Mouse models and quantitative trait loci of susceptibility to bleomycin- and radiation-induced pulmonary fibrosis

Radiotherapy involving the thoracic cavity and chemotherapy with the drug bleomycin are both dose limited by the development of pulmonary fibrosis. From evidence that there is variation in the population in susceptibility to pulmonary fibrosis, and animal data, it was hypothesized that individual variation in susceptibility to bleomycin-induced, or radiation-induced, pulmonary fibrosis is, in part, genetically controlled. In this thesis a three generation mouse genetic model of C57BL/6J (fibrosis prone) and C3Hf/Kam (fibrosis resistant) mouse strains and F1 and F2 (F1 intercross) progeny derived from the parental strains was developed to investigate the genetic basis of susceptibility to fibrosis. In the bleomycin studies the mice received 100 mg/kg (125 for females) of bleomycin, via mini osmotic pump. The animals were sacrificed at eight weeks following treatment or when their breathing rate indicated respiratory distress. In the radiation studies the mice were given a single dose of 14 or 16 Gy (Co$\sp{60})$ to the whole thorax and were sacrificed when moribund. The phenotype was defined as the percent of fibrosis area in the left lung as quantified with image analysis of histological sections. Quantitative trait loci (QTL) mapping was used to identify the chromosomal location of genes which contribute to susceptibility to bleomycin-induced pulmonary fibrosis in C57BL/6J mice compared to C3Hf/Kam mice and to determine if the QTL's which influence susceptibility to bleomycin-induced lung fibrosis in these progenitor strains could be implicated in susceptibility to radiation-induced lung fibrosis. For bleomycin, a genome wide scan revealed QTL's on chromosome 17, at the MHC, (LOD = 11.7 for males and 7.2 for females) accounting for approximately 21% of the phenotypic variance, and on chromosome 11 (LOD = 4.9), in male mice only, adding 8% of phenotypic variance. The bleomycin QTL on chromosome 17 was also implicated for susceptibility to radiation-induced fibrosis (LOD = 5.0) and contributes 7% of the phenotypic variance in the radiation study. In conclusion, susceptibility to both bleomycin-induced and radiation-induced pulmonary fibrosis are heritable traits, and are influenced by a genetic factor which maps to a genomic region containing the MHC. ^

Genetic analysis of tumor progression susceptibility in the mouse skin model

In the field of chemical carcinogenesis the use of animal models has proved to be a useful tool in dissecting the multistage process of tumor formation. In this regard the outbred SENCAR mouse has been the strain of choice in the analysis of skin carcinogenesis given its high sensitivity to the chemically induced acquisition of premalignant lesions, papillomas, and the later progression of these lesions into squamous cell carcinomas (SCC).^ The derivation of an inbred strain from the SENCAR stock called SSIN, that in spite of a high sensitivity to the development of papillomas lack the ability to transform these premalignant lesions into SCC, suggested that tumor promotion and progression were under the genetic control of different sets of genes.^ In the present study the nature of susceptibility to tumor progression was investigated. Analysis of F1 hybrids between the outbred SENCAR and SSIN mice suggested that there is at least one dominant gene responsible for susceptibility to tumor progression.^ Later development of another inbred strain from the outbred SENCAR stock, that had sensitivity to both tumor promotion and progression, allowed the formulation of a more accurate genetic model. Using this newly derived line, SENCAR B/Pt. and SSIN it was determined that there is one dominant tumor progression susceptibility gene. Linkage analysis showed that this gene maps to mouse chromosome 14 and it was possible to narrow the region to a 16 cM interval.^ In order to better characterize the nature of the progression susceptibility differences between these two strains, their proliferative pattern was investigated. It was found that SENCAR B/Pt, have an enlarged proliferative compartment with overexpression of cyclin D1, p16 and p21. Further studies showed an aberrant overexpression of TGF-$\beta$ in the susceptible strain, an increase in apoptosis, p53 protein accumulation and early loss of connexin 26. These results taken together suggest that papillomas in the SENCAR B/Pt. mice have higher proliferation and may have an increase in genomic instability, these two factors would contribute to a higher sensitivity to tumor progression. ^

Molecular mechanisms of chondrocyte-specific expression on the mouse pro$\alpha$1(II) collagen gene

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro$\alpha1$(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a $\beta$-galactosidase reporter gene. A construction containing a 3000-bp promoter and a 3020-bp intron 1 fragment directed high levels of $\beta$-galactosidase expression specifically to chondrocytes. Successive deletions of intron 1 delineated a 48-bp fragment which targeted $\beta$-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. When the Col2a1 promoter was replaced with a minimal $\beta$-globin promoter, the 48-bp intron 1 sequence was still able to target expression of the transgene to chondrocytes, specifically. Therefore a 48-bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression to a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression. Nuclear proteins present selectively in mouse primary chondrocytes and rat chondrosarcoma cells bind to the three putative HMG (High-Mobility-Group) domain protein binding sites in this 48-bp sequence and the chondrocyte-specific proteins likely bind the DNA through minor groove. Together, my results indicate that a 48-bp sequence in Col2a1 intron 1 controls chondrocyte-specific expression in vivo and suggest that chondrocytes contain specific nuclear proteins involved in enhancer activity. ^

Functional studies of the homeobox gene {\it Goosecoid\/} during mouse embryogenesis

A fundamental question in developmental biology is to understand the mechanisms that govern the development of an adult individual from a single cell. Goosecoid (Gsc) is an evolutionarily conserved homeobox gene that has been cloned in vertebrates and in Drosophila. In mice, Gsc is first expressed during gastrulation stages where it marks anterior structures of the embryo, this pattern of expression is conserved among vertebrates. Later, expression is observed during organogenesis of the head, limbs and the trunk. The conserved pattern of expression of Gsc during gastrulation and gain of function experiments in Xenopus suggested a function for Gsc in the development of anterior structures in vertebrates. Also, its expression pattern in mouse suggested a role in morphogenesis of the head, limbs and trunk. To determine the functional requirement of Gsc in mice a loss of function mutation was generated by homologous recombination in embryonic stem cells and mice mutant for Gsc were generated.^ Gsc-null mice survived to birth but died hours after delivery. Phenotypic analysis revealed craniofacial and rib cage abnormalities that correlated with the second phase of Gsc expression in the head and trunk but no anomalies were found that correlated with its pattern of expression during gastrulation or limb development.^ To determine the mode of action of Gsc during craniofacial development aggregation chimeras were generated between Gsc-null and wild-type embryos. Chimeras were generated by the aggregation of cleavage stage embryos, taking advantage of two different Gsc-null alleles generated during gene targeting. Chimeras demonstrated a cell-autonomous function for Gsc during craniofacial development and a requirement for Gsc function in cartilage and mesenchymal tissues.^ Thus, during embryogenesis in mice, Gsc is not an essential component of gastrulation as had been suggested in previous experiments. Gsc is required for craniofacial development where it acts cell autonomously in cartilage and mesenchymal tissues. Gsc is also required for proper development of the rib cage but it is dispensable for limb development in mice. ^

myogenin: Human candidate gene and knockout mouse effect

The myogenin gene encodes an evolutionarily conserved basic helix-loop-helix transcription factor that regulates the expression of skeletal muscle-specific genes and its homozygous deletion results in mice who die of respiratory failure at birth. The histology of skeletal muscle in the myogenin null mice is reminiscent of that found in some severe congenital myopathy patients, many of whom also die of respiratory complications and provides the rationale that an aberrant human myogenin (myf4) coding region could be associated with some congenital myopathy conditions.^ With PCR, we found similarly sized amplimers for the three exons of the myogenin gene in 37 patient and 40 control samples. In contrast to the GeneBank sequence for human myogenin, we report several differences in flanking and coding regions plus an additional 659 and 498 bps in the first and second introns, respectively, in all patients and controls. We also find a novel (CA)-dinucleotide repeat in the second intron. No causative mutations were detected in the myogenin coding regions of genomic DNA from patients with severe congenital myopathy.^ Severe congenital myopathies in humans are often associated with respiratory complications and pulmonary hypoplasia. We have employed the myogenin null mouse, which lacks normal development of skeletal muscle fibers as a genetically defined severe congenital myopathy mouse model to evaluate the effect of absent fetal breathing movement on pulmonary development.^ Significant differences are observed at embryonic days E14, E17 and E20 of lung:body weight, total DNA and histologically, suggesting that the myogenin null lungs are hypoplastic. RT-PCR, in-situ immunofluorescence and EM reveal pneumocyte type II differentiation in both null and wild lungs as early as E14. However, at E14, myogenin null lungs have decreased BrdU incorporation while E17 through term, augmented cell death is detected in the myogenin null lungs, not seen in wild littermates. Absent mechanical forces appear to impair normal growth, but not maturation, of the developing lungs in myogenin null mouse.^ These investigations provide the basis for delineating the DNA sequence of the myogenin gene and and highlight the importance of skeletal muscle development in utero for normal lung organogenesis. My observation of no mutations within the coding regions of the human myogenin gene in DNA from patients with severe congenital myopathy do not support any association with this condition. ^

Estrogen action during early mouse mammary gland development

Estrogens have been implicated in the normal and neoplastic development of the mammary gland. Although estradiol is essential for early mammary differentiation, its role in postnatal ductal morphogenesis is poorly defined. We have found that neonatal estradiol exposure promotes precocious ductal outgrowth and terminal end bud formation in 21 day-old female mice. In contrast to this precocious phenotype, day 21 estradiol-treated epithelium, transplanted into control host fatpads, grows more slowly than control epithelium. Western and immunohistochemical (IHC) analyses indicate that neonatally-estrogenized glands have significantly less total ER than controls at days 7 and 21, and significantly more stromal ER at day 35. Estrogen receptor α (ER) is present in the gland when treatment is initiated at day 1. We propose that the premature activation of ER by neonatal estradiol exposure, during this critical perinatal period, is a key factor in the alteration of mammary growth and ER expression. ^ To address the role of ER function in mammary morphogenesis, we have developed an in vitro system to study the effect of estradiol exposure in vivo. Keratin and ER-positive mammary epithelial cell lines from 7, 21 and 35 day-old oil or estradiol treated mice have been established. Cell lines derived from estradiol-treated mice grow significantly slower than cells from control glands. Although the level of ER expressed by each cell line is correlated to its rate of growth, epithelial growth in vitro is estradiol-independent and antiestrogen-insensitive. Estradiol-induced transcription from an ERE-reporter in transiently-transfected cell lines confirms the functionality of the ER detected by western and IHC. However, there are no differences in estradiol-stimulated transcription between cell lines. ^ In conclusion, neonatal estradiol treatment alters the pattern of ER expression in mammary epithelial and stromal cells in vivo, and the growth of mammary epithelial cells in vivo and in vitro. When grown outside of the estrogenized host, exposed epithelium grows more slowly than the control. Therefore, an extra-epithelial factor is necessary for enhanced epithelial growth. Our model, which couples an in vivo-in vitro approach, can be used in the future to identify factors involved in the period of early mammary outgrowth and carcinogen susceptibility. ^

Use of a hypomorphic allele of myogenin to analyze Myogenin-dependent processes in mouse development

Myogenin is a muscle-specific transcription factor essential for skeletal muscle differentiation. A severe reduction in the number of fused myotubes is seen in myogenin-null mice, and the expression of genes characteristic of differentiated skeletal muscle is reduced. Additionally, sternebrae defects are seen in myogenin-null mice, a secondary defect in the sternal cartilage precursors. Very little is known about the quantitative requirement for myogenin in muscle differentiation and thoracic skeletal development in vivo. In this thesis I describe experiments utilizing a mouse line harboring a hypomorphic allele of myogenin, generated by gene targeting techniques in embryonic stem cells. The nature of the hypomorphism was due to lowered levels of myogenin from this allele. In embryos homozygous for the hypomorphic allele, normal sternum formation and extensive muscle differentiation was observed. However, muscle hypoplasia and reduced muscle-specific gene expression were apparent in these embryos, and the mice were not viable after birth. These results suggest skeletal muscle differentiation is highly sensitive to the absolute amounts of myogenin, and reveal distinct threshold requirements for myogenin in skeletal muscle differentiation, sternum formation, and viability in vivo. The hypomorphic allele was utilized as a genetically sensitized background to identify other components of myogenin-mediated processes. Using a candidate gene approach I crossed null mutations in MEF2C and MRF4 into the hypomorphic background and examined whether these mutations affected muscle differentiation and skeleton formation in the myogenin hypomorph. Although MEF2C mutation did not affect any phenotypes seen in the hypomorphic background, MRF4 was observed to be an essential component of myogenin-mediated processes of thoracic skeletal development. Additionally, the hypomorphic allele was very sensitive to genetic effects, suggesting the existence of mappable genetic modifiers of the hypomorphic allele of myogenin. ^

MOUSE MAMMARY TUMOR VIRUS EPITOPES EXPRESSED ON TUMOR SPECIFIC TRANSPLANTATION ANTIGENS OF CHEMICALLY INDUCED C3H/HEJ SARCOMAS (TSTA, FIBROSARCOMA, CARCINOGENESIS, MMTV, METHYLCHOLANTHRONE)

This dissertation addressed the hypothesis that the unique tumor specific transplantation antigens (TSTA) of chemically induced sarcomas express epitopes encoded by endogenous viral genes. TSTA from two 3-methylcholanthrene-induced, C3H/HeJ fibrosarcomas (MCA-F and MCA-D) were serologically assessed for viral epitopes in an enzyme-linked immunospecific assay (ELISA) and by immunoaffinity chromatography. Initial evidence with an anti-TSTA antiserum suggested that TSTA were associated with mouse mammary tumor virus (MMTV) peptides, but not peptides from murine leukemia virus (MuLV). TSTA extracted from MCA-F, was assessed with specific anti viral antibodies at three levels of purification for its association with MuLV peptides (gp 70 and p 15E) and MMTV peptides (gp52, gp36 and p27). The results demonstrate that purified preparations enriched for TSTA activity are devoid of MuLV epitopes, but enriched for a subset of MMTV epitopes. Immunoaffinity supports constructed with anti-MMTV antibodies retained TSTA from partially purified MCA-F or MCA-D extracts. Immunoaffinity chromatography with antibodies against individual MMTV peptides demonstrated that the MCA-F TSTA was specifically retained by anti-gp36 and anti-p27 supports, but not by anti-gp52 supports nor a support made with bovine serum albumin. Analysis of the affinity purified TSTA preparations by HPGPC and SDS-PAGE revealed only a few components. Application of the anti-gp36 and anti-p27 retained materials to HPGPC and subsequent in vivo analysis demonstrated that the TSTA migrated in a low and a high molecular weight region. These results suggest that TSTA specificity in C3H/HeJ mice, results from MMTV recombinant proteins. ^

High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations

Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.