Generation of collagen XIX mutant mouse strains
The mouse α1(XIX) chain is 1,136 residues long and consists of a discontinuous collagenous region flanked by a cysteine-rich noncollagenous (NC) amino terminal (NC6) and a 19-residue carboxyl peptide (NC1) ( 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 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.
Figure 1. Col19a1 gene targeting. (A) Schematic representation of the α1(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 (more ...)
The null mutation (N19) was generated by inserting the PGK-neo
cassette in place of exon 4 ( 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 transmission of the mutant allele was followed by Southern blot analysis ( B). Northern hybridizations failed to detect Col19a1
transcripts in homozygous mutant tissues ( C). Moreover, sequencing of RT-PCR–amplified products across and downstream of the targeted genomic region excluded the existence 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transcripts (unpublished data).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 of the expected 165-kD collagenase-sensitive product in the former compared with the latter specimen ( 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 (Δ19) was created by inserting the PGK-neo
cassette in place of exons 38–40 ( 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 were generated from two independently derived clones and the progeny was genotyped by Southern blot analysis using a diagnostic restriction enzyme cleavage site ( E). The deletion of exons 39–40 maintains the frame of the Col19a1
transcript, and thus it is predicted to yield an internally deleted α1(XIX) chain that should participate in homotrimer formation. Sequencing of RT-PCR–amplified products confirmed that the mutant transcript is in frame (unpublished data), whereas immunoblots identified a collagenase-sensitive product in the mutant tissue slightly smaller than the wild-type 165-kD species ( D). Finally, PCR amplification estimated that the mutant and wild-type Col19a1
alleles are expressed at comparable levels in the heterozygous Δ19 mouse ( F). Therefore, the Δ19 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 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 esophagus (megaesophagus) with retention of ingesta, immediately above the diaphragm level ( 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 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 ( B).
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 (more ...)
Abnormal LES physiology in collagen XIX null mice
Megaesophagus is a distinguishing feature of severe human achalasia, an esophageal motility disorder characterized by elevated 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, 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 (). In marked contrast, mutant muscle strips failed to produce significant relaxation in response to EFS; furthermore, L-NA virtually eliminated any residual relaxation (). 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 ( A). It also documented severely impaired or absent relaxation upon swallowing; even when present, relaxation was abnormally brief ( 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.
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) (more ...)
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 (more ...)
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 (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. 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 ( 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 ( B). No remarkable differences were also observed with additional neurospecific markers, such as vasoactive intestinal peptide and choline acetyltransferase (unpublished data).
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 μm. (B) Numbers of c-Kit– and nNOS-positive (more ...)
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 ( B). Similarly, administration of a β-adrenoreceptor agonist to Col19a1−/− mice caused normal LES relaxation ( 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, α-actinin, and vinculin failed to identify variations between wild-type and mutant SMC (unpublished data).
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 ( A). Electron microscopy confirmed this result by showing a thicker BM around the mutant SMC ( B). It also revealed that intercellular spacing of the mutant smooth muscle is appreciably greater than the wild-type control ( C). Additional abnormalities include convoluted SMC profiles, excessive extracellular accumulation of collagen fibrils, and highly irregular intercellular space ( C). Consistent with progressive degeneration of matrix organization, these morphological abnormalities were more pronounced in adult than newborn mutant mice ( C). Collectively, these analyses suggested a specialized and highly restricted role of collagen XIX in organizing the BM zone of the LES.
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) (more ...)
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 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 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 the first week of postnatal life ( A). The same analysis documented that the postnatal front of myogenin
expression in the collagen XIX–deficient esophagus remains at the same level as at birth ( 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 the entire abdominal segment of the Col19a1−/−
esophagus ( C). These results conclusively established a causal relationship between extracellular deposition of collagen XIX and developmentally programmed activation of MRF-driven smooth muscle transdifferentiation.
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 (−/−) (more ...)
The Δ19 mutation perturbs only LES function
The milder phenotype of the Δ19 mutation afforded the opportunity to further explore the role of collagen XIX in skeletal myogenesis and sphincteric muscle relaxation. Heterozygous and homozygous Δ19 mice were born at the expected frequency and were apparently unaffected by the deposition into the ECM of abnormal α1(XIX) homotrimers. 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 Δ19 heterozygotes and homozygotes were analyzed for possible manifestations in the gastroesophageal system.
Immunohistological examination of gastroesophageal tissues from four adult Δ19/+ mice and four Δ19/Δ19 littermates revealed normal transdifferentiation of the muscle layer in the abdominal segment of the esophagus ( B). By contrast, the EFS assay documented reduced or absent relaxation of LES muscle strips in half of the eight heterozygous and eight homozygous Δ19 specimens examined ( C). Moreover, LES samples from randomly chosen Δ19/+ or Δ19/Δ19 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 regulated process of skeletal myogenesis.