Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation

T-box gene family members have important roles during murine embryogenesis, gastrulation, and organogenesis. Although relatively little is known about how T-box genes are regulated, published gene expression studies have revealed dynamic and specific patterns in both embryonic and extraembryonic tissues of the mouse conceptus. Mutant alleles of the T-box gene Brachyury (T) have identified roles in formation of mesoderm and its derivatives, such as somites and the allantois. However, given the cell autonomous nature of T gene activity and conflicting results of gene expression studies, it has been difficult to attribute a primary function to T in normal allantoic development. We report localization of T protein by sectional immunohistochemistry in both embryonic and extraembryonic tissues during mouse gastrulation, emphasizing T localization within the allantois. T was detected in all previously reported sites within the conceptus, including the primitive streak and its derivatives, nascent embryonic mesoderm, the node and notochord, as well as notochord-associated endoderm and posterior neurectoderm. In addition, we have clarified T within the allantois, where it was first detected in the proximal midline of the late allantoic bud (approximately 7.5 days postcoitum, dpc) and persisted within an expanded midline domain until 6-somite pairs (s; approximately 8.5 dpc). Lastly, we have discovered several novel T sites, including the developing heart, visceral endoderm, extraembryonic ectoderm, and its derivative, chorionic ectoderm. Together, these data provide a unified picture of T in the mammalian conceptus, and demonstrate T's presence in unrelated cell types and tissues in highly dynamic spatiotemporal patterns in both embryonic and extraembryonic tissues.     

Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues

A new polyclonal antibody to the humanerythrocyte urea transporter UT-B detects a broad band between 45 and65 kDa in human erythrocytes and between 37 and 51 kDa in raterythrocytes. In human erythrocytes, the UT-B protein is the Kidd (Jk)antigen, and Jk(a+b+) erythrocytes express the 45- to 65-kDa band.However, in Jk null erythrocytes [Jk(ab)], only a faint band at55 kDa is detected. In kidney medulla, a broad band between 41 and 54 kDa, as well as a larger band at 98 kDa, is detected. Human and ratkidney show UT-B staining in nonfenestrated endothelial cells indescending vasa recta. UT-B protein and mRNA are detected in rat brain,colon, heart, liver, lung, and testis. When kidney medulla or liverproteins are analyzed with the use of a native gel, only a singleprotein band is detected. UT-B protein is detected in cultured bovineendothelial cells. We conclude that UT-B protein is expressed in morerat tissues than previously reported, as well as in erythrocytes.

     

Localization of L-arginine-glycine amidinotransferase protein in rat tissues by immunofluorescence microscopy

Creatine is a major component of energy metabolism and enzymes involved in its synthesis have therefore been of considerable interest. L-arginine-glycine amidinotransferase, commonly called transamidinase, catalyzes the first reaction in the biosynthesis of creatine. This first reaction is believed to occur in the kidney because of the high concentration of transamidinase in that tissue. Transamidinase activity is also found in many other tissues of the rat, but its role in these tissues is not known. Immunochemical studies with antisera and monoclonal antibodies were used to confirm and refine our understanding of the presence of transamidinase in rat tissues. Immunofluorescence histochemistry was performed to localize transamidinase immunoreactivity within specific tissues including cells in the proximal tubules of the kidney, hepatocytes of the liver, and alpha cells of the pancreatic islet. Immunochemical studies with monoclonal antibodies confirm localization of transamidinase immunoreactivity in the proximal tubules of the kidney. The localization of such immunoreactivity in specialized cells yields insight into possible physiological role(s) of transamidinase in the rat.     

Lipoprotein receptors in extraembryonic tissues of the chicken

Yolk is the major source of nutrients for the developing chicken embryo, but molecular details of the delivery mechanisms are largely unknown. During oogenesis in the chicken, the main yolk components vitellogenin and very low density lipoprotein (VLDL) are taken up into the oocytes via a member of the low density lipoprotein receptor gene family termed LR8 (Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T., and Schneider, W. J. (1994) EMBO J. 13, 5165-5175). This endocytosis is accompanied by partial degradation of the yolk precursor protein moieties; however, fragmentation does not abolish binding of VLDL to LR8. The receptor exists in two isoforms that differ by a so-called O-linked sugar domain; the shorter form (LR8-) is the major form in oocytes, and the longer protein (LR8+) predominates in somatic cells. Here we show that both LR8 isoforms are expressed at ratios that vary with embryonic age in the extraembryonic yolk sac, which mobilizes yolk for utilization by the embryo, and in the allantois, the embryo's catabolic sink. Stored yolk VLDL interacts with LR8 localized on the surface of the yolk sac endodermal endothelial cells (EEC), is internalized, and degraded, as demonstrated by the catabolism of fluorescently labeled VLDL in cultured EEC. Addition to the incubation medium of the 39-kDa receptor-associated protein, which inhibits all known LR8/ligand interactions, blocks the uptake of VLDL by EEC. The levels of endogenous receptor-associated protein correspond to those of LR8+ but not LR8-, suggesting that it may play a role in the modulation of surface presentation of LR8+. Importantly, EEC express significant levels of microsomal triglyceride transfer protein and protein disulfide isomerase, key components required for lipoprotein synthesis. Because the apolipoprotein pattern of VLDL isolated from the yolk sac-efferent omphalomesenteric vein is very different from that of yolk VLDL, these data strongly suggest that embryo plasma VLDL is resynthesized in the EEC. LR8 is a key mediator of a two-step pathway, which affects the uptake of VLDL from the yolk sac and the subsequent delivery of its components to the growing embryo.     

Immunocytochemical detection of oncomodulin in tumor tissue

Using the rat hepatoma calcium-binding protein, oncomodulin, from Morris hepatoma 5123tc, an antiserum has been raised in rabbits useful for immunostaining of this tumor protein. The peroxidase-antiperoxidase (PAP) technique has been used to demonstrate oncomodulin in sections of Morris rat hepatomas 5123D, 5123tc, 7288Ctc, and 7777 fixed with Bouin's or Carnoy's fixatives, or using freeze substitution. Oncomodulin-specific staining was also shown by a hepatoma metastasis in lung. Optimal conditions for the indirect fluorescent antibody technique were established to demonstrate oncomodulin in virally transformed NRK cells (ASV-NRK). In both tumors and cultured neoplastic cells staining appeared which suggested that oncomodulin might occur in nucleus and cytoplasm. Normal untransformed tissues and uninfected cells did not show oncomodulin staining.     

Lanthanide-binding properties of rat oncomodulin

Oncomodulin, the parvalbumin-like calcium-binding protein frequently expressed in tumor tissue, was isolated from Morris hepatoma 5123tc and studied using the luminescent lanthanide ions, Eu3+ and Tb3+. Titrations of the apoprotein - whether monitored by indirect excitation of bound Tb3+, by direct laser excitation of bound Eu3+, or by quenching of the intrinsic tyrosine fluorescence - all indicated the presence of two high-affinity binding sites for lanthanide ions, as in parvalbumin. Moreover, the appearance of the Eu3+ 7F0----5D0 excitation spectrum of Eu2-oncomodulin was found to be highly pH-dependent, as previously observed with parvalbumin. At pH 5.0, it consists of a single peak centered at 5796 A, having a linewidth of approximately 6 A. At higher pH values, this spectrum is replaced by a broader, more symmetric peak at 5782 A. Oncomodulin, however, was found to differ from parvalbumin in at least one important respect: In contrast to the muscle-associated protein, the affinities of the CD site in oncomodulation for Tb3+ and Ca2+ were found to be rather similar, with KCa/KTb approximately equal to 11 +/- 2.     

Localization of pan-cadherin immunoreactivity in adult rat tissues

Cadherins, being responsible for selective cell recognition and normal tissue integrity in adults, regulate morphogenesis in a variety of organs during development. In this study, anti-rat pan-cadherin antibody, specific to all subgroups of the cadherin family, was used to map the distribution of the pan-cadherin immunoreactivity in adult rat organs. Pan-cadherin immunoreactivity positive tissues were: secretory cells of the adenohypophysis, autonomic nerve, corneal epithelium, oesophageal nerve plexus, stomach and pyloric glandular cells, epithelium of the ileum and its nerve plexus, alveolar cells of the lung, proximal convoluted tubules of the kidney, islet cells of Langerhans, and the acinar cells of the exocrine pancreas. For the first time, positive pan-cadherin immunoreactivity was demonstrated in the epithelial cells of the corpus ciliaris and in the nerve plexus of corpus cavernosum of the penis. In conclusion, our results suggest that cells in many tissues and organs of the adult rat synthesize cadherins.     

Localization of lipoprotein lipase mRNA in selected rat tissues

Measurements of enzymatic activity have demonstrated that lipoprotein lipase (LPL), the principal enzyme responsible for hydrolysis of circulating triglyceride, is present in a number of tissues including brain, kidney, and adrenal gland. To determine the sites of synthesis of LPL in these tissues, in situ hybridization studies were performed using a non-sense 35S-labeled RNA probe produced from a 624-bp mouse LPL cDNA fragment. Control studies were performed with a sense RNA strand. Using 5-10-micron sections of 5-day-old rat brain, strong hybridization was found in pyramidal neurons of the hippocampus. Positive hybridization, indicating the presence of LPL mRNA, was also found in brain cortex and in the intermediate lobe of adult rat pituitary gland. Specific areas of adrenal and kidney medulla showed hybridization with the probe. LPL mRNA is, therefore, present in a number of specific regions of the body. LPL in these areas may not be important in regulating circulating levels of lipoproteins, but may be essential for cellular uptake, binding, and transfer of free fatty acids or other lipophilic substances.     

Prostaglandin synthesis and metabolism in the human uterus and midtrimester fetal tissues

Prostaglandin (PG) synthesis and metabolism was studied in human fetal kidney, lung, small intestine, heart, brain and liver (gestational ages: 10, 12, 14, 18 and 23 weeks) and pregnant uterus (4-40 weeks of pregnancy). PG synthesis was increased in the myometrium during pregnancy while the capacity of metabolism did not change. PG synthesis increased in lung and kidney (4-fold), brain (20-fold) and small intestine (2-fold) but not in heart or liver. Metabolic activity increased only in fetal kidney and lung.     

Localization of androgen and estrogen receptors in rat and primate tissues

There is now evidence that estrogens and androgens are exerting their effects in different tissues throughout the body. In order to determine the sites of action of these steroids, studies have been performed to identify at the cellular level the localization of androgen receptor (AR) and the two estrogen receptor (ER) subtypes, ERalpha and ERbeta, specially in the rat, monkey and human. In the prostate, AR was observed in the secretory and stromal cells. In the testis, Sertoli, Leydig and myoid cells were labelled. In the epididymis and seminal vesicles, both epithelial and stromal cells contained AR. In the ovary, AR was detected in granulosa and interstitial cells. In the uterus, epithelial, stromal and muscle cells were all immunopositive for AR. In the central nervous system, AR-containing neurons were found to be widely distributed throughout the brain. In the mammary gland, epithelial cells in acini and ducts and stromal cells were demonstrated to express AR. In the skin, AR was detected in keratinocytes, sebaceous and sweat glands, and hair follicles. In addition, AR was also found in anterior pituitary, thyroid, adrenal cortex, liver, kidney tubules, urinary bladder, cardiac and striated muscle, and bone. The ER subtypes are in general differentially expressed. While ERalpha has been predominantly found in anterior pituitary, uterus, vagina, testis, liver and kidney, ERbeta is predominant in thyroid, ovary, prostate, skin, bladder, lungs, gastro-intestinal tract, cartilage and bone. In tissues which contain both receptor subtypes, such as ovary, testis and various regions of the brain, a cell-specific localization for each ER subtype has been generally observed. Altogether, the recent results on the cellular localization of sex steroid receptors will certainly contribute to a better understanding of the specific role of these steroids in different target organs.     

Sulfated mucopolysaccharides of midgestation embryonic and extraembryonic tissues of the mouse

Incorporation of [³⁵S]sulfate into sulfated mucopolysaccharides has been characterized in midgestation mouse embryo, yolk sac, trophoblast, and decidua. Enzymatic analysis indicated that chondroitin sulfates contained approximately half of the label in embryo, trophoblast, and decidua, but less than 20% in yolk sac. While the labeled chondroitin sulfate fraction of trophoblast and decidua was mainly chondroitin-4-sulfate, only embryo contained a significant proportion of labeled chondroitin-6-sulfate. The relative incorporation into embryo chondroitin-6-sulfate was also substantially higher than that observed in four adult soft tissues. Labeled dermatan sulfate was absent from the embryo and yolk sac, but small amounts might have been synthesized by the placenta. Nitrous acid degradation studies revealed that essentially all the chondroitinase resistant MPS was N-sulfated, i.e., heparan sulfate and/or heparin. Electrophoretic profiles indicate that the bulk of the N-sulfated material resembles heparan sulfate rather than heparin. Electrophoretic heterogeneity and slow migration rates relative to standard markers suggest that the majority of labeled chondroitin sulfates may be undersulfated. The different mucopolysaccharide patterns in the various tissues may reflect their specialized properties and functions.     

Gap junctional communication in the extraembryonic tissues of the gastrulating mouse embryo

We characterized gap junctional communication in the extraembryonic tissues of the 7.5-d gastrulating mouse embryo. At this stage of development, the extraembryonic tissues form a large part of the conceptus, and link the embryo proper to the maternal tissue. Using Lucifer yellow injections, cells in most extraembryonic tissues were observed to be very well dye coupled, the only exception being the peripheral regions of the ectoplacental cone. Of particular interest was the fact that no dye coupling was detected between the three major extraembryonic tissues. Thus, the extraembryonic ectoderm (EEC), the extraembryonic endoderm (EEN), and the ectoplacental cone (EPC) corresponded to separate communication compartments, with the EPC being further subdivided into three compartments. Interestingly, the EEN was observed to exhibit a very low level of dye coupling with the adjacent visceral embryonic endoderm (EN), and consistent with the latter dye coupling results was the finding that the EEN was ionically coupled to the EN, but not with any other extraembryonic tissues. However, in the EPC, ionic coupling studies show that the central region was well coupled ionically to the EEC, but only weakly coupled to the peripheral EPC. These findings, in conjunction with our previous study (1988. J. Cell Biol. 107:241-255), demonstrate that the 7.5-d mouse conceptus is subdivided into at least nine major Lucifer yellow-delineated communication compartments, with ionic coupling across some of these compartments effectively unifying the embryo into two large domains corresponding to the embryo proper and the major extraembryonic tissues.     

Localization of catalytic and regulatory subunits of cyclic AMP-dependent protein kinases in mitochondria from various rat tissues.

Observation and quantification of the catalytic subunit C of cyclic AMP-dependent protein kinases by immuno-gold electron microscopy suggested a high concentration of cyclic AMP-dependent protein kinases in mitochondria from liver, kidney, heart and skeletal muscle, pancreas, parotid gland and brain cells. The position of gold particles pointed to a localization in the inner membrane/matrix space. A similar distribution was obtained by immunolocalization of the cyclic AMP-dependent protein kinase regulatory subunits RI and RII in liver, pancreas and heart cells. The results indicated the presence of both the type I and the type II cyclic AMP-dependent protein kinases in mitochondria of hepatocytes, and the preferential occurrence of the type I protein kinase in mitochondria from exocrine pancreas and heart muscle. The immunocytochemical results were confirmed by immunochemical determination of cyclic AMP-dependent protein kinase subunits in fractionated tissues. Determinations by e.l.i.s.a. of the C-subunit in parotid gland cell fractions indicated about a 4-fold higher concentration of C-subunit in the mitochondria than in a crude 1200 g supernatant. Immunoblot analysis of subfractions from liver mitochondria supported the localization in situ of cyclic AMP-dependent protein kinases in the inner membrane/matrix space and suggested that the type I enzyme is anchored by its regulatory subunit to the inner membrane. In accordance with the immunoblot data, the specific activity of cyclic AMP-dependent protein kinase measured in the matrix fraction was about twice that measured in whole mitochondria. These findings indicate the importance of cyclic AMP-dependent protein kinases in the regulation of mitochondrial functions.     

Human tumor cell lines express low levels of oncomodulin

Different human carcinoma cell lines were screened for the presence of Ca2(+)-binding oncomodulin. A specific polyclonal antibody was raised against a synthetic peptide (amino acids 99-108) of oncomodulin coupled to hemocyanin. Extracts of tumor cell lines (several human, one rat) were analyzed for the presence of oncomodulin by immunoblotting. A strong immunoreaction of oncomodulin was obtained in chemically transformed rat fibroblasts (T14c) in contrast to all human tumor cell lines investigated, where no immunoreaction was obtained. These results suggest that oncomodulin cannot be used in diagnostics of human tumors.     

Allelic expression of the putative tumor suppressor gene p73 in human fetal tissues and tumor specimens

p73, a proposed tumor suppressor, shares significant amino acid sequence homology with p53. However, p73 is rarely mutated in tumors but it has been suggested that p73 is monoallelically expressed in some tissues. This latter feature would predispose p73 to gene inactivation because a single genetic 'hit' or the loss of the expressed parental allele would leave the cell without p73 activity. We examined the allelic expression of p73 in normal fetal tissues and in ovarian cancer and Wilms' tumor. We found that p73 was biallelically expressed in all fetal tissues, except in brain, where differential expression of the two parental alleles was observed. Biallelic expression of p73 was also observed in paired samples of ovary cancer and Wilms' tumor. Loss of heterozygosity of p73 occurred at relatively low rates in tumors: one of 11 informative samples (9.1%) of ovarian cancer and two of 19 (10.1%) Wilms' tumors. These data demonstrate that p73 is biallelically expressed in most tissues, thus excluding genomic imprinting as a molecular mechanism to predispose to allelic inactivation of p73 in human tumors.