ResearchPad - xenopus https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Early correction of synaptic long-term depression improves abnormal anxiety-like behavior in adult GluN2B-C456Y-mutant mice]]> https://www.researchpad.co/article/elastic_article_13831 Mice that carry a heterozygous, autism spectrum disorder-risk C456Y mutation in the NMDA receptor (NMDAR) subunit GluN2B show decreased protein levels, hippocampal NMDAR currents, and NMDAR-dependent long-term depression and have abnormal anxiolytic-like behavior. Early, but not late, treatment of the young mice with the NMDAR agonist D-cycloserine rescues these phenotypes.

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<![CDATA[Distinctive single-channel properties of α4β2-nicotinic acetylcholine receptor isoforms]]> https://www.researchpad.co/article/5c8acc7cd5eed0c48498f842

Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4β2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4β2)2β2- and LS-(α4β2)2α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)β2 subunit binding interfaces. However LS-(α4β2)2α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4β2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4β2)2β2-nAChR exhibit a single conductance state, whereas LS-(α4β2)2α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4β2)2α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site’s role in mediating LS-(α4β2)2α4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(α4β2)2α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)β2 subunit interfaces, rather than independent induction of high conductance channel openings.

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<![CDATA[GluClR-mediated inhibitory postsynaptic currents reveal targets for ivermectin and potential mechanisms of ivermectin resistance]]> https://www.researchpad.co/article/5c59fee7d5eed0c484135792

Glutamate-gated chloride channel receptors (GluClRs) mediate inhibitory neurotransmission at invertebrate synapses and are primary targets of parasites that impact drastically on agriculture and human health. Ivermectin (IVM) is a broad-spectrum pesticide that binds and potentiates GluClR activity. Resistance to IVM is a major economic and health concern, but the molecular and synaptic mechanisms of resistance are ill-defined. Here we focus on GluClRs of the agricultural endoparasite, Haemonchus contortus. We demonstrate that IVM potentiates inhibitory input by inducing a tonic current that plateaus over 15 minutes and by enhancing post-synaptic current peak amplitude and decay times. We further demonstrate that IVM greatly enhances the active durations of single receptors. These effects are greatly attenuated when endogenous IVM-insensitive subunits are incorporated into GluClRs, suggesting a mechanism of IVM resistance that does not affect glutamate sensitivity. We discovered functional groups of IVM that contribute to tuning its potency at different isoforms and show that the dominant mode of access of IVM is via the cell membrane to the receptor.

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<![CDATA[The interplay between voluntary food intake, dietary carbohydrate-lipid ratio and nutrient metabolism in an amphibian, (Xenopus laevis)]]> https://www.researchpad.co/article/5c141e67d5eed0c484d26705

Digestion of food and metabolism of frogs are little studied at the moment, and such processes could be very particular in the case of amphibians, given their ectothermic and carnivorous nature which may lead them to use nutrients through specific biochemical pathways. In the present study, 24 adult Xenopus laevis (six replicates with two frogs per treatment) were randomly assigned to two diets with different carbohydrate:fat ratio (4.5:1 and 2.1:1), changing the dietary glucogenic and lipogenic proportions. Food intake (FI) per unit metabolic body weight (MBW) as well as macronutrient digestibility were calculated, and circulating blood acylcarnitines and amino acids measured, in order to evaluate the effect of the diet treatments. Results demonstrated that food intake regulated most of the changes in the parameters evaluated; significant differences were obtained in crude protein and fat digestibilities through the effect of FI/MBW (p<0.05), whereas diet treatment had a significant effect on the levels of malonyl-CoA. Food intake also significantly impacted malonyl, isovaleryl, hydroxyisovaleryl and long chain fatty acid concentrations; significant (p<0.05) interactions between those metabolites were detected owing to diet. The findings obtained suggest that food intake was the main factor controlling digestion and metabolism in X. laevis, with frogs preferring to utilise protein and fat as primary sources for energy production in the citric acid cycle, reflecting characteristics of a strict carnivore physiological model.

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<![CDATA[Requirement of the 3′-UTR-dependent suppression of DAZL in oocytes for pre-implantation mouse development]]> https://www.researchpad.co/article/5b28b933463d7e146ff345d1

Functional oocytes are produced through complex molecular and cellular processes. In particular, the contribution of post-transcriptional gene regulation mediated by RNA-binding proteins (RBPs) is crucial for controlling proper gene expression during this process. DAZL (deleted in azoospermia-like) is one of the RBPs required for the sexual differentiation of primordial germ cells and for the progression of meiosis in ovulated oocytes. However, the involvement of DAZL in the development of follicular oocytes is still unknown. Here, we show that Dazl is translationally suppressed in a 3′-UTR-dependent manner in follicular oocytes, and this suppression is required for normal pre-implantation development. We found that suppression of DAZL occurred in postnatal oocytes concomitant with the formation of primordial follicles, whereas Dazl mRNA was continuously expressed throughout oocyte development, raising the possibility that DAZL is dispensable for the survival and growth of follicular oocytes. Indeed, follicular oocyte-specific knockout of Dazl resulted in the production of normal number of pups. On the other hand, genetically modified female mice that overexpress DAZL produced fewer numbers of pups than the control due to defective pre-implantation development. Our data suggest that post-transcriptional suppression of DAZL in oocytes is an important mechanism controlling gene expression in the development of functional oocytes.

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<![CDATA[A Markovian Entropy Measure for the Analysis of Calcium Activity Time Series]]> https://www.researchpad.co/article/5989d9f2ab0ee8fa60b6ef3c

Methods to analyze the dynamics of calcium activity often rely on visually distinguishable features in time series data such as spikes, waves, or oscillations. However, systems such as the developing nervous system display a complex, irregular type of calcium activity which makes the use of such methods less appropriate. Instead, for such systems there exists a class of methods (including information theoretic, power spectral, and fractal analysis approaches) which use more fundamental properties of the time series to analyze the observed calcium dynamics. We present a new analysis method in this class, the Markovian Entropy measure, which is an easily implementable calcium time series analysis method which represents the observed calcium activity as a realization of a Markov Process and describes its dynamics in terms of the level of predictability underlying the transitions between the states of the process. We applied our and other commonly used calcium analysis methods on a dataset from Xenopus laevis neural progenitors which displays irregular calcium activity and a dataset from murine synaptic neurons which displays activity time series that are well-described by visually-distinguishable features. We find that the Markovian Entropy measure is able to distinguish between biologically distinct populations in both datasets, and that it can separate biologically distinct populations to a greater extent than other methods in the dataset exhibiting irregular calcium activity. These results support the benefit of using the Markovian Entropy measure to analyze calcium dynamics, particularly for studies using time series data which do not exhibit easily distinguishable features.

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<![CDATA[An Intact Brachyury Function Is Necessary to Prevent Spurious Axial Development in Xenopus laevis]]> https://www.researchpad.co/article/5989daf2ab0ee8fa60bc1b3d

We have previously shown that the member of the HES family hairy2 induces the ectopic expression of dorsal markers when it is overexpressed in the ventral side of Xenopus embryos. Intriguingly, hairy2 represses the mesoderm transcription factor brachyury (bra) throughout its domain in the marginal zone. Here we show that in early gastrula, bra and hairy2 are expressed in complementary domains. Overexpression of bra repressed hairy2. Interference of bra function with a dominant-negative construct expanded the hairy2 domain and, like hairy2 overexpression, promoted ectopic expression of dorsal axial markers in the ventral side and induced secondary axes without head and notochord. Hairy2 depletion rescued the ectopic dorsal development induced by interference of bra function. We concluded that an intact bra function is necessary to exclude hairy2 expression from the non-organiser field, to impede the ectopic specification of dorsal axial fates and the appearance of incomplete secondary axes. This evidence supports a previously unrecognised role for bra in maintaining the dorsal fates inhibited in the ventral marginal zone, preventing the appearance of trunk duplications.

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<![CDATA[Ventx Factors Function as Nanog-Like Guardians of Developmental Potential in Xenopus]]> https://www.researchpad.co/article/5989da54ab0ee8fa60b8e709

Vertebrate development requires progressive commitment of embryonic cells into specific lineages through a continuum of signals that play off differentiation versus multipotency. In mammals, Nanog is a key transcription factor that maintains cellular pluripotency by controlling competence to respond to differentiation cues. Nanog orthologs are known in most vertebrates examined to date, but absent from the Anuran amphibian Xenopus. Interestingly, in silico analyses and literature scanning reveal that basal vertebrate ventral homeobox (ventxs) and mammalian Nanog factors share extensive structural, evolutionary and functional properties. Here, we reassess the role of ventx activity in Xenopus laevis embryos and demonstrate that they play an unanticipated role as guardians of high developmental potential during early development. Joint over-expression of Xenopus ventx1.2 and ventx2.1-b (ventx1/2) counteracts lineage commitment towards both dorsal and ventral fates and prevents msx1-induced ventralization. Furthermore, ventx1/2 inactivation leads to down-regulation of the multipotency marker oct91 and to premature differentiation of blastula cells. Finally, supporting the key role of ventx1/2 in the control of developmental potential during development, mouse Nanog (mNanog) expression specifically rescues embryonic axis formation in ventx1/2 deficient embryos. We conclude that during Xenopus development ventx1/2 activity, reminiscent of that of Nanog in mammalian embryos, controls the switch of early embryonic cells from uncommitted to committed states.

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<![CDATA[Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins]]> https://www.researchpad.co/article/5989d9eeab0ee8fa60b6d8b4

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

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<![CDATA[Diurnal Variation of Tight Junction Integrity Associates Inversely with Matrix Metalloproteinase Expression in Xenopus laevis Corneal Epithelium: Implications for Circadian Regulation of Homeostatic Surface Cell Desquamation]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdb77e

Background and Objectives

The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs) are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium.

Methodology/Principal Findings

Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2), membrane type 1-MMP (MT1-MMP) and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohistochemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime.

Conclusions/Significance

MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and activation, tight junction protein cleavage, and subsequent surface cell desquamation and renewal may be orchestrated by nocturnal circadian signals.

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<![CDATA[Defining Synphenotype Groups in Xenopus tropicalis by Use of Antisense Morpholino Oligonucleotides]]> https://www.researchpad.co/article/5989da1cab0ee8fa60b7d2a1

To identify novel genes involved in early development, and as proof-of-principle of a large-scale reverse genetics approach in a vertebrate embryo, we have carried out an antisense morpholino oligonucleotide (MO) screen in Xenopus tropicalis, in the course of which we have targeted 202 genes expressed during gastrula stages. MOs were designed to complement sequence between −80 and +25 bases of the initiating AUG codons of the target mRNAs, and the specificities of many were tested by (i) designing different non-overlapping MOs directed against the same mRNA, (ii) injecting MOs differing in five bases, and (iii) performing “rescue” experiments. About 65% of the MOs caused X. tropicalis embryos to develop abnormally (59% of those targeted against novel genes), and we have divided the genes into “synphenotype groups,” members of which cause similar loss-of-function phenotypes and that may function in the same developmental pathways. Analysis of the expression patterns of the 202 genes indicates that members of a synphenotype group are not necessarily members of the same synexpression group. This screen provides new insights into early vertebrate development and paves the way for a more comprehensive MO-based analysis of gene function in X. tropicalis.

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<![CDATA[Remodeling of the Metabolome during Early Frog Development]]> https://www.researchpad.co/article/5989dae5ab0ee8fa60bbd305

A rapid series of synchronous cell divisions initiates embryogenesis in many animal species, including the frog Xenopus laevis. After many of these cleavage cycles, the nuclear to cytoplasmic ratio increases sufficiently to somehow cause cell cycles to elongate and become asynchronous at the mid-blastula transition (MBT). We have discovered that an unanticipated remodeling of core metabolic pathways occurs during the cleavage cycles and the MBT in X.laevis, as evidenced by widespread changes in metabolite abundance. While many of the changes in metabolite abundance were consistently observed, it was also evident that different female frogs laid eggs with different levels of at least some metabolites. Metabolite tracing with heavy isotopes demonstrated that alanine is consumed to generate energy for the early embryo. dATP pools were found to decline during the MBT and we have confirmed that maternal pools of dNTPs are functionally exhausted at the onset of the MBT. Our results support an alternative hypothesis that the cell cycle lengthening at the MBT is triggered not by a limiting maternal protein, as is usually proposed, but by a decline in dNTP pools brought about by the exponentially increasing demands of DNA synthesis.

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<![CDATA[Circadian Genes, xBmal1 and xNocturnin, Modulate the Timing and Differentiation of Somites in Xenopus laevis]]> https://www.researchpad.co/article/5989dae6ab0ee8fa60bbdb1a

We have been investigating whether xBmal1 and xNocturnin play a role in somitogenesis, a cyclic developmental process with an ultradian period. Previous work from our lab shows that circadian genes (xPeriod1, xPeriod2, xBmal1, and xNocturnin) are expressed in developing somites. Somites eventually form the vertebrae, muscles of the back, and dermis. In Xenopus, a pair of somites is formed about every 50 minutes from anterior to posterior. We were intrigued by the co-localization of circadian genes in an embryonic tissue known to be regulated by an ultradian clock. Cyclic expression of genes involved in Notch signaling has been implicated in the somite clock. Disruption of Notch signaling in humans has been linked to skeletal defects in the vertebral column. We found that both depletion (morpholino) and overexpression (mRNA) of xBMAL1 protein (bHLH transcription factor) or xNOCTURNIN protein (deadenylase) on one side of the developing embryo led to a significant decrease in somite number with respect to the untreated side (p<0.001). These manipulations also significantly affect expression of a somite clock component (xESR9; p<0.05). We observed opposing effects on somite size. Depletion of xBMAL1 or xNOCTURNIN caused a statistically significant decrease in somite area (quantified using NIH ImageJ; p<0.002), while overexpression of these proteins caused a significant dose dependent increase in somite area (p<0.02; p<0.001, respectively). We speculate that circadian genes may play two separate roles during somitogenesis. Depletion and overexpression of xBMAL1 and NOCTURNIN both decrease somite number and influence expression of a somite clock component, suggesting that these proteins may modulate the timing of the somite clock in the undifferentiated presomitic mesoderm. The dosage dependent effects on somite area suggest that xBMAL1 and xNOCTURNIN may also act during somite differentiation to promote myogenesis.

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<![CDATA[Brg1 chromatin remodeling ATPase balances germ layer patterning by amplifying the transcriptional burst at midblastula transition]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be03ab

Zygotic gene expression programs control cell differentiation in vertebrate development. In Xenopus, these programs are initiated by local induction of regulatory genes through maternal signaling activities in the wake of zygotic genome activation (ZGA) at the midblastula transition (MBT). These programs lay down the vertebrate body plan through gastrulation and neurulation, and are accompanied by massive changes in chromatin structure, which increasingly constrain cellular plasticity. Here we report on developmental functions for Brahma related gene 1 (Brg1), a key component of embyronic SWI/SNF chromatin remodeling complexes. Carefully controlled, global Brg1 protein depletion in X. tropicalis and X. laevis causes embryonic lethality or developmental arrest from gastrulation on. Transcriptome analysis at late blastula, before development becomes arrested, indicates predominantly a role for Brg1 in transcriptional activation of a limited set of genes involved in pattern specification processes and nervous system development. Mosaic analysis by targeted microinjection defines Brg1 as an essential amplifier of gene expression in dorsal (BCNE/Nieuwkoop Center) and ventral (BMP/Vent) signaling centers. Moreover, Brg1 is required and sufficient for initiating axial patterning in cooperation with maternal Wnt signaling. In search for a common denominator of Brg1 impact on development, we have quantitatively filtered global mRNA fluctuations at MBT. The results indicate that Brg1 is predominantly required for genes with the highest burst of transcriptional activity. Since this group contains many key developmental regulators, we propose Brg1 to be responsible for raising their expression above threshold levels in preparation for embryonic patterning.

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<![CDATA[Arginine and Lysine Transporters Are Essential for Trypanosoma brucei]]> https://www.researchpad.co/article/5989d9d2ab0ee8fa60b64b44

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.

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<![CDATA[The Principal Forces of Oocyte Polarity Are Evolutionary Conserved but May Not Affect the Contribution of the First Two Blastomeres to the Blastocyst Development in Mammals]]> https://www.researchpad.co/article/5989dad4ab0ee8fa60bb748d

Oocyte polarity and embryonic patterning are well-established features of development in lower species. Whether a similar form of pre-patterning exists in mammals is currently under hot debate in mice. This study investigated this issue for the first time in ovine as a large mammal model. Microsurgical trisection of unfertilized MII-oocytes revealed that cortical cytoplasm around spindle (S) contained significant amounts of total maternal mRNAs and proteins compared to matched cytoplast hemispheres that were located either near (NS) or far (FS) -to-spindle. RT-qPCR provided striking examples of maternal mRNA localized to subcellular substructures S (NPM2, GMNN, H19, PCAF, DNMT3A, DNMT1, and STELLA), NS (SOX2, NANOG, POU5F1, and TET1), and FS (GCN) of MII oocyte. Immunoblotting revealed that specific maternal proteins DNMT3A and NANOG were asymmetrically enriched in MII-spindle-half of the oocytes. Topological analysis of sperm entry point (SEP) revealed that sperm preferentially entered via the MII-spindle-half of the oocytes. Even though, the topological position of first cleavage plane with regard to SEP was quite stochastic. Spatial comparison of lipid content revealed symmetrical distribution of lipids between 2-cell blastomeres. Lineage tracing using Dil, a fluorescent dye, revealed that while the progeny of leading blastomere of 2-cell embryos contributed to more cells in the developed blastocysts compared to lagging counterpart, the contributions of leading and lagging blastomeres to the embryonic-abembryonic parts of the developed blastocysts were almost unbiased. And finally, separated sister blastomeres of 2-cell embryos had an overall similar probability to arrest at any stage before the blastocyst (2-cell, 4-cell, 8-cell, and morula) or to achieve the blastocyst stage. It was concluded that the localization of maternal mRNAs and proteins at the spindle are evolutionarily conserved between mammals unfertilized ovine oocyte could be considered polar with respect to the spatial regionalization of maternal transcripts and proteins. Even though, the principal forces of this definitive oocyte polarity may not persist during embryonic cleavages.

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<![CDATA[Neofunctionalization in Vertebrates: The Example of Retinoic Acid Receptors]]> https://www.researchpad.co/article/5989db03ab0ee8fa60bc760b

Understanding the role of gene duplications in establishing vertebrate innovations is one of the main challenges of Evo-Devo (evolution of development) studies. Data on evolutionary changes in gene expression (i.e., evolution of transcription factor-cis-regulatory elements relationships) tell only part of the story; protein function, best studied by biochemical and functional assays, can also change. In this study, we have investigated how gene duplication has affected both the expression and the ligand-binding specificity of retinoic acid receptors (RARs), which play a major role in chordate embryonic development. Mammals have three paralogous RAR genes—RARα, β, and γ—which resulted from genome duplications at the origin of vertebrates. By using pharmacological ligands selective for specific paralogues, we have studied the ligand-binding capacities of RARs from diverse chordates species. We have found that RARβ-like binding selectivity is a synapomorphy of all chordate RARs, including a reconstructed synthetic RAR representing the receptor present in the ancestor of chordates. Moreover, comparison of expression patterns of the cephalochordate amphioxus and the vertebrates suggests that, of all the RARs, RARβ expression has remained most similar to that of the ancestral RAR. On the basis of these results together, we suggest that while RARβ kept the ancestral RAR role, RARα and RARγ diverged both in ligand-binding capacity and in expression patterns. We thus suggest that neofunctionalization occurred at both the expression and the functional levels to shape RAR roles during development in vertebrates.

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<![CDATA[The Testis-Specific Factor CTCFL Cooperates with the Protein Methyltransferase PRMT7 in H19 Imprinting Control Region Methylation ]]> https://www.researchpad.co/article/5989da9fab0ee8fa60ba533c

Expression of imprinted genes is restricted to a single parental allele as a result of epigenetic regulation—DNA methylation and histone modifications. Igf2/H19 is a reciprocally imprinted locus exhibiting paternal Igf2 and maternal H19 expression. Their expression is regulated by a paternally methylated imprinting control region (ICR) located between the two genes. Although the de novo DNA methyltransferases have been shown to be necessary for the establishment of ICR methylation, the mechanism by which they are targeted to the region remains unknown. We demonstrate that CTCFL/BORIS, a paralog of CTCF, is an ICR-binding protein expressed during embryonic male germ cell development, coinciding with the timing of ICR methylation. PRMT7, a protein arginine methyltransferase with which CTCFL interacts, is also expressed during embryonic testis development. Symmetrical dimethyl arginine 3 of histone H4, a modification catalyzed by PRMT7, accumulates in germ cells during this developmental period. This modified histone is also found enriched in both H19 ICR and Gtl2 differentially methylated region (DMR) chromatin of testis by chromatin immunoprecipitation (ChIP) analysis. In vitro studies demonstrate that CTCFL stimulates the histone-methyltransferase activity of PRMT7 via interactions with both histones and PRMT7. Finally, H19 ICR methylation is demonstrated by nuclear co-injection of expression vectors encoding CTCFL, PRMT7, and the de novo DNA methyltransferases, Dnmt3a, -b and -L, in Xenopus oocytes. These results suggest that CTCFL and PRMT7 may play a role in male germline imprinted gene methylation.

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<![CDATA[Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes]]> https://www.researchpad.co/article/5989da22ab0ee8fa60b7f592

Bax promotes mitochondrial permeabilization during apoptosis via a phase-transition-like event in the membrane and oligomerization of a catalyst molecule that facilitates Bax pore formation.

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<![CDATA[Ligand Binding at the 4-4 Agonist-Binding Site of the 42 nAChR Triggers Receptor Activation through a Pre-Activated Conformational State]]> https://www.researchpad.co/article/5989da98ab0ee8fa60ba2aa8

The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant subtype in the brain and exists in two functional stoichiometries: (α4)3(β2)2 and (α4)2(β2)3. A distinct feature of the (α4)3(β2)2 receptor is the biphasic activation response to the endogenous agonist acetylcholine, where it is activated with high potency and low efficacy when two α4-β2 binding sites are occupied and with low potency/high efficacy when a third α4-α4 binding site is occupied. Further, exogenous ligands can bind to the third α4-α4 binding site and potentiate the activation of the receptor by ACh that is bound at the two α4-β2 sites. We propose that perturbations of the recently described pre-activation step when a third binding site is occupied are a key driver of these distinct activation properties. To investigate this, we used a combination of simple linear kinetic models and voltage clamp electrophysiology to determine whether transitions into the pre-activated state were increased when three binding sites were occupied. We separated the binding at the two different sites with ligands selective for the α4-β2 site (Sazetidine-A and TC-2559) and the α4-α4 site (NS9283) and identified that when a third binding site was occupied, changes in the concentration-response curves were best explained by an increase in transitions into a pre-activated state. We propose that perturbations of transitions into a pre-activated state are essential to explain the activation properties of the (α4)3(β2)2 receptor by acetylcholine and other ligands. Considering the widespread clinical use of benzodiazepines, this discovery of a conserved mechanism that benzodiazepines and ACh potentiate receptor activation via a third binding site can be exploited to develop therapeutics with similar properties at other cys-loop receptors.

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