Esophageal muscle physiology and morphogenesis require assembly of a collagen XIX-rich basement membrane zone. J Cell Biol

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Abstract
Collagen XIX is an extremely rare extracellular matrix component that localizes to basement membrane zones and is transiently expressed by differentiating muscle cells. Characterization of mice harboring null and structural mutations of the collagen XIX (Col19a1) gene has revealed the critical contribution of this matrix protein to muscle physiology and differentiation. The phenotype includes smooth muscle motor dysfunction and hypertensive sphincter resulting from impaired swallowing-induced, nitric oxide-dependent relaxation of the sphincteric muscle. Muscle dysfunction was correlated with a disorganized matrix and a normal complement of enteric neurons and interstitial cells of Cajal. Mice without collagen XIX exhibit an additional defect, namely impaired smooth-to-skeletal muscle cell conversion in the abdominal segment of the esophagus. This developmental abnormality was accounted for by failed activation of myogenic regulatory factors that normally drive esophageal muscle transdifferentiation. Therefore, these findings identify collagen XIX as the first structural determinant of sphincteric muscle function, and as the first extrinsic factor of skeletal myogenesis in the murine esophagus.

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The Journal of Cell Biology, Volume 166, Number 4, August 16, 2004 591–600
http://www.jcb.org/cgi/doi/10.1083/jcb.200402054
JCB
Article
591
Esophageal muscle physiology and morphogenesis
require assembly of a collagen XIX–rich basement
membrane zone
Hideaki Sumiyoshi,
1,2
Niv Mor,
1
Sui Y. Lee,
1
Stephen Doty,
1
Scott Henderson,
3
Shizuko Tanaka,
1
Hidekatsu Yoshioka,
2
Satish Rattan,
4
and Francesco Ramirez
1
1
Research Division of the Hospital for Special Surgery and Department of Physiology and Biophysics at the Weill College
of Medicine of Cornell University, New York, NY 10019
2
Department of Anatomy, Biology, and Medicine, Oita Medical University, Oita 879-5593, Japan
3
Department of Molecular, Cellular, and Developmental Biology, Mount Sinai School of Medicine, New York, NY 10029
4
Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA 19107
ollagen XIX is an extremely rare extracellular matrix
component that localizes to basement membrane
zones and is transiently expressed by differentiating
muscle cells. Characterization of mice harboring null and
structural mutations of the collagen XIX (
Col19a1
) gene has
revealed the critical contribution of this matrix protein to
muscle physiology and differentiation. The phenotype in-
cludes smooth muscle motor dysfunction and hypertensive
sphincter resulting from impaired swallowing-induced, nitric
oxide–dependent relaxation of the sphincteric muscle.
Muscle dysfunction was correlated with a disorganized
C
matrix and a normal complement of enteric neurons and
interstitial cells of Cajal. Mice without collagen XIX exhibit
an additional defect, namely impaired smooth-to-skeletal
muscle cell conversion in the abdominal segment of the
esophagus. This developmental abnormality was accounted
for by failed activation of myogenic regulatory factors that
normally drive esophageal muscle transdifferentiation.
Therefore, these findings identify collagen XIX as the first
structural determinant of sphincteric muscle function, and
as the first extrinsic factor of skeletal myogenesis in the
murine esophagus.
Introduction
The ECM consists of a highly heterogeneous mixture of
interacting proteins that form a complex array of supra-
molecular structures, and that bind cell surface receptors and
soluble signaling molecules (Ramirez and Rifkin, 2003).
The collagens represent the largest family of structural ECM
components with 27 designated trimers that are broadly divided
into fibrillar and nonfibrillar collagen types (Myllyharju and
Kivirikko, 2004). The latter group includes collagens that
are components of or associated with basement membranes
(BMs), highly specialized macroaggregates that subserve the
dual function of organizing multicellular structures and of
instructing tissue differentiation, maintenance, and remodeling
(Timpl, 1996; Ortega and Werb, 2002). Naturally occurring
human mutations and genetically engineered mice have
implied specialized roles of this particular class of non-
fibrillar collagens at distinct anatomical locations. Illustrative
examples of these tissue-restricted pathologies include glo
merulonephrites (minor collagens IV), skeletal myopathies
(collagens VI and XV), skin blistering (collagens VII and
XVII), and vitreoretinal degeneration (collagen XVIII) (Chris-
tiano et al., 1993; Zhou et al., 1993; Mochizuki et al., 1994;
McGrath et al., 1995; Jobsis et al., 1996; Eklund et al.,
2001; Vanegas et al., 2001; Fukai et al., 2002; Marneros et
al., 2004).
Collagen XIX is the latest example of a nonfibrillar collagen
type that localizes to BM zones and that may potentially
have an anatomically restricted function. Collagen XIX is
deposited at extremely low amounts (
10
6
% of dry tissue
Address correspondence to Francesco Ramirez, Laboratory of Genetics
and Organogenesis, Hospital for Special Surgery at the Weill College of
Medicine of Cornell University, 535 East 70th St., New York, NY
10021. Tel.: (212) 7874-7554. Fax: (212) 774-7864.
email: ramirezf@hss.edu
Key words: achalasia; basement membrane; collagen; muscle transdiffer-
entiation; nitrergic neurotransmission
Abbreviations used in this paper: BM, basement membrane; EFS, electrical
field stimulation; ES, embryonic stem; ICC-IM, intramuscular interstitial
cells of Cajal; LES, lower esophageal sphincter; MRF, myogenic regulatory
factor; NANC, nonadrenergic noncholinergic; NC, noncollagenous;
NO, nitric oxide; NOS, nitric oxide synthase; SMC, smooth muscle cell.
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592 The Journal of Cell Biology
|
Volume 166, Number 4, 2004
weight) in the BM zones of vascular, neural, and mesenchy-
mal tissues (Myers et al., 1997). Collagen XIX forms higher
order aggregates that may conceivably modulate cell–matrix
interactions, cell–cell communications, and/or local concen-
trations of signaling molecules (Myers et al., 2003). Em-
bryonic expression of the collagen XIX (
Col19a1
) gene is
transient and confined almost exclusively to differentiating
muscles (Sumiyoshi et al., 2001). Onset of
Col19a1
expres-
sion in myotomes and myotome derivatives occurs soon af-
ter activation of the myogenic regulatory factor (MRF) gene
Myf5,
and declines concomitantly to the accumulation of
myogenin
transcripts (Sumiyoshi et al., 2001).
The above findings are consistent with the notion that
collagen XIX may be involved in muscle differentiation and
function (Sumiyoshi et al., 2001). Unfortunately, experi-
mental validation of this postulate has been hampered by the
paucity of collagen XIX in tissues. Therefore, the present
work was undertaken to overcome this problem using ge-
netic means. Toward this end, we created a null mutation
(N19) and a structural mutation (
19) of the
1(XIX) col-
lagen chain by targeting different regions of the
Col19a1
gene in mouse embryonic stem (ES) cells. Characterization
of the resulting mouse phenotypes demonstrated that col-
lagen XIX plays a dual role in muscle physiology and differ-
entiation. Specifically, we found that proper assembly of the
collagen XIX–rich BM zone is a prerequisite for nitric oxide
(NO)–dependent relaxation of the lower esophageal sphinc-
ter (LES) muscle, and that collagen XIX deposition into the
matrix of the developing esophagus is an extrinsic determi-
nant of skeletal myogenesis in this organ.
Results
Generation of collagen XIX mutant mouse strains
The mouse
1(XIX) chain is 1,136 residues long and con-
sists of a discontinuous collagenous region flanked by a cys-
teine-rich noncollagenous (NC) amino terminal (NC6) and
a 19-residue carboxyl peptide (NC1) (Fig. 1 A; Sumiyoshi et
al., 1997). Recent ultrastructural analyses have shown that
the NC interruptions impart flexibility to the otherwise rigid
triple helical (collagenous) domain; they have also docu-
mented that interactions amongst globular NC6 domains
are responsible for the formation of collagen XIX oligomers
(Myers et al., 2003). Therefore, two different mutations
were engineered in the mouse in order to compare and con-
trast the phenotypic consequences of assembling BM zones
devoid of collagen XIX or containing structurally abnormal
collagen XIX trimers.
Figure 1. Col19a1 gene targeting. (A) Schematic representation of the 1(XIX) collagen chain where the gray bars correspond to exon 4 and
exons 38–40 sequences in the NC6 and NC3 domains, respectively. Targeting strategies are shown on the left for the N19 allele (N) and on the
right for the 19 allele (). (B) Southern hybridization of SacI-digested wild-type (/) and mutant (N/N) DNA to the upstream probe (P).
(C) Northern hybridizations of wild-type (/) and mutant (N/N) RNA to Col19a1 and actin probes. (D) Collagen XIX immunoblot of wild-type
(/) and mutant tissues from nullizygous (N/N) mice or mice producing internally deleted 1(XIX) chains (/). Samples were electrophoresed
under reducing (2 and 6) and nonreducing (1 and 5) conditions; collagenase- and mock-treated samples are in lanes 4 and 3, respectively.
Migration of protein markers are shown on the left; asterisk indicates an unspecific collagenase-resistant product, whereas the arrowhead
points to a probable proteolytic product of collagen XIX. Parallel Coomassie blue staining documented equal protein loading in each lane
(not depicted). (E) Southern hybridization of SacI-digested wild-type (/) and mutant (/) DNA to the downstream probe (P). (F) RT-PCR
amplification of collagen XIX transcripts from heterozygous 19 () mice for 25, 30, and 35 cycles (lanes 1, 2, and 3, respectively).
Page 2
Collagen XIX in muscle development and function |
Sumiyoshi et al. 593
The null mutation (N19) was generated by inserting the
PGK-
neo
cassette in place of exon 4 (Fig. 1 A, left). Exon 4
codes for 32 internal amino acids of the 268-residue NC6
peptide and includes split codons for the first and last resi-
dues (Sumiyoshi et al., 1997). After homologous recombina-
tion in ES cells, chimeric animals from two correctly tar-
geted ES clones were generated and germ line transmission
of the mutant allele was followed by Southern blot analysis
(Fig. 1 B). Northern hybridizations failed to detect
Col19a1
transcripts in homozygous mutant tissues (Fig. 1 C). More-
over, sequencing of RT-PCR–amplified products across and
downstream of the targeted genomic region excluded the
existence of shorter, in-frame
Col19a1
transcripts (unpub-
lished data). Immunoblots of partially purified collagen
preparations from homozygous mutant and wild-type tissues
corroborated the mRNA data by documenting absence of
the expected 165-kD collagenase-sensitive product in the
former compared with the latter specimen (Fig. 1 D). Un-
fortunately, the same antibodies proved unsuitable to con-
firm loss of collagen XIX in tissues. This last point notwith-
standing, we concluded that the N19 allele does indeed
represent a null mutation.
The structural mutation (
19) was created by inserting
the PGK-
neo
cassette in place of exons 38–40 (Fig. 1 A,
right). Exons 38–40 code for the 20-residue NC3 interrup-
tion of the helical domain and for one and six collagenous
tripeptides located amino- and carboxyl-terminal of it, re-
spectively (Sumiyoshi et al., 1997). Chimeric animals were
generated from two independently derived clones and the
progeny was genotyped by Southern blot analysis using a di-
agnostic restriction enzyme cleavage site (Fig. 1 E). The de-
letion of exons 39–40 maintains the frame of the
Col19a1
transcript, and thus it is predicted to yield an internally de-
leted
1(XIX) chain that should participate in homotrimer
formation. Sequencing of RT-PCR–amplified products con-
firmed that the mutant transcript is in frame (unpublished
data), whereas immunoblots identified a collagenase-sensi-
tive product in the mutant tissue slightly smaller than the
wild-type 165-kD species (Fig. 1 D). Finally, PCR amplifi-
cation estimated that the mutant and wild-type
Col19a1
al-
leles are expressed at comparable levels in the heterozygous
19 mouse (Fig. 1 F). Therefore, the
19 allele represents a
structural mutation that eliminates one of the flexible points
in the triple helix. Characterization of the two collagen XIX
mutations initially focused on the more severe phenotype of
the nullizygous mouse.
Collagen XIX null mice display altered
esophageal morphology
Heterozygous N19 mice were born at the expected Mende-
lian frequency; they were morphologically normal, viable,
and fertile. Homozygous N19 mice were born at the ex-
pected frequency as well, but the vast majority of them
(
95%) died within the first 3 wk of postnatal life, showing
signs of malnourishment. Postmortem inspection of new-
born homozygous mutants did not detect gross anatomical
abnormalities, except for the smaller size of the internal or-
gans. On the other hand, necroscopy of the few
Col19a1
/
mice that survived past weaning stage revealed a dilated
esophagus (megaesophagus) with retention of ingesta, im-
mediately above the diaphragm level (Fig. 2 A). Based on
these observations, we reevaluated the pattern of
Col19a1
expression in the embryonic digestive system, and found it
to coincide with the formation and growth of the gastro-
esophageal junction. Specifically, in situ hybridizations re-
vealed high
Col19a1
expression in the lower-third portion of
the embryonic esophagus destined to become the abdominal
segment; thereafter
Col19a1
activity becomes gradually re-
stricted to the mature LES, while decreasing in the muscle
layer of the proximal stomach (Fig. 2 B).
Abnormal LES physiology in collagen XIX null mice
Megaesophagus is a distinguishing feature of severe human
achalasia, an esophageal motility disorder characterized by ele-
vated basal tone and impaired swallowing-induced relaxation
of the LES (Goyal, 2001). This phenotypic trait, coincident
with high and persistent
Col19a1
expression in the LES,
Figure 2. Analysis of gastroesophageal region. (A) Representative
megaesophagus of a 3-mo-old Col19a1 null mouse (N/N); note the
esophageal enlargement that begins immediately above the diaphragm.
(B) In situ hybridizations of the gastroesophageal region from E13.5,
E16.5, and P16 wild-type mice to the antisense Col19a1 probe.
Sense probe yielded no signal above background (not depicted).
Arrowhead and arrow indicate the internal LES and the proximal
stomach, respectively. Bars, 0.5 mm.
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594 The Journal of Cell Biology
|
Volume 166, Number 4, 2004
prompted us to examine whether early demise of homozygous
mutants may be in part accounted for by sphincteric mus-
cle dysfunction. Accordingly, the physiology of the collagen
XIX–deficient LES muscle was assessed in vitro by monitor-
ing basal muscle tone in response to electrical field stimulation
(EFS), and in vivo by examining swallowing-induced LES re-
laxation using intraluminal esophageal manometry.
Mechanical responses to nonadrenergic noncholinergic
(NANC) nerve stimulation with EFS of sphincteric muscle
strips from wild-type and nullizygous adult mice were mea-
sured in the absence and in the presence of L-NA, an inhibi-
tor of NO synthase (NOS; Mashimo et al., 1996). As ex-
pected, we found that EFS elicited frequency-dependent
relaxation of wild-type LES strips followed by pronounced
rebound contraction, and that muscle relaxation was signifi-
cantly reduced by L-NA treatment (Fig. 3, A and B). In
marked contrast, mutant muscle strips failed to produce sig-
nificant relaxation in response to EFS; furthermore, L-NA
virtually eliminated any residual relaxation (Fig. 3, A and B).
Comparable results were obtained in intact animals. Intralu-
minal pressure recorded at the LES level of adult
Col19a1
null mice in fact showed significantly higher basal tone
(three- to eightfold) than wild-type animals (Fig. 4 A). It
also documented severely impaired or absent relaxation
upon swallowing; even when present, relaxation was abnor-
mally brief (Fig. 4 A). The results of the manometric tests
were remarkably similar to those reported for achalasic pa-
tients (Richter, 2001). Altogether, the in vitro and in vivo
experiments indicated that NO-dependent neurotransmis-
sion is perturbed in the collagen XIX–deficient LES.
Nitrergic nerves and ICC-IM are present
in the mutant LES
It has been proposed that c-Kit–positive intramuscular inter-
stitial cells of Cajal (ICC-IM) transduce inhibitory signals
from nerve terminals to sphincteric smooth muscle cells
Figure 3. LES in vitro physiology. (A) Typical tracings to illustrate LES responses to 10- and 20-Hz EFS (20 V, 0.5-ms pulse duration, 4-s train)
in control (top) and L-NA–treated (bottom) samples from 3-mo-old wild-type (/) and collagen XIX null (N/N) animals. (B) Summary of
quantitative relaxation and contraction data of wild-type (/) and collagen XIX null (N/N) animals (n 6) in the presence and absence
of L-NA. (C) Typical tracings to illustrate the same responses as in A using heterozygous (/) and homozygous 19 (/) mice.
Page 4
Collagen XIX in muscle development and function |
Sumiyoshi et al. 595
(SMCs; Ward et al., 1998). Therefore, antibodies against
c-Kit and nNOS were used to assess whether or not loss of
nitrergic nerves and/or ICC-IM may account for sphincteric
muscle dysfunction in
Col19a1
/
mice. Immunofluores-
cence staining of wild-type and homozygous mutant LES
muscle showed more nNOS-positive cells than c-Kit–posi-
tive ICC-IM in both samples; moreover, the two cell types
were often seen in close association (Fig. 5 A). Quantitative
analysis of the confocal images demonstrated the presence of
statistically comparable numbers of each cell type in the
wild-type and mutant tissues (Fig. 5 B). No remarkable dif-
ferences were also observed with additional neurospecific
markers, such as vasoactive intestinal peptide and choline
acetyltransferase (unpublished data).
Altered matrix in collagen XIX–deficient LES tissue
Loss of collagen XIX could in principle alter the architecture
of the smooth muscle matrix or the intrinsic ability of SMC
to relax upon nerve stimulation. The latter possibility was
assessed in the collagen XIX–deficient LES muscle by in
vitro and in vivo assays. An exogenously applied NO donor
caused relaxation of mutant LES muscle strips precontracted
with bethanechol (Fig. 4 B). Similarly, administration of a
-adrenoreceptor agonist to
Col19a1
/
mice caused nor-
mal LES relaxation (Fig. 4 C). Therefore, these results dem-
onstrated integrity of the SMC membrane and of the intra-
cellular signal transduction machinery responsible for LES
relaxation in
Col19a1
null mice. Along these lines, immuno-
fluoresence staining of connexin 43, smooth muscle actin,
-actinin, and vinculin failed to identify variations between
wild-type and mutant SMC (unpublished data).
Figure 4. LES in vivo physiology. (A) Typical tracings to illustrate
swallow-induced changes in LES pressure of 3-mo-old wild-type (/)
and collagen XIX null (N/N) mice. Contrast the normal basal tone
and LES relaxation in wild-type mice with the higher basal tone with
frank contraction (one example in the middle tracing) or extremely
high basal tone with incomplete relaxation (another example in the
right tracing) in mutant mice. (B) Measurements of LES muscle tension
in the presence of cumulative concentrations of bethanechol (left)
and sodium nitroprusside (right) in tissue strips from 3-mo-old wild-
type (blue) and null (red) mice. (C) Isoproterenol hydrochloride
(0.4 g/Kg; i.v.) treatment of a 3-mo-old Col19a1
/
mouse.
Figure 5. Cellular analyses of mutant LES. (A) Confocal micro-
graphs of wild-type (/) and collagen XIX null (N/N) LES muscles
stained for nNOS and c-Kit with the superimposed images shown at
the bottom. Bar, 50 m. (B) Numbers of c-Kit– and nNOS-positive
cells in 0.235-mm
2
fields from wild-type and mutant tissues (n 4).
An average of 44.5 and 40 c-Kit–positive cells and 79 and 74
nNOS-positive cells were counted in wild-type and mutant tissues,
respectively. Bars indicate SEM.
Page 5
596 The Journal of Cell Biology
|
Volume 166, Number 4, 2004
Immunostaining of collagen XIX–deficient LES tissue
with antibodies against nidogen-1 documented a largely pre-
served BM, which, however, stained more intensively than
the wild-type counterpart (Fig. 6 A). Electron microscopy
confirmed this result by showing a thicker BM around the
mutant SMC (Fig. 6 B). It also revealed that intercellular
spacing of the mutant smooth muscle is appreciably greater
than the wild-type control (Fig. 6 C). Additional abnormali-
ties include convoluted SMC profiles, excessive extracellular
accumulation of collagen fibrils, and highly irregular inter-
cellular space (Fig. 6 C). Consistent with progressive degen-
eration of matrix organization, these morphological abnor-
malities were more pronounced in adult than newborn
mutant mice (Fig. 6 C). Collectively, these analyses sug-
gested a specialized and highly restricted role of collagen
XIX in organizing the BM zone of the LES.
Impaired muscle transdifferentiation in the collagen
XIX null esophagus
Necroscopic examination of the esophagus of adult
Col19a1
/
mice revealed the presence of another pheno-
typic manifestation indicative of a morphogenetic defect.
The muscle layer of the murine esophagus undergoes a
transdifferentiation process from smooth to skeletal muscle
that begins at about embryonic day 15.5 (E15.5) and that
ends approximately at postnatal day 21 (P21; Patapoutian et
al., 1995; Kablar et al., 2000). This poorly understood de-
velopmental program is accompanied by rostrocaudal ex-
pression of MRF genes, and varies in timing and extent de-
pending on the mouse strain (Patapoutian et al., 1995;
Kablar et al., 2000; unpublished data).
Progression of muscle transdifferentiation in the wild-type
129/Sv mouse was monitored by following the expression of
myogenin
, an MRF that instructs skeletal muscle differentia-
tion (Molkentin and Olson, 1996). In situ hybridizations at
different prenatal and postnatal stages of esophageal de-
velopment revealed that the front of myogenin expression
reaches diaphragm level at birth, and gradually progresses
into the abdominal segment of the esophagus during the
first week of postnatal life (Fig. 7 A). The same analysis doc-
umented that the postnatal front of myogenin expression in
the collagen XIX–deficient esophagus remains at the same
level as at birth (Fig. 7 B). Immunostaining of the wild-type
and mutant specimens for skeletal and smooth muscle–spe-
cific proteins demonstrated that loss of MRF gene expres-
sion translates into failed muscle transdifferentiation in the
entire abdominal segment of the Col19a1
/
esophagus
(Fig. 7 C). These results conclusively established a causal
relationship between extracellular deposition of collagen
XIX and developmentally programmed activation of MRF-
driven smooth muscle transdifferentiation.
The 19 mutation perturbs only LES function
The milder phenotype of the 19 mutation afforded the op-
portunity to further explore the role of collagen XIX in skel-
etal myogenesis and sphincteric muscle relaxation. Het-
erozygous and homozygous 19 mice were born at the
expected frequency and were apparently unaffected by the
deposition into the ECM of abnormal 1(XIX) homotri-
mers. However, a significant number of adult (8–12-mo-old)
heterozygous and homozygous mutant mice were noted to
display evident signs of compromised fitness, such as weight
loss, lethargy, and patchy hair loss. Therefore, adult 19
heterozygotes and homozygotes were analyzed for possible
manifestations in the gastroesophageal system.
Immunohistological examination of gastroesophageal tis-
sues from four adult 19/ mice and four 19/19 litter-
mates revealed normal transdifferentiation of the muscle
layer in the abdominal segment of the esophagus (Fig. 6 B).
By contrast, the EFS assay documented reduced or absent
relaxation of LES muscle strips in half of the eight hetero-
zygous and eight homozygous 19 specimens examined
(Fig. 3 C). Moreover, LES samples from randomly chosen
Figure 6. BM in the mutant LES. (A) Light microscopy of represen-
tative LES tissues from 7-mo-old wild-type (/) and N19/N19
(N/N) mice immunostained with antibodies against nidogen-1,
showing positive staining of the BM around SMC (arrowhead). Bar,
0.01 mm. (B) Electron microscopy of 7-mo-old wild-type (/) and
N19/N19 (N/N) LES documenting the thicker BM in the mutant sample
(arrowhead). Bar, 0.5 m. (C) Electron microscopy of smooth muscle
in the medial portion of the gastroesophageal junction of P5 and
1-yr-old wild-type and collagen XIX null (N/N) mice. Intercellular
spacing in neonatal mice ranges from 30 to 316 nm in wild-type vs.
55 to 708 nm in mutant mice, whereas in adult animals ranges from
94 nm to 1.0 m in wild-type vs. 99 nm to 3.3 m in mutant mice.
Red and yellow arrowheads point to spacing between smooth
muscle cells (SMC) and collagen fibrils, respectively. Bar, 1 m.
Page 6
Collagen XIX in muscle development and function | Sumiyoshi et al. 597
19/ or 19/19 mice often displayed altered nidogen-1
immunostaining (unpublished data). Therefore, we concluded
that the structural and compositional integrity of the BM
zone are both prerequisites for proper LES function, and
that only the latter is required for the developmentally regu-
lated process of skeletal myogenesis.
Discussion
Compositional diversification of BMs at distinct anatomical lo-
cations is a major determinant of the functional specificity of
these highly specialized and widely distributed ECM structures
(Timpl, 1996). A growing body of genetic evidence indicates
that the functional integrity of individual BM zones also re-
quires the deposition of collagen molecules that connect them
to the underlying connective tissue stroma (Ortega and Werb,
2002). Illustrative examples include the role of BM-associated
collagens XV and XVIII in providing integrity to skeletal mus-
cle, microvessels, and vitreous (Eklund et al., 2001; Fukai et al.,
2002). The present paper adds collagen XIX to the list of BM-
associated collagens by implicating this molecule in the organi-
zation of the pericellular matrix of the sphincteric smooth mus-
cle. It has also unraveled an unexpected role of this rare col-
lagen type in esophageal morphogenesis.
Structural role of collagen XIX in LES physiology
Activation of inhibitory NANC nerves is critical for LES re-
laxation upon swallowing (Goyal and Hirano, 1996). Al-
though NO is widely recognized as the major inhibitory neu-
rotransmitter of NANC nerves, the mechanism and factors
responsible for conveying NO signals to SMC remain ill-
defined. Conflicting models postulate that the highly labile
NO either diffuses freely in the extracellular space between
nerve varicosities and SMC or is transduced to the target
muscle cells by ICC-IM (Ward et al., 1998; Sivarao et al.,
2001). The achalasia-like manifestations of mice lacking col-
lagen XIX or producing structurally abnormal collagen XIX
trimers imply that NO-dependent smooth muscle relaxation
requires a properly organized LES matrix as well. That LES
muscle relaxation is impaired in spite of a seemingly normal
complement of ICC-IM and nitrergic nerves and of func-
tionally viable SMC further supports this conclusion.
The hypertensive and nonrelaxing LES of the Col19a1 mu-
tant mice resembles the clinical manifestations of human
Figure 7. Esophageal muscle transdifferentiation. (A) In situ hybridizations of gastroesophageal specimens from P0 and P8 wild-type mice
to myogenin and Col19a1 antisense probes. (B) In situ hybridizations of P14 wild-type (/) and collagen XIX null (/) gastroesophageal
specimens to myogenin antisense probe. In both panels, sense probes yielded no signals above background (not depicted). (C) Immunostaining
of the abdominal segments of esophagi from 18-mo-old wild-type (/) or collagen XIX null (N/N) mice, and from 1-yr-old mice heterozygous
(/) or homozygous (/) for the collagen XIX deletion using antibodies against smooth muscle actin (top) or skeletal muscle myosin (bottom).
The lumina of the esophagus and stomach are indicated by eL and sL, respectively. In A and B, the yellow arrowhead points to the LES; in C,
the red arrowhead indicates the normal transition point between skeletal and smooth muscle. Bars, 0.5 mm.
Page 7
598 The Journal of Cell Biology | Volume 166, Number 4, 2004
patients with achalasia (Goyal, 2001; Richter, 2001). The eti-
ology of this primary esophageal motor disorder is heteroge-
neous and may include genetic, infectious, autoimmune, and
degenerative factors. Loss of nitrergic nerves in achalasia is
widely believed to cause hypertensive LES due to unopposed
cholinergic excitation, a notion indirectly supported by the
basal LES hypertension in nNOS
/
mice (Goyal, 2001;
Richter, 2001; Sivarao et al., 2001). Similarly, impaired LES
relaxation to swallowing in nNOS
/
mice underscores the
prominent contribution of nitrergic neurotransmission to in-
hibitory neurotransmission (Sivarao et al., 2001). The mano-
metric data from Col19a1
/
and nNOS
/
mice are virtually
identical and as such, they emphasize functional equivalency
between NO release from nitrergic varicosities and ECM or-
ganization. By contrast, c-Kit mutant (W/W
v
) mice, which
lack ICC-IM, have a hypotensive LES with normal NANC
relaxation (Sivarao et al., 2001). Hence, the data from the
Col19a1 and nNOS null mice concur in strongly suggesting
that ICC-IM play a lesser role than previously suggested in
sphincteric muscle relaxation.
The structural role of the collagen XIX–rich BM zone may
extend beyond supporting NO-dependent relaxation of the
LES to organizing the neuromuscular junction of the sphinc-
teric muscle. In this respect, an analogy could be drawn with
perlecan in clustering acetylcholinesterase to the synaptic basal
lamina of the neuromuscular junction (Arikawa-Hirasawa et
al., 2002). Our postulate is based on the intriguing observa-
tion that the NOS inhibitor L-NA affects the relaxation of
mutant muscles relatively less than wild-type muscles (Fig. 3).
Irrespective of the underlying mechanism, our work conclu-
sively proves that collagen XIX is a new contributing factor to
sphincteric muscle physiology.
Instructive role of collagen XIX in
esophageal development
Collagenous and elastic macroaggregates have been tradi-
tionally viewed as the main structural determinants of con-
nective tissue architecture. However, there is emerging evi-
dence that they also participate in modulating a variety of
cellular activities and signaling events (Ortega and Werb,
2002; Ramirez and Rifkin, 2003). For example, proteolytic
products of collagens XV and XVIII—also known as restin
and endostatin—have been reported to control programs as
diverse as angiogenesis, neuronal cell migration, and epithe-
lial cell morphogenesis (O’Reilly et al., 1997; Sasaki et al.,
1998; Ackley et al., 2001; Karihaloo et al., 2001). Moreover,
failed regression of hyaloid vessels in the eyes of collagen
XVIII-deficient mice has been interpreted to imply that this
BM-stabilizing molecule promotes programmed cell death
and macrophage activation during tissue remodeling (Fukai
et al., 2002). Failed muscle transdifferentiation in Col19a1
/
mice similarly implicates this collagen type in modulating a
specific morphogenetic process.
Developmentally programmed cell transdifferentiation is
a rare phenomenon in vertebrates that has been described for
a few contractile cell types, including the mouse esophagus
and the chick iris (Volpe et al., 1993; Patapoutian et al.,
1995; Link and Nishi, 1998a; Kablar et al., 2000). Transdif-
ferentiation in both organ systems involves the conversion of
smooth muscle to skeletal muscle. Co-culture experiments
have suggested that activin and follistatin coordinate mus-
cle transdifferentiation in the chick iris (Link and Nishi,
1998b). That normal development of striated muscles and
nicotinic receptor clusters take place in Mash1
/
mice,
which lack enteric neurons, has indicated that skeletal myo-
genesis in the mouse esophagus occurs independently of in-
nervation (Sang et al., 1999).
The biological mechanism responsible for skeletal myogen-
esis in the mature muscle layer of the mouse esophagus is
controversial. In the original description of the phenomenon,
Patapoutian et al. (1995) reported that smooth-to-skeletal
muscle conversion is preceded by MyoD and myogenin expres-
sion. Kablar et al. (2000) subsequently used transgenic and
knock-in mice to document that initiation and progression of
muscle transdifferentiation depend on Myf5 expression. The
apparent discrepancy between these two reports may reflect
the fact that each followed muscle transdifferentiation in dif-
ferent esophageal segments (i.e., abdominal vs. thoracic/cervi-
cal). Others have argued that smooth and skeletal muscles
originate from distinct precursor cells already present at early
embryonic stages (Zhao and Dhoot, 2000a,b; Rishniw et al.,
2003). However, this argument is not supported by evidence
of significant SMC apoptosis during esophageal development
(Patapoutian et al., 1995; Kablar et al., 2000).
Our findings implicate the collagen XIX–rich matrix as
the first extrinsic factor to guide skeletal myogenesis in the
developing mouse esophagus. Interestingly, the same mor-
phogenetic defect was not observed in the C57/Bl/6J genetic
background, implying that collagen XIX action is modu-
lated by modifier gene products. On the other hand, associa-
tion of an achalasia-like phenotype with the same physiolog-
ical and morphological manifestations in both C57/Bl/6J
and 129T2/SvEmsJ mutant mice indicated that distinct
mechanisms are responsible for the genesis of LES dysfunc-
tion and failed muscle transdifferentiation. Along these
lines, normal esophageal muscle transdifferentiation in mice
producing abnormal collagen XIX trimers demonstrated
that a specific peptide sequence (rather than the whole mole-
cule) is involved in muscle transdifferentiation. One attrac-
tive mechanism is that the collagen XIX–rich matrix may
control the distribution and/or activity of growth factors
that ultimately trigger MRF gene expression in the abdomi-
nal esophagus. The elegant work of Myers et al. (2003) sup-
ports this hypothesis. These investigators have shown that
collagen XIX forms higher order aggregates in which indi-
vidual molecules extend radially from a globular core of in-
teracting NC6 domains. Therefore, they have argued that
this configuration, together with the NC6 heparin-bind-
ing site, may contribute to localize and concentrate signal-
ing molecules within the BM zone. Such a model is analo-
gous to the recently reported involvement of extracellular
microfibrils in limb patterning and lung morphogenesis
through the modulation of TGF/BMP signaling (Arteaga-
Solis et al., 2001; Neptune et al., 2003). Alternatively, the
flexibility of the collagen XIX monomers and the presence of
a Tsp-N module in NC6 may regulate skeletal myogenesis
by mediating critical cell–matrix and/or cell–cell interactions
(Myers et al., 2003).
Although the precise mechanism underlying the role of
collagen XIX in muscle transdifferentiation remains undeter-
Page 8
Collagen XIX in muscle development and function | Sumiyoshi et al. 599
mined, our work has yielded a number of interesting obser-
vations and plausible predictions. First, impaired muscle
transdifferentiation is only seen in the absence of collagen XIX
deposition and is associated with failed myogenin activation.
This finding could be interpreted to indicate that collagen
XIX lies upstream of the MRF(s) driving this morphogenetic
pathway. Second, early embryonic expression of Col19a1 is in
the region that will eventually become the abdominal segment
of the adult esophagus, and that fails to transdifferentiate in
collagen XIX null mice. Therefore, it is plausible to argue that
SMCs become fated for cell conversion soon after onset of
Col19a1 expression in the lower-third portion of the primitive
esophagus. Third, persistency of the smooth muscle pheno-
type in null mice is confined to the abdominal segment and
conversely, Col19a1 gene activity is absent in the upper two
thirds of the wild-type esophagus. A likely explanation of
these observations is that distinct factors and different mecha-
nisms drive muscle transdifferentiation along the esophageal
axis. Implicitly, this last prediction may reconcile the contro-
versy about the identity of the MRF(s) driving esophageal
transdifferentiation (Patapoutian et al., 1995; Kablar et al.,
2000). The availability of the Col19a1 null mouse provides
the opportunity to test these predictions and further charac-
terize this poorly understood biological phenomenon.
Materials and methods
Generation of Co119a1 mutant mice
Targeting vectors were engineered using Coll9a1 fragments isolated from a
129T2/SvEmsJ mouse genomic library by inserting the PGK-neo cassette
within the HindIII site of exon 4 (N19) or in place of the 3-kb-long XbaI
fragment containing exons 38–40 (19; Sumiyoshi et al., 1997). Mainte-
nance, transfection, and selection of mouse ES cells, as well as generation
of chimeric animals were performed as described previously (Andrikopou-
los et al., 1995). Electroporated ES cells and mutant mice were genotyped
by Southern blot analysis using diagnostic restriction enzyme sites and
probes outside and inside of the targeted region. Northern blot hybridiza-
tion and sequencing of RT-PCR products were used to confirm the identity
and relative expression levels of the mutant transcript. RT-PCR analysis of
19/ transcripts was performed after 25, 30, or 35 cycles in order to
compare the relative representation of wild-type and mutant products dur-
ing the linear phase of the amplification. Mutant mice were generated in
the Mount Sinai Mouse Genetics Shared Resource Facility (New York, NY)
and bred onto the pure 129T2/SvEmsJ genetic background.
Immunoblot analysis
Crude collagen XIX fraction was extracted from adult brains of wild-type
and mutant mice as described previously (Sumiyoshi et al., 1997). The fi-
nal 5 M NaCl precipitate was resuspended in 50 mM Tris-HCl, pH 7.5,
100 mM NaCl, and 0.5% NP-40; 100 g of the crude collagen XIX prep-
aration was then separated on 4.5% SDS-PAGE and electroblotted onto a
PVDF membrane in the presence of 15 mM sodium-borate buffer. Immu-
noblots were probed with rabbit pAbs raised against a recombinant fusion
protein produced by the pMAL-c2 expression plasmid (New England
Biolabs, Inc.) and containing the sequence encoding aa 1004–1036 of col-
lagen XIX. Immunocomplexes were detected using the ECL blotting system
(Amersham Biosciences) with secondary goat anti–rabbit IgG conjugated
with HRP (Santa Cruz Biotechnology, Inc.). For collagenase digestion,
7.5 U bacterial collagenase type III (Advance Biofactures) was added to
100 g of crude collagen XIX extract, and was incubated for 2 h at 37C in
50 mM Tris-HCl, pH 7.2, and 20 mM CaCl
2
.
Immunohistochemistry, electron microscopy,
and in situ hybridizations
Esophagi were removed along with the LES from necropsied animals, fixed
in 4% PFA, embedded in paraffin, serially sectioned, and immunostained
with alkaline phosphatase conjugated to mAbs against smooth muscle ac-
tin or skeletal fast myosin (Sigma-Aldrich). Rabbit pAbs against nidogen-1
were provided by Dr. Ulrike Mayer (University of Manchester, Manches-
ter, UK; Fox et al., 1991). For immunofluorescence staining, frozen LES
sections were incubated overnight at 4C with rabbit anti-nNOS antibody
(1:500; BD Biosciences) and goat anti-cKit antibody (1:500; Santa Cruz
Biotechnology, Inc.). After removal of unbound antibodies, sections were
incubated with FITC-conjugated donkey anti–rabbit IgG and Texas red–
conjugated donkey anti–goat IgG (1:200; Jackson ImmunoResearch Labo-
ratories) for 2 h at RT. Slides were examined using a confocal laser
scanning microscope (TCS-SP (UV); Leica) equipped with a four-channel
spectrophotometer scan head and four lasers (Ar-UV, Argon, Krypton, and
HeNe). Sections were illuminated simultaneously with the 488- and
568-nm laser lines and the AOTF was adjusted such that no signal
“cross-talk” occurred between channels. Gastroesophageal junctions were
collected from 4-mo-old animals, fixed overnight in 4% PFA, processed
through paraffin, and sectioned at 5–7-m thickness. Sections were depar-
affinized and treated with 100 g/ml protease XXIV (P8038; Sigma-
Aldrich) for 10 min at 37C (Willem et al., 2002). Antibodies against ni-
dogen-1 (1:1,000) were applied for 2 h at RT in a humid chamber; the
streptavidin-HRP technique (LSAB 2 system; DakoCytomation) was used to
localize the antibody and hematoxylin was used as a counterstain. Sam-
ples of the same tissues used for immunohistochemistry were processed for
electron microscopy by fixation in 2% PFA plus 0.5% glutaraldehyde in
0.05 M cacodylate buffer, pH 7.2, for 4 h at 4C. Tissues were rinsed, post-
fixed for 1 h in 2% osmium tetroxide at RT, dehydrated, and embedded in
Epon 812. Thin sections were stained with uranyl acetate for 20 min and
with lead citrate for 5 min. Photographs were taken on a transmission elec-
tron microscope (CM-12; Philips) at 80 kV or using a transmission electron
microscope (H-7000; Hitachi) operated at 75 kV. Confocal and electron
microscopy were performed at the Mount Sinai Microscopy Shared Re-
source Facility (New York, NY) and at the Hospital for Special Surgery An-
alytical Microscopy Core Facility (New York, NY). In situ hybridizations
were performed on serial tissue sections using Col19a1 and myogenin
probes as described previously (Sumiyoshi et al., 2001).
Physiological tests
Mechanical responses of LES strips to EFS were measured using standard
organ bath techniques in the presence or absence of 1 mM N-nitro-
L argi-
nine (L-NA; Mashimo et al., 1996). LES tone was also measured in the pres-
ence of increasing concentrations of bethanechol (0.1–300 M) followed
by sodium nitroprusside (0.1–100 M) as described previously (Chakder et
al., 1997). Intraluminal esophageal manometry was performed as de-
scribed by Sivarao et al. (2001) using a custom-designed catheter assembly
(Dentsleeve; Dentsleeve Pty Ltd.) that consists of silicon tubing made of
three individual channels of a 0.3-mm inside and 0.6-mm outside diameter
each. Swallows were induced by instilling a 1020-l bolus of water on
the tongue of the animals. To determine the integrity of the sphincteric
muscle, isoprotenerol hydrochloride (0.4 g/Kg) was injected i.v.
We thank Y. Nimomiya for generous support throughout the work, U.
Mayer for providing critical reagents and protocols, S. Digavalli for invalu-
able assistance in the physiological tests, V. William and N. Tulchin for
technical assistance, and K. Johnson for typing the manuscript.
This work was supported by grants from the National Institutes of
Health (AR-38648, CA-095823, and DK-35385), the St. Giles Foundation,
and the Japanese Ministry of Education, Science and Culture (Grant-in-aid
for Scientific Research 11470312 and 11877275). The authors declare
they have no competing financial interests.
This work is dedicated to the memory of Rupert Timpl.
Submitted: 9 February 2004
Accepted: 22 June 2004
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Page 10
    • "Animals CD1 and C57BL/6 mice were obtained from Charles River Laboratories . Collagen XIX–null mice (col19a1 −/− ; previously referred to as N19) were generated by deleting the fourth exon of collagen XIX (Sumiyoshi et al., 2004). Col19a1 −/− mutant mice were backcrossed for more than 10 generations on C57BL/6 mice. "
    [Show abstract] [Hide abstract] ABSTRACT: Inhibitory synapses comprise only ∼20% of the total synapses in the mammalian brain but play essential roles in controlling neuronal activity. In fact, perturbing inhibitory synapses is associated with complex brain disorders, such as schizophrenia and epilepsy. Although many types of inhibitory synapses exist, these disorders have been strongly linked to defects in inhibitory synapses formed by Parvalbumin-expressing interneurons. Here, we discovered a novel role for an unconventional collagen—collagen XIX—in the formation of Parvalbumin + inhibitory synapses. Loss of this collagen results not only in decreased inhibitory synapse number, but also in the acquisition of schizophrenia-related behaviors. Mechanistically, these studies reveal that a proteolytically released fragment of this collagen, termed a matricryptin, promotes the assembly of inhibitory nerve terminals through integrin receptors. Collectively, these studies not only identify roles for collagen-derived matricryptins in cortical circuit formation, but they also reveal a novel paracrine mechanism that regulates the assembly of these synapses.
    Full-text · Article · Mar 2016
    • "In adults, its distribution is more restricted: vessels, skeletal muscle, spleen, prostate, kidney, liver, colon, placenta, breast, neurons and skin [10, 11]. It is involved in embryogenesis, in muscle differentiation [12] and in the development of esophagus [13]. Mice lacking collagen XIX are not viable and die early, due to abnormalities in muscle development. "
    [Show abstract] [Hide abstract] ABSTRACT: Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvβ3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3β activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.
    Full-text · Article · Dec 2015
    • "The changes in isometric tension of isolated smooth muscle strips of LES placed in an organ bath was measured in response to EFS and drugs as described by Sumiyoshi et al. (2004). The esophagus and stomach were removed immediately after sacrifice and placed in oxygenated (95% O 2 /5% CO 2 ) Krebs' physiological solution (KPS). "
    [Show abstract] [Hide abstract] ABSTRACT: Postnatal maturation of esophageal musculature involves proximal-to-distal replacement of smooth muscle with skeletal muscle by elusive mechanisms. We report that this process is impaired in mice lacking the cell surface receptor Cdo and identify the underlying developmental mechanism. A myogenic transition zone containing proliferative skeletal muscle precursor cells migrated in a proximal-distal direction, leaving differentiated myofibers in its wake. Distal to the transition zone, smooth muscle fascicles underwent a morphogenetic process whereby they changed their orientation relative to each other and to the lumen. Consequently, a path was cleared for the transition zone, and smooth muscle ultimately occupied only the distal-most esophagus; there was no loss of smooth muscle. Cdo(-/-) mice were specifically defective in fascicular reorientation, resulting in an aberrantly proximal skeletal-smooth muscle boundary. Furthermore, Cdo(-/-) mice displayed megaesophagus and achalasia, and their lower esophageal sphincter was resistant to nitric oxide-induced relaxation, suggesting a developmental linkage between patterning and sphincter function. Collectively, these results illuminate mechanisms of esophageal morphogenesis and motility disorders.
    Full-text · Article · Apr 2013
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