Published August 9, 2004

JCB
ArticleEsophageal 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 Ramirez1
1

C

ollagen XIX is an extremely rare extracellular matrix
component that localizes to basement membranezones and is transiently expressed by differentiatingmuscle cells. Characterization of mice harboring null andstructural mutations of the collagen XIX (Col19a1) gene hasrevealed the critical contribution of this matrix protein tomuscle physiology and differentiation. The phenotype includes smooth muscle motor dysfunction and hypertensive
sphincter resulting from impaired swallowing-induced, nitricoxide–dependent relaxation of the sphincteric muscle.
Muscle dysfunction was correlated with a disorganizedmatrix and a normal complement of enteric neurons andinterstitial cells of Cajal. Mice without collagen XIX exhibit
an additional defect, namely impaired smooth-to-skeletal
muscle cell conversion in the abdominal segment of theesophagus. 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 firststructural determinant of sphincteric muscle function, andas the first extrinsic factor of skeletal myogenesis in themurine esophagus.

Introduction
The ECM consists of a highly heterogeneous mixture ofinteracting proteins that form a complex array of supramolecular structures, and that bind cell surface receptors andsoluble 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 andKivirikko, 2004). The latter group includes collagens thatare components of or associated with basement membranes(BMs), highly specialized macroaggregates that subserve thedual function of organizing multicellular structures and of
instructing tissue differentiation, maintenance, and remodeling(Timpl, 1996; Ortega and Werb, 2002). Naturally occurring
Address correspondence to Francesco Ramirez, Laboratory of Genetics
and Organogenesis, Hospital for Special Surgery at the Weill College ofMedicine 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 transdifferentiation; nitrergic neurotransmission© The Rockefeller University Press, 0021-9525/2004/08/591/10 $8.00
The Journal of Cell Biology, Volume 166, Number 4, August 16, 2004 591–600
http://www.jcb.org/cgi/doi/10.1083/jcb.200402054

human mutations and genetically engineered mice have
implied specialized roles of this particular class of nonfibrillar 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) (Christiano 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 etal., 2004).
Collagen XIX is the latest example of a nonfibrillar collagentype that localizes to BM zones and that may potentiallyhave an anatomically restricted function. Collagen XIX is
deposited at extremely low amounts (106% of dry tissueAbbreviations used in this paper: BM, basement membrane; EFS, electricalfield stimulation; ES, embryonic stem; ICC-IM, intramuscular interstitialcells 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.

591

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The Journal of Cell Biology

Research Division of the Hospital for Special Surgery and Department of Physiology and Biophysics at the Weill Collegeof Medicine of Cornell University, New York, NY 10019
2
Department of Anatomy, Biology, and Medicine, Oita Medical University, Oita 879-5593, Japan3
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

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weight) in the BM zones of vascular, neural, and mesenchymal tissues (Myers et al., 1997). Collagen XIX forms higher
order aggregates that may conceivably modulate cell–matrix
interactions, cell–cell communications, and/or local concentrations of signaling molecules (Myers et al., 2003). Embryonic expression of the collagen XIX (Col19a1) gene istransient and confined almost exclusively to differentiatingmuscles (Sumiyoshi et al., 2001). Onset of Col19a1 expression in myotomes and myotome derivatives occurs soon after 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 thatcollagen XIX may be involved in muscle differentiation andfunction (Sumiyoshi et al., 2001). Unfortunately, experimental validation of this postulate has been hampered by thepaucity of collagen XIX in tissues. Therefore, the present
work was undertaken to overcome this problem using genetic means. Toward this end, we created a null mutation(N19) and a structural mutation (19) of the 1(XIX) collagen chain by targeting different regions of the Col19a1
gene in mouse embryonic stem (ES) cells. Characterizationof the resulting mouse phenotypes demonstrated that collagen XIX plays a dual role in muscle physiology and differ-entiation. Specifically, we found that proper assembly of thecollagen XIX–rich BM zone is a prerequisite for nitric oxide(NO)–dependent relaxation of the lower esophageal sphincter (LES) muscle, and that collagen XIX deposition into thematrix of the developing esophagus is an extrinsic determinant 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 consists of a discontinuous collagenous region flanked by a cysteine-rich noncollagenous (NC) amino terminal (NC6) anda 19-residue carboxyl peptide (NC1) (Fig. 1 A; Sumiyoshi etal., 1997). Recent ultrastructural analyses have shown that
the NC interruptions impart flexibility to the otherwise rigid
triple helical (collagenous) domain; they have also documented 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 contrast the phenotypic consequences of assembling BM zones
devoid of collagen XIX or containing structurally abnormal
collagen XIX trimers.

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Figure 1. Col19a1 gene targeting. (A) Schematic representation of the 1(XIX) collagen chain where the gray bars correspond to exon 4 andexons 38–40 sequences in the NC6 and NC3 domains, respectively. Targeting strategies are shown on the left for the N19 allele (N) and on theright 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).

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Collagen XIX in muscle development and function | Sumiyoshi et al. 593

Collagen XIX null mice display alteredesophageal morphology
Heterozygous N19 mice were born at the expected Mendelian frequency; they were morphologically normal, viable,
and fertile. Homozygous N19 mice were born at the expected 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 newborn homozygous mutants did not detect gross anatomical
abnormalities, except for the smaller size of the internal organs. On the other hand, necroscopy of the few Col19a1/
mice that survived past weaning stage revealed a dilated

Figure 2. Analysis of gastroesophageal region. (A) Representative
megaesophagus of a 3-mo-old Col19a1 null mouse (N/N); note theesophageal 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.

esophagus (megaesophagus) with retention of ingesta, immediately above the diaphragm level (Fig. 2 A). Based onthese 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 gastroesophageal junction. Specifically, in situ hybridizations revealed high Col19a1 expression in the lower-third portion of
the embryonic esophagus destined to become the abdominal
segment; thereafter Col19a1 activity becomes gradually restricted 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 humanachalasia, an esophageal motility disorder characterized by elevated basal tone and impaired swallowing-induced relaxationof the LES (Goyal, 2001). This phenotypic trait, coincident
with high and persistent Col19a1 expression in the LES,

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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 residues (Sumiyoshi et al., 1997). After homologous recombination in ES cells, chimeric animals from two correctly targeted ES clones were generated and germ line transmissionof 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). Moreover, sequencing of RT-PCR–amplified products across anddownstream of the targeted genomic region excluded theexistence of shorter, in-frame Col19a1 transcripts (unpublished data). Immunoblots of partially purified collagen
preparations from homozygous mutant and wild-type tissues
corroborated the mRNA data by documenting absence ofthe expected 165-kD collagenase-sensitive product in theformer compared with the latter specimen (Fig. 1 D). Unfortunately, the same antibodies proved unsuitable to confirm loss of collagen XIX in tissues. This last point notwithstanding, 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 interruption of the helical domain and for one and six collagenous
tripeptides located amino- and carboxyl-terminal of it, respectively (Sumiyoshi et al., 1997). Chimeric animals weregenerated from two independently derived clones and theprogeny was genotyped by Southern blot analysis using a diagnostic restriction enzyme cleavage site (Fig. 1 E). The deletion of exons 39–40 maintains the frame of the Col19a1
transcript, and thus it is predicted to yield an internally deleted 1(XIX) chain that should participate in homotrimer
formation. Sequencing of RT-PCR–amplified products confirmed that the mutant transcript is in frame (unpublisheddata), whereas immunoblots identified a collagenase-sensitive product in the mutant tissue slightly smaller than thewild-type 165-kD species (Fig. 1 D). Finally, PCR amplification estimated that the mutant and wild-type Col19a1 alleles 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 ofthe nullizygous mouse.

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prompted us to examine whether early demise of homozygous
mutants may be in part accounted for by sphincteric muscle dysfunction. Accordingly, the physiology of the collagen
XIX–deficient LES muscle was assessed in vitro by monitoring basal muscle tone in response to electrical field stimulation(EFS), and in vivo by examining swallowing-induced LES relaxation 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 measured in the absence and in the presence of L-NA, an inhibitor of NO synthase (NOS; Mashimo et al., 1996). As expected, we found that EFS elicited frequency-dependent
relaxation of wild-type LES strips followed by pronounced
rebound contraction, and that muscle relaxation was significantly reduced by L-NA treatment (Fig. 3, A and B). In
marked contrast, mutant muscle strips failed to produce significant 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. Intraluminal 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 abnormally brief (Fig. 4 A). The results of the manometric tests
were remarkably similar to those reported for achalasic patients (Richter, 2001). Altogether, the in vitro and in vivo
experiments indicated that NO-dependent neurotransmission 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 interstitial cells of Cajal (ICC-IM) transduce inhibitory signals
from nerve terminals to sphincteric smooth muscle cells

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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 ofquantitative relaxation and contraction data of wild-type (/) and collagen XIX null (N/N) animals (n  6) in the presence and absenceof L-NA. (C) Typical tracings to illustrate the same responses as in A using heterozygous (/) and homozygous 19 (/) mice.

Published August 9, 2004

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 ofnitrergic nerves and/or ICC-IM may account for sphinctericmuscle dysfunction in Col19a1/ mice. Immunofluorescence staining of wild-type and homozygous mutant LES
muscle showed more nNOS-positive cells than c-Kit–positive 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 ofstatistically comparable numbers of each cell type in thewild-type and mutant tissues (Fig. 5 B). No remarkable differences were also observed with additional neurospecificmarkers, 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 architectureof the smooth muscle matrix or the intrinsic ability of SMC

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Figure 4. LES in vivo physiology. (A) Typical tracings to illustrateswallow-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 withfrank contraction (one example in the middle tracing) or extremely
high basal tone with incomplete relaxation (another example in theright 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 wildtype (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 micrographs 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-mm2 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.

to relax upon nerve stimulation. The latter possibility wasassessed in the collagen XIX–deficient LES muscle by invitro and in vivo assays. An exogenously applied NO donorcaused relaxation of mutant LES muscle strips precontractedwith bethanechol (Fig. 4 B). Similarly, administration of a-adrenoreceptor agonist to Col19a1/ mice caused normal LES relaxation (Fig. 4 C). Therefore, these results demonstrated integrity of the SMC membrane and of the intracellular signal transduction machinery responsible for LES
relaxation in Col19a1 null mice. Along these lines, immunofluoresence staining of connexin 43, smooth muscle actin,
-actinin, and vinculin failed to identify variations between
wild-type and mutant SMC (unpublished data).

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gested a specialized and highly restricted role of collagen
XIX in organizing the BM zone of the LES.

Immunostaining of collagen XIX–deficient LES tissue
with antibodies against nidogen-1 documented a largely preserved 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 themutant SMC (Fig. 6 B). It also revealed that intercellularspacing of the mutant smooth muscle is appreciably greaterthan the wild-type control (Fig. 6 C). Additional abnormalities include convoluted SMC profiles, excessive extracellularaccumulation of collagen fibrils, and highly irregular intercellular space (Fig. 6 C). Consistent with progressive degeneration of matrix organization, these morphological abnormalities were more pronounced in adult than newborn
mutant mice (Fig. 6 C). Collectively, these analyses sug-

The 19 mutation perturbs only LES function
The milder phenotype of the 19 mutation afforded the opportunity to further explore the role of collagen XIX in skeletal myogenesis and sphincteric muscle relaxation. Heterozygous and homozygous 19 mice were born at the
expected frequency and were apparently unaffected by thedeposition into the ECM of abnormal 1(XIX) homotrimers. However, a significant number of adult (8–12-mo-old)heterozygous and homozygous mutant mice were noted todisplay 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 tissues from four adult 19/ mice and four 19/19 littermates 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 heterozygous and eight homozygous 19 specimens examined(Fig. 3 C). Moreover, LES samples from randomly chosen

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Figure 6. BM in the mutant LES. (A) Light microscopy of representative 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 (/) andN19/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 and1-yr-old wild-type and collagen XIX null (N/N) mice. Intercellularspacing 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 from94 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 smoothmuscle cells (SMC) and collagen fibrils, respectively. Bar, 1 m.

Impaired muscle transdifferentiation in the collagen
XIX null esophagus
Necroscopic examination of the esophagus of adult
Col19a1/ mice revealed the presence of another phenotypic manifestation indicative of a morphogenetic defect.
The muscle layer of the murine esophagus undergoes atransdifferentiation 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 etal., 1995; Kablar et al., 2000). This poorly understood developmental program is accompanied by rostrocaudal expression of MRF genes, and varies in timing and extent depending 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 differentiation (Molkentin and Olson, 1996). In situ hybridizations at
different prenatal and postnatal stages of esophageal development revealed that the front of myogenin expression
reaches diaphragm level at birth, and gradually progresses
into the abdominal segment of the esophagus during thefirst week of postnatal life (Fig. 7 A). The same analysis documented that the postnatal front of myogenin expression in
the collagen XIX–deficient esophagus remains at the samelevel as at birth (Fig. 7 B). Immunostaining of the wild-type
and mutant specimens for skeletal and smooth muscle–specific proteins demonstrated that loss of MRF gene expression translates into failed muscle transdifferentiation in theentire 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 MRFdriven smooth muscle transdifferentiation.

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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, andthat only the latter is required for the developmentally regulated process of skeletal myogenesis.

Discussion
Compositional diversification of BMs at distinct anatomical locations is a major determinant of the functional specificity ofthese highly specialized and widely distributed ECM structures(Timpl, 1996). A growing body of genetic evidence indicatesthat the functional integrity of individual BM zones also requires the deposition of collagen molecules that connect themto the underlying connective tissue stroma (Ortega and Werb,
2002). Illustrative examples include the role of BM-associatedcollagens XV and XVIII in providing integrity to skeletal muscle, microvessels, and vitreous (Eklund et al., 2001; Fukai et al.,2002). The present paper adds collagen XIX to the list of BMassociated collagens by implicating this molecule in the organization of the pericellular matrix of the sphincteric smooth mus-cle. It has also unraveled an unexpected role of this rare collagen type in esophageal morphogenesis.
Structural role of collagen XIX in LES physiology
Activation of inhibitory NANC nerves is critical for LES relaxation upon swallowing (Goyal and Hirano, 1996). Although NO is widely recognized as the major inhibitory neurotransmitter of NANC nerves, the mechanism and factors
responsible for conveying NO signals to SMC remain illdefined. 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 collagen 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 functionally viable SMC further supports this conclusion.
The hypertensive and nonrelaxing LES of the Col19a1 mutant mice resembles the clinical manifestations of human

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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) Immunostainingof 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.

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Instructive role of collagen XIX inesophageal development
Collagenous and elastic macroaggregates have been traditionally viewed as the main structural determinants of connective tissue architecture. However, there is emerging evidence that they also participate in modulating a variety ofcellular 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 epithelial 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 thisBM-stabilizing molecule promotes programmed cell deathand 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 isa rare phenomenon in vertebrates that has been described fora 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). Transdifferentiation in both organ systems involves the conversion ofsmooth muscle to skeletal muscle. Co-culture experiments

have suggested that activin and follistatin coordinate muscle transdifferentiation in the chick iris (Link and Nishi,
1998b). That normal development of striated muscles andnicotinic receptor clusters take place in Mash1/ mice,
which lack enteric neurons, has indicated that skeletal myogenesis in the mouse esophagus occurs independently of innervation (Sang et al., 1999).
The biological mechanism responsible for skeletal myogenesis in the mature muscle layer of the mouse esophagus iscontroversial. In the original description of the phenomenon,
Patapoutian et al. (1995) reported that smooth-to-skeletal
muscle conversion is preceded by MyoD and myogenin expression. Kablar et al. (2000) subsequently used transgenic andknock-in mice to document that initiation and progression ofmuscle transdifferentiation depend on Myf5 expression. The
apparent discrepancy between these two reports may reflect
the fact that each followed muscle transdifferentiation in different esophageal segments (i.e., abdominal vs. thoracic/cervical). 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 evidenceof 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 thedeveloping mouse esophagus. Interestingly, the same morphogenetic defect was not observed in the C57/Bl/6J genetic
background, implying that collagen XIX action is modulated by modifier gene products. On the other hand, association of an achalasia-like phenotype with the same physiological and morphological manifestations in both C57/Bl/6J
and 129T2/SvEmsJ mutant mice indicated that distinct
mechanisms are responsible for the genesis of LES dysfunction and failed muscle transdifferentiation. Along theselines, normal esophageal muscle transdifferentiation in mice
producing abnormal collagen XIX trimers demonstrated
that a specific peptide sequence (rather than the whole molecule) is involved in muscle transdifferentiation. One attractive mechanism is that the collagen XIX–rich matrix may
control the distribution and/or activity of growth factorsthat ultimately trigger MRF gene expression in the abdominal esophagus. The elegant work of Myers et al. (2003) supports this hypothesis. These investigators have shown thatcollagen XIX forms higher order aggregates in which individual molecules extend radially from a globular core of interacting NC6 domains. Therefore, they have argued that
this configuration, together with the NC6 heparin-binding site, may contribute to localize and concentrate signaling molecules within the BM zone. Such a model is analogous to the recently reported involvement of extracellular
microfibrils in limb patterning and lung morphogenesis
through the modulation of TGF/BMP signaling (ArteagaSolis et al., 2001; Neptune et al., 2003). Alternatively, theflexibility of the collagen XIX monomers and the presence ofa Tsp-N module in NC6 may regulate skeletal myogenesisby mediating critical cell–matrix and/or cell–cell interactions(Myers et al., 2003).
Although the precise mechanism underlying the role ofcollagen XIX in muscle transdifferentiation remains undeter-

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patients with achalasia (Goyal, 2001; Richter, 2001). The etiology of this primary esophageal motor disorder is heterogeneous and may include genetic, infectious, autoimmune, anddegenerative factors. Loss of nitrergic nerves in achalasia iswidely believed to cause hypertensive LES due to unopposedcholinergic excitation, a notion indirectly supported by thebasal LES hypertension in nNOS/ mice (Goyal, 2001;
Richter, 2001; Sivarao et al., 2001). Similarly, impaired LES
relaxation to swallowing in nNOS/ mice underscores theprominent contribution of nitrergic neurotransmission to inhibitory neurotransmission (Sivarao et al., 2001). The manometric data from Col19a1/ and nNOS/ mice are virtually
identical and as such, they emphasize functional equivalency
between NO release from nitrergic varicosities and ECM organization. By contrast, c-Kit mutant (W/Wv ) mice, whichlack ICC-IM, have a hypotensive LES with normal NANC
relaxation (Sivarao et al., 2001). Hence, the data from theCol19a1 and nNOS null mice concur in strongly suggestingthat ICC-IM play a lesser role than previously suggested insphincteric 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 sphincteric muscle. In this respect, an analogy could be drawn withperlecan in clustering acetylcholinesterase to the synaptic basal
lamina of the neuromuscular junction (Arikawa-Hirasawa etal., 2002). Our postulate is based on the intriguing observation that the NOS inhibitor L-NA affects the relaxation ofmutant muscles relatively less than wild-type muscles (Fig. 3).
Irrespective of the underlying mechanism, our work conclusively proves that collagen XIX is a new contributing factor tosphincteric muscle physiology.

Published August 9, 2004

Collagen XIX in muscle development and function | Sumiyoshi et al. 599

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). Maintenance, transfection, and selection of mouse ES cells, as well as generationof chimeric animals were performed as described previously (Andrikopoulos et al., 1995). Electroporated ES cells and mutant mice were genotyped
by Southern blot analysis using diagnostic restriction enzyme sites andprobes outside and inside of the targeted region. Northern blot hybridization and sequencing of RT-PCR products were used to confirm the identity
and relative expression levels of the mutant transcript. RT-PCR analysis of19/ transcripts was performed after 25, 30, or 35 cycles in order tocompare the relative representation of wild-type and mutant products during 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 final 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 preparation was then separated on 4.5% SDS-PAGE and electroblotted onto a
PVDF membrane in the presence of 15 mM sodium-borate buffer. Immunoblots 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 collagen XIX. Immunocomplexes were detected using the ECL blotting system(Amersham Biosciences) with secondary goat anti–rabbit IgG conjugatedwith HRP (Santa Cruz Biotechnology, Inc.). For collagenase digestion,
7.5 U bacterial collagenase type III (Advance Biofactures) was added to100 g of crude collagen XIX extract, and was incubated for 2 h at 37C in
50 mM Tris-HCl, pH 7.2, and 20 mM CaCl2.

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 immunostainedwith alkaline phosphatase conjugated to mAbs against smooth muscle actin 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 4C 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 wereincubated with FITC-conjugated donkey anti–rabbit IgG and Texas red–conjugated donkey anti–goat IgG (1:200; Jackson ImmunoResearch Laboratories) 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, andHeNe). 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 werecollected from 4-mo-old animals, fixed overnight in 4% PFA, processed
through paraffin, and sectioned at 5–7-m thickness. Sections were deparaffinized and treated with 100 g/ml protease XXIV (P8038; SigmaAldrich) for 10 min at 37C (Willem et al., 2002). Antibodies against nidogen-1 (1:1,000) were applied for 2 h at RT in a humid chamber; thestreptavidin-HRP technique (LSAB 2 system; DakoCytomation) was used to
localize the antibody and hematoxylin was used as a counterstain. Samples of the same tissues used for immunohistochemistry were processed forelectron microscopy by fixation in 2% PFA plus 0.5% glutaraldehyde in
0.05 M cacodylate buffer, pH 7.2, for 4 h at 4C. Tissues were rinsed, postfixed 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 andwith lead citrate for 5 min. Photographs were taken on a transmission electron 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 Resource Facility (New York, NY) and at the Hospital for Special Surgery Analytical Microscopy Core Facility (New York, NY). In situ hybridizations
were performed on serial tissue sections using Col19a1 and myogeninprobes 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 arginine (L-NA; Mashimo et al., 1996). LES tone was also measured in the presence of increasing concentrations of bethanechol (0.1–300 M) followedby sodium nitroprusside (0.1–100 M) as described previously (Chakder etal., 1997). Intraluminal esophageal manometry was performed as described by Sivarao et al. (2001) using a custom-designed catheter assembly(Dentsleeve; Dentsleeve Pty Ltd.) that consists of silicon tubing made ofthree 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 sphinctericmuscle, 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 invaluable assistance in the physiological tests, V. William and N. Tulchin fortechnical 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 declarethey 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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