Embryonic lethal mutation in mouse collagen I gene causes rupture of blood vessels and is associated with erythropoietic and mesenchymal cell death

The role of collagen I for midgestation development was studied in homozygous Mov 13 embryos, which cannot synthesize alpha 1(1) mRNA as a result of insertional mutagenesis and most of which die between day 12 and 14 of gestation. No type I collagen was detected in mutant embryos, while the distribution of other collagens, laminin, and fibronectin was not affected. Mutant embryos develop normally up to day 12 of gestation, suggesting that collagen I has no essential role in the early phase of morphogenesis. The first pathological events were detected in hemopoietic cells of the liver, followed by necroses of mesenchymal cells in other parts of the embryo. The sudden death is caused by the rupture of a major blood vessel, indicating an important role for collagen I in establishing the mechanical stability of the circulatory system. Our results furthermore suggest that complex cell interactions in embryonic development such as those in early hemopoiesis may depend on the presence of collagen type I.     

Use of the collagen I-deficient Mov13 mouse mutant to analyse epithelial-mesenchymal tissue interaction

The collagen I-deficient mouse mutant (Mov13 — an embryonic recessive lethal) was used to investigate the function of this major constituent of the extracellular matrix (ECM) in organ development. All epithelial-mesenchymal organs tested as explants (lung, kidney, pancreas, salivary glands, skin) developed normally and, in particular, showed typical branching morphogenesis in the absence of collagen I. It is concluded that the ECM of these organs can organize for proper developmental function in the absence of the major interstitial collagen, but a possible morphogenetic function of other fibrillar collagens (types III and V) cannot be excluded. The only insufficiencies in the mutant were seen in the cornea where deposition and organization of the collagenous stroma was highly inadequate; but even there, development and migration of cells proceeded normally. In summary, the results indicate that ‘cellular’ development in epithelial-mesenchymal organs (including growth, morphogenesis, and differentiation) does not depend on collagen I.     

Human-mouse interspecies collagen I heterotrimer is functional during embryonic development of Mov13 mutant mouse embryos.

To investigate whether the human pro alpha 1(I) collagen chain could form an in vivo functional interspecies heterotrimer with the mouse pro alpha 2(I) collagen chain, we introduced the human COL1A1 gene into Mov13 mice which have a functional deletion of the endogenous COL1A1 gene. Transgenic mouse strains (HucI and HucII) carrying the human COL1A1 gene were first generated by microinjecting the COL1A1 gene into wild-type mouse embryos. Genetic evidence indicated that the transgene in the HucI strain was closely linked to the endogenous mouse COL1A1 gene and was X linked in the HucII transgenic strain. Northern (RNA) blot and S1 protection analyses showed that the transgene was expressed in the appropriate tissue-specific manner and as efficiently as the endogenous COL1A1 gene. HucII mice were crossed with Mov13 mice to transfer the human transgene into the mutant strain. Whereas homozygous Mov13 embryos die between days 13 and 14 of gestation, the presence of the transgene permitted apparently normal development of the mutant embryos to birth. This indicated that the mouse-human interspecies collagen I heterotrimer was functional in the animal. The rescue was, however, only partial, as all homozygotes died within 36 h after delivery, with signs of internal bleeding. This could have been due to a functional defect in the interspecies hybrid collagen. Extensive analysis failed to reveal any biochemical or morphological abnormalities of the collagen I molecules in Mov13-HucII embryos. This may indicate that there was a subtle functional defect of the interspecies hybrid protein which was not revealed by our analysis or that another gene has been mutated by the retroviral insertion in the Mov13 mutant strain.     

Corneal morphogenesis in the Mov13 mutant mouse is characterized by normal cellular organization but disordered and thin collagen

This paper compares corneal development in the normal and in the Mov13 mutant mouse homozygote which does not synthesize type I collagen. During the period 12-14 days of development, there is no obvious difference between cellular organization in the normal and the mutant corneas or, indeed, elsewhere in the eye. In particular, there is normal colonization of the mutant cornea by the mesenchymal cells which will form the endothelium and the fibroblasts. In the early stages of stromal deposition (less than 14 days), when relatively little collagen is normally laid down, mutant and wild-type corneas differ only in that mutant collagen fibrils are less uniform than normal ones. Later development in the Mov13 mutant cannot usually be studied because almost all mutant embryos are dead by 14 days, but we now have two homozygous embryos from a single, 16-day litter. Their stromas obviously differed from those of their normal littermates: there was markedly less collagen in the mutant cornea and the collagen that was deposited lacked orthogonal organization. Fibril morphology also differed: the diameters of fibrils in the normal corneas peaked sharply at about 20 nm, whereas the diameters of mutant fibrils were spread over the range 5-15 nm, with only a small percentage overlapping the normal distribution. These results suggest that type I collagen is of negligible importance in controlling the cellular organization of the cornea, but has a dominant role in the formation of normal 20 nm fibrils and of normal stromal organization. They also show that, as collagen production is markedly lower in the mutant than in the wild-type cornea, the production of other collagens cannot compensate in any way for the lack of type I collagen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death

Thirteen mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (M-MuLV) at a single locus (Mov locus) have been derived previously. Experiments were performed to investigate whether homozygosity at the Mov loci would be compatible with normal development. Animals heterozygous at an Mov locus were mated, and the genotype of the offspring was analyzed. From parents heterozygous at the loci Mov1 to Mov12, respectively, homozygous offspring were obtained with the expected Mendelian frequency. In contrast, no homozygous offspring or embryos older than day 15 of gestation were obtained from parents heterozygous at the Mov13 locus. When pregnant Mov13 females at day 13 and day 14 of gestation were analyzed, approximately 25% of the embryos were degenerated. Genotyping revealed that these degenerated embryos were invariably homozygous and the normal appearing embryos were either heterozygous or negative for M-MuLV. These results suggest that integration of M-MuLV at the Mov13 locus leads to insertion mutagenesis, resulting in embryonic arrest between day 12 and day 13 of gestation. It is possible that the Mov13 locus represents a gene or gene complex involved in the early embryonic development of the mouse.     

Fibronectin binding site in type I collagen regulates fibronectin fibril formation

Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral insertion, were used to study the role of type I collagen in the process of fibronectin fibrillogenesis. While Mov13 cells produced a sparse matrix containing short fibronectin fibrils, transfection with a wild type pro alpha 1(I) collagen gene resulted in the production of an extensive matrix containing fibronectin fibrils of normal length. To study the amino acids involved in the fibronectin-collagen interaction, mutations were introduced into the known fibronectin binding region of the pro alpha 1(I) collagen gene. Substitution of Gln and Ala at positions 774 and 777 of the alpha 1(I) chain for Pro resulted in the formation of short fibronectin fibrils similar to what was observed in untransfected Mov13 cells. Type I collagen carrying these substitutions bound weakly to fibronectin- sepharose and could be eluted off with 1 M urea. The effect of this mutation on fibronectin fibrillogenesis could be rescued by adding either type I collagen or a peptide fragment (CB.7) which contained the wild type fibronectin binding region of the alpha 1(I) chain to the cell culture. These results suggest that fibronectin fibrillogenesis in tissue culture is dependent on type I collagen synthesis, and define an important role for the fibronectin binding site in this process.     

Actinotrichia collagens and their role in fin formation

The skeleton of zebrafish fins consists of lepidotrichia and actinotrichia. Actinotrichia are fibrils located at the tip of each lepidotrichia and play a morphogenetic role in fin formation. Actinotrichia are formed by collagens associated with non-collagen components. The non-collagen components of actinotrichia (actinodins) have been shown to play a critical role in fin to limb transition. The present study has focused on the collagens that form actinotrichia and their role in fin formation. We have found actinotrichia are formed by Collagen I plus a novel form of Collagen II, encoded by the col2a1b gene. This second copy of the collagen II gene is only found in fishes and is the only Collagen type II expressed in fins. Both col1a1a and col2a1b were found in actinotrichia forming cells. Significantly, they also expressed the lysyl hydroxylase 1 (lh1) gene, which encodes an enzyme involved in the post-translational processing of collagens. Morpholino knockdown in zebrafish embryos demonstrated that the two collagens and lh1 are essential for actinotrichia and fin fold morphogenesis. The col1a1 dominant mutant chihuahua showed aberrant phenotypes in both actinotrichia and lepidotrichia during fin development and regeneration. These pieces of evidences support that actinotrichia are composed of Collagens I and II, which are post-translationally processed by Lh1, and that the correct expression and assembling of these collagens is essential for fin formation. The unique collagen composition of actinotrichia may play a role in fin skeleton morphogenesis.     

Dysmorphogenesis of the inner ear: disruption of extracellular matrix (ECM) formation by an L-proline analog in otic explants

L-azetidine-2-carboxylic acid (LACA), a l-proline analog, disrupts collagen secretion by cells and prevents normal morphogenesis of in vitro developing organ rudiments. Otic explants derived from 10.5-through 14-day-old mouse embryos were continuously exposed to LACA in the nutrient medium at concentrations of 75, 150, and 300 micrograms/ml. LACA disrupted normal in vitro otic morphogenesis in inner ears explanted from embryos of 10.5 through 13 days' gestation. Development of 14-day-old otic explants were not affected by LACA at the concentrations tested. There was a direct correlation between the embryonic age of the explant when exposed to LACA, and the severity of otic dysmorphogenesis. The younger explants (10.5-to 12-day-old) developed abnormalities of both vestibular and auditory structures, but with increasing embryonic age of the explants (12-to 13.5-day-old) abnormalities were confined more to the auditory portion of the inner ear. Disruption of collagen secretion of connective tissue cells of the otic explants are a major teratogenic action of LACA on inner ear development. Disrupted collagen secretion alters otic extracellular matrix production, which in turn affects the tissue interactions that regulate the progressive expression of otic morphogenesis and differentiation.     

Developmental modulation of embryonic cardiac myocyte adhesion to cardiac collagens in vitro.

The goal of this investigation is to identify molecules that mediate embryonic cardiac myocyte adhesion during chick cardiac morphogenesis. The assay used employs culturing embryonic myocytes on substrata containing embryonic heart proteins separated by molecular weight. This assay shows that embryonic myocytes from 10- to 14-day-old embryos will bind to 140,000 and 128,000 Da proteins present in embryonic hearts and do not require Mg2+ or Ca2+ for adhesion. Myocytes from embryos younger than 10 days or older than 14 days display little or no binding. Embryonic heart fibroblasts collected at these same ages do not bind to these proteins. The 140- and 128-kDa proteins were found to copurify in extraction procedures for procollagens. Amino acid analysis shows that both proteins contain high glycine and hydroxyproline, indicating that they are collagens. However, glycine and imino acid levels are low relative to other known collagens, indicating a nonhelical domain present in each molecule and most closely resembled levels present in procollagens. Immunoblots show that antisera to chick collagen type I recognizes the 128-kDa protein while anti-collagen type III recognizes the 140-kDa protein. Monoclonal antibodies to the amino terminal propeptide of collagen type I recognize the 128-kDa protein in immunoblotting procedures. Embryonic chick myocytes bind to 140/128 kDa proteins present in extracts of sympathetic trunk, although they do not bind to 140/128 kDa proteins in embryonic tendon. The findings thereby indicate that forms of type III and type I collagens in embryonic heart support direct adhesion of embryonic myocytes for a restricted period of cardiac myogenesis and that these proteins differ from collagen types I and III present in other tissues and from fully processed collagen types I and III.     

Type I collagen mutation alters the strength and fatigue behavior of Mov13 cortical tissue

Despite advances in understanding the molecular basis of Osteogenesis Imperfecta, the mechanisms by which type I collagen mutations compromise whole bone function are not well understood. Previously, we have shown that a heterozygous type I collagen mutation is associated with increased brittleness of long bones from Mov13 transgenic mice, a model of the mild form of Osteogenesis Imperfecta. In the current study, we investigated tissue-level damage processes by testing the hypothesis that the fatigue properties of Mov13 tissue were significantly compromised relative to littermate controls. We also quantified tissue structure and mineral content to explain variations in the fatigue behavior. Micro-beam specimens were machined from the anterior and posterior quadrants of Mov13 and control femurs and subjected to cyclic bending at one of four stress levels. Mov13 tissue exhibited a 22–25% reduction in tissue bending strength and a similar reductions in fatigue life and the stress level at which damage was apparent. These results provided tissue-level evidence that damage accumulation mechanisms were significantly compromised in Mov13 cortical tissue. Given that significant alterations in tissue structure were observed in Mov13 femurs, the results of this study support the idea that Mov13 femurs were brittle because alterations in tissue structure associated with the mutation interfered with normal damage processes. These results provide new insight into the pathogenesis of Osteogenesis Imperfecta and are consistent with bone behaving as a damaging composite material, where damage accumulation is central to bone fracture.     

Developmental modulation of embryonic cardiac myocyte adhesion to cardiac collagens in vitro

The goal of this investigation is to identify molecules that mediate embryonic cardiac myocyte adhesion during chick cardiac morphogenesis. The assay used employs culturing embryonic myocytes on substrata containing embryonic heart proteins separated by molecular weight. This assay shows that embryonic myocytes from 10- to 14-day-old embryos will bind to 140,000 and 128,000 Da proteins present in embryonic hearts and do not require Mg²⁺ or Ca²⁺ for adhesion. Myocytes from embryos younger than 10 days or older than 14 days display little or no binding. Embryonic heart flbroblasts collected at these same ages do not bind to these proteins. The 140- and 128-kDa proteins were found to copurify in extraction procedures for procollagens. Amino acid analysis shows that both proteins contain high glycine and hydroxyproline, indicating that they are collagens. However, glycine and imino acid levels are low relative to other known collagens, indicating a nonhelical domain present in each molecule and most closely resembled levels present in procollagens. Immunoblots show that antisera to chick collagen type I recognizes the 128-kDa protein while anti-collagen type III recognizes the 140-kDa protein. Monoclonal antibodies to the amino terminal propeptide of collagen type I recognize the 128-kDa protein in immunoblotting procedures. Embryonic chick myocytes bind to 140/128 kDa proteins present in extracts of sympathetic trunk, although they do not bind to 140/128 kDa proteins in embryonic tendon. The findings thereby indicate that forms of type III and type I collagens in embryonic heart support direct adhesion of embryonic myocytes for a restricted period of cardiac myogenesis and that these proteins differ from collagen types I and III present in other tissues and from fully processed collagen types I and III.     

Association of collagen with preimplantation and peri-implantation mouse embryos

By immunofluorescence analyses, we have determined that Type III procollagen, Type III collagen, and B and C chains of basement membrane collagen are associated with preimplantation mouse embryos. Type III collagen and procollagen appear to be associated with embryos at the 4-cell stage and beyond, whereas antibodies to B and C collagen chains bind to 2-cell and later embryos. All of these collagen types are detected in increasing amounts as embryos develop in a defined medium, indicating that the embryo is capable of their synthesis. By the blastocyst stage, the collagens are primarily localized intercellularly. Cells of the inner cell mass (ICM) also bind collagen antibodies. When isolated ICMs become two-layered, both the inner presumptive ectoderm layer and the outer primitive endoderm layer react with antibodies to Type III collagen and procollagen. The endoderm cells also react avidly with antibodies to B- and C-chain collagens. Preimplantation embryos and ICMs fail to react with antibodies to Types I and II collagen. During peri-implantation stages, blastocysts continue to react with antibodies to Type III and basement membrane collagens. There is no obvious relationship between the intensity of immunofluorescence and the change in the blastocyst surface from nonadhesive to adhesive. Furthermore, blastocysts prevented from undergoing implantation-related events in utero and in vitro react extensively with collagen antibodies. Blastocyst surface collagens might, nevertheless, play a role in implantation by undergoing organizational changes.     

Changes in the types of collagen synthesized during chondrogenesis of the mouse otic capsule

We have investigated the temporal relationship between the morphological differentiation of the mouse otic capsule and the pattern of collagen synthesis by mouse otocyst-mesenchyme complexes labeled in vitro. In 10.5- to 12-day embryos the mesenchyme surrounding the otocyst was loosely organized except for a few lateroventral condensations; explants from these embryos synthesized only small amounts of collagen. Collagen synthesis by whole explants increased by more than 50% between 12 and 13 days concomitant with metachromatic staining of the lateral periotic mesenchyme. Cartilage specific type II collagen was the predominant collagen synthesized by these explants as confirmed by SDS-PAGE, densitometry, CNBr cleavage, and V8 protease digestion. This biochemical expression of the cartilage phenotype preceded morphologic recognition of otic capsular cartilage by almost 2 days. Type II collagen synthesis continued to increase and predominate through Day 16 of gestation by which time the otic labyrinth was surrounded by mature cartilage. The minor cartilage collagen chains, 1 alpha, 2 alpha, and 3 alpha, first appeared on different days of gestation. The 1 alpha, and 3 alpha chains were synthesized by explants from 11-day embryos while the 2 alpha chain appeared during Day 13, just before overt differentiation of mature cartilage. These results suggested that the 1 alpha, 2 alpha, and 3 alpha chains may not form heterotrimers containing all three chains and that synthesis of the 2 alpha chain may be associated with stabilization of the cartilaginous matrix. Comparison of these data with the patterns of collagen production by mutant, diseased, or experimentally manipulated inner ear tissues may provide insights into the molecular basis of chondrogenic tissue interactions.     

Collagens support embryo attachment and outgrowth in vitro: effects of the Arg-Gly-Asp sequence

Collagen types I through VI support attachment and outgrowth of mouse blastocysts in vitro. We found that embryos acquire the ability to attach to collagens type II and VI relatively early in their developmental program. The time at which half of the embryos displayed outgrowth formation and the morphology of outgrowths formed on these two collagen types are similar to those observed for laminin, fibronectin, and hyaluronate. Embryos acquire the ability to outgrow on the other collagen types at a later time in culture. Both "native" and denatured collagens support embryo attachment and outgrowth, indicating that this activity is intrinsic to the primary collagens' structure. A synthetic peptide containing the sequence Arg-Gly-Asp inhibits embryo outgrowth on collagen type II and denatured collagen type IV, whereas a peptide containing the related sequence, Arg-Gly-Glu, has relatively little effect on embryo outgrowth. In contrast, embryo attachment to collagen types I, V, and VI was not inhibited specifically by the Arg-Gly-Asp peptide sequence. Consequently, it appears that embryos use multiple adhesion systems to attach to collagens. Among these are adhesion systems that have a peptide recognition specificity similar to that of fibronectin receptors. These studies indicate that embryo interactions with collagens may be one aspect of the tissue invasion processes that take place during implantation.     

Global defects in collagen secretion in a Mia3/TANGO1 knockout mouse

Melanoma inhibitory activity member 3 (MIA3/TANGO1) [corrected] is an evolutionarily conserved endoplasmic reticulum resident transmembrane protein. Recent in vitro studies have shown that it is required for the loading of collagen VII, but not collagen I, into COPII-coated transport vesicles. In this paper, we show that mice lacking Mia3 are defective for the secretion of numerous collagens, including collagens I, II, III, IV, VII, and IX, from chondrocytes, fibroblasts, endothelial cells, and mural cells. Collagen deposition by these cell types is abnormal, and extracellular matrix composition is compromised. These changes are associated with intracellular accumulation of collagen and the induction of a strong unfolded protein response, primarily within the developing skeleton. Chondrocyte maturation and bone mineralization are severely compromised in Mia3-null embryos, leading to dwarfism and neonatal lethality. Thus, Mia3's role in protein secretion is much broader than previously realized, and it may, in fact, be required for the efficient secretion of all collagen molecules in higher organisms.