Vesikeln defekt sind, akkumuliert ubiquityliertes SNC1 an der Plasmamembran. Die Ubiquitylierungs-Reaktion muß daher an der Plasmamembran erfolgen. Eine der Ubiquitylierungsstellen, Lysin-63, befindet sich in der Coiled-Coil-Domäne der SNAREs. Der Austausch des Lysins durch ein Arginin an dieser Stelle führt dazu, dass SNC1 nicht mehr in das Lumen der Vakuole lokalisiert wird. Stattdessen verbleibt das Protein in der Membran der Vakuole. Die zweite Ubiquitylierungsstelle konnte noch nicht identifiziert werden. Das Ubiquitin Bindeprotein DDI1 interagiert mit SNC1/SNC2, und beeinflußt die Verfügbarkeit des SNAREs für den trans-SNARE Komplex. Ob DDI1 über die interne UBA (ubiquitin-associated)-Domäne mit ubiquityliertem SNC1/SNC2 interagiert, ist noch unbekannt.
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ABSTRACT: Claudin proteins are the major components of tight junctions connecting adjacent cells, where they regulate a variety of cellular activities. In the present paper we identified two Xenopus claudin5 genes (cldn5a and 5b), which are expressed early in the developing cardiac region. Precocious cldn5 expression was observed in explants of non-heart-forming mesoderm under inhibition of the canonical Wnt pathway. Cardiogenesis was severely perturbed by antisense oligonucleotides against cldn5 or by Cldn5 proteins lacking the cytoplasmic domain. Results of light- and electron-microscopic observations suggested that cldn5a and 5b are required for Xenopus heart tube formation through epithelialization of the precardiac mesoderm.Embryologia 09/2010; 52(7):665-75. · 2.21 Impact Factor
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ABSTRACT: Claudins are the structural and molecular building blocks of tight junctions. Individual cells express more than one claudin family member, which suggests that a combinatorial claudin code that imparts flexibility and dynamic regulation of tight junction function could exist. Although we have learned much from manipulating claudin expression and function in cell lines, loss-of-function and gain-of-function experiments in animal model systems are essential for understanding how claudin-based boundaries function in the context of a living embryo and/or tissue. These in vivo manipulations have pointed to roles for claudins in maintaining the epithelial integrity of cell layers, establishing micro-environments and contributing to the overall shape of an embryo or tissue. In addition, loss-of-function mutations in combination with the characterization of mutations in human disease have demonstrated the importance of claudins in regulating paracellular transport of solutes and water during normal physiological states. In this review, we will discuss specific examples of in vivo studies that illustrate the function of claudin family members during development and in disease.Clinical Genetics 04/2010; 77(4):314-25. · 4.25 Impact Factor
Gene expression pattern analysis of the tight junction protein, Claudin, in
the early morphogenesis of Xenopus embryos
Makiko Fujitaa, Mari Itoha,b, Mikihito Shibataa,b, Sumiko Tairaa,b, Masanori Tairaa,b,*
aDepartment of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
bCore Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Hongo 7-3-1, Bunkyo-ku,
Tokyo 113-0033, Japan
Received 19 July 2002; received in revised form 4 September 2002; accepted 4 September 2002
To study how epithelial layers are formed during early development in Xenopus embryos, we have focused on Claudin, the major
component of the tight junction. So far, 19 claudin genes have been found in the mouse, expressed in different epithelial tissues. However,
though a number of cytological studies have been done for the roles of Claudins, their expression patterns and functions during early
embryogenesis are largely unknown. We found three novel Xenopus claudin genes, which are referred to as claudin-4L1, -4L2, and -7L1. At
the early gastrula stage, claudin-4L1, -4L2, and -7L1 mRNAs were detected in the ectoderm and in the mesoderm. At the late gastrula stage,
claudin mRNAs were detected in the ectoderm through the involuting archenteron roof. At the neurula stage, claudin-4L1/4L2 and -7L1
mRNAs were differentially expressed in the neural groove and the epidermal ectoderm. At the tailbud stage, the claudin mRNAs were found
in the branchial arches, the otic vesicles, the sensorial layer of the epidermis, and along the dorsal midline of the neural tube. In addition,
claudin-4L1/4L2 mRNAs were detected in the pronephros and the endoderm, whereas claudin-7L1 mRNA was observed in the epithelial
layer of the epidermis. q 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Xenopus laevis; Early development; Expression pattern; Morphogenetic movement; Gastrulation; Neurulation; Neural plate; Epithelial layer;
Sensorial layer; Branchial arches; Otic vesicle; Pronephros; Claudin
1. Results and discussion
Seven cDNA clones that were homologous to mouse
Claudins at the amino acid sequence level were found in
expressed sequence tags (ESTs) of a Xenopus anterior endo-
mesoderm cDNA library (Shibata et al., 2001). We deter-
mined the nucleotide sequences of these clones, and found
three novel Xenopus claudin genes (Fig. 1a). They encode
214, 213 and 213 amino acids and were named as claudin-4-
like 1 (claudin-4L1), claudin-4-like 2 (claudin-4L2), and
claudin-7-like 1 (claudin-7L1) owing to their similarity to
mouse claudin genes (Fig. 1a,b). They have four putative
transmembrane regions, which are highly homologous to
those of mouse Claudin-1 to -8 (Morita et al., 1999), and
the tyrosine–valine (YV) motif at the COOH-terminus,
which is common to previously known Claudins and is
thought to be recognized by tight junction associated mole-
cules ZO-1, -2, and -3 through their PDZ domains (Haskins
et al., 1998).
Claudin-4L1 and -4L2 have 66% identity to mouse Clau-
din-4 and -6, respectively, while 4L1 and 4L2 have 189
amino acids (88%) in common. The level of amino acid
homology suggests that Claudin-4L1 and -4L2 are most
closely related to mouse Claudin-4. However, the evolu-
tionary scheme made using the UPGMA analysis showed
that 4L1 and 4L2 are also highly related to mouse Claudin-
6 (Fig. 1c). 4L1 has 92.5% amino acid and 88.5% DNA
identity to the recently reported Xenopus claudin (Xcla)
(Brizuela et al., 2001). 4L1 and Xcla may correspond to
A and B genes of Xenopus pseudotetraploid. Claudin-7L1
was highly homologous to both human and mouse Claudin-
7 and -1.
To characterize expression patterns of claudin-4L1,
-4L2, and -7L1 during Xenopus embryogenesis, whole-
mount in situ hybridization was performed. To examine
the inside of embryos, embryos were either hemisectioned
sagittally then subjected to whole-mount in situ hybridiza-
tion, or sectioned after whole-mount in situ hybridization.
Claudin-4L1, -4L2, and -7L1 were expressed in the animal
half at the blastula stage (data not shown). At the gastrula
stage (stage 11), claudin expression was found to cover the
Mechanisms of Development 119S (2002) S27–S30
0925-4773/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved.
* Corresponding author. Tel.: 181-3-5841-4434; fax: 181-3-5841-4434.
E-mail address: email@example.com (M. Taira).
entire surface ectoderm, but was not found in the yolk plug
endoderm (Fig. 2a–c). Sagittal sections of stained embryos
showed strong claudin expression in the epithelial layer
and weak expression in the sensorial layer of the ectoderm
(4L1, Fig. 2d,e; 4L2 and 7L1, data not shown). Hemisec-
tions showed that the claudin genes were expressed in the
ectoderm including the blastocoel roof and the mesoderm
area at the early gastrula stage (stage 10.25) (Fig. 2f–h). At
the late gastrula stage (stage 12), claudin expression in the
ectoderm continued in the involuting archenteron roof
while expression in the involuting mesoderm decreased
and disappeared (Fig. 2i–n). Thus, claudin-4L1, -4L2, and
-7L1 showed similar expression patterns during the
At the neurula stage, expression of claudin-4L1 and -4L2
decreased in the epidermis and became localized to the
neural plate and later to the neural groove area (Fig.
3a,b,d,e,g,h). Sections showed that expression of claudin-
4L1 and -4L2 in the epithelial layer of epidermal ectoderm
decreased and disappeared as the stage proceeded, while
expression in the sensorial layer became evident (Fig.
4a,b). In contrast, claudin-7L1 was expressed in the entire
epidermis (both epithelial and sensorial layers of the ecto-
derm), whereas the expression in the neural plate was
reduced (Figs. 3c,f and 4e). Expression of claudin-7L1 in
the neural groove area was located to a more distal area
(Figs. 3i and 4f) compared to that of claudin-4L1 and -4L2,
which were along the midline of the neural groove (Figs.
3g,h and 4b). In late neurula to tailbud stage embryos,
claudin-4L1 and -4L2 were expressed along the dorsal
midline of the neural tube, in the otic placode and vesicle,
the branchial arches, the pronephros (Fig. 3j,k,m,n,p,q),
and the sensorial layer of the epidermis and the ventral
region of the endoderm (Fig. 4c,d). Claudin-7L1 was
expressed along the dorsal midline of the neural tube, in
the otic placode and vesicle, the branchial arches (Fig.
3l,o,r), and both the epithelial and sensorial layers of the
epidermis (Fig. 4g,h).
Expression patterns of claudin-4L1 and -4L2, as well as
reported Xcla, were very much alike during early develop-
ment. Claudin-7L1 showed a similar expression pattern to
claudin-4L1 and -4L2 during the gastrula stage but clearly
different patterns during the neurula and tailbud stages. It
should be noted that claudin-4L1, -4L2, and -7L1 are
expressed in tissues showing morphogenetic movements,
such as epiboly and convergent extension, in which migra-
tion study in mice has shown that claudin-1 is required for
pus,tight junctions as well as gap junctions are implicated in
the determination of left–right asymmetry as assayed by
modulating the activities of Xcla and connexin gene, respec-
be interesting to analyze the role of each claudin gene in the
2. Materials and methods
pBluescript SK (2) plasmid cDNAs were isolated from
an anterior endomesoderm cDNA library of lZAP II
clones (Shibata et al., 2001). Inserts were sequenced on
both strands with LONG READER 4200 (LI-COR) with
the T3 or T7 primer. Manipulation of Xenopus embryos
was as described previously (Shibata et al., 2001).
Embryos were staged according to Nieuwkoop and Faber
(1967). Whole-mount in situ hybridization of albino
M. Fujita et al. / Mechanisms of Development 119S (2002) S27–S30
Fig. 1. Structural analysis of Xenopus claudin cDNAs. (a) Seven cDNA
clones encoding Xenopus claudin genes. Boxes, coding region; arrows,
sequenced region. (b) Alignment of the deduced amino acid sequences of
Xenopus Claudin-4L1, -4L2, and -7L1. Boxes, highly homologous residues;
lines, putative transmembrane region; asterisk, tyrosine–valine (YV)
sequence, a supposed PDZ domain-binding site.(c) Evolutionary relation-
ships obtained using the UPGMA analysis. Mouse and Xenopus Claudin
proteins were deduced from the corresponding nucleotide sequences. The
GenBank accession numbers for sequences used are: mouse claudin 1
(NM016674), claudin 4 (AF087822), claudin 6 (BC005718), claudin 7
(BC008104), and Xenopus Cla (AF224712). The nucleotide sequences of
Xenopus claudin4L1, claudin4L2, and claudin7L1 have been deposited in
DDBJ/EMBL/GenBank under accession numbers AB072908, AB072909,
and AB072910, respectively.
embryos with DIG-RNA probes was performed with an
automated ISH system (AIH-101, Aloka) according to
the method of Harland (1991). Stained embryos were
embedded in paraffin and sectioned. Nuclei were stained
This work was supported in part by a Grant in-Aid for
Scientific Research from the Ministry of Education,
Science, Sports and Culture of Japan; by the Toray Science
M. Fujita et al. / Mechanisms of Development 119S (2002) S27–S30
Fig. 3. Expression of Xenopus claudin-4L1, -4L2, and -7L1 genes in neurula to early tailbud stage embryos. (a–c) early neurula. (d–i) midneurula. (j–o) late
neurula. (p–r) tailbud stage embryo. (a–l) dorsal view. Anterior is up. (m–r) lateral view. Anterior to the left. br, Branchial arch; ng, neural groove; op, otic
placode; ov, otic vesicle; pn, pronephros.
Fig. 2. Expression of Xenopus claudin-4L1, -4L2, and -7L1 genes in gastrula embryos. (a–c) Vegetal view of midgastrulae. (d,e) An animal pole area of sagittal
section of early gastrula (-4L1) in bright field (d) and DAPI staining (e). The blastocoel was collapsed in this section. Sagittal hemisections of early gastrulae
(f–h) or late gastrulae (i–k). (l–n) Enlargement of upper part of the archenteron of i–k, respectively. ac, archenteron; bc, blastocoel; el, epithelial layer of
ectoderm; sl, sensorial layer of ectoderm; arrowhead, blastopore; small arrows in f–h, mesodermal expression; large arrows in l–n, expression in the arch-
enteron roof; dotted line in i–k indicates the blastocoel. Developmental stages (st.) are shown above each column of pictures.
Foundation, Japan; and by a grant from the Japan Society
for the Promotion of Science Fellowship (M.S.).
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M. Fujita et al. / Mechanisms of Development 119S (2002) S27–S30
Fig. 4. Expression of Xenopus claudin genes examined by transverse sections of embryos. Expression of either Claudin-4L1 or -4L2 was shown, because
Claudin-4L1 and -4L2 showed similar expression patterns. (a) claudin-4L1 at stage 15. (b) claudin-4L2 at stage 17/18. (c) claudin-4L2 at stage 21. (e–g)
claudin-7L1 at stage 15 (e), stage 17/18 (f), and stage 21 (g). (d,h) Enlargement of the ventral region of c and g, respectively. Dorsal is up. Upper panels, bright
field; lower panels, DAPI staining. el, epithelial layer of epidermal ectoderm; end, endoderm; sl, sensorial layer of epidermal ectoderm.