Extracellular domain of CD98hc is required for early murine development

Background

The multifunctional protein CD98 heavy chain (CD98hc, Slc3a2) associates with integrin β1 through its cytoplasmic and transmembrane domains and the CD98hc-mediated integrin signaling is required for maintenance of ES cell proliferation. CD98hc-null mice exhibit early post-implantation lethality similar to integrin β1-null mice, supporting the importance of its interaction with integrin β1. On the other hand, the extracellular domain of CD98hc interacts with L-type amino acid transporters (LATs) and is essential for appropriate cell surface distribution of LATs. LATs mediate the transport of amino acids and other molecules such as thyroid hormone. In this respect, CD98hc may also affect development via these transporters.

Results

In this study, mice were generated from embryonic stem (ES) cell line (PST080) harboring a mutant CD98hc allele (CD98hc^Δ/+). Expression of the CD98hc mutant allele results in ΔCD98hc-β geo fusion protein where extracellular C-terminal 102 amino acids of CD98hc are replaced with β geo. Analyses of PST080 ES cells as well as reconstituted frog oocytes demonstrated that ΔCD98hc-β geo fusion protein preserved its ability to interact with integrin β1 although this mutant protein was hardly localized on the cell surface. These findings suggest that ΔCD98hc-β geo protein can mediate integrin signaling but cannot support amino acid transport through LATs. CD98hc^Δ/+ mice were normal. Although some of the implantation sites lacked embryonic component at E9.5, all the implantation sites contained embryonic component at E7.5. Thus, CD98hc^Δ/Δ embryos are likely to die between E7.5 and E9.5.

Conclusions

Considering that CD98hc complete knockout (CD98hc^-/-) embryos are reported to die shortly after implantation, our findings suggest potential stage-specific roles of CD98hc in murine embryonic development. CD98hc may be essential for early post-implantation development by regulating integrin-dependent signaling, while the other function of CD98hc as a component of amino acid transporters may be required for embryonic development at later stages.     

Inflammasome components caspase-1 and adaptor protein apoptosis-associated speck-like proteins are important in resistance to Cryptosporidium parvum

Cryptosporidium spp. are opportunistic protozoan parasites that infect epithelial cells in the intestinal tract and cause a flu-like diarrheal illness. Innate immunity is key to limiting the expansion of parasitic stages early in infection. One mechanism in which it does this is through the generation of early cytokines, such as IL-18. The processing and secretion of mature IL-18 (and IL-1β) is mediated by caspase-1 which is activated within an inflammasome following the engagement of inflammasome-initiating sensors. We examined how the absence of caspase-1 and caspase-11, the adapter protein Asc, and other inflammasome components affects susceptibility to cryptosporidial infection by these and other key cytokines in the gut. We found that Casp-11^?/?Casp-1^?/? knockout mice have increased susceptibility to Cryptosporidium parvum infection as demonstrated by the 35-fold higher oocyst production (at peak infection) compared to wild-type mice. Susceptibility correlated with a lack of IL-18 in caspase-1 and caspase1/11 knockout mice, whereas IL-18 is significantly elevated in wildtype mice. IL-1β was not generated in any significant amount following infection nor was any increased susceptibility observed in IL-1β knockout mice. We also show that the adapter protein Asc is important to susceptibility, and that the caspase-1 canonical inflammasome signaling pathway is the dominant pathway in C. parvum resistance.     

Polyadenylation of RNA in immature oocytes and early cleavage of Urechis caupo

The poly(A) content of RNA extracted from four stages of immature oocytes, mature oocytes, and cleavage embryos through the eight-cell stage was determined by hybridization with [³H]-poly(U). During oogenesis the poly(A) content per cell gradually increases from 0.007 pg of poly(A)/cell in the 10- to 39-μm oocytes to 0.20 pg of poly(A)/cell in the 125-μm mature oocytes. After fertilization there is an additional increase to approximately 1.1 pg of poly(A)/embryo at the two-cell stage which is followed by a slight decline between the two- and eight-cell stages. Most of the increase in poly(A) after fertilization occurs in a 45-min interval coincident with the appearance of the polar bodies. The size distribution of the poly(A) in RNA from the different stages of development was determined based on the length of RNase-resistant poly(U) obtained from poly(U)-poly(A) hybrids. The size distribution of the poly(A) sequences is constant through each stage of development which indicates that the increase in the poly(A) content of the cells is the result of polyadenylation of new RNA sequences.     

Differential responses of bovine oocytes and preimplantation embryos to heat shock

The authors sought to determine whether developmental differences in the magnitude of embryonic mortality caused by heat stress in vivo are caused by changes in resistance of embryos to elevated temperature. In this regard, responses of oocytes, two-cell embryos, four- to eight-cell embryos, and compacted morulae to heat shock were compared. An additional goal was to define further the role of cumulus cells and glutathione in thermoprotection of oocytes. In experiment 1, heat shock (41°C for 12 hr) decreased the number of embryos developing to the blastocyst stage for two-cell (26% vs. 0%) and four- to eight-cell (25% vs. 10%) embryos but did not affect morulae (37% vs. 42%). In experiment 2, exposure of two-cell embryos to 41°C for 12 hr reduced the number of four- to eight-cell embryos present 24 hr after the end of heat shock (88% vs. 62%). In experiment 3, heat shock reduced the number of two-cell embryos developing to blastocyst (49% vs. 8%) but did not affect subsequent development of oocytes when heat shock occurred during the first 12 hr of maturation (46% vs. 41% development to blastocyst); membrane integrity was not altered. In experiment 4, oocytes were cultured with an inhibitor of glutathione synthesis, _DL-buthionine-[S,R]-sulfoximine (BSO), for 24 hr and exposed to 41°C for the first 12 hr of maturation. Percentages of blastocysts were 35% (39°C), 18% (41°C), 17% (39°C+BSO), and 11% (41°C+BSO). For experiment 5, oocytes were either denuded or left with cumulus intact and were then radiolabeled with [³⁵S]methionine and [³⁵S]cysteine at 39°C or 41°C for 12 hr. Exposure of oocytes to 41°C for 12 hr reduced overall synthesis of ³⁵S-labeled TCA-precipitable intracellular proteins (18,160 vs. 14,594 dpm/oocyte), whereas presence of cumulus increased synthesis (9,509 vs. 23,246). Analysis by two-dimensional SDS PAGE and fluorography revealed that heat shock protein 68 (HSP68) and two other putative heat shock proteins, P71 and P70, were synthesized by all oocytes regardless of treatment. Heat shock did not alter the synthesis of HSP68 or P71 but decreased amounts of newly synthesized P70. Cumulus cells increased synthesis of P71 and P70. Results indicate there is a biphasic change in resistance to elevations in temperature as oocytes mature, become fertilized, and develop. Resistance declines from the oocyte to the two-cell stage and then increases. Evidence suggests a role for cumulus cells in increasing HSP70 molecules and protein synthesis. Data also indicate a role for glutathione in oocyte function. Mol Reprod Dev 46:138–145, 1997. © 1997 Wiley-Liss, Inc.     

DNA polymerase β decrement triggers death of olfactory bulb cells and impairs olfaction in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) involves the progressive degeneration of neurons critical for learning and memory. In addition, patients with AD typically exhibit impaired olfaction associated with neuronal degeneration in the olfactory bulb (OB). Because DNA base excision repair (BER) is reduced in brain cells during normal aging and AD, we determined whether inefficient BER due to reduced DNA polymerase‐β (Polβ) levels renders OB neurons vulnerable to degeneration in the 3xTgAD mouse model of AD. We interrogated OB histopathology and olfactory function in wild‐type and 3xTgAD mice with normal or reduced Polβ levels. Compared to wild‐type control mice, Polβ heterozygous (Polβ^+/?), and 3xTgAD mice, 3xTgAD/Polβ^+/? mice exhibited impaired performance in a buried food test of olfaction. Polβ deficiency did not affect the proliferation of OB neural progenitor cells in the subventricular zone. However, numbers of newly generated neurons were reduced by approximately 25% in Polβ^+/? and 3xTgAD mice, and by over 60% in the 3xTgAD/Polβ^+/? mice compared to wild‐type control mice. Analyses of DNA damage and apoptosis revealed significantly greater degeneration of OB neurons in 3xTgAD/Polβ^+/? mice compared to 3xTgAD mice. Levels of amyloid β‐peptide (Aβ) accumulation in the OB were similar in 3xTgAD and 3xTgAD/Polβ^+/? mice, and cultured Polβ‐deficient neurons exhibited increased vulnerability to Aβ‐induced death. Olfactory deficit is an early sign in human AD, but the mechanism is not yet understood. Our findings in a new AD mouse model demonstrate that diminution of BER can endanger OB neurons, and suggest a mechanism underlying early olfactory impairment in AD.     

Time-lapse cinematography study of the germinal vesicle behaviour in mouse primary oocytes treated with activators of protein kinases A and C

Previous studies have shown that early embryos contain information that can alter the developmental fate of adjacent cells and transferred nuclei. In this report we show that a specific combination of cells from early murine embryos, a single blastomere from an eight-cell embryo placed under the zona pellucida with a two-cell embryo, results in a difference in incorporation of ³H-uridine and expression of two protein bands between the chimeric treatment group and the nonchimeric controls, a single blastomere from an eight-cell embryo in a separate zona pellucida and a two-cell embryo. The incorporation of ³H-uridine in the chimeric group and nonchimeric control group was significantly different at 45 hours after chimerization (P < .02). A stage-specific protein band (52k) on a polyacrylamide gel detected with fluorography was found to be qualitatively different (present more often; P < .01) and another stage-specific protein band (48k) was found to be quantitatively different (more protein; P equals; .07) in the chimeric treatment vs. the nonchimeric controls at 45 hours after chimerization. These results suggest communication between the cells resulting in a change in their incorporation of uridine and protein synthetic profiles.     

Cell contact induces multiple types of electrical excitability from ascidian two-cell embryos that are cleavage arrested and contain all cell fate determinants

Ascidian early embryonic cells undergo cell differentiation without cell cleavage, thus enabling mixture of cell fate determinants in single cells, which will not be possible in mammalian systems. Either cell in a two-cell embryo (2C cell) has multiple fates and develops into any cell types in a tadpole. To find the condition for controlled induction of a specific cell type, cleavage-arrested cell triplets were prepared in various combinations. They were 2C cells in contact with a pair of anterior neuroectoderm cells from eight-cell embryos (2C-aa triplet), with a pair of presumptive notochordal neural cells (2C-AA triplet), with a pair of presumptive posterior epidermal cells (2C-bb triplet), and with a pair of presumptive muscle cells (2C-BB triplet). The fate of the 2C cell was electrophysiologically identified. When two-cell embryos had been fertilized 3 h later than eight-cell embryos and triplets were formed, the 2C cells became either anterior-neuronal, posterior-neuronal or muscle cells, depending on the cell type of the contacting cell pair. When two-cell embryos had been fertilized earlier than eight-cell embryos, most 2C cells became epidermal. When two- and eight-cell embryos had been simultaneously fertilized, the 2C cells became any one of three cell types described above or the epidermal cell type. Differentiation of the ascidian 2C cell into major cell types was reproducibly induced by selecting the type of contacting cell pair and the developmental time difference between the contacting cell pair and 2C cell. We discuss similarities between cleavage-arrested 2C cells and vertebrate embryonic stem cells and propose the ascidian 2C cell as a simple model for toti-potent stem cells.     

Transforming growth factor-β in the early mouse embryo: Implications for the regulation of muscle formation and implantation

In a search for functions of transforming growth factor-β during early embryonic development we used two different experimental approaches. In the first we made use of embryonic stem (ES) cells. ES cells in culture differentiate to derivatives of all three germ layers and mimic some aspects of organogenesis when grown as aggregates in suspension to form embryoid bodies. Differentiation procedes further when the embryold bodies attach to suitable substrates. Muscle and neuronal cells are among the most readily identified cell types then formed. We examined the effect of all-trans retinoic acid (RA) and members of the transforming growth factor-β family(TGF-βl, TGF-β2) under these conditions in an assay where single aggregates formed in hanging microdrops in medium supplemented with serum depleted of lipophilic substances which would include retinoids. Endoderm-like cells formed under all conditions tested. RA at concentrations of 10⁸ M and 10⁷ M induced the formation of neurons but in the absence of RA or at concentrations up to 10?⁹ M, neurons were not observed. Instead, beating muscle formed in about one-third of the plated aggregates; this was greatly reduced when RA concentrations increased above 10?⁹ M. Immunofluorescent staining for muscle specific myosin showed that two muscle cell types could be distinguished: elongated, non-contractile myoblasts and mononucleate flat cells. The mononucleate flat cells appeared to correspond with rhythmically contracting muscle. The number of non-contractile myoblasts increased 3-fold over controls in the presence of 10?⁹ M RA. TGF-βs increased the number of contractile and non-contractile muscle cells by a factor 3 to 7 over controls, depending on the TGF-β isoform added and the muscle cell type formed. TGF-β2 also invariably increased the rate at which contracting muscle cells were first observed in replated aggregates. The stimulatory effect of TGF-βs on the formation of mononucleate flat cells was completely abrogated by RA at 10?⁹ M while the number of myoblasts under similar conditions was unchanged. These data suggest that a complex interplay between retinoids and TGF-β isoforms may be involved in regulation of differentiation in early myogenesis. In the second approach, neutralizing polyclonal rabbit antibodies specific for TGF-β2 were injected into the cavity of mouse blastocysts 3.5 days post coitum (pc). After 1 day in culture, embryos were transferred to pseudopregnant females. The number of decidua, embryos and resorptions were counted at day 8.5–9.5 pc. Control antibody injected embryos implanted with high efficiency (87%) compared with anti-TGF-β2 injected embryos which implanted with an efficiency of only 43%. If empty decidua (resorptions) were included, the overall recovery was 71% and 32% for control and experimental embryos, respectively. Embryos that were recovered showed no overt macroscopic abnormalities. These results together impiy functions for TGF-βs in implantation as well as in later development of the embryo. © 1993Wiley-Liss, Inc.     

Effects of dexamethasone on the expression of transforming growth factor-β in mouse embryonic palatal mesenchymal cells

The central role of TGF-β in the development of the embryonic palate has been well characterized. TGF-β inhibits mesenchymal cell proliferation, induces medial edge epithelial cell differentiation, and modulates the expression of extracellular matrix proteins as well as the proteases that act upon them. Mechanisms by which TGF-β expression itself is regulated are less well understood. Glucocorticoids are recognized in several cellular systems as able to regulate the expression of TGF-β. This study was therefore designed to examine whether glucocorticoids affect the expression of TGF-β isoforms in embryonic palatal cells. Based on flow cytometric analysis and viability determination, confluent primary cultures of mouse embryonic palate mesenchymal (MEPM) cells exposed to up to 10⁻⁶ M dexamethasone (dex) exhibited no signs of cytotoxicity after 24 hours of exposure. Northern blot analyses revealed that dexamethasone reduced steady-state mRNA levels of TGF-β3 in a dose-dependent manner as early as 4 hours after treatment but had little effect on TGF-β1 and TGF-β2 expression up to 24 hours of dex exposure. Dex also reduced the synthesis of both latent and mature forms of TGF-β protein by approximately four-fold as determined by the mink lung epithelial cell growth inhibition bioassay. Assessment of the ratio of mature to latent protein found in conditioned medium of control compared to dex-treated cultures indicated that dexamethasone may reduce the activation of latent TGF-β to mature biologically active TGF-β. Dexamethasone inhibited the proliferation of MEPM cells despite the down-regulation of TGF-β suggesting that dex-induced growth inhibition of MEPM cells is not mediated by TGF-β. These data suggest that dex modulates TGF-β signaling pathways directly by down-regulating TGF-β expression and possibly indirectly by altering the availability of mature TGF-β necessary to exert its biological effects in the developing palate. © 1996 Wiley-Liss, Inc.     

The db mutation improves memory in younger mice in a model of Alzheimer's disease

Alzheimer's disease (AD) is the most common age-related neurodegenerative disease, while obesity is a major global public health problem associated with the metabolic disorder type 2 diabetes mellitus (T2DM). Chronic obesity and T2DM have been identified as invariant risk factors for dementia and late-onset AD, while their impacts on the occurrence and development of AD remain unclear. As shown in our previous study, the diabetic mutation (db, Lepr^db/db) induces mixed or vascular dementia in mature to middle-aged APP^ΔNL/ΔNL x PS1^P264L/P264L knock-in mice (db/AD). In the present study, the impacts of the db mutation on young AD mice at 10 weeks of age were evaluated. The db mutation not only conferred young AD mice with severe obesity, impaired glucose regulation and activated mammalian target of rapamycin (mTOR) signaling pathway in the mouse cortex, but lead to a surprising improvement in memory. At this young age, mice also had decreased cerebral Aβ content, which we have not observed at older ages. This was unlikely to be related to altered Aβ synthesis, as both β- and γ-secretase were unchanged. The db mutation also reduced the cortical IL-1β mRNA level and IBA1 protein level in young AD mice, with no significant effect on the activation of microglia and astrocytes. We conclude that the db mutation could transitorily improve the memory of young AD mice, a finding that may be partially explained by the relatively improved glucose homeostasis in the brains of db/AD mice compared to their counterpart AD mice, suggesting that glucose regulation could be a strategy for prevention and treatment of neurodegenerative diseases like AD.     

Biglycan and decorin differentially regulate signaling in the fetal membranes

Preterm birth is the leading cause of newborn mortality in the United States and about one third of cases are caused by preterm premature rupture of fetal membranes, a complication that is frequently observed in patients with Ehlers–Danlos Syndrome. Notably, a subtype of Ehlers–Danlos Syndrome is caused by expression of abnormal biglycan and decorin proteoglycans. As compound deficiency of these two small leucine-rich proteoglycans is a model of preterm birth, we investigated the fetal membranes of Bgn^−/−; Dcn^−/− double-null and single-null mice. Our results showed that biglycan signaling supported fetal membrane remodeling during early gestation in the absence of concomitant changes in TGFβ levels. In late gestation, biglycan signaling acted in a TGFβ-dependent manner to aid in membrane stabilization. In contrast, decorin signaling supported fetal membrane remodeling at early stages of gestation in a TGFβ-dependent manner, and fetal membrane stabilization at later stages of gestation without changes in TGFβ levels. Furthermore, exogenous soluble decorin was capable of rescuing the TGFβ signaling pathway in fetal membrane mesenchymal cells. Collectively, these findings provide novel targets for manipulation of fetal membrane extracellular matrix stability and could represent novel targets for research on preventive strategies for preterm premature rupture of fetal membranes.     

Microglia in juvenile neuronal ceroid lipofuscinosis are primed toward a pro‐inflammatory phenotype

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a lysosomal storage disease caused by an autosomal recessive mutation in CLN3. Regions of microglial activation precede and predict areas of neuronal loss in JNCL; however, the functional role of activated microglia remains to be defined. The inflammasome is a key molecular pathway for activating pro‐IL‐1β in microglia, and IL‐1β is elevated in the brains of JNCL patients and can induce neuronal cell death. Here, we utilized primary microglia isolated from CLN3^Δex7/8 mutant and wild‐type (WT) mice to examine the impact of CLN3 mutation on microglial activation and inflammasome function. Treatment with neuronal lysates and ceramide, a lipid intermediate elevated in the JNCL brain, led to inflammasome activation and IL‐1β release in CLN3^Δex7/8 microglia but not WT cells, as well as increased expression of additional pro‐inflammatory mediators. Similar effects were observed following either TNF‐α or IL‐1β treatment, suggesting that CLN3^Δex7/8 microglia exist in primed state and hyper‐respond to several inflammatory stimuli compared to WT cells. CLN3^Δex7/8 microglia displayed constitutive caspase‐1 activity that when blocked led to increased glutamate release that coincided with hemichannel opening. Conditioned medium from activated CLN3^Δex7/8 or WT microglia induced significant cell death in CLN3^Δex7/8 but not WT neurons, demonstrating that intrinsically diseased CLN3^Δex7/8 neurons are less equipped to withstand cytotoxic insults generated by activated microglia. Collectively, aberrant microglial activation may contribute to the pathological chain of events leading to neurodegeneration during later stages of JNCL.

     

Interleukin-1β inhibits normal hematopoietic expansion and promotes acute myeloid leukemia progression via the bone marrow niche

Enhanced interleukin-1β (IL-1β) signaling is a common event in patients with acute myeloid leukemia (AML). It was previously demonstrated that chronic IL-1β exposure severely impaired hematopoietic stem cell (HSC) self-renewal capability in mice and promoted leukemia cell growth in primary AML cells. However, the role of IL-1β in the murine bone marrow (BM) niche remains unclear. Here, we explored the role of IL-1β in the BM niche in Il-1r1^−/− mice, chronic IL-1β exposure mice and mixed lineage leukemia-AF9 fusion gene (MLL-AF9)–induced AML mice models. We demonstrated that IL-1R1 deficiency did not affect the function of HSCs or niche cells under steady-state conditions or during transplantation. Chronic exposure to IL-1β decreased the expansion of Il-1r1^−/− hematopoietic cells in Il-1r1^+/+ recipient mice. These results indicated that IL-1β exposure impaired the ability of niche cells to support hematopoietic cells. Furthermore, we revealed that IL-1R1 deficiency in niche cells prolonged the survival of MLL-AF9–induced AML mice. The results of our study suggest that inhibition of the IL-1β/IL-1R1 signaling pathway in the niche might be a non–cell-autonomous therapy strategy for AML.     

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.