IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION : I. During Mouse Fetal Liver Development

Globin mRNA levels in 11–15-day mouse fetal liver cells have been estimated by in situ hybridization of a highly labeled DNA copy (cDNA) of adult globin messenger RNAs (mRNAs) (globin cDNA) to fixed preparations of cells. Under the conditions employed, no significant in situ hybridization occurred to lymphoma cells (L 51787), mouse L cells, or hepatocytes; whereas reticulocytes from phenyl hydrazine-treated mice showed extensive in situ hybridization. The proportion of fetal liver cells showing predominantly cytoplasmic in situ hybridization increased from about 30% at the 11th day of development to 80–85% by days 13–15. Unlike more mature cells, proerythroblasts did not show in situ hybridization, except to a slight extent at later stages of development. These studies therefore indicate that globin mRNAs begin to accumulate during or shortly after the proerythroblastbasophilic erythroblast transition. The fact that certain immature erythroid cells from 14-day fetal liver contain substantial amounts of globin mRNAs has been confirmed by comparing the hybridization in solution of globin cDNA to cytoplasmic RNA extracted from total fetal liver cells or from immature erythroid cells obtained by treatment of fetal liver cells with an antiserum raised against erythrocytes.     

Structure of recombinant plasmids containing synthetic human foetal globin gene sequences

Summary In vitro synthesized duplex DNA complementary to human foetal globin messenger RNA was integrated into bacterial plasmids and amplified by transformation of Escherichia coli. Recombinants carrying globin DNA were identified by hybridization of foetal globin messenger RNA to bacterial DNA in situ and by liquid hybridization of purified plasmids to specific globin complementary DNA probes. Heteroduplex mapping revealed either a simple insertion loop at the position of the EcoRI site of the parental plasmid or substitution loops due to insertion of globin DNA sequences combined with deletions of the parental plasmid DNA. We provide evidence to suggest that these deletions are the result of a site-specific nicking activity of the EcoRI preparations used in the formation of recombinant plasmids.     

Gestational high-fat diet impaired demethylation of Pparα and induced obesity of offspring

Gestational and postpartum high-fat diets (HFDs) have been implicated as causes of obesity in offspring in later life. The present study aimed to investigate the effects of gestational and/or postpartum HFD on obesity in offspring. We established a mouse model of HFD exposure that included gestation, lactation and post-weaning periods. We found that gestation was the most sensitive period, as the administration of a HFD impaired lipid metabolism, especially fatty acid oxidation in both foetal and adult mice, and caused obesity in offspring. Mechanistically, the DNA hypermethylation level of the nuclear receptor, peroxisome proliferator-activated receptor-α (Pparα), and the decreased mRNA levels of ten-eleven translocation 1 (Tet1) and/or ten-eleven translocation 2 (Tet2) were detected in the livers of foetal and adult offspring from mothers given a HFD during gestation, which was also associated with low Pparα expression in hepatic cells. We speculated that the hypermethylation of Pparα resulted from the decreased Tet1/2 expression in mothers given a HFD during gestation, thereby causing lipid metabolism disorders and obesity. In conclusion, this study demonstrates that a HFD during gestation exerts long-term effects on the health of offspring via the DNA demethylation of Pparα, thereby highlighting the importance of the gestational period in regulating epigenetic mechanisms involved in metabolism.     

Developmental pattern and molecular identification of globin chains in Xenopus laevis

Summary High-resolution electrophoresis of larval and adult hemoglobins of Xenopus laevis reveals stage-specific differences in the number and mobility of the globin chains. To establish the relationship between the globin chains and the previously described globin genes, the corresponding mRNAs were hybrid-selected from total erythroblast RNA by representative cDNA clones, and translated in vitro. Electrophoretic separation of the translation products allowed identification of a major and a minor -globin chain in the larval and adult stages. This also holds for the adult -chains, however in the larval stage a difference in abundance is only detectable in the -mRNAs, but not in the translation products, because they comigrate. The fact that major and minor globin chains can be assigned to genes, which are located in two clusters, suggests that the related genes are expressed coordinately, but at different levels. Analysis of the globin patterns during development reveals that transition from the larval to the adult globin chains coincides with metamorphosis. Moreover, there is evidence of two globin chains that are only expressed in early larval stages and hence might be related to additional larval -globin genes of as yet unknown genomic location.     

Inhibition of infection‐mediated preterm birth by administration of broad spectrum chemokine inhibitor in mice

Preterm birth (PTB) is the single most important cause of perinatal and infant mortality worldwide. Maternal infection can result in PTB. We investigated the ability of a Broad Spectrum Chemokine Inhibitor (BSCI) to prevent infection‐induced PTB in mice. PTB was initiated in pregnant mice by intraperitoneal injection of lipopolysaccharide (LPS; 50 μg). Half the mice received BSCI (10 mg/kg) 24 hrs prior to and immediately before LPS administration. The impact of LPS alone or LPS plus BSCI was assessed on (i) injection‐to‐delivery interval, foetal survival rate, placental and neonates' weight; (ii) amniotic fluid and maternal plasma cytokine levels (by Luminex assay); foetal and maternal tissue cytokine gene expression levels (by Real‐Time RT‐PCR); (iii) immune cells infiltration into the uterine tissue (by stereological immunohistochemistry). Pre‐treatment with BSCI (i) decreased LPS‐induced PTB (64% versus 100%, P < 0.05); (ii) significantly attenuated cytokine/chemokine expression in maternal tissues (plasma, liver, myometrium, decidua); (iii) significantly decreased neutrophil infiltration in the mouse myometrium. BSCI‐treated mice in which PTB was delayed till term had live foetuses with normal placental and foetal weight. BSCI represents a promising new class of therapeutics for PTB. In a mouse model of preterm labour, BCSI suppresses systemic inflammation in maternal tissues which resulted in the reduced incidence of LPS‐mediated PTB. These data provide support for efforts to target inflammatory responses as a means of preventing PTB.     

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.     

Cloning of a new mouse foetal beta-globin mRNA sequence.

A novel globin cDNA recombinant (pFG5) has been isolated from a 14-15 day Porton mouse foetal liver cDNA library. It codes for a beta-like globin mRNA expressed in foetal liver-derived erythroblasts and erythrocytes but not in adult reticulocytes nor in yolk sac derived nucleated erythrocytes. It is also found in Friend cells induced to differentiate by DMSO. The nucleotide sequence of pFG5 confirms that it does not code for the beta major or beta minor globin chains nor the embryonic epsilon Y2 globin chain; but it is identical to the published partial sequence of the epsilon Y3 globin gene over the region of overlap (78 nucleotides).     

ANALYSIS OF PROLIFERATION AND DIFFERENTIATION OF FOETAL GRANULOCYTE-MACROPHAGE PROGENITOR CELLS IN HAEMOPOIETIC TISSUE*

Analysis of in vitro colony formation in agar cultures of foetal haemopoietic tissues of eight mammalian species has shown that granulocyte-macrophage progenitor cells are present in foetal liver, yolk sac, marrow and spleen in numbers approaching the incidence in adult marrow. Such characteristics as buoyant density, growth rate and differentiation served to distinguish foetal from adult colony forming cells (CFCs). Cell cycle analysis performed by exposing haemopoietic cells to high doses of tritiated thymidine in vitro showed that foetal CFC proliferation in species of short gestation (rabbit, rat, mouse) approached or exceeded that observed in adult marrow. In contrast, in species of long gestation (human, monkey, calf, lamb, guinea-pig) a period of variable duration was observed when foetal liver CFCs entered a non-cycling G₀ or blocked G₁ phase. In these species foetal liver CFCs were found to be proliferating actively early in gestation and following the non-cycling phase again re-entered a proliferative state associated with onset of active granulopoiesis in foetal marrow and possible migration of CFC from liver to marrow. These results indicate the existence of granulocyte-macrophage progenitor populations displaying foetal characteristics and adapted to particular stages of haemopoietic development, a situation which closely parallels that reported for erythropoiesis.     

Maternal–foetal genomic conflict and speciation: no evidence for hybrid placental dysplasia in crosses between two house mouse subspecies

Interspecific hybridization between closely related mammalian species, including various species of the genus Mus, is commonly associated with abnormal growth of the placenta and hybrid foetuses, a phenomenon known as hybrid placental dysplasia (HPD). The role of HPD in speciation is anticipated but still poorly understood. Here, we studied placental and foetal growth in F₁ crosses between four inbred mouse strains derived from two house mouse subspecies, Mus musculus musculus and Mus musculus domesticus. These subspecies are in the early stage of speciation and still hybridize in nature. In accordance with the maternal–foetal genomic conflict hypothesis, we found different parental influences on placental and foetal development, with placental weight most affected by the father's body weight and foetal weight by the mother's body weight. After removing the effects of parents’ body weight, we did not find any significant differences in foetal or placental weights between intra‐subspecific and inter‐subspecific F₁ crosses. Nevertheless, we found that the variability in placental weight in inter‐subspecific crosses is linked to the X chromosome, similarly as for HPD in interspecific mouse crosses. Our results suggest that maternal–foetal genomic conflict occurs in the house mouse system, but has not yet diverged sufficiently to cause abnormalities in placental and foetal growth in inter‐subspecific crosses. HPD is thus unlikely to contribute to speciation in the house mouse system. However, we cannot rule out that it might have contributed to other speciation events in the genus Mus, where differences in the levels of polyandry exist between the species.     

The expression of viral and globin genes during differentiation of the friend cell

A complementary DNA probe has been prepared from the Friend murine erythroleukaemia virus complex (FV) released from Friend cells treated with dimethylsulphoxide (DMSO). The complementary DNA (cDNA) forms a hybrid specifically with the viral RNA genome. The availability of this viral probe together with mouse globin cDNA has made it possible to study the expression of both viral and globin genes in the Friend cell during differentiation using molecular hybridisation techniques. These specific probes have been used in an attempt to determine whether any connection exists between expression of Friend virus sequences and erythroid differentiation as measured by globin gene expression. A titration technique has been used to quantitate the levels of Friend viral- and globin-specific sequences in various Friend cell lines which differ in their ability to release Friend virus in response to DMSO although all produce haemoglobin under the same conditions. The results show: (a) that Friend cell lines unable to release virus nevertheless have a large pool of entire virus specific sequences in the polysomes; (b) an increase in virus release induced with DMSO is normally associated with a modest increase in viral sequence in the polysomes; (c) most cell lines show an early accumulation of viral and a later increase in globin mRNA sequences; (d) in an exceptional virus-negative, BUdR-resistant cell clone (B8/3), the accumulation of globin mRNA takes place very rapidly but there is no concomitant increase in viral RNA during differentiation.     

alpha-Fetoprotein biosynthesis and hepatocellular differentiation

In anemia of the Belgrade rat ($\text{b}\text{b}$) reticulocytes contain less than half of the normal amount of mRNA for seven adult rat globin chains. cDNA hybridization measurements of the relative sizes of polysomal and nonpolysomal pools of globin mRNAs in these cells show that 45% of all globin mRNA molecules are not used at any given time in protein synthesis. This implies a translational control which ensures a production of globin chains in a correct ratio despite a severe mRNA unbalance.     

mRNA surveillance mitigates genetic dominance in Caenorhabditis elegans

Nonsense mutant mRNAs are unstable in all eucaryotes tested, a phenomenon termed nonsense-mediated mRNA decay (NMD) or mRNA surveillance. Functions of the seven smg genes are required for mRNA surveillance in Caenorhabditis elegans. In Smg(+) genetic backgrounds, nonsense-mutant mRNAs are unstable, while in Smg(−) backgrounds such mRNAs are stable. Previous work has demonstrated that the elevated level of nonsense-mutant mRNAs in Smg(−) animals can influence the phenotypic effects of heterozygous nonsense mutations. Certain nonsense alleles of a muscle myosin heavy chain gene are recessive in Smg(+) backgrounds but strongly dominant in Smg(−) backgrounds. Such alleles probably express disruptive myosin polypeptide fragments whose abundance is elevated in smg mutants due to elevation of mRNA levels. We report here that mutations in a variety of C. elegans genes are strongly dominant in Smg(−), but recessive or only weakly dominant in Smg(+) backgrounds. We isolated 32 dominant visible mutations in a Smg(−) genetic background and tested whether their dominance requires a functional NMD system. The dominance of 21 of these mutations is influenced by NMD. We demonstrate, furthermore, that in the case of myosin, the dominant-negative effects of nonsense alleles are likely to be due to expression of N-terminal nonsense-fragment polypeptides, not to mistranslation of the nonsense codons. mRNA surveillance, therefore, may mitigate potentially deleterious effects of many heterozygous germline and somatic nonsense or frameshift mutations. We also provide evidence that smg-6, a gene previously identified as being required for NMD, performs essential function(s) in addition to its role in NMD. Received: 10 June 1998 / Accepted: 21 July 1998     

Albumin and apolipoprotein H mRNAs in human plasma as potential clinical biomarkers of liver injury: analyses of plasma liver-specific mRNAs in patients with liver injury

Context: Plasma liver-specific mRNAs are useful biomarkers of hepatotoxicity in rats.

Objective: To investigate the potential application of liver-specific mRNAs as biomarkers for liver injury in humans.

Methods: We determined the plasma levels of liver-specific mRNAs by real-time qRT-PCR in healthy donors and patients with liver injury.

Results: Plasma levels of albumin (ALB) and apolipoprotein H (APOH) mRNAs increased in patients with elevated serum alanine aminotransferase. These mRNAs also increased in plasma after transcatheter arterial chemoembolization, which induces specific injury to liver.

Conclusions: We demonstrated the potential application of plasma ALB and APOH mRNAs as clinical biomarkers for liver injury.     

Erythropoiesis and unexpected expression pattern of globin genes in the salamander Hynobius retardatus

Transition of hemoglobin (Hb) from larval to adult types during the metamorphosis in a salamander Hynobius retardatus has been reported to occur almost independently of thyroid activity, in contrast to the case with many amphibians. In order to obtain further information on the mechanism of the transition in H. retardatus, larval and adult globin cDNAs were cloned, and the globin gene expression was analyzed in normally developing and metamorphosis-arrested animals. Northern hybridization and RT-PCR revealed that larval globin genes were initially expressed 5 days before hatching, and unexpectedly remained expressed even in juveniles 2 years old. The adult globin gene was expressed 19 days after hatching, much earlier than the initiation of morphological metamorphosis. Furthermore, the pattern of globin gene expression in metamorphosis-arrested larvae was almost identical to that in normal controls, suggesting that the transition occurs independently of thyroid hormones. In larvae recovering from anemia, precocious Hb transition, which occurs in Xenopus laevis and Rana catesbeiana, did not occur in H. retardatus. In situ hybridization convincingly demonstrated that the erythropoietic sites are the ventral blood island and the dorsolateral plate at the prehatching stage. During the ontogeny they changed to the liver, kidney, and spleen and were finally restricted to the spleen. Single erythroid cells expressed concurrently larval and adult globin genes, as demonstrated by double in situ hybridization. Thus the transition occurred within a single erythroid cell population, a unique characteristic of H. retardatus. Received: 5 August 1999 / Accepted: 14 October 1999     

Changes in globin messenger RNA content during erythroid cell differentiation.

Previous studies have shown that mouse fetal erythroid precursor cells isolated by an immunological technique synthesize little or no globin and contain little, if any, globin mRNA, as assayed in a cell-free system (translatable mRNA). After culture for 10 hours in the presence of erythropoietin, there is a marked increase in globin synthesis and in translatable globin mRNA. The present studies were designed to measure directly the content of globin mRNA sequences during erythroid cell differentiation, by molecular hybridization with 3H-labeled DNA complementary to globin mRNA. The results indicate that few, if any, globin mRNA sequences are present in the total RNA of erythroid precursor cells. There is little or no pool of untranslated globin mRNA in these cells. After 10 hours of culture with erythropoietin, there is an increase in globin mRNA content, as ;easured by a change in the Cot1/2 values obtained by cDNA: mRNA hybridization with (Co) representing the concentration of RNA. Between 0 and 22 hours of culture, there is a 250-fold rise, and between 22 and 44 hours, a further 2-fold increase in globin mRNA content. During the 44 hours in culture, the number of cells in culture increases 2- to 3-fold. The number of globin mRNA molecules rises in erythroid precursor cells to an average value of 1800 molecules/cell during 22 hours of culture. In cultures without added erythropoietin, the absolute number of cells decreases, however, cells presumably induced to differentiate by exposure to erythropoietin in vivo continue to differentiate in vitro, accumulating globin mRNA and initiating globin synthesis.