DEVELOPMENTAL DYNAMICS 220:155–162 (2001)

BRIEF COMMUNICATION

Embryonic Expression of Type XIX Collagen Is Transientand Confined to Muscle Cells
HIDEAKI SUMIYOSHI,1 FRIEDRICH LAUB,1 HIDEKATSU YOSHIOKA,2 AND FRANCESCO RAMIREZ1*1Brookdale Center in the Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine-New York
University, New York, New York
2Oita Medical University, Hasama-machi, Oita, JapanABSTRACT
Type XIX collagen is a poorly
characterized extracellular matrix component
thought to be involved in the formation of specialized basement membrane zones. Here we examined the developmental expression of themouse gene (Col19a1) by in situ hybridization.
Col19a1 expression during embryogenesis commences at ⬃E9.5 in the myotome and with a pattern that closely follows the myogenic regulatory
factor myf-5. Like myf-5, Col19a1 transcription
gradually decreases in differentiating skeletal
muscle progenitors and concomitantly to increased myogenin gene expression. Transient expression of Col19a1 in muscular tissues is confined to a few sites of the developing embryo,such as limbs, tongue, and the smooth muscle
layers of the stomach and esophagus. Additional
non-muscular sites of Col19a1 activity include
the skin of the E16.5 embryos and the cerebral
cortex and hippocampus of the new born brain.
Unlike all other tissues, expression of Col19a1 inthe central nervous system gradually increases
after birth. © 2001 Wiley-Liss, Inc.
Key words: basement
membranes;
collagen;extracellular matrix; myogenesis;
esophageal transdifferentiation
INTRODUCTION
Type XIX is the latest addition to the collagen superfamily of extracellular matrix proteins, whose various
members are segregated into different groups according to structural and/or functional considerations(Brown and Timpl, 1995). Originally identified fromcDNA cloning experiments, the existence of type XIX
collagen was later confirmed by biochemical and immunohistochemical studies (Yoshioka et al., 1992; Myerset al., 1993, 1997; Sumiyoshi et al., 1997). Information
regarding the nature and possible function of type XIX
collagen aggregates is currently lacking. Structuralconsiderations have placed type XIX collagen among
the so-called fibril-associated collagens with interrupted triple helix, or FACIT (types IX, XII, XIV, and© 2001 WILEY-LISS, INC.

XVI) (Yoshioka et al., 1992; Myers et al., 1994; Inoguchi et al., 1995; Khaleduzzaman et al., 1997). Immunohistochemical work, on the other hand, has locatedtype XIX collagen preferentially in basement membrane zones of several organ systems, thus suggestingthat it may belong to the multiplexin group (collagenswith multiple triple-helix domains and interruptions)(Myers et al., 1997).
Type XIX collagen transcripts have been detected atvery low levels in mouse embryonic tissues by the RTPCR technique, and in human rhabdomyosarcoma celllines by Northern analysis (Myers et al., 1994; Sumiyoshi et al., 1997). A recent report has raised theintriguing possibility that type XIX collagen may also
be involved in the initial stages of skeletal muscle celldifferentiation. Human rhadomyosarcoma cells cultured under conditions that stimulate myoblast differentiation were in fact shown to selectively up-regulate
the production of skeletal muscle myosin heavy chainand ␣-actinin, and of type XIX collagen as well (Myerset al., 1999). Unfortunately, the significance of this invitro observation could not be placed into a physiologiccontext due to the lack of information about the developmental expression of the type XIX collagen gene. The
present study was designed to fill this gap in knowledge, in addition to providing useful information for
the future characterization of type XIX collagen(Col19a1) null mice.
The results of our in situ hybridizations demonstratethat type XIX collagen is preferentially and transientlyexpressed in differentiating muscle cells. Embryonicaccumulation of Col19a1 transcripts in myotomes andmyotome derivatives closely resembles that of myf-5, inthat they both decline concomitantly to the increase inmyogenin gene activity. Col19a1 is also expressed insome smooth muscle cells, such as those of the devel-Grant sponsor: NIH; Grant number: AR 38468.
*Correspondence to: Francesco Ramirez, Ph.D., Department of Biochemistry and Molecular Biology, Mount Sinai School of MedicineNew York University, One Gustave L. Levy Place, Box 1020, NewYork, NY 10029. E-mail: ramirf01@doc.mssm.edu
Received 26 October 2000; Accepted 3 November, 2000
Published online 5 January 2001

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Fig. 1. A: Whole-mount in situ hybridization of E10.5 embryo to the Col19a1 probe. B: (Top to bottom) insitu hybridization of cross-sections from a E11.5 embryo to Col19a1, myf-5, and Col2a1 probes. C: In situ
hybridization of cross-sections from E8.5 (top) and E9.5 (bottom) embryos to Col19a1 (left) or myf-5 (right)
probes. Arrows point to Col19a1 (A) and myf-5 expression (C) in the myotome.

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Fig. 2. Serial cross-sections from E11.5, E13.5, and E16.5 mouse embryos hybridized to the Col19a1,myf-5 and myogenin probes. Arrows indicate unspecific signal produced by blood cells.

oping stomach and esophagus, as well as in embryonicskin and adult brain.
RESULTS AND DISCUSSION
Whole mount in situ hybridization of mid-gestation(E10.5) embryos identified the strongest Col19a1 signal in the somites (Fig. 1A). Hybridizations of crosssections of E11.5 embryos confirmed the restricted distribution of Col19a1 transcripts in the myotomes,
within the broader area of expression of myogenic regulatory factor (MRF) myf-5 (Smith et al., 1994) anddistinct from type II collagen (Fig. 1B). Similarly, hybridizations of E8.5 and E95 embryos documented thelater appearance of Col19a1 transcripts compared tomyf-5 (Fig. 1C). Comparison of cross-sections of E11.5
to E16.5 embryos hybridized to Col19a1, myf-5, andmyogenin probes documented the gradual decrease in
the intensity of the first two signals, and the steadyincrease of myogenin accumulation in differentiatingmuscles (Fig. 2). Comparison of sagittal sections of

E16.5 embryos confirmed the overall decline of Col19a1
and myf-5 gene activity compared to myogenin (Fig. 3).
The difference in intensity and distribution between
the Col19a1 and myogenin signals was most evident in
the muscles of the E16.5 limbs and tongue (Fig. 4A,B).
Col19a1 transcripts in the tongue display a characteristic pattern that mirrors the histological organizationof this muscle (Fig. 4C).
Col19a1 is also actively transcribed in some smoothmuscle cells. Illustrative examples include the outermuscular layer of the developing stomach and theforming aductory muscles around the hair follicles of
the jaw of E13.5 and E16.5 embryos, respectively (Fig.
5A,B). Expression in the stomach remains detectable
only for a few days after birth (data not shown).
Col19a1 expression was also observed in the post-natalesophagus. Consistent with the transdifferentiation ofsmooth muscle to skeletal muscle (Patapoutian et al.,1995), we found that rostro-to-caudal progression of
myogenin expression in the esophagus is completed

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Fig. 3. Serial sagittal sections from an E16.5 mouse embryo hybridized to the Col19a1, myf-5, andmyogenin probes. Arrows indicate unspecific signal.

within the first week of post-natal life (Fig. 5C,D). By
contrast, Col19a1 transcripts were only detected in thecaudal portion of the esophagus and before onset ofmyogenin expression (Fig. 5E). The region of overlap
between the Col19a1 and myogenin hybridizations
could be appreciated by comparing the patterns in theabdominal esophagus of P6 and P12 mice, respectively(Fig. 5F). Finally, the analysis failed to detect substantial Col19a1 accumulation in the smooth muscle cells of
the developing intestine, uterus, and blood vessels(data not shown).
The above data demonstrate that type XIX collagenis transiently expressed in muscles, irrespective of theembryonic origin of the cells. There are, however, a few
discrepancies between the results of our in situ hybridizations and the immunohistolocalization in humanadult tissues (Myers et al., 1997). The most evident ofthem includes the reported presence of type XIX molecules around all vessels and the stromal smooth muscle cells of the intestine (Myers et al., 1997). The discrepancy may simply reflect differences between thetwo experimental approaches; alternatively, it may in-dicate that type XIX collagen deposition in these particular matrices is a post-natal event.
Robust expression of the Col19a1 gene during theearly stages of skeletal muscle cell differentiation
contrasts with the reported absence of type XIX proteins in mature muscles (Myers et al., 1997), unless
one argues for a transient role of this collagen matrix
during myogenesis. For example, activation of type
XIX collagen synthesis in proliferating myoblasts
soon after induction by MRFs may provide the cellswith a competent extracellular environment in support of the differentiation process. Consistent withthis hypothesis, onset of Col19a1 expression was detected after myf-5, thus suggesting that this gene
may be a downstream target of MRFs. Along theselines, we have identified two potential MRF-bindingsites (E-box; Firulli and Olson, 1997) within the0.7-kb upstream sequence of the Col19a1 gene, and acluster of six E-boxes 200 bp downstream of exon 1(data not shown).
In contrast to the esophageal musculature of theembryo, which is entirely composed of differentiatedCOLLAGEN EXPRESSION DURING MYOGENESIS

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Fig. 4. In situ hybridizations of Col19a1 (left) and myogenin (right) probes to tissue sections from E16.5
mouse limbs (A) and tongue (B). Note the distribution of the Col19a1 signal at the periphery of the skeletal
muscles in the limb, and in alternate rows of positive cells in the tongue. The bright (left) and dark (right) fieldviews of the tongue in C correlate the Col19a1 signal to a specific arrangement of muscle cells.

smooth muscle, the adult layer is made of striatedmuscle as a result of the conversion of smooth muscle toskeletal muscle (Patapoutian et al., 1995). Col19a1 expression is restricted to the muscular layer of dia-phragm-level esophagus and appears prior to myogenin
expression and, thus, before overt muscle transdifferentiation. It is tempting to speculate that an extracellular matrix rich in type XIX collagen may endow spe-

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Fig. 5. Col19a1 hybridization in the forming stomach (A) of the E13.5
embryo and in the jaw (B) of the E16.5 embryo. C–E: Hybridization ofmyogenin (C and D) and Col19a1 (E) probes in longitudinal sections ofthe esophagus and stomach of 2- (C and E) and 6-day-old (D) mice.Let-ters indicate esophagus (E) and stomach (S). F: Closer view of theoverlapping zone of hybridization of myogenin (top) and Col19a1 (bottom) in the abdominal sections of the esophagus from P16 and P12 mice,respectively. Arrows indicate unspecific signal.

cialized information to the transdifferentiating cells of
the abdominal esophagus.
Our analysis also documented the presence of low
amounts of Col19a1 transcripts in a few non-muscular
tissues, such as skin and brain (Fig 6). Col19a1 mRNA
accumulation in the skin is maximal between E11.5
and E13.5 (Fig. 6A) and later decreases to undetectablelevels (data not shown). Expression in the nervous
system is clearly seen at the base of the forebrain of theP12 mouse (Fig. 6B). Col19a1 signals are still high inthe 5-week-old mouse and mostly confined in the gyrus
dentatus of the hippocampus (Fig. 6C). Northern analysis of Col19a1 mRNA accumulation in the brain during the first 2 weeks of post-natal life documented thegradual increase in mRNA accumulation from P0 toP12 (Fig. 6D). Col19a1 expression in the skin is consistent with the immunolocalization of the protein at thedermal-epidermal junction of adult human skin (Myerset al., 1997).
In conclusion, the results of the present study are in
overall agreement with the previous notion that type

XIX collagen is a minorly expressed embryonic geneproduct (Sumiyoshi et al., 1997; Myers et al., 1997).
The major new finding reported here is the discovery
that expression of Col19a1 during embryonic development is transient and confined to muscle cells. Unfortunately, there are no available antibodies againstmouse type XIX collagen that could correlate themRNA data at the protein level. With this limitation inmind, we note that Col19a1 mRNA accumulation inmuscles is at least consistent with the hypothesis thattype XIX collagen-rich matrices may play specialized
roles during morphogenesis. The postulate is based on
the finding that transient Col19a1 expression includes
myoblasts that are undergoing differentiation; a specific organization of cells in the tongue; and the abdominal portion of the esophagus prior to muscle transdifferentiation. Ongoing generation of Col19a1 null mice
will ultimately test the validity of our hypothesis, andfirmly establish the contribution of type XIX collagen to
the assembly and function of the vertebrate connectivetissue.

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Fig. 6. A: Col19a1 hybridization of a cross-section at the hind limb
level of a E13.5 embryo showing an intense positive signal in the skin(arrows). B,C: Col19a1 hybridization in the P12 (B) and 5-week-old (C)forebrain showing a diffuse signal in the former (arrows), which becomes
gradually restricted to the hippocampal region of the latter (arrows). D:

EXPERIMENTAL PROCEDURESIn Situ and Northern Hybridizations
Radiolabeled ⬃500 bp long cDNAs for the 5⬘ untranslated regions Col19a1 and Col2a1 were used for in situ
hybridization along with similar probes for mouse
myf-5 and myogenin (Wright et al., 1989; Ott et al.,
1991; Andrikopoulos et al., 1992; Sumiyoshi et al.,1997). The MRF cDNAs were generously provided Dr.
D. Sassoon, Mount Sinai School of Medicine, New York.
After linearization at appropriate restriction sites,
sense and antisense probes were generated by in vitrotranscription with T3 and T7 polymerases in the presence of [35S]-UTP. In situ hybridizations were performed essentially as previously described (Wright etal., 1989; Andrikopoulos et al., 1992) and slides wereexposed at 4°C for 5– 8 days. Total RNA purificationfrom staged mouse embryos and Northern blot hybridizations were performed as previously described (Sumiyoshi et al., 1997). The relative intensity of the hybridizing bands was quantified by the public domain NIH
Image program (http://rsb.info.nih.gov//nih.image).

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Northern blot hybridizations to Col19a1 (top) and GAPDH (bottom)
probes documenting the gradual increase of the Col19a1 mRNA during
the first 2 weeks of post-natal (PO-P12) life and in the 2-month-old mouse(Ad). Bottom: Graphic representation of Col19a1 expression in the PO toP12 brain relative to the GAPDH signal.

ACKNOWLEDGMENTS
The authors thank Dr. David Sassoon for suggestions and reagents and Ms. Karen Johnson for typing
the manuscript.
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