Ubiquinone is necessary for mouse embryonic development but is not essential for mitochondrial respiration

Ubiquinone (UQ) is a lipid found in most biological membranes and is a co-factor in many redox processes including the mitochondrial respiratory chain. UQ has been implicated in protection from oxidative stress and in the aging process. Consequently, it is used as a dietary supplement and to treat mitochondrial diseases. Mutants of the clk-1 gene of the nematode Caenorhabditis elegans are fertile and have an increased life span, although they do not produce UQ but instead accumulate a biosynthetic intermediate, demethoxyubiquinone (DMQ). DMQ appears capable to partially replace UQ for respiration in vivo and in vitro. We have produced a vertebrate model of cells and tissues devoid of UQ by generating a knockout mutation of the murine orthologue of clk-1 (mclk1). We find that mclk1-/- embryonic stem cells and embryos accumulate DMQ instead of UQ. As in the nematode mutant, the activity of the mitochondrial respiratory chain of -/- embryonic stem cells is only mildly affected (65% of wild-type oxygen consumption). However, mclk1-/- embryos arrest development at midgestation, although earlier developmental stages appear normal. These findings indicate that UQ is necessary for vertebrate embryonic development but suggest that mitochondrial respiration is not the function for which UQ is essential when DMQ is present.     

Progressive recruitment of muscle fibers is not necessary for the slow component of VO2 kinetics.

The "slow component" of O2 uptake (VO2) kinetics during constant-load heavy-intensity exercise is traditionally thought to derive from a progressive recruitment of muscle fibers. In this study, which represents a reanalysis of data taken from a previous study by our group (Grassi B, Hogan MC, Greenhaff PL, Hamann JJ, Kelley KM, Aschenbach WG, Constantin-Teodosiu D, Gladden LB. J Physiol 538: 195-207, 2002) we evaluated the presence of a slow component-like response in the isolated dog gastrocnemius in situ (n=6) during 4 min of contractions at approximately 60-70% of peak VO2. In this preparation all muscle fibers are maximally activated by electrical stimulation from the beginning of the contraction period, and no progressive recruitment of fibers is possible. Muscle VO2 was calculated as blood flow multiplied by arteriovenous O2 content difference. The muscle fatigued (force decreased by approximately 20-25%) during contractions. Kinetics of adjustment were evaluated for 1) VO2, uncorrected for force development; 2) VO2 normalized for peak force; 3) VO2 normalized for force-time integral. A slow component-like response, described in only one muscle out of six when uncorrected VO2 was considered, was observed in all muscles when VO2/peak force and VO2/force-time were considered. The amplitude of the slow component-like response, expressed as a fraction of the total response, was higher for VO2/peak force (0.18+/-0.06, means+/-SE) and for VO2/force-time (0.22+/-0.05) compared with uncorrected VO2 (0.04+/-0.04). A progressive recruitment of muscle fibers may not be necessary for the development of the slow component of VO2 kinetics, which may be caused by the metabolic factors that induce muscle fatigue and, as a consequence, reduce the efficiency of muscle contractions.     

The FgfrL1 receptor is required for development of slow muscle fibers

FgfrL1, which interacts with Fgf ligands and heparin, is a member of the fibroblast growth factor receptor (Fgfr) family. FgfrL1-deficient mice show two significant alterations when compared to wildtype mice: They die at birth due to a malformed diaphragm and they lack metanephric kidneys. Utilizing gene arrays, qPCR and in situ hybridization we show here that the diaphragm of FgfrL1 knockout animals lacks any slow muscle fibers at E18.5 as indicated by the absence of slow fiber markers Myh7, Myl2 and Myl3. Similar lesions are also found in other skeletal muscles that contain a high proportion of slow fibers at birth, such as the extraocular muscles. In contrast to the slow fibers, fast fibers do not appear to be affected as shown by expression of fast fiber markers Myh3, Myh8, Myl1 and MylPF. At early developmental stages (E10.5, E15.5), FgfrL1-deficient animals express slow fiber genes at normal levels. The loss of slow fibers cannot be attributed to the lack of kidneys, since Wnt4 knockout mice, which also lack metanephric kidneys, show normal expression of Myh7, Myl2 and Myl3. Thus, FgfrL1 is specifically required for embryonic development of slow muscle fibers.     

Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors

In the embryonic mouse retina, retinoic acid (RA) is unevenly distributed along the dorsoventral axis: RA-rich zones in dorsal and ventral retina are separated by a horizontal RA-poor stripe that contains the RA-inactivating enzyme CYP26A1. To explore the developmental role of this arrangement, we studied formation of the retina and its projections in Cyp26a1 null-mutant mice. Expression of several dorsoventral markers was not affected, indicating that CYP26A1 is not required for establishing the dorsoventral retina axis. Analysis of the mutation on a RA-reporter mouse background confirmed, as expected, that the RA-poor stripe was missing in the retina and its projections at the time when the optic axons first grow over the diencephalon. A day later, however, a gap appeared both in retina and retinofugal projections. As explanation, we found that CYP26C1, another RA-degrading enzyme, had emerged centrally in a narrower domain within the RA-poor stripe. While RA applications increased retinal Cyp26a1 expression, they slightly reduced Cyp26c1. These observations indicate that the two enzymes function independently. The safeguard of the RA-poor stripe by two distinct enzymes during later development points to a role in maturation of a significant functional feature like an area of higher visual acuity that develops at its location.     

Vitamin E is essential for mouse placentation but not for embryonic development itself

Vitamin E (alpha-tocopherol) was discovered 80 years ago to be an indispensable nutrient for reproduction in the female. However, it has not been clarified when or where vitamin E is required during pregnancy. We examined the role of alpha-tocopherol in pregnancy using alpha-tocopherol transfer protein (Ttpa)-deficient mice fed specific alpha-tocopherol diets that led to daily, measurable change in plasma alpha-tocopherol levels from nearly normal to almost undetectable levels. A dietary supplement of alpha-tocopherol to pregnant Ttpa-/- (homozygous null) mice was shown to be essential for maintenance of pregnancy from 6.5 to 13.5 days postcoitum but found not to be crucial before or after this time span, which corresponds to initial development and maturation of the placenta. In addition, exposure to a low alpha-tocopherol environment after initiation of placental formation might result in necrosis of placental syncytiotrophoblast cells, followed by necrosis of fetal blood vessel endothelial cells. When Ttpa(-/-)-fertilized eggs were transferred into Ttpa+/+ (wild-type) recipients, plasma alpha-tocopherol concentrations in the Ttpa-/- fetuses were below the detection limit but the fetuses grew normally. These results indicate that alpha-tocopherol is indispensable for the proliferation and/or function of the placenta but not necessary for development of the embryo itself.     

Endothelial heparan sulfate is necessary but not sufficient for control of vascular smooth muscle cell growth

The state of the endothelial cell (EC) determines the nature of its control of vascular smooth muscle cell (vSMC) biology. Conditioned medium from postconfluent ECs inhibits vSMC proliferation, whereas subconfluent conditioned medium from the same ECs has a stimulatory effect. We and others have identified confluent endothelial cells' production of heparan sulfate proteoglycans (HSPG) as critical to vSMC growth control. The question that arises is whether the stimulation that is observed with subconfluent cells is from (1) aberrant HSPG production, (2) elaboration of noninhibitory species of HSPG, or (3) production of other factors, such as mitogens, which counteract the inhibitory HSPG to stimulate vSMCs. We studied the relative effects of conditioned medium produced by both subconfluent and postconfluent EC cultures on vSMC growth. Conditioned medium was fractionated into nonproteoglycan (non-PG) and proteoglycan (PG) components by anion-exchange chromatography. The PG fractionation profile and the antiproliferative activity of the HSPGs isolated from both subconfluent and postconfluent EC-conditioned media were similar. However, the HSPG fraction alone could not approach the inhibitory potential of unfractionated conditioned medium from postconfluent EC cultures. Non-PG proteins produced by the endothelial cultures had no effect on vSMC growth on their own. Yet, when they were mixed together with HSPG fractions, from either subconfluent or postconfluent EC cultures, the full growth effects were returned. Non-PG protein fractions from postconfluent cultures with HSPG fractions gave maximal inhibition of vSMC growth, whereas non-PG protein fractions from subconfluent EC cultures with HSPG fractions produced the maximal stimulation. Thus, whereas the net stimulatory or inhibitory effect on vSMC growth of EC-conditioned medium is density dependent, this effect does not result from a difference in the antiproliferative heparan sulfate component but rather from non-PG proteins that interact with the heparan sulfates.     

The glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension

Glucocorticoids are used as a treatment for a variety of conditions and hypertension is a well-recognized side effect of their use. The mechanism of glucocorticoid-induced hypertension is incompletely understood and has traditionally been attributed to promiscuous activation of the mineralocorticoid receptor by cortisol. Multiple lines of evidence, however, point to the glucocorticoid receptor as an important mediator as well. We have developed a mouse model of glucocorticoid-induced hypertension, which is dependent on the glucocorticoid receptor. To determine the site(s) of glucocorticoid receptor action relevant to the development of hypertension, we studied glucocorticoid-induced hypertension in a mouse with a tissue-specific knockout of the glucocorticoid receptor in the distal nephron. Although knockout mice had similar body weight, nephron number and renal histology compared to littermate controls, their baseline blood pressure was mildly elevated. Nevertheless, distal nephron glucocorticoid receptor knockout mice and controls had a similar hypertensive response to dexamethasone. Urinary excretion of electrolytes, both before and after administration of glucocorticoid was also indistinguishable between the two groups. We conclude that the glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension in our model.     

Jak2 and Tyk2 are necessary for lineage-specific differentiation, but not for the maintenance of self-renewal of mouse embryonic stem cells

As the LIF-induced Jak1/STAT3 pathway has been reported to play a crucial role in self-renewal of mESCs, we sought to determine if Jak2, which is also expressed in mESCs, might also be involved in the pathway. By employing an RNAi strategy, we established both Jak2 and Jak2/Tyk2 knockdown mESC clones. Both Jak2 and Jak2/Tyk2 knockdown clones maintained the undifferentiated state as wild-type controls, even in a very low concentration of LIF. However, we observed not only faster onset of differentiation but also differential expression of tissue-specific lineage genes for ectodermal and mesodermal, but not endodermal origins from embryoid bodies generated from both types of knockdown clones compared to the wild-type. Furthermore, the reduced level of Jak2 caused differentiation of mESCs in the presence of LIF when the Wnt pathway was activated by LiCl treatment. Taken together, we demonstrated that Jak2 and Tyk2 are not involved in LIF-induced STAT3 pathway for self-renewal of mESCs, but play a role in early lineage decision of mESCs to various differentiated cell types.     

IL-9 promotes but is not necessary for systemic anaphylaxis

Anaphylaxis represents an extreme form of allergic reaction, consisting of a sensitization phase during which allergen-specific IgE are produced and an acute effector phase triggered by allergen-induced degranulation of mast cells. We studied the role of IL-9, a Th2 cytokine implicated in asthma, in different models of murine anaphylaxis. Using a passive model of systemic anaphylaxis, in which anti-DNP IgE Abs were administered before challenge with DNP-BSA, we found that IL-9-transgenic mice or wild-type mice treated with IL-9 for 5 days were highly sensitive to fatal anaphylaxis. This effect was reproduced in both anaphylaxis-susceptible and -resistant backgrounds (FVB/N or [FVB/N x BALB/c] F(1) mice, respectively) and correlated with increased serum concentrations of mouse mast cell protease-1 level, a protein released upon mast cells degranulation. By contrast, IL-9 did not increase the susceptibility to passive cutaneous anaphylaxis. IL-9 expression also increased the susceptibility to fatal anaphylaxis when mice were sensitized by immunization against OVA before challenge with the same Ag. In this model, serum from sensitized, IL-9-transgenic mice was more potent in transferring susceptibility to OVA challenge into naive mice, indicating that IL-9 also promotes the sensitization stage. Finally, using IL-9R-deficient mice, we found that despite its anaphylaxis-promoting activity, IL-9 is dispensable for development of both passive and active anaphylaxis, at least in the C57BL/6 mouse background. Taken together, the data reported in this study indicate that IL-9 promotes systemic anaphylaxis reactions, acting at both the sensitization and effector stages, but is not absolutely required for this process.     

Tmem26 is dynamically expressed during palate and limb development but is not required for embryonic survival

The Tmem26 gene encodes a novel protein that we have previously shown to be regulated by hedgehog signalling in the mouse limb. We now report that Tmem26 expression is spatially and temporally restricted in other regions of the mouse embryo, most notably the facial primordia. In particular, Tmem26 expression in the mesenchyme of the maxillary and nasal prominences is coincident with fusion of the primary palate. In the secondary palate, Tmem26 is expressed in the palatal shelves during their growth and fusion but is downregulated once fusion is complete. Expression was also detected at the midline of the expanding mandible and at the tips of the eyelids as they migrate across the cornea. Given the spatio-temporally restricted expression of Tmem26, we sought to uncover a functional role in embryonic development through targeted gene inactivation in the mouse. However, ubiquitous inactivation of Tmem26 led to no overt phenotype in the resulting embryos or adult mice, suggesting that TMEM26 function is dispensable for embryonic survival.     

Localization of phospholamban in slow but not fast canine skeletal muscle fibers. An immunocytochemical and biochemical study

Phospholamban, originally described as a cardiac sarcoplasmic reticulum protein, was localized in cryostat sections of three adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with highly specific phospholamban antibodies. Only some myofibers were strongly labeled with phospholamban antibodies. The labeling of myofibers with phospholamban antibodies was compared to the distribution of Type I (slow) and Type II (fast) myofibers as determined by staining adjacent sections cytochemically for the alkali-stable myosin ATPase, a specific marker for Type II myofibers. All the skeletal myofibers labeled for phospholamban above background levels corresponded to Type I (slow) myofibers. The presence of phospholamban in microsomal fractions isolated from canine superficial digitalis flexor (89 +/- 3% Type I) and extensor carpi radialis skeletal muscle (14 +/- 6% Type I) was confirmed by immunoblotting. Antiserum to cardiac phospholamban bound to proteins of apparent Mr values of 25,000 (oligomeric phospholamban) and 5,000-6,000 (monomeric phospholamban) in sarcoplasmic reticulum vesicles from both muscles. Quantification of phospholamban in sarcoplasmic reticulum vesicles from cardic, slow, and fast skeletal muscle tissues following phosphorylation with [gamma-32P] ATP suggested that superficial digitalis flexor and extensor carpi radialis skeletal muscle contained about 16 and 3%, respectively, as much phospholamban as cardiac muscle per unit of sarcoplasmic reticulum. The presence of phospholamban in both Type I (slow) and cardiac muscle fibers supports the possibility that the Ca2+ fluxes across the sarcoplasmic reticulum in both fiber types are similarly regulated, and is consistent with the idea that the relaxant effect of catecholamines on slow skeletal muscle is mediated in part by phosphorylation of phospholamban.     

Chondroitin sulfate synthase-2 is necessary for chain extension of chondroitin sulfate but not critical for skeletal development

Chondroitin sulfate (CS) is a linear polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid residues, modified with sulfated residues at various positions. Based on its structural diversity in chain length and sulfation patterns, CS provides specific biological functions in cell adhesion, morphogenesis, neural network formation, and cell division. To date, six glycosyltransferases are known to be involved in the biosynthesis of chondroitin saccharide chains, and a hetero-oligomer complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is known to have the strongest polymerizing activity. Here, we generated and analyzed CSS2(-/-) mice. Although they were viable and fertile, exhibiting no overt morphological abnormalities or osteoarthritis, their cartilage contained CS chains with a shorter length and at a similar number to wild type. Further analysis using CSS2(-/-) chondrocyte culture systems, together with siRNA of CSS1, revealed the presence of two CS chain species in length, suggesting two steps of CS chain polymerization; i.e., elongation from the linkage region up to Mr ～10,000, and further extension. There, CSS2 mainly participated in the extension, whereas CSS1 participated in both the extension and the initiation. Our study demonstrates the distinct function of CSS1 and CSS2, providing a clue in the elucidation of the mechanism of CS biosynthesis.     

Deglycosylation is necessary but not sufficient for activation of proconcanavalin A.

Concanavalin A (ConA), one of the most studied plant lectins, is formed in jack bean (Canavalia ensiformis) seeds. ConA is synthesized as an inactive glycoprotein precursor proConA. Different processing events such as endoproteolytic cleavages, ligation of peptides and deglycosylation of the precursor are required to generate the different polypeptides constitutive of mature ConA. Among these events, deglycosylation of the prolectin appears as a key step in the lectin activation. The detection of deglycosylated proConA in immature jack bean seeds indicates that endoproteolytic cleavages are not prerequisite for its deglycosylation. Both the structure of the lectin precursor N-glycans Man8-9GlcNAc2 and the capacity of Endo H to cleave these oligosaccharide from native proConA in vitro favoured Endo H-type glycosidases as candidates for proConA deglycosylation in planta. Evidence for pH-dependent changes in the prolectin folding were obtained from analysis of the N-glycan accessibility and activation of the deglycosylated lectin precursor in acidic conditions. These data are consistent with the observation that both deglycosylation and acidification of the pH are the minimum requirements to convert the inactive precursor into an active lectin.     

O-GlcNAcase is essential for embryonic development and maintenance of genomic stability

Dysregulation of O-GlcNAc modification catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, type 2 diabetes, and Alzheimer's disease. Here we found that natural aging in wild-type mice was marked by a decrease in OGA and OGT protein levels and an increase in O-GlcNAcylation in various tissues. Genetic disruption of OGA resulted in constitutively elevated O-GlcNAcylation in embryos and led to neonatal lethality with developmental delay. Importantly, we observed that serum-stimulated cell cycle entry induced increased O-GlcNAcylation and decreased its level after release from G2/M arrest, indicating that O-GlcNAc cycling by OGT and OGA is required for precise cell cycle control. Constitutively, elevated O-GlcNAcylation by OGA disruption impaired cell proliferation and resulted in mitotic defects with downregulation of mitotic regulators. OGA loss led to mitotic defects including cytokinesis failure and binucleation, increased lagging chromosomes, and micronuclei formation. These findings suggest an important role for O-GlcNAc cycling by OGA in embryonic development and the regulation of the maintenance of genomic stability linked to the aging process.     

Cortical map plasticity improves learning but is not necessary for improved performance

Cortical map plasticity is believed to be a key substrate of perceptual and skill learning. In the current study, we quantified changes in perceptual ability after pairing tones with stimulation of the cholinergic nucleus basalis to induce auditory cortex map plasticity outside of a behavioral context. Our results provide evidence that cortical map plasticity can enhance perceptual learning. However, auditory cortex map plasticity fades over weeks even though tone discrimination performance remains stable. This observation is consistent with recent reports that cortical map expansions associated with perceptual and motor learning are followed by a period of map renormalization without a decrement in performance. Our results indicate that cortical map plasticity enhances perceptual learning, but is not necessary to maintain improved discriminative ability.     

E2-c-Cbl recognition is necessary but not sufficient for ubiquitination activity

The E2 ubiquitin-conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING (really interesting new gene) domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. Here, we found that neither structural changes nor subtle differences in the E2-E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5B∼Ub thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7∼Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required but not sufficient for transfer of ubiquitin to potential targets.     

Endogenous thrombospondin-1 is not necessary for proliferation but is permissive for vascular smooth muscle cell responses to platelet-derived growth factor.

We have reexamined the role of endogenous thrombospondin-1 (TSP1) in growth and motility of vascular smooth muscle cells (SMCs). Based on the ability of aortic-derived SMCs isolated from TSP1 null mice and grown in the absence of exogenous TSP1 to grow at comparable rates and to a slightly higher density than equivalent cells from wild-type mice, TSP1 is not necessary for their growth. Low concentrations of exogenous TSP1 stimulate growth of TSP1 null SMCs, but higher doses of TSP1 or its C-terminal domain are inhibitory. However, SMCs from TSP1 null mice are selectively deficient in chemotactic and proliferative responses to platelet-derived growth factor and in outgrowth in three-dimensional cultures. Recombinant portions of the N- and C-terminal domains of TSP1 stimulate SMC chemotaxis through different integrin receptors. Based on these data, the relative deficiency in SMC outgrowth during an ex vivo angiogenic response of muscle tissue from TSP1 null mice is probably due to restriction of platelet-derived growth factor dependent SMC migration and/or proliferation.     

NODAL in the uterus is necessary for proper placental development and maintenance of pregnancy

Preterm birth is the single leading cause of perinatal mortality in developed countries, affecting approximately 12% of pregnancies and accounting for 75% of neonatal loss in the United States. Despite the prevalence and severity of premature delivery, the causes and mechanisms that underlie spontaneous and idiopathic preterm birth remain unknown. Our inability to elucidate these fundamental causes has been attributed to a poor understanding of the signaling pathways associated with the premature induction of parturition and a lack of suitable animal models available for preterm birth research. In this study, we describe the generation and analysis of a novel conditional knockout of the transforming growth factor beta (TGFB) superfamily member, Nodal, from the maternal reproductive tract of mice. Strikingly, uterine Nodal knockout females exhibited a severe malformation of the maternal decidua basalis during placentation, leading to significant intrauterine growth restriction, and ultimately preterm birth and fetal loss on Day 17.5 of gestation. Using several approaches, we characterized aberrant placental development and demonstrated that reduced proliferation combined with increased apoptosis resulted in a diminished decidua basalis and compromised maternal-fetal interface. Last, we evaluated various components of the established parturition cascade and determined that preterm birth derived from the maternal Nodal knockout occurs prior to PTGS2 (COX-2) upregulation at the placental interface. Taken together, the results presented in this study highlight an in vivo role for maternal NODAL during placentation, present an interesting link between disrupted decidua basalis formation and premature parturition, and describe a potentially valuable model toward elucidating the complex processes that underlie preterm birth.