Mechanical strain regulation of the Chicken glypican-4 gene ...

AJP-Regu Articles in PresS. Published on June 13, 2002 as DOI 10.1152/ajpregu.00088.2002
Mechanical strain regulation of the Chicken glypican-4 gene expression
in the avian eggshell gland
Irena Lavelin, Noam Meiri, Miriam Einat, Olga Genina, and Mark Pines
Institute of Animal Science, Agricultural Research Organization, The Volcani Center,
Bet Dagan 50250 Israel.
Short title: Glypican-4 in avian eggshell gland
Correspondence to:
Dr. Mark Pines
Beth Israel Deaconess Medical Center
Division of Bone and Mineral Metabolism
330 Brookline Ave. HIM 948, Boston MA 02215
Tel - 617-667-4459
Fax- 617-667-4432
E-mail – pines@agri.huji.ac.il
Copyright 2002 by the American Physiological Society.
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ABSTRACT
Comparison of RNA fingerprinting of the avian eggshell gland (ESG) without and with
an egg revealed up-regulation of a 382-bp cDNA fragment that showed high homology
to the mammalian glypican 4 (GPC-4). The gene sequence revealed a conserved glypican
signature, a GPI-anchorage site and cystein residues most of which were conserved.
GPC-4 was expressed in the ESG in a circadian fashion only during the period of
eggshell calcification, when maximal mechanical strain was imposed. Removal of the
egg just before to its entry into the ESG, with consequent elimination of the mechanical
strain caused reduction in the gene expression. Artificial application of the mechanical
strain induced expression of the GPC-4 gene that was related to the level of the strain.
GPC-4 expression was strain-dependent in other parts of the oviduct. In the ESG, GPC-4
was expressed exclusively by the glandular epithelium and not by the pseudostratified
epithelium facing the lumen.
In summary, we cloned the avian homologue of GPC-4, established its pattern of
expression in the avian ESG and demonstrated for the first time that this gene is
regulated by mechanical strain.
Key words: Heparan sulfate proteoglycans; calcification, eggshell gland
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Abbreviations
ESG - avian eggshell gland
GPC-4 - glypican 4
OPN- osteopontin
ECM - extracellular matrix
GPI- glycosyl phosphatidyl inositol
RACE- Rapid amplification of cDNA ends
SSRE- shear stress response element
PE- pseudostratified epithelium facing the lumen
GE- inner glandular epithelium
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INTRODUCTION
During the past few years, the effect of mechanical force on the regulation of cell
functions has been extensively studied (8). Various stresses or strains, such as hydrostatic
or hydrodynamic pressure, tensile or biaxial stretching, fluid shear stress and
hypergravity have been applied to various cells in vitro to examine their response to the
mechanical stimuli. The most commonly used cell types were bone (31), cartilage (19),
smooth muscle (38), endothelial cells (7), and cardiomyocytes (47) that are usually
subjected to high fluid shear stresses or pressure loads. The applied forces caused a
variety of physiological responses such as increased bone resorption by osteoclasts (23),
changes in matrix protein synthesis by chondrocytes and cardiac fibroblasts (19,27),
pulmonary epithelial cell differentiation (36), changes in smooth muscle contractility
(38) and increased migration and tube formation by coronary endothelial cells (50).
Most, if not all, of these experiments were performed in vitro and were associated with
pathophysiological states such as hemodynamic overload of heart and blood vessels,
osteoarthrosis, osteoporosis, angiogenesis due to cancer, etc. The mechanism probably
involved multiple signal transduction pathways (1).
Recently we demonstrated that the entry of an egg into the eggshell gland (ESG) of the
laying hen imposes a mechanical strain that regulates the expression of a set of genes
associated with the eggshell formation (24,25). Thus, the avian ESG is a unique in vivo
biological system in which the mechanical forces are coupled to a physiological readout
and are imposed in a circadian fashion during the daily egg cycle. Using this system we
previously demonstrated a strain-dependent up-regulation of genes encoding for the �1
subunit of the Na-K ATPase (25) and osteopontin (OPN) (24,33), that are involved in ion
transport and calcification processes in eggshell and bone, respectively.
The eggshell is formed during the passage of the egg through the oviduct, where the
various layers of the eggshell are assembled sequentially. After fertilization of the ovum,
the egg spends 2-3 h in the magnum where the proteins for embryo consumption are
secreted and 1-2 h in the isthmus where the two egg membranes are built. Then the egg
enters the ESG, where the calcium deposition occurs, for an additional 18-20 h. During
the transport of calcium for shell formation from the plasma to the lumen of the ESG,
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about 10% of the total body calcium is secreted within 18-29 h. This makes eggshell
formation one of the most rapid biomineralization processes known (10).
In the present study we applied the RNA fingerprinting technique in an effort to identify
additional genes that might be involved in shell formation and regulated by mechanical
strain. The unique circadian pattern of eggshell calcification allows us to compare gene
expression in two different physiological states: A– no calcification and no mechanical
strain, B– peak of calcification and maximal mechanical strain. By this technique we
identified a 382-bp cDNA that was up-regulated at the time of maximal mechanical
strain, and that exhibited 80% homology with the mouse heparin-sulfate proteoglycan,
glypican 4 (GPC-4).
Proteoglycans are proteins containing glycosaminoglycan side chains that exist in the
extracellular matrix (ECM) and on the surface of many types of cell. These molecules are
thought to play an important role in cell growth and morphogenesis, and in cancer
development (18,35). The most abundant proteoglycans are those that bear
glycosaminoglycan chains consisting of heparan-sulfate. One family of the heparansulfate
proteoglycans comprises the cell-surface glypicans and includes six members, to
date (14). All known members of this family have similar core protein sizes and spacing,
and a glycosaminoglycans attachment consensus sequence close to the C-terminus of the
proteins; they all exhibit conservation of cystein residues, and all are linked to the cell
surface by a glycosyl phosphatidyl inositol (GPI) anchor (12). Members of this family
have been associated with the Simpson-Golabi-Behmel syndrome, that is characterized
by pre- and postnatal overgrowth (32) and with embryonic medullary renal dysplasia (5),
and they modulate the action of stimulatory and inhibitory growth factors during
morphogenesis (16). All structural features of the vertebrate glypicans are also
represented in the product of dally, a drosophila melanogaster locus regulating cell
division patterning in the developing central nervous system (21). Taken together, these
observations implicate glypicans in control of cell division and patterning during
development. The gene for the human GPC-4 has been localized to Xq26 in close
proximity to GPC-3 (43) and its mRNA is ubiquitously expressed in many tissues such as
bone marrow stromal cells and hematopoietic progenitor cells (37), kidney (16) and
embryonic brain (45). To date, no specific function has been associated with the GPC-4
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gene product beyond its ability to serve as co-receptor to the fibroblast growth factor
receptors.
In the present study we evaluated the expression of the GPC-4 gene in relation to avian
eggshell formation and demonstrated for the first time that GPC-4 gene expression is
regulated by mechanical strain.
METHODS
Animals and mechanical strain induction
Female Loman chickens, 4-8-months –old, were used for all experiments. Samples of
magnum, isthmus and ESG at various stages of the egg cycle, and other tissues were
collected. Samples were either frozen in liquid nitrogen for RNA extraction, or fixed
overnight at 4oC in 4% paraformaldehyde for in situ hybridization. Mechanical strain
was induced by insertion into the ESG of endotracheal tubes that after inflation
resembled the shape of an egg, with volumes of 60 or 20 cm3 , as previously described
(24,25).
Preparation of RNA, and RNA Fingerprinting
Total RNA was extracted with TRI REAGENT (MRC, Inc., Ohio, USA) according to the
company’s recommended protocol. The mRNA was prepared from total RNA with the
mRNA Isolation Kit (Boehringer Mannheim, Ottweiler, Germany) according to the
manufacturer’s instructions. Total RNA was fingerprinted with the Delta RNA
fingerprinting kit (Clontech, EMC, USA) using dATP from the Radiochemical Centre
(Amersham, England). In brief: first-strand cDNA was synthesized by using 2 µg of total
RNA as a template, oligo(dT) as a primer and MoMLV-RT reverse transcriptase (Life
Technologies, Inc). Two dilutions of each cDNA template (corresponding to 5 and 20 ng
of reverse transcribed RNA) were used for the PCR reaction. In addition to the template,
each PCR reaction contained 50 µM dNTPs, 1 µM primers, 50 nM [ 33 P] dATP (1,000-
3,000 Ci/mmol), and 1µl Taq DNA polymerase (Perkin-Elmer, N.J., USA). The PCR
primers used were a pairwise combination of arbitrary "P" and oligo(dT) "T" primers (see
Perking Elmer’s reference manual for oligonucleotide sequences). Thermal cycling was
performed according to the following program: one cycle of 94, 40 and 68°C, each for 5
min; two cycles of 94°C for 2 min, 40°C for 5 min and 68°C for 5 min; 22 cycles of 94°C
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for 1 min, 60°C for 1 min and 68°C for 2 min. PCR products were electrophoresed on an
8% acrylamide/8 M urea gel and run in 0.1 M Tris borate/2 mM EDTA (TBE) buffer (pH
8.3). The gels were dried under vacuum and exposed to x-ray films. In a typical RNA
fingerprint, about 80-100 bands were evident in each amplification. Differentially
expressed cDNAs were eluted from the gel, re-amplified, subcloned into the pGEM-T
Easy cloning vector and sequenced. Nucleotide sequences were subjected to FASTA
searches for sequence homologies.
Reverse Transcription (RT)-PCR and PCR Cloning
RNA from ESG was reverse transcribed to single-stranded cDNAs with the aid of
oligo(dT) primers and MoMLV-RT. A PCR master mix was added, to yield the following
concentrations for the cDNA reaction: 1 µM of specific primers, 200 µM dNTPs, 2.5
units of Taq DNA polymerase (Perkin-Elmer, N.J., USA), and Taq buffer containing 1.5
mM MgCl2. PCR was performed through 30 cycles (94°C for 15 sec, 60°C for 30 sec,
and 72°C for 30 sec). Amplification products arising from RT-PCR were electrophoresed
on a 2% agarose gel and visualized by ethidium bromide staining. A range of forward and
reverse primers based on the mouse K-glypican cDNA sequence (accession number
X83577) were used for PCR cloning of the chicken GPC-4. Forward 5’-
GAAAAGTTGCTCGGAAGTGC-3’ and reverse 5’-AAAAGGCTTCAGCTGCTCTG-
3’ primers produced a new 483-bp cDNA fragment. The forward primer pair based on the
sequence received by PCR (5’- GGTACTACGTGGGTGGCAAT-3’) and a reverse
primer pair based on the sequence received by RNA fingerprinting (5’-
GCTGTTCAAGGACCTCTTCG-3’) produced a 319-bp fragment which connected two
previous cDNA fragments to one 969-bp fragment.
To identify GPC-4 and GAPdH mRNAs by RT-PCR in various avian tissues, the
following forward (F) and reverse (R) primers were used.
For GPC-4: F: 5-GAAACGCCGTTGTAGAGCTT-3, and
R: 5-GCATGCTGTTCTCCTGCATA-3;
and for GAPdH:
F: 5-CCATCACAGCCACACAGAAG-3, and
R: 5-CGCATCAAAGGTGGAAGAAT-3.
The expected amplification products were 646 bp for the GPC-4 and 343 bp for GAPdH.
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Rapid amplification of cDNA ends (RACE)
Rapid amplification of cDNA ends (RACE), to complete the 3’ region of the chicken GPC-
4, was performed with the SMART(TM) RACE cDNA amplification kit (Clontech)
according to the manufacturer’s manual. In brief, the first strand cDNA was synthesized
from chicken ESG mRNA by means of the MMLV reverse transcriptase with the aid of
SMART II oligonucleotide and 3'-RACE cDNA synthesis primers, at 42 0 C for 60 min.
PCR was then carried out with a 5’ gene-specific primer – 5’-
GCTGGAGGGGCCTTTTAACATTGAGTC-3’ – synthesized according to the cloned
969-bp fragment and the mix of two universal adapter primers provided with the kit.
Cycling conditions were: five cycles of 94 0 C for 15 s and 72 0 C for 3 min; five cycles of
94 0 C for 15 s, 70 0 C for 30 s and 72 0 C for 3 min; and 30 cycles of 94 0 C for 15 s, 68 0 C for
30 s and 72 0 C for 3 min. A nested PCR reaction was carried out under similar conditions,
with the kit primer (Nested Universal Primer) and a specific nested GPC-4 5’ primer (5'-
GAACAGCATGCAAGTGTCTCA-3'). Figure 1 describes the strategy in the assembly of
the chicken GPC-4. The obtained PCR product was separated on 1% agarose and purified,
cloned into the pGEM-T easy vector and sequenced from both directions with T7, SP6 and
specific primers. The cDNA and deduced amino acid sequences were compared with the
DNA and protein databases at the National Center for Biotechnology Information.
Preparation of riboprobe and in situ hybridization.
A fragment of chicken GPC-4 cDNA (646bp) was synthesized by reverse transcription
(RT)-PCR from the chicken ESG (see under "RT-PCR"). This fragment shared no
homology with any other known chicken cDNA and was subcloned into the pGEM-T
Easy cloning vector to produce a template for riboprobe synthesis. The sense and
antisense riboprobes were synthesized by in vitro transcription with T7 and SP6 RNA
polymerase, respectively, in the presence of linearized plasmid DNA and DIG RNA
labeling mix (Boehringer Mannheim, Ottweiler, Germany). Serial 5-µm sections of the
ESG or magnum were hybridized with a digoxigenin-labeled chicken GPC-4 probe as
described previously (25). No hybridization was observed with the sense riboprobe,
which was used as a negative control. Hybridization with avian �-actin probe was
performed in order to rule out the possibility that changes in GPC-4 expression were due
to variation in tissue processing and/or RNA preservation.
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RESULTS
Identification of the chicken GPC-4 gene in chicken ESG
RNA fingerprinting was used to identify genes involved in the eggshell calcification.
Differential screening was performed between cDNA derived from ESG biopsies from
two different states of the daily egg cycle: 1- no egg resides in the ESG, no calcification
and no mechanical strain; 2- the egg resides in the ESG, at the peaks of eggshell
calcification and of mechanical strain. We isolated and sequenced 14 clones that
appeared to be up-regulated at the time of eggshell calcification. One of these clones – a
382-bp cDNA sequence – showed 80% similarity to the mouse K-glypican (GenBank,
accession number X83577). This sequence was extended to both the 3’ and 5’ directions,
resulting in a complete 3’ region of the gene, and a 5’ region with a missing part
estimated to comprise the first 71 bp of the open reading frame, according to the
mammalian homologues (Fig. 2). The amplified PCR fragments were subcloned, and 11
randomly selected subclones were analysed by sequencing, the obtained sequence was
additionally verified by RT-PCR. Analysis of the obtained sequence revealed 77 and 78%
similarity to the mouse and human GPC-4 genes, respectively, with two stretches
showing 92% homology. Multiple alignment of the predicted protein product of the GPC-
4 gene showed 74 and 75% identity to the amino acid sequences of mouse and human
GPC-4, respectively. Of most importance is the conservancy within the sequence of
motifs that is characteristic of all members of the glypican family; these motifs includ the
glypican signature region and the GPI binding site (Fig.2). Out of the 13 cystein residues
known to be conserved among all mammalian glypicans, 12 were found to be conserved
also in the avian GPC-4. One cystein residue at position 224 corresponding to the mouse
sequence was replaced by glycine as confirmed by RT-PCR analysis of cDNA obtained
from different avian tissues.
Temporal and spatial expression of the chicken GPC-4 gene during eggshell formation
Temporal and spatial localization of the avian GPC-4 in the ESG during the eggshell
formation cycle was performed by in situ hybridization (Fig. 3). Very low levels of
expression of the GPC-4 gene was observed prior to the entry of the egg into the ESG,
while the egg was still located in the magnum or isthmus (Fig. 3A), or 1 h after the egg
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entered the ESG (Fig. 3B). Two hours after the egg entered the ESG (Fig. 3C) some of
the epithelium cells started to express the GPC-4 gene. Gradually, more epithelium cells
expressed the GPC-4 gene, and 8 h after the entry of the egg into the ESG (Fig. 3D) – at
the time when the shell is being rapidly formed – most of the cells expressed the GPC-4
gene. A high level of expression was observed during all the time of massive
calcification (Fig. 3E – 12 h after the entry of the egg into the ESG). One hour before
oviposition (Fig. 3F), at the phase of eggshell completion, a massive reduction in the
gene expression was observed.
The epithelium of the ESG consists of two different cell types: the pseudostratified
epithelium (PE) facing the lumen and inner glandular epithelium (GE). At higher
magnification, the GPC-4 gene was found to be expressed exclusively by the GE cells
and not by the PE cells, regardless of the time the egg had resided in the ESG (Fig. 4).
Effect of mechanical stress on the GPC-4 gene expression
Insertion of an egg-shaped endotracheal tube into the ESG 3 h after oviposition, at a time
when the GPC-4 gene is not naturally expressed (Fig. 5A) caused gene induction. The
expression of the GPC-4 gene was strain-dependent: insertion of a small-size tube (with
a volume of 20 cm 3 ) caused a lower induction of GPC-4 gene expression (Fig. 5B) than
observed with the larger tube (60 cm 3 ; Fig. 5C). Moreover, removing the mechanical
strain by expulsion of the egg close to the time of its entry into the ESG and thus causing
premature oviposition, attenuated GLC-4 gene expression (Fig. 6A), compared with the
level of expression in the control laying hen (Fig. 6B). No change in �-actin gene
expression was observed in ESG sections 3 and 12 h after oviposition or after mechanical
strain application at the time of major increase in the CPC-4 expression, ruling out the
possibility of variation in tissue processing and RNA preservation (Fig. 7).
Expression of the GPC-4 gene depends on mechanical strain, not only in the ESG but
also in other parts of the oviduct. In the magnum, the gene is expressed only when the
egg resides within this part of the oviduct and mechanical strain is imposed (Fig. 8). No
effect of mechanical strain was observed in the sexually immature pre-laying pullet (data
not shown).
Expression GPC-4 gene in various tissues
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The expression of the GPC-4 gene in a variety of tissues was examined by RT-PCR (Fig.
9). In addition to the high levels of the GPC-4 gene that were expressed in the ESG , high
levels were also observed in the liver, pancreas and kidneys. The expression of the GPC-
4 gene in these tissues was unaffected by the daily egg cycle (data not shown).
DISCUSSION
The avian GPC-4 gene exhibits high homology with the mammalian ones and presents
highly conserved sequences such as the glypican signature and the GPI-binding site in
the same locations as the mammalian homologues. Moreover, the conserved locations of
the cystein residues suggest similar three-dimensional structures (Fig. 2). To date, GPC-4
is the second member of the glypican family identified in the chicken [the first being
GPC-1 (29)], which suggests that, similarly to mammals, avian species may contain
more members of this family. In the present study we demonstrated for the first time that
the avian GPC-4 gene is expressed in the ESG in a circadian fashion and is probably
regulated by mechanical strain. This hypothesis is supported by the following
observations: A- The gene is expressed in the ESG only when an egg resides in the ESG
and imposes a mechanical strain (Fig. 3); B- Removal of the mechanical strain caused
reduction in the gene expression (Fig. 6); C- Artificial application of the mechanical
strain caused induction of the GPC-4 expression that was related to the level of the strain
(Fig. 5); and D- the GPC-4 gene was induced by mechanical strain in other parts of the
oviduct too (Fig. 8). In the ESG the GPC-4 was expressed only by the GE cells and not
by the cells facing the lumen (Fig. 4). Previously (24) we demonstrated that OPN
expressed by the PE cells was regulated by the mechanical strain while calbindin, a
calcium-binding protein expressed by the GE cells in a circadian fashion, was not.
Moreover, the α1 subunit of the Na-K ATPase that was expressed by both cell types was
regulated by mechanical strain only in the PE cells, whereas in the GE cells it was
regulated by the calcium flux (25). Thus GPC-4 is the first example of a mechanicalstrain-dependent
gene expressed by the GE cells. This suggests that the mechanical strain
signal that is first sensed by the cells facing the lumen had to be transduced to the inner
cell layer either directly or mediated by factor(s) from the pseudostratified epithelium.
The expression of other proteoglycan genes such as versican, biglycan, perlecan and
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decorin, some of which are differentially regulated in various tissues, is also known to be
affected by mechanical strain (26). For example, biglycan is activated in smooth muscle
cells (26) and attenuated in lung epithelium (46) in response to mechanical strain.
Mechanical signals activate many different signal transduction pathways, but currently
no single transcriptional regulatory element or combination of elements can account for
cell-specific mechanically induced responses. It is still to be determined if the expression
of the GPC-4 gene in other tissues such as liver and kidney (Fig. 9) is also regulated by
mechanical stimulus, as in other parts of the oviduct (Fig. 8).
Although the precise mechanism by which mechanical strain regulates gene expression
has not yet been elucidated, it is interesting to note that OPN (44,49), the α1 subunit of
Na, K ATPase (22,48) and the glypican genes (3,20), all of which were activated in the
ESG by the same mechanical strain, share some potential transcription factor binding
motif such as Sp1. These Sp1 binding sites have been found in shear stress response
element (SSRE) (34). In our search for mechanical strain-dependent genes in the ESG we
found that Rho A, a small GTP-binding protein known to be involved in mechanicalstrain
signal transduction was also differentially displayed at the time of eggshell
formation (data not shown). Rho A has been reported to play a role in mechanical-stressinduced
responses in cardiac myocytes (1) and aortic smooth muscle cells (30), and has
been implicated as a downstream target of the integrin-dependent signal pathway (9,15).
Rho A has been shown to activate the expression of various transcription factors, such as
MyoD (6), c-fos (42), and of various other genes, via the serum response factor, SRF
(41,4) which requires Sp1 factor binding sites (39). The involvement of Rho A in the
strain-dependent transcriptional activation of the GPC-4 in the ESG can only be
speculated upon, but the correlation between the temporal and spatial activation of these
two genes, supports this hypothesis.
The avian ESG is a tissue specialised in the massive calcium transport needed for
eggshell formation, and is the source of the organic matrix of the shell. The role of GPC-
4 in the process of eggshell formation is not known. One possibility is that the function
of GPC-4 in the ESG is related to its ability to serve as a co-receptor and/or a modulator
of the activity of various growth factors (17). Alternatively, like other proteoglycans (40)
and glypicans (12,28,45), GPC-4 may be shed from the cell surface and its role might be
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to become part of the assembly of proteoglycans that contribute to the biochemical
properties of the mature product (13). Avian-specific glypican antibodies would enable
its presence in the eggshell to be verified. It is interesting to note that calcium flux due to
increase loading such as hypertension was observed in other tissues as well (11).
In a sexually immature pre-laying hen, before the onset of reproduction, the GPC-4 gene
in the ESG was found to be silent and could not be induced by a mechanical strain. These
results suggest a priming mechanism of the gene that occurs during the transition from a
non-laying to a laying state, only after which can the GPC-4 gene be induced by the
mechanical strain. Hormones such as estrogen, progesterone, etc., that are involved in
maturation of the laying hen may, therefore, be involved in this process.
In summary, in this study we demonstrated that the avian homologue of the mammalian
GPC-4 is expressed in the ESG in a circadian fashion and is regulated by mechanical
strain.
ACKNOWLEDGEMENT
This study is a contribution from the Agricultural Research Organization, The Volcani
Center, Bet Dagan, Israel.
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18

LEGENDS TO FIGURES
Fig. 1. The assembly of the PCR products of the chicken GPC-4.
Fig. 2. Predicted amino acid sequence of the coding region of chicken GPC-4. Amino
acids are denoted by the single-letter code. Gray background represents identity in the
amino acids between the chicken and the mammalian genes. Open circles indicate cystein
residues that are conserved in all GPC members. The N-terminal signal and C-terminal
hydrophobic region are boxed. Glypican signature region is represented by a solid line.
Fig.3. In situ hybridization of ESG biopsies, taken at various intervals after oviposition
with the avian GPC-4 probe. (A) The egg resides in the isthmus; (B) 1 h, (C) 2 h, (D) 8 h,
(E) 12 h and (F) 17 h after the entry of the egg into the ESG. Magnification 20x.
Fig. 4. GPC-4 gene expression in the ESG. (A) Hematoxylin-Eosin staining. (B) In situ
hybridization with chicken GPC-4 specific probe. PE - pseudostratified epithelium, GE –
glandular epithelium.
Fig 5. Effect of mechanical strain on GPC-4 gene expression in the ESG. Mechanical
strain was applied when there were no egg in the ESG and the GPC-4 gene was not
expressed. (A) Control, no strain; (B) 3 h after mechanical strain induction; inflated
volume was 20 cm 3 , (C) 3 h after mechanical strain induction; inflated volume was 60
cm 3 . Magnification 20x.
Fig.6. Effect of mechanical strain withdrawal on GPC-4 gene expression. Premature
forced oviposition was performed in order to remove the mechanical strain imposed by
the resident egg at the time of maximal mechanical strain induction. (A) 2 h after
expulsion of the egg from the ESG; (B) Control, the egg is in the ESG, 9 h after
oviposition.
19

Fig.7. In situ hybridization of ESG biopsies with the avian �-actin probe. (A) The egg
resides in the isthmus; (B) 3 h, (C) 12 h after the entry of the egg into the ESG. D- ESG
after mechanical-strain induction.
Fig.8. Effect of mechanical strain on GPC-4 gene expression in the magnum. (A) 3 h
after oviposition, the egg resided in the magnum; (B) 12 h after oviposition, the egg
resided in the ESG. PE- pseudostratified epithelium, GE– glandular epithelium.
Magnification 50x.
Fig.9. GPC-4 gene expression in chicken tissues. RNA was isolated from various tissues
and GPC-4 expression was evaluated by RT-PCR.
20

1.
2.
3.
4.
81 bp
PCR
482 bp
PCR
969 bp
382 bp
382 bp
1593 bp
Figure 1
RACE
570 bp

o o
Chicken GPC4 ---------------------------KSCSEVRRLYAAKGFSQSEAPSHEISGDHLKVC
Human GPC4 MARFGLPALLCTLAVLSAALLAAELKSKSCSEVRRLYVSKGFNKNDAPLHEINGDHLKIC
Mouse GPC4 MARLGLLALLCTLAALSASLLAAELKSKSCSEVRRLYVSKGFNKNDAPLYEINGDHLKIC 60
N-term. signal
oo
Chicken GPC4 SQAYTCCTQEMEERYSQLSKHDFRNAVVELSNHLQTMFSSRYKKFDEFFKELLENAEKSL
Human GPC4 PQGSTCCSQEMEEKYSLQSKDDFKSVVSEQCNHLQAVFASRYKKFDEFFKELLENAEKSL
Mouse GPC4 PQDYTCCSQEMEEKYSLQSKDDFKTVVSEQCNHLQAIFASRYKKFDEFFKELLENAEKSL 120
Chicken GPC4 NDMFVRTYGRLYMQNSELFKDLFVELKRYYVGGNVNLEEMLNDFWARLLERMFRLVNPQY
Human GPC4 NDMFVKTYGHLYMQNSELFKDLFVELKRYYVVGNVNLEEMLNDFWARLLERMFRLVNSQY
Mouse GPC4 NDMFVKTYGHLYMQNSELFKDLFVELKRYYVAGNVNLEEMLNDFWARLLERMFRLVNSQY 180
o
Chicken GPC4 HFTDEYLECVSKYTEQLKPFGDVPRKLKLQVTRAFVAARTFAQGLAVARDVISKVSAVNP
Human GPC4 HFTDEYLECVSKYTEQLKPFGDVPRKLKLQVTRAFVAARTFAQGLAVAGDVVSKVSVVNP
Mouse GPC4 HFTDEYLECVSKYTEQLKPFGDVPRKLKLQVTRAFVAARTFAQGLAVARDVVSKVSVVNP 240
o o o o o o
Chicken GPC4 TPQGTQALLKMMYCPHCRGLTSVKPCYNYCFNVMRGCLANQGDLDAEWNIFMGSMLLVAE
Human GPC4 TAQCTHALLKMIYCSHCRGLVTVKPCYNYCSNIMRGCLANQGDLDFEWNNFIDAMLMVAE
Mouse GPC4 TAQCTHALLKMIYCSHCRGLVTVKPCYNYCSNIMRGCLANQGDLDFEWNNFIDAMLMVAE 300
Glypican signature
o
Chicken GPC4 RLEGPFNIESVMDPIDVKISDAIMNMQENSMQVSQKVFQGCGQPKTLAQGRTARSISESA
Human GPC4 RLEGPFNIESVMDPIDVKISDAIMNMQDNSVQVSQKVFQGCGPPKPLPAGRISRSISESA
Mouse GPC4 RLEGPFNIESVMDPIDVKISDAIMNMQDNSVQVSQKVFQGCGPPKPLPAGRISRSISESA 360
o
Chicken GPC4 FSARFRPYNPEERPTTAAGTSLDRLVTDVKEKLKQAKKFWSSLPGNICSDEKISVGTGNE
Human GPC4 FSARFRPHHPEERPTTAAGTSLDRLVTDVKDKLKQAKKFWSSLPSNVCNDERMAAGNGNE
Mouse GPC4 FSARFRPYHPEQRPTTAAGTSLDRLVTDVKEKLKQAKKFWSSLPSTVCNDERMAAGNENE 420
Chicken GPC4 NECWNGSAKSRYDFAVTGNGLASQVNNPEVEVDITKPDMVIRRQIMVLRVMTNKLKNAYS
Human GPC4 DDCWNGKGKSRYLFAVTGNGLANQGNNPEVQVDTSKPDILILRQIMALRVMTSKMKNAYN
Mouse GPC4 DDCWNGKGKSRYLFAVTGNGLANQGNNPEVQVDTSKPDILILRQIMALRVMTSKMKNAYN 480
Chicken GPC4 GNDVDFIDVSEESSGEESGSGCEFQQCSTEFEFNATEVTGSSNKSDKKVNTSAAPSSGLS
Human GPC4 GNDVDFFDISDESSGEGSGSGCEYQQCPSEFDYNATDHAGKS-ANEKADSAG-VRPGAQA
Mouse GPC4 GNDVDFFDISDESSGEGSGSGCEYQQCPSEFEYNATDHSGKS-ANEKADSAGGAHAETKP 540
GPI-modification site
Chicken GPC4 QAALFLSVLVLAMQRQWR
Human GPC4 YLLTVFCILFLVMQREWR
Mouse GPC4 YLLAALCILFLAVQGEWR 558
C-term hydrophobic region
Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9