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.     

Morphofunctional transformations of the yolk syncytial layer during zebrafish development

The yolk syncytial layer (YSL) is a provisory extraembryonic structure of teleost fishes and representatives of some other taxa with meroblastic cleavage. The YSL of teleosts is a symplast with polymorphous polyploid nuclei. It is known to perform nutritional, morphogenetic, immune, and, probably, other functions. Data about the YSL organization, functioning and regulation is fragmentary. Although gene expression patterns and other aspects of YSL functioning have been studied in Danio rerio, the morphology of its YSL has not been described in detail. The study of zebrafish YSL structure on sequential developmental stages is necessary to recognize specific features of this important polyfunctional system in this model organism and to extend our knowledge about provisory systems. The thickness of the YSL and the distribution of its nuclei are not uniform on each stage and change during development. During oblong and sphere stages the internal YSL (I‐YSL) is filled with yolk inclusions; interphase yolk syncytial nuclei (YSN) and mitotic asters can be seen. During doming and epiboly the external YSL (E‐YSL) is thicker than I‐YSL. On the subsequent stages the YSL is thickened caudally. The dorsal YSL part is thickened during early segmentation stages and becomes the thinnest YSL region later. The anterior part of the YSL is thin, but enlarges during larval period. The YSN of different size and diverse forms, from regular to lobed, are present and form clusters. The number of irregular‐shaped nuclei increases during development. The YSL thickens in the end of endotrophic and in the course of endo‐exotrophic period, and its cytoplasm contains numerous yolk inclusions. After yolk exhaustion the YSL is flat. As the YSL degrades , the YSN become pycnotic, and the YSL remnant probably is cleared by phagocytes. J. Morphol. 275:206–216, 2014. © 2013 Wiley Periodicals, Inc.     

Differential gene expression during early murine yolk sac development

The visceral yolk sac (VYS), composed of extraembryonic mesoderm and visceral endoderm, is the initial site of blood cell development and serves important nutritive and absorptive functions. In the mouse, the visceral endoderm becomes a morphologically distinct tissue at the time of implantation (E4.5), while the extraembryonic mesoderm arises during gastrulation (E6.5–8.5). To isolate genes differentially expressed in the developing yolk sac, polymerase chain reaction (PCR) methods were used to construct cDNA from late primitive streak to neural plate stage (E7.5) murine VYS mesoderm and VYS endoderm tissues. Differential screening led to the identification of six VYS mesoderm-enriched clones: ribosomal protein L13a, the heat shock proteins hsc 70 and hsp 86, guanine-nucleotide binding protein-related gene, cellular nucleic acid binding protein, and ã-enolase. One VYS endoderm-specific cDNA was identified as apolipoprotein C2. In situ hybridization studies confirmed the differential expression of these genes in E7.5 yolk sac tissues. These results indicate that representative cDNA populations can be obtained from small numbers of cells and that PCR methodologies permit the study of gene expression during early mammalian postimplantation development. While all of the mesoderm-enriched genes were ubiquitously expressed in the embryo proper, apolipoprotein C2 expression was confined to the visceral endoderm. These results are consistent with the hypothesis that at E7.5, the yolk sac endoderm provides differentiated liver-like functions, while the newly developing extraembryonic mesoderm is still a largely undifferentiated tissue. © 1995 wiley-Liss, Inc.     

Apolipoprotein C-II and lipoprotein lipase show a temporal and geographic correlation with surfactant lipid synthesis in preparation for birth

Background

Fatty acids are precursors in the synthesis of surfactant phospholipids. Recently, we showed expression of apolipoprotein C-II (apoC-II), the essential cofactor of lipoprotein lipase (LPL), in the fetal mouse lung and found the protein on the day of the surge of surfactant synthesis (gestation day 17.5) in secretory granule-like structures in the distal epithelium. In the present study, we will answer the following questions: Does apoC-II protein localization change according to the stage of lung development, thus according to the need in surfactant? Are LPL molecules translocated to the luminal surface of capillaries? Do the sites of apoC-II and LPL gene expression change according to the stage of lung development and to protein localization?

Results

The present study investigated whether the sites of apoC-II and LPL mRNA and protein accumulation are regulated in the mouse lung between gestation day 15 and postnatal day 10. The major sites of apoC-II and LPL gene expression changed over time and were found mainly in the distal epithelium at the end of gestation but not after birth. Accumulation of apoC-II in secretory granule-like structures was not systematically observed, but was found in the distal epithelium only at the end of gestation and soon after birth, mainly in epithelia with no or small lumina. A noticeable increase in surfactant lipid content was measured before the end of gestation day 18, which correlates temporally with the presence of apoC-II in secretory granules in distal epithelium with no or small lumina but not with large lumina. LPL was detected in capillaries at all the developmental times studied.

Conclusions

This study demonstrates that apoC-II and LPL mRNAs correlate temporally and geographically with surfactant lipid synthesis in preparation for birth and suggests that fatty acid recruitment from the circulation by apoC-II-activated LPL is regionally modulated by apoC-II secretion. We propose a model where apoC-II is retained in secretory granules in distal epithelial cells until the lumina reaches a minimum size, and is then secreted when the rate of surfactant production becomes optimal.

     

Apolipoprotein E polymorphism in the Finnish population: gene frequencies and relation to lipoprotein concentrations

ApoE phenotypes were determined in 615 unrelated Finnish individuals. The apoE gene frequencies observed (epsilon 2, 0.041; epsilon 3, 0.733; epsilon 4, 0.227) differ significantly from those in other populations. The frequency of the allele epsilon 2 was lower and that of epsilon 4 higher than in all other studied populations. Plasma lipids and apolipoproteins A-I, A-II and B were recorded in 207 of the typed subjects. By comparison with the most frequent homozygous apoE 3/3 phenotype, it was found that total cholesterol, LDL-cholesterol, and apoB concentrations were all markedly higher in apoE 4/4 and to a lesser degree in apoE 4/3 phenotypic groups. On the other hand, these lipid and apolipoprotein levels tended to be lower in E-2 heterozygotes. These data confirm and extend, in a different ethnic group, previous results of an effect of apoE genes on plasma lipoprotein concentrations. The data suggest that the apoE gene locus may be one factor responsible for the high LDL cholesterol concentrations in the Finnish population.     

Structure of the yolk syncytial layer in Teleostei and analogous structures in animals of the meroblastic type of development

The structure of the yolk syncytial layer (YSL) of the larvae of two cyprinids, Danio rerio and Cyprinus carpio koi, has been studied by transmission electron microscopy and by the histological methods. The structure of the YSL of these taxonomically related species of Teleostei is characterized by both similarity and dissimilarity in particular features, related to the overall shape of YSL, functional regionalization, and programmed death. Original and published data on the morphofunctional structure of the YSL have been discussed for representatives of Teleostei, Myxini, Chondrichthyes, Lepisosteiformes and Cephalopoda.     

Localized <Emphasis Type="Italic">rbp4</Emphasis>expression in the yolk syncytial layer plays a role in yolk cell extension and early liver development

Background  

The number of genes characterized in liver development is steadily increasing, but the origin of liver precursor cells and the molecular control of liver formation remain poorly understood. Existing theories about formation of zebrafish visceral organs emphasize either their budding from the endodermal rod or formation of independent anlage followed by their later fusion, but none of these is completely satisfactory in explaining liver organogenesis in zebrafish.     

Apolipoprotein E polymorphism and its association with serum lipid levels in Brazilian children

The influence of apolipoprotein E (APOE) genotypes on plasma lipid levels was determined in 414 Brazilian healthy children of mixed ethnicity, age 5 to 15 years (mean 8.9+/-2.9). The APOE*3 allele was the most frequent (77%), followed by APOE*4 (17%) and APOE*2 (6%). The pattern of lipid differences among genotypes was similar in both boys and girls. We did not detect an increase in cholesterol levels with the presence of the APOE*4 allele. Because a growing body of evidence suggests that the effect of *4 depends on its interaction with diet, the frequency of *4 might be more variable in children than in adults as well as among populations. Children carrying a *2 allele had lower total cholesterol (TC) and LDL-cholesterol levels (138.47+/-24.32 and 77.72+/-19.42, respectively) compared with *3/*3 children (158.33+/-25.28 and 97.05+/-21.82, respectively). Mean TC/HDLC ratio was also lower in children with the APOE*2 allele (3.27+/-0.66 versus 3.64+/-0.76). In this highly admixed population sample, the *2 anti-atherogenic lipid pattern is already detected in children.     

Apolipoprotein E gene polymorphism and serum lipid levels in the Guangxi Hei Yi Zhuang and Han populations

Hei Yi Zhuang is an isolated subgroup of the Zhuang minority in China. Little is known about the distribution of apolipoprotein (apo) E genetic variations and its role in lipid metabolism in this population. The present study was undertaken to compare the effect of apoE gene polymorphism on serum lipid levels between the Guangxi Hei Yi Zhuang and Han populations. A total of 873 subjects of Hei Yi Zhuang and 867 participants of Han Chinese were surveyed by a stratified randomized cluster sampling. Genotyping of apoE was performed using polymerase chain reaction and restriction fragment length polymorphism. The frequencies of 2, 3, and 4 alleles were 15.23%, 79.84%, and 4.93% in Hei Yi Zhuang, and 9.23%, 81.43%, and 9.34% in Han (P < 0.001); respectively. The frequencies of 2/ 2, 2/ 3, 2/ 4, 3/ 3, 3/ 4, and 4/ 4 genotypes were 4.70%, 17.86%, 3.21%, 68.16%, 5.50%, and 0.57% in Hei Yi Zhuang, and 2.54%, 9.23%, 4.15%, 70.70%, 12.23%, and 1.15% in Han (P < 0.001); respectively. Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and apoB levels were lower in Hei Yi Zhuang than in Han (P < 0.01-0.001), but high-density lipoprotein cholesterol (HDL-C) levels and the ratio of apoA-I to apoB were higher in Hei Yi Zhuang than in Han (P < 0.001 for each). There were significant differences in TC, HDL-C, LDL-C, and apoB levels among the six genotypes in both ethnic groups (P < 0.01-0.001). Hyperlipidemia was positively correlated with age, body mass index, hypertension, alcohol consumption, and apoE allele in both populations (P < 0.05-0.001). TC, LDL-C, and apoB levels were positively correlated, and HDL-C levels were negatively associated with apoE genotypes in both ethnic groups (P < 0.001 for all). The differences in the lipid profiles between Hei Yi Zhuang and Han Chinese might partly attribute to the differences in apoE genotypic and allelic frequencies.     

Intra- and extracellular lipid composition and associated gene expression patterns during pollen development in Brassica napus

Pollen development in angiosperms is regulated by the interaction of products contributed by both the gametophytic (haploid) and sporophytic (diploid) genomes. In entomophilous species, lipids are major products of both sporophytic and gametophytic metabolism during pollen development. Mature pollen grains of Brassica napus are shown to contain three major acyl lipid pools as follows: (i) the extracellular tryphine mainly consisting of medium-chain neutral esters; (ii) the intracellular membranes, particularly endoplasmic reticulum, mainly containing phospholipids; and (iii) the intracellular storage lipids, which are mostly triacylglycerols. This paper reports on the kinetics of accumulation of these lipid classes during pollen maturation and the expression patterns of several lipid biosynthetic genes and their protein products that are differentially regulated in developing microspores/ pollen grains (gametophyte) and tapetal cells (sporophyte) of B. napus. Detailed analysis of three members of the stearoyl-ACP desaturase (sad) gene family by Northern blotting, in situ hybridization and RT-PCR showed that the same individual genes were expressed both in gametophytic and sporophytic tissues, although under different temporal regulation. In the tapetum, maximal expression of two marker genes for lipid biosynthesis (sad and ear) occurred at a bud length of 2–3 mm, and the corresponding gene products SAD and EAR were detected by Western blotting in 3–4 mm buds, coinciding with the maximal rates of tapetal lipid accumulation. These lipids are released following tapetal cell disintegration and are relocated to form the major structural component of the extracellular tryphine layer that coats the mature pollen grain. In contrast, in developing microspores/pollen grains, maximal expression of the lipid marker genes sad, ear, acp and cyb5 was at the 3–5 mm bud stages, with the SAD and EAR gene products detected in 4–7 mm buds. This pattern of expression coincided with accumulation of the intracellular storage and membrane lipid components of pollen. These results suggest that, although the same genes may be expressed in the sporophytic tapetal cells and in gametophytic tissues, they are regulated differentially leading to the production of the various contrasting lipidic structures that are assembled together to give rise to a viable, fertile pollen grain.     

Expression of the Prader-Willi gene Necdin during mouse nervous system development correlates with neuronal differentiation and p75NTR expression

The expression pattern of Necdin, a gene involved in the etiology of Prader-Willi syndrome and a member of the MAGE family of genes, is described during mouse nervous system development. Using RNA in situ hybridization, immunohistochemical staining, and colocalization with neuronal differentiation markers, we found that Necdin RNA and protein are expressed within post-mitotic neurons at all stages studied. From E10 to E12, Necdin is detected in all developing neurons, in both central and peripheral nervous system, with the highest expression levels in the diencephalon and the hindbrain. After E13, Necdin is expressed in specific structures of the nervous system, in particular the hypothalamus, the thalamus, and the pons, suggesting a specific developmental role therein. In addition, Necdin expression is also detected in non-neural tissues, such as the somites, the developing limb buds, the first branchial arches, the tong, and the axial muscles. Recently, Necdin and other MAGE proteins were found to interact in vitro with the intracellular domain of the p75NTR neurotrophin receptor, but this interaction has not been validated in vivo. We report here that the spatial and temporal expression of p75NTR is included in Necdin expression domain. These results are in agreement with Necdin proposed role on cell cycle arrest, inhibition of apoptosis and facilitation of neuronal differentiation in vitro, and with hypothalamic cellular deficiencies reported in mice with abrogation of the Necdin gene. Furthermore, they are also consistent with the putative role of Necdin in signaling events promoted by p75NTR during mouse nervous system development.     

Heat production and lipid metabolism in broiler and layer chickens during embryonic development

We compared heat production (HP) and lipid metabolism in broiler and layer chickens (Gallus gallus) during embryonic development. To investigate HP and respiratory quotient (RQ), oxygen (O2) consumption and carbon dioxide (CO2) production were measured using an open-circuit calorimeter system. HP consistently had a tendency (P = 0.06) to be lower in broilers than in layers during embryonic development, and HP gradually decreased with developmental stage in both strains. RQ values of both strains were approximately 0.7 at every embryonic stage investigated. These results suggest that chicken embryos mainly use lipid for energy, and the RQ was significantly lower in broilers than in layers during embryonic development. Consumption of the yolk sac as a lipid source was faster in broilers than in layers. Plasma D-3-hydroxybutyrate (D3HB) and glycerol concentrations, associated with fatty acid oxidation, were lower in broiler than layer embryos. These results demonstrate that HP and lipid metabolism are different between the strains during embryonic development, and may be one factor for the growth difference between broiler and layer embryos.     

Metabolic fates of yolk lipid and individual fatty acids during embryonic development of the coot and moorhen

There is currently little information regarding the metabolic fates of yolk lipid and individual fatty acids during embryonic development of free-living avian species. Here we report the pattern of lipid utilization during embryonic development of the coot (Fulica atra) and the moorhen (Gallinula chloropus), two related species producing precocial offspring from eggs with a distinctive fatty acid composition and with an incubation period similar to that of the chicken. By the time of hatching, the proportions of the initial yolk lipid that had been transferred to the embryo were 88.2% and 79.8% for the coot and moorhen respectively. During the whole incubation period, 42.9% and 40.0% of the initial yolk lipid of the coot and moorhen respectively were lost from the system due to oxidation for energy, equating to 47.8% and 50.0% respectively of the actual amount of lipid transferred over this time. Thus, the lipid received by the embryos of both species is partitioned almost equally between the alternative fates of energy metabolism and incorporation into tissue lipids. In the coot, this 50:50 split between oxidation and tissue formation was maintained during the hatching process. The proportions of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) in the yolk lipids of these species were 2.5-3.5 times higher than in eggs of domestic poultry. In contrast to the situation in the chicken, there was no preferential uptake of 22:6n-3 from the yolk during coot and moorhen development. The fatty acid compositions of the whole body lipids of the coot and moorhen hatchlings were almost identical to those of the initial yolks indicating that, unlike the chicken, these species display relatively little overall biomagnification of 20:4n-6 and 22:6n-6 during development. It is suggested that the yolk fatty acid profiles of the coot and moorhen are particularly well matched to the requirements of the embryo, reducing the need for selective uptake of 22:6n-3 and for the overall biomagnification of 22:6n-3 and 20:4n-6.     

HLA class I expression in metastatic melanoma correlates with tumor development during autologous vaccination

Our knowledge of the mechanisms underlying tumor-specific immune response and tumor escape has considerably increased. HLA class I antigen defects remain an important tumor escape mechanism since they influence the interactions between tumor cells and specific T and NK cells in the course of malignant disease. We have studied here HLA class I expression in six subcutaneous metastases obtained from a melanoma patient immunized with an autologous melanoma cell vaccine (M-VAX). We report in this paper that HLA class I antigen expression on these metastatic lesions strongly correlated with the course of the disease. The three metastases that were partially regressing at the time of their excision showed a strong HLA class I expression, whereas the progressing ones showed a very weak or negative staining with most of the anti-HLA class I mAbs used. Real-time quantitative PCR of the samples obtained from microdissected tumor tissue revealed a significant difference in the mRNA levels of HLA-ABC heavy chain and beta2m between the two types of metastases, i.e., lower levels in progressing metastases and high levels in regressing ones, confirming the immunohistological findings. This is, to our knowledge, the first report where the clinical outcome of different HLA class I positive and negative melanoma metastases can be clearly correlated with the regression and progression of the disease, respectively.