DNAase-I-hypersensitive sites in the mouse albumin gene

We have analyzed the DNAase I sensitivity of the mouse alpha-fetoprotein and albumin structural genes from fetal liver, adult liver and kidney. The albumin gene shows distinct hypersensitive sites in adult liver in addition to an overall DNAase I sensitivity, but is only slightly nuclease-sensitive in fetal liver. The alpha-fetoprotein gene does not show hypersensitive sites but displays an overall DNAase I sensitivity in fetal liver; however, it is nuclease-insensitive in adult liver. Both genes are insensitive to DNAase I in kidney. The presence of DNAase-I-hypersensitive sites in the albumin structural gene correlates with extensive demethylation of the gene in adult liver.     

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.     

Sequence content of alpha-fetoprotein, albumin and fibrinogen polypeptide mRNAs in different organs, developing tissues and in liver during carcinogenesis in rats

To investigate the variable gene activities of alpha-fetoprotein, albumin and fibrinogen polypeptides as markers of 'liver specific proteins' in different developing organs or tissues, we have used specific complementary DNA probes to detect and to quantitate alpha-fetoprotein, albumin and fibrinogen polypeptide mRNA, respectively, in RNA fractions, prepared from various tissues of rats at different stages of fetal and postnatal development and from hepatomas induced by diethylnitrosamine. The results indicate that there is no consistent relationship between sequence content of alpha-fetoprotein, albumin and fibrinogen polypeptide mRNA in different developing tissues. Intestines which are like the liver also of endodermal origin do not contain alpha-fetoprotein, albumin and fibrinogen polypeptide mRNAs, while kidneys which are mesodermal in origin were found to be alpha-fetoprotein, albumin and fibrinogen polypeptide mRNA producers in neonatal life. In yolk sac, only alpha-fetoprotein and fibrinogen polypeptide mRNA could be detected. In the liver, the increased level of albumin and fibrinogen polypeptide mRNA during fetal and neonatal development is accompanied with a diminished amount of alpha-fetoprotein mRNA. The neosynthesis of alpha-fetoprotein mRNA in the liver during carcinogenesis occurred without a decreased content of albumin and fibrinogen polypeptide mRNAs. These findings suggest that complex mechanisms of gene regulation are involved in variable gene activities of alpha-fetoprotein, albumin and fibrinogen polypeptides in cells of different organs or tissues developed from a single cell.     

Effects of laminin, fibronectin and type IV collagen on liver cell cultures

The effects on mouse liver cells of laminin, fibronectin and type IV collagen, all of which are the main matrix of the basement membrane, were studied. Laminin, a glycoprotein isolated from cultures of rat yolk sac carcinoma cells, promoted the attachment of mouse fetal liver cells to laminin-coated dishes, but did not have a strong influence upon the attachment of normal adult liver cells. On the other hand, fibronectin which was purified from mouse plasma promoted the attachment of adult liver cells but not that of fetal liver cells. The number of neonatal liver cells attached to the surfaces coated was intermediate between those of fetal and adult liver cells in each matrix. DNA synthesis and cell proliferation during the culture of full-term fetal liver cells in laminin-coated dishes were higher than those in fibronectin- or type IV collagen-coated dishes. The amount of alpha-fetoprotein secreted in the laminin-coated dishes was more than in other groups. No differences in secretion of albumin into media, however, were observed in either group. These results suggest that laminin may be necessary for cell growth, tissue organization and cell differentiation during the normal development of liver in vivo.     

Expression of mRNAs for alpha-fetoprotein (AFP) and albumin and incorporation of AFP and docosahexaenoic acid in baboon fetuses.

alpha-Fetoprotein and albumin, two members of a multigene family, reversibly bind fatty acids with high affinity. The origin of alpha-fetoprotein (AFP) and albumin present in fetal tissues other than the liver and yolk sac is a subject of controversy. In this work, we have searched for the presence of the albumin and AFP mRNA molecules in different fetal organs of the baboon (Papio cinocephalus), using a highly sensitive gel-blot hybridization assay with human albumin and AFP cDNA probes. Large amounts of albumin and AFP mRNA molecules were found in the fetal liver; significant quantities were also present in the gastrointestinal tract and in the kidney. No detectable levels were found in the other tissues examined (brain, skin, spleen, pancreas, muscle, heart, thymus, placenta, and amnion). After injection of radiolabeled AFP into pregnant baboons, all fetal tissues took up the protein. White adipose tissue, kidney, intestine, lung, liver, and cerebral cortex showed a great uptake of exogenous AFP. [14C]Docosahexaenoic acid (22:6, n-3), injected at the same time, was actively transferred from the maternal compartment across the placenta and incorporated into cellular lipids by all fetal tissues and particularly by liver (around 70% of total incorporation). The levels of [14C]docosahexaenoic acid per gram of tissue increased in the order: maternal blood less than placenta less than fetal liver, indicating a selective accumulation of this fatty acid by the fetus. These results indicate that intracellular AFP in non-hepatic tissues of the developing baboon is, for the most part, of plasma origin.     

Immunocytochemical localization of oncodevelopmental proteins in human germ cell and hepatic tumors.

In a combined tissue and serum study alpha-1-antitrypsin (AAT) and alpha-fetoprotein are demonstrated in parallel within tumor tissue inclusions in both endodermal sinus (yolk sac) tumors and malignant hepatomas, and AAT is demonstrated as a marker in both neoplastic and preneoplastic liver lesions occurring in oral contraceptive users, all in association with normal serum AAT phenotype. The tumor inclusions in the first two instances differ immunocytochemically from AAT liver cell globules found in inherited AAT deficiency, which are unreactive for alpha-fetoprotein. It is concluded that unlike the molecular basis of storage associated with AAT phenotypic variation, the tumor inclusions reflect a separate, nongenetic mechanism of AAT storage, which may be epigenetic in nature. AAT and alpha-fetoprotein both are synthesized normally in yolk sac and fetal liver, a parallelism which disappears soon after birth. The reexpression of both proteins in two distinct tumor types arising from endodermal origins (yolk sac and liver), suggests that these markers may represent reemerging fetal gene products, a phenomenon previously proposed only for alpha-fetoprotein, a prototypic "oncofetal antigen."     

Developmental changes in the methylation of the rat albumin and alpha-fetoprotein genes

We have analyzed methylation of the rat albumin and alpha-fetoprotein (AFP) genes by hydridizing labeled cDNA clones to HpaII and MspI digests of DNA from different stages of development. These CCGG-cutting enzymes distinguish 5-methylcystosine in mCCGG (sensitive to HpaII) and CmCGG (sensitive to MspI). In the liver, the albumin gene is heavily methylated at 18 days gestation and uniformly demethylated in the adult. The AFP gene is also heavily methylated at 18 days gestation, and develops demethylated regions at the 3' half of the gene in the adult. These methylation changes are not observed in other embryonic or adult tissues. We also evaluated expression of these genes by measuring their corresponding mRNAs. The albumin gene is actively transcribed in 18-day fetal liver, when it is heavily methylated, as well as in adult liver, when it is unmethylated. In contrast, the AFP gene is transcribed only in fetal liver, even though it is less methylated in adult liver. These findings suggest that specific methylation changes are associated with changes in gene expression, but that this association is not adequately described by the simple hypothesis that methylation turns genes off.     

The ontogeny of expression of murine metallothionein: comparison with the alpha-fetoprotein gene

The ontogeny of expression of mouse metallothionein was studied by RNA dot and Northern blot hybridization using a cloned cDNA probe. In some instances the synthesis of metallothionein was analyzed by cell-free translation of RNA as well as pulse-labeling of proteins in short-term organ cultures followed by polyacrylamide gel electrophoresis. Interesting parallels between metallothionein and alpha-fetoprotein gene expression during development were noted. Like alpha-fetoprotein mRNA ( Dziadek and Andrews, 1983), metallothionein mRNA was found to be abundant in developing liver as well as in visceral yolk sac endoderm. In addition, metallothionein mRNA was abundant in parietal yolk sac. During liver development metallothionein and alpha-fetoprotein mRNAs were abundant by Day 12 of gestation, increasing to maximal levels on Day 16 and decreasing during late fetal and neonatal life to basal levels in adult. Metallothionein mRNA increased in maternal liver and was also abundant in certain hepatomas. Synthesis of metallothionein and levels of metallothionein mRNA in visceral yolk sac increased from Day 9 of gestation to maximal levels on Days 11-12 and then decreased abruptly after Day 15. RNA from differentiated teratocarcinoma cells with primitive, parietal or visceral endoderm characteristics each contained high levels of metallothionein mRNA, whereas, levels of this mRNA varied widely among embryonal carcinoma stem cell lines. alpha-Fetoprotein mRNA was not detected in embryonal carcinoma cells but was expressed in visceral endoderm-like differentiated cells. These results indicate that parietal and visceral endoderm cells actively express the metallothionein gene and further suggest that expression may be initiated at the earlier stage of primitive endoderm.     

Apolipoprotein B-related gene expression and ultrastructural characteristics of lipoprotein secretion in mouse yolk sac during embryonic development.

In mice, the yolk sac appears to play a crucial role in nourishing the developing embryo, especially during embryonic days (E) 7;-10. Lipoprotein synthesis and secretion may be essential for this function: embryos lacking apolipoprotein (apo) B or microsomal triglyceride transfer protein (MTP), both of which participate in the assembly of triglyceride-rich lipoproteins, are apparently defective in their ability to export lipoproteins from yolk sac endoderm cells and die during mid-gestation. We therefore analyzed the embryonic expression of apoB, MTP, and alpha-tocopherol transfer protein (alpha-TTP), which have been associated with the assembly and secretion of apoB-containing lipoproteins in the adult liver, at different developmental time points. MTP expression or activity was found in the yolk sac and fetal liver, and low levels of activity were detected in E18.5 placentas. alpha-TTP mRNA and protein were detectable in the fetal liver, but not in the yolk sac or placenta. Ultrastructural analysis of yolk sac visceral endoderm cells demonstrated nascent VLDL within the luminal spaces of the rough endoplasmic reticulum and Golgi apparatus at E7.5 and E8.5. The particles were reduced in diameter at E13.5 and reduced in number at E18.5;-19.The data support the hypothesis that the yolk sac plays a vital role in providing lipids and lipid-soluble nutrients to embryos during the early phases (E7;-10) of mouse development. secretion in mouse yolk sac during embryonic development.     

Pattern of serum protein gene expression in mouse visceral yolk sac and foetal liver.

We have shown by molecular hybridisation that the mRNAs for albumin, transferrin, apolipoprotein-A1, and alpha 1-antitrypsin are expressed at high levels in mouse visceral yolk sac. In contrast, the mRNAs for contrapsin (a plasma protease inhibitor) and the major urinary proteins (MUPs) are not detected in the visceral yolk sac at any stage of embryonic development. Contrapsin and MUP mRNAs both appear late in liver development. These differences in expression suggest that the visceral yolk sac is more similar to the foetal than adult mouse liver in its pattern of gene expression. However, the developmental time course of expression of these mRNAs is different between the foetal liver and the yolk sac. Evidence is also presented that the visceral yolk sac synthesises and secretes other apolipoproteins in addition to apolipoprotein-A1. These results suggest that the visceral yolk sac and foetal liver, two tissues with different embryological lineages, perform similar functions but are independently programmed for expression of the same set of serum protein genes.