Differentiation Events Research Articles

OR07-5 PARP1 Contributes to the Glucocorticoid Receptor Transcriptional Response And Influences the Skeletal Muscle Phenotype

Abstract Background NAD+-dependent PARP1 (Poly-(ADP-ribosyl)polymerase 1) generates the post-translational modification ADP-Ribosylation (ADPR). Given PARP1-ADPR determines cellular NAD+ availability, which in turn impacts a range of hormonally governed actions, we sought to identify the molecular roles of PARP1 in relation to glucocorticoids in the endocrine sensitive tissue skeletal muscle. Importance: Activity of the glucocorticoid receptor (GR) is dictated by molecular partners whose actions skew the frequency with which GR produces meaningful transcriptional outcomes. As a result, our molecular understanding of this steroid hormone receptor remains incomplete. Hypothesis PARP1 through ADPR actions impacts skeletal muscle phenotype and co-regulates glucocorticoid-mediated transcriptional responses. Experimental Design: Murine muscle myoblasts (C2C12) or primary murine muscle cultures were treated with PARP inhibitors (PJ34(10uM) or BYK204165(1uM)) or transfected with silencing RNA targeting PARP1. Cells were treated with ± dexamethasone (1uM). Major Results Analysis of differentiating muscle revealed PARP1 and ADPR are dynamic at days 0-5 (measured by Western immunoblot. p<0.001; n=6). Chemical inhibition of PARP1 significantly decreased ADPR (Western immunoblot. p<0.001; n=9) but did not prevent myotube formation. To identify if initial ADPR dynamics held consequence for fully differentiated myotubes, cells were treated with a single dose of the PARP1 inhibitor BYK204165 at induction of differentiation. Myotube lysates were collected 5 days later and were subjected to unlabelled SWATH-MS. This measured the abundance of 180 proteins as significantly different in PARP inhibitor-treated cells (n=7). Analyses of these proteins revealed over-representation of pathways governing muscle development (FDR 8.99-08), contraction (FDR 9.47-08, myofiber assembly (FDR 8.06-05), and energy metabolism (FDR 2.93-06). Indicating PARP1-ADPR regulates myogenesis and plays a role in the striated muscle contractile response. ChIP assays for PARP1 binding in cells showed a (30-fold enrichment ±3.2SEM) at GR response elements and detectable ADPR was reduced when exposed to dexamethasone (Western immunoblot n=5). These data support ADPR as responsive to glucocorticoid exposure and PARP1 influences GR recruitment to the genome. RNAseq of siPARP1 (n=4) versus scrambled myoblasts (n=5) identified differential expression of 165 genes. Gene ontology revealed over-representation in siPARP1 cells of those pathways regulating muscle differentiation, including actin-binding, cytoskeletal structure, and NAD+ binding genes. SiRNA of PARP1 had no influence over the ability of glucocorticoids to activate archetype target genes (DUSP1, MURF1, ATROGIN1 & GILZ), but differentially impacted the expression of 434 other genes. Conclusions PARP1 mediated ADPR is critical to the 'early phase' events of muscle cell differentiation. PARP1 influences short-term transcriptional behaviors of the glucocorticoid receptor; in turn, glucocorticoids themselves impact PARP1-genome interaction events. These findings are important for the understanding of general GR mechanisms and have relevance for the widespread functional decline observed during tissue glucocorticoid excess. Presentation: Saturday, June 11, 2022 12:30 p.m. - 12:45 p.m.

Involvement of a Basic Helix-Loop-Helix Gene BHLHE40 in Specification of Chicken Retinal Pigment Epithelium.

The first event of differentiation and morphogenesis in the optic vesicle (OV) is specification of the neural retina (NR) and retinal pigment epithelium (RPE), separating the inner and outer layers of the optic cup, respectively. Here, we focus on a basic helix-loop-helix gene, BHLHE40, which has been shown to be expressed by the developing RPE in mice and zebrafish. Firstly, we examined the expression pattern of BHLHE40 in the developing chicken eye primordia by in situ hybridization. Secondly, BHLHE40 overexpression was performed with in ovo electroporation and its effects on optic cup morphology and expression of NR and RPE marker genes were examined. Thirdly, we examined the expression pattern of BHLHE40 in LHX1-overexpressed optic cup. BHLHE40 expression emerged in a subset of cells of the OV at Hamburger and Hamilton stage 14 and became confined to the outer layer of the OV and the ciliary marginal zone of the retina by stage 17. BHLHE40 overexpression in the prospective NR resulted in ectopic induction of OTX2 and repression of VSX2. Conversely, BHLHE40 was repressed in the second NR after LHX1 overexpression. These results suggest that emergence of BHLHE40 expression in the OV is involved in initial RPE specification and that BHLHE40 plays a role in separation of the early OV domains by maintaining OTX2 expression and antagonizing an NR developmental program.

Integrin Alpha v Beta 6 (αvβ6) and Its Implications in Cancer Treatment.

Integrins are necessary for cell adhesion, migration, and positioning. Essential for inducing signalling events for cell survival, proliferation, and differentiation, they also trigger a variety of signal transduction pathways involved in mediating invasion, metastasis, and squamous-cell carcinoma. Several recent studies have demonstrated that the up- and down-regulation of the expression of αv and other integrins can be a potent marker of malignant diseases and patient prognosis. This review focuses on an arginine-glycine-aspartic acid (RGD)-dependent integrin αVβ6, its biology, and its role in healthy humans. We examine the implications of αVβ6 in cancer progression and the promotion of epithelial-mesenchymal transition (EMT) by contributing to the activation of transforming growth factor beta TGF-β. Although αvβ6 is crucial for proper function in healthy people, it has also been validated as a target for cancer treatment. This review briefly considers aspects of targeting αVβ6 in the clinic via different therapeutic modalities.

Temporal evolution of 142Nd signatures in SW Greenland from high precision MC-ICP-MS measurements

Measurements of 142Nd isotope signatures in Archean rocks are a powerful tool to investigate the earliest silicate differentiation events on Earth. Here, we introduce a new analytical protocol that allows high precision radiogenic and mass-independent Nd isotope measurements by MC-ICP-MS. To validate our method, we have measured well-characterized ∼3.72 to ∼3.8 Ga samples from the Eoarchean Itsaq Gneiss Complex and associated supracrustal belts, as well as Mesoarchean greenstones and a Proterozoic dike in SW Greenland, including lithostratigraphic units that were previously analyzed for 142-143Nd isotope systematics, by both TIMS and MC-ICP-MS. Our μ142Nd values for ∼3.72 to ∼3.8 Ga rocks from the Isua region range from +9.2 ± 2.6 to +13.2 ± 1.1 ppm and are in good agreement with previous studies. Using coupled 142,143Nd/144Nd isotope systematics from our data for ∼3.8 Ga mafic-ultramafic successions from the Isua region, we can confirm previous age constraints on the earliest silicate differentiation events with differentiation age of 4.390−0.060+0.045 Ga. Moreover, we can resolve a statistically significant decrease of 142Nd/144Nd isotope compositions in the ambient mantle of SW Greenland that already started to commence by Eoarchean time, between ∼3.8 Ga (μ142Nd = +13.0 ± 1.1) and ∼ 3.72 Ga (μ142Nd = +9.8 ± 1.0). Even lower but homogeneous μ142Nd values of +3.8 ± 1.1 are found in ∼3.4 Ga mantle-derived rocks from the Ameralik dike swarms. Our study reveals that ε143Nd(i) and εHf(i) values of Isua rocks scatter more than it would be expected from a single stage differentiation event as implied from nearly uniform μ142Nd values, suggesting that the previously described decoupling of Hf and Nd isotopes is not a primordial magma ocean signature. Instead, we conclude that some of second stage processes like younger mantle depletion events or recycling of subducted material affected the 147Sm143Nd isotope systematics. The preservation of pristine whole-rock isochrons largely rules out a significant disturbance by younger alteration events. Based on isotope and trace element modelling, we argue that the temporal evolution of coupled 142,143Nd/144Nd isotope compositions in the ambient mantle beneath the Isua rocks is best explained by the progressive admixture of material to the Isua mantle source that must have had present-day-like μ142Nd compositions. In contrast, Mesoarchean mafic rocks from the ∼3.08 Ga Ivisaartoq greenstone belt and the 2.97 Ga inner Ameralik Fjord region as well as a 2.0 Ga Proterozoic dike within that region all have higher μ142Nd values as would be expected from our simple replenishment model. This argues for reworking of older Isua crustal material that carried elevated μ142Nd compositions.

Phenotypic selection during laboratory evolution of yeast populations leads to a genome-wide sustainable chromatin compaction shift.

In a previous study, we have shown how microbial evolution has resulted in a persistent reduction in expression after repeatedly selecting for the lowest PGAL1-YFP-expressing cells. Applying the ATAC-seq assay on samples collected from this 28-day evolution experiment, here we show how genome-wide chromatin compaction changes during evolution under selection pressure. We found that the chromatin compaction was altered not only on GAL network genes directly impacted by the selection pressure, showing an example of selection-induced non-genetic memory, but also at the whole-genome level. The GAL network genes experienced chromatin compaction accompanying the reduction in PGAL1-YFP reporter expression. Strikingly, the fraction of global genes with differentially compacted chromatin states accounted for about a quarter of the total genome. Moreover, some of the ATAC-seq peaks followed well-defined temporal dynamics. Comparing peak intensity changes on consecutive days, we found most of the differential compaction to occur between days 0 and 3 when the selection pressure was first applied, and between days 7 and 10 when the pressure was lifted. Among the gene sets enriched for the differential compaction events, some had increased chromatin availability once selection pressure was applied and decreased availability after the pressure was lifted (or vice versa). These results intriguingly show that, despite the lack of targeted selection, transcriptional availability of a large fraction of the genome changes in a very diverse manner during evolution, and these changes can occur in a relatively short number of generations.

A TRiP RNAi screen to identify molecules necessary for Drosophila photoreceptor differentiation.

Drosophila rhabdomeric terminal photoreceptor differentiation is an extended process taking several days to complete. Following ommatidial patterning by the morphogenetic furrow, photoreceptors are sequentially recruited and specified, and terminal differentiation begins. Key events of terminal differentiation include the establishment of apical and basolateral domains, rhabdomere and stalk formation, inter-rhabdomeral space formation, and expression of phototransduction machinery. While many key regulators of these processes have been identified, the complete network of transcription factors to downstream effector molecules necessary for regulating each of these major events remains incomplete. Here, we report an RNAi screen to identify additional molecules and cellular pathways required for photoreceptor terminal differentiation. First, we tested several eye-specific GAL4 drivers for correct spatial and temporal specificity and identified Pph13-GAL4 as the most appropriate GAL4 line for our screen. We screened lines available through the Transgenic RNAi Project and isolated lines that when combined with Pph13-GAL4 resulted in the loss of the deep pseudopupil, as a readout for abnormal differentiation. In the end, we screened 6,189 lines, representing 3,971 genes, and have identified 64 genes, illuminating potential new regulatory molecules and cellular pathways for the differentiation and organization of Drosophila rhabdomeric photoreceptors.

Switching cell fate by the actin–auxin oscillator in Taxus: cellular aspects of plant cell fermentation

Key messagePaclitaxel synthesis in Taxus cells correlates with a cell-fate switch that leads to vacuoles of a glossy appearance and vermiform mitochondria. This switch depends on actin and apoplastic respiratory burst.Plant cell fermentation, the production of valuable products in plant cell culture, has great potential as sustainable alternative to the exploitation of natural resources for compounds of pharmaceutical interest. However, the success of this approach has remained limited, because the cellular aspects of metabolic competence are mostly unknown. The production of the anti-cancer alkaloid Paclitaxel has been, so far, the most successful case for this approach. In the current work, we map cellular aspects of alkaloid synthesis in cells of Taxus chinensis using a combination of live-cell imaging, quantitative physiology, and metabolite analysis. We show evidence that metabolic potency correlates with a differentiation event giving rise to cells with large vacuoles with a tonoplast that is of a glossy appearance, agglomerations of lipophilic compounds, and multivesicular bodies that fuse with the plasma membrane. Cellular features of these glossy cells are bundled actin, more numerous peroxisomes, and vermiform mitochondria. The incidence of glossy cells can be increased by aluminium ions, and this increase is significantly reduced by the actin inhibitor Latrunculin B, and by diphenylene iodonium, a specific inhibitor of the NADPH oxidase Respiratory burst oxidase Homologue (RboH). It is also reduced by the artificial auxin Picloram. This cellular fingerprint matches the implications of a model, where the differentiation into the glossy cell type is regulated by the actin–auxin oscillator that in plant cells acts as dynamic switch between growth and defence.

A multi-omics approach to visualize early neuronal differentiation from hESCs in 4D

SummaryNeuronal differentiation of pluripotent stem cells is an established method to study physiology, disease, and medication safety. However, the sequence of events in human neuronal differentiation and the ability of in vitro models to recapitulate early brain development are poorly understood. We developed a protocol optimized for the study of early human brain development and neuropharmacological applications. We comprehensively characterized gene expression and epigenetic profiles at four timepoints, because the cells differentiate from embryonic stem cells towards a heterogeneous population of progenitors, immature and mature neurons bearing telencephalic signatures. A multi-omics roadmap of neuronal differentiation, combined with searchable interactive gene analysis tools, allows for extensive exploration of early neuronal development and the effect of medications.

Integrating machine learning to construct aberrant alternative splicing event related classifiers to predict prognosis and immunotherapy response in patients with hepatocellular carcinoma.

Introduction: In hepatocellular carcinoma (HCC), alternative splicing (AS) is related to tumor invasion and progression. Methods: We used HCC data from a public database to identify AS subtypes by unsupervised clustering. Through feature analysis of different splicing subtypes and acquisition of the differential alternative splicing events (DASEs) combined with enrichment analysis, the differences in several subtypes were explored, cell function studies have also demonstrated that it plays an important role in HCC. Results: Finally, in keeping with the differences between these subtypes, DASEs identified survival-related AS times, and were used to construct risk proportional regression models. AS was found to be useful for the classification of HCC subtypes, which changed the activity of tumor-related pathways through differential splicing effects, affected the tumor microenvironment, and participated in immune reprogramming. Conclusion: In this study, we described the clinical and molecular characteristics providing a new approach for the personalized treatment of HCC patients.

Inhibitory Effects of Hydrolysable Tannins on Lipid Accumulation in 3T3-L1 Cells.

Obesity is currently the most common cause of metabolic diseases including type 2 diabetes and hyperlipidemia. Obesity results from excess lipid accumulation in adipose tissue. Several studies have investigated the inhibitory effects of natural plant-derived products on adipocyte differentiation and lipid accumulation. In this study, we examined the effect of hydrolysable tannins composed of gallic acid and glucose on adipocyte differentiation in 3T3-L1 cells. 1,2,3,4,6-Penta-O-galloyl-β-D-glucose (PGG) (1), a representative gallotannin, inhibited lipid accumulation in 3T3-L1 cells, whereas ellagitannins (tellimagrandin I, eugeniin and casuarictin) did not. The expression of adipocyte differentiation-related genes, including peroxisome proliferator activator γ2 (Pparγ2), CCAAT/enhancer binding protein α (C/EBPα) and adipocyte fatty acid binding protein (aP2), was significantly suppressed in PGG (1)-treated 3T3-L1 cells beginning at day 2 after induction of differentiation. While PGG (1) did not directly reduce Pparγ2 expression, it reduced the expression of its target genes in mature adipocytes. In addition, PGG (1) treatment inhibited mitotic clonal expansion, one of earliest events of adipocyte differentiation. These findings indicate that PGG (1) has an inhibitory effect on adipocyte differentiation through the suppression of mitotic clonal expansion.

Histological Evaluation of the Effect of Local Application of Punica granatum Seed Oil on Bone Healing.

Background Bone healing is a complex and dynamic process that represents a well-orchestrated series of biological events of cellular recruitment, proliferation, and differentiation. The use of medicinal plants in bone healing has attracted increasing interest because of their lower side effects. Punica granatum seed oil (PSO) contains high levels of phenolic compounds, promotes osteoblast function, and plays an important role in bone remodeling. A gelatin sponge (Spongostan) is a hemostatic agent that is extensively applied as scaffolds in engineering and as drug carriers in the medical field. This study aimed to evaluate the effectiveness of PSO for bone healing enhancement. Twenty adult male New Zealand rabbits, weighing an average of 1.5–2 kg, were used in this study. Three intrabony holes were created in the tibiae of each animal, which were filled with a gelatin sponge (GS group) and combined gelatin sponge and PSO (GS/PSO group). Holes without material application were designated as the control group (C group). The animals were sacrificed at the healing duration (2–4 weeks) to prepare bone specimens for histological and histomorphometric analyses. Results Histological findings indicated that the bone defects in the GS/PSO group showed more bone formation, mineralization, and maturation compared with the C and GS groups. Multiple group differences for bone cells showed a highly significant difference among all groups in the 2- and 4-week healing periods except for the C/GS and GS/GS/PSO groups at 4-weeks duration. Furthermore, highly significant results were obtained between both durations regarding the trabecular area, trabecular number, and bone marrow area. Conclusion The study revealed that the combined application of GS and PSO was more effective in enhancing bone regeneration and accelerating bone healing compared with the other groups.

Modeling the effect of environmental cytokines, nutrient conditions and hypoxia on CD4+ T cell differentiation.

Upon antigen stimulation and co-stimulation, CD4+ T lymphocytes produce soluble factors that promote the activity of other immune cells against pathogens or modified tissues; this task must be performed in presence of a variety of environmental cytokines, nutrient, and oxygen conditions, which necessarily impact T cell function. The complexity of the early intracellular processes taking place upon lymphocyte stimulation is addressed by means of a mathematical model based on a network that integrates variable microenvironmental conditions with intracellular activating, regulatory, and metabolic signals. Besides the phenotype subsets considered in previous works (Th1, Th2, Th17, and Treg) the model includes the main early events in differentiation to the T FH phenotype. The model describes how cytokines, nutrients and oxygen availability regulate the differentiation of naïve CD4+ T cells into distinct subsets. Particularly, it shows that elevated amounts of an all-type mixture of effector cytokines under optimal nutrient and oxygen availability conduces the system towards a highly-polarized Th1 or Th2 state, while reduced cytokine levels allow the expression of the Th17, Treg or T FH subsets, or even hybrid phenotypes. On the other hand, optimal levels of an all-type cytokine mixture in combination with glutamine or tryptophan restriction implies a shift from Th1 to Th2 expression, while decreased levels of the Th2-inducing cytokine IL-4 leads to the rupture of the Th1-Th2 axis, allowing the manifestation of different (or hybrid) subsets. Modeling proposes that, even under reduced levels of pro-inflammatory cytokines, the sole action of hypoxia boost Th17 expression.

Hypoxia-Inducible Factors Signaling in Osteogenesis and Skeletal Repair.

Sufficient oxygen is required to maintain normal cellular and physiological function, such as a creature’s development, breeding, and homeostasis. Lately, some researchers have reported that both pathological hypoxia and environmental hypoxia might affect bone health. Adaptation to hypoxia is a pivotal cellular event in normal cell development and differentiation and in pathological settings such as ischemia. As central mediators of homeostasis, hypoxia-inducible transcription factors (HIFs) can allow cells to survive in a low-oxygen environment and are essential for the regulation of osteogenesis and skeletal repair. From this perspective, we summarized the role of HIF-1 and HIF-2 in signaling pathways implicated in bone development and skeletal repair and outlined the molecular mechanism of regulation of downstream growth factors and protein molecules such as VEGF, EPO, and so on. All of these present an opportunity for developing therapies for bone regeneration.

Anther development-The long road to making pollen.

Anthers express the most genes of any plant organ, and their development involves sequential redifferentiation of many cell types to perform distinctive roles from inception through pollen dispersal. Agricultural yield and plant breeding depend on understanding and consequently manipulating anthers, a compelling motivation for basic plant biology research to contribute. After stamen initiation, two theca form at the tip, and each forms an adaxial and abaxial lobe composed of pluripotent Layer 1-derived and Layer 2-derived cells. After signal perception or self-organization, germinal cells are specified from Layer 2-derived cells, and these secrete a protein ligand that triggers somatic differentiation of their neighbors. Historically, recovery of male-sterile mutants has been the starting point for studying anther biology. Many genes and some genetic pathways have well-defined functions in orchestrating subsequent cell fate and differentiation events. Today, new tools are providing more detailed information; for example, the developmental trajectory of germinal cells illustrates the power of single cell RNA-seq to dissect the complex journey of one cell type. We highlight ambiguities and gaps in available data to encourage attention on important unresolved issues.

Population Structure of Finless Porpoise (Neophocaena phocaenoides) Discovered off Coastal Waters, Republic of Korea.

The finless porpoise (Neophocaena phocaenoides Cuvier, 1829) is distributed in the coastal waters of Asia, throughout Indonesia to the east, and as far north as the Taiwan Strait. The finless porpoise has been declared critically endangered by the WWF (World Wide Fund for Nature), and in 2017 was rated vulnerable on the IUCN Red Threatened Species List. Since this species is distributed near the coast and has many interactions with humans, effective conservation of the species requires further studies into their genetic diversity and population. In this study, 45 samples were obtained from bycatch or stranded individuals in the East, South, and West Seas, where Korean porpoises were mainly distributed from 2017–2021. We compared 473 bp mtDNA sequences from the control region. Pairwise fixation indices (FST) revealed that the two populations differed significantly (FST = 0.4557, p = 0.000). In contrast to high levels of genetic differentiation, gene flow was identified as medium levels (Nm of 0.04–0.71). Our data suggest that finless porpoises may have undergone a historic differentiation event, and that finless porpoises in the three regions could be divided into two populations: West and East/South.

Expression of microRNA let-7 in cleavage embryos modulates cell fate determination and formation of mouse blastocysts†.

After fertilization, the zygote undergoes cell division. Up to the 8-cell stage, the blastomeres of mouse preimplantation embryos are morphologically identical. The first cell differentiation starts in the morula leading to the formation of trophectoderm cells and inner cell mass cells of the blastocyst. The regulation of the differentiation event and the formation of blastocysts are not fully known. Lethal-7 (let-7) is a family of evolutionarily conserved microRNAs. Here, we showed that the expression of let-7a and let-7g decreased drastically from the 1-cell stage to the 2-cell stage, remained low up to the 8-cell stage and slightly increased after the morula stage of mouse embryos. The expression of let-7 in the inner cell mass was higher than that in the trophectoderm. Forced expression of let-7a in embryos at the 1-cell and 4-cell stage inhibited blastocyst formation and downregulated the expression of CDX2 but maintained that of OCT4 in the trophectoderm. Forced expression of other let-7 isoforms exhibited similar inhibitory action on blastulation. On the other hand, inhibition of let-7a at the 4-cell stage and the 8-cell stage enhanced blastocyst formation. Co-injection of green fluorescent protein (GFP) mRNA (lineage tracer) with either precursor of let-7a (pre-let-7a) or scramble control into one blastomere of 2-cell embryos showed that ~75% of the resulting blastocysts possessed GFP+ cells in their inner cell mass only. The biased development towards the inner cell mass with forced expression of let-7 was reproduced in 2-cell chimeric embryos consisting of one wildtype blastomere and one GFP mRNA-injected blastomere from another 2-cell embryo carrying a doxycycline-inducible let-7g gene. Bioinformatics analysis indicated that Tead4 was a potential target of let-7. Let-7 bound to the 3'UTR of Tead4 and let-7 forced expression downregulated the expression of Tead4 in mouse blastocysts. Co-injection of Tead4 mRNA partially nullified the modulatory roles of let-7a in the inner cell mass cell fate. In conclusion, a high level of let-7 at the 2-cell stage favored the formation of the inner cell mass, whereas a low level of let-7 at the 4-cell to 8-cell stage enhanced blastocyst formation. Tead4 mediated the action of let-7 on the inner cell mass cell-fate determination.

Evidence for Protracted Intracrustal Reworking of Palaeoarchaean Crust in the Pilbara Craton (Mount Edgar Dome, Western Australia)

Abstract ~3.5-2.8 Ga granitoids from the Pilbara Craton in Western Australia are one of the most ancient and best-preserved records of early processes of continental crust generation. A number of recent studies have focused on the nature of the mantle source from which Pilbara granitoids derived, yet no consensus has been reached on whether the mantle was chondritic or depleted in the Eo/Palaeoarchaean. Here we present integrated whole-rock (major and trace elements) and zircon (U-Pb and Lu-Hf isotopes) data for 10 granitoids sampled across the Mount Edgar Dome, which recorded four main magmatic events between 3.47 and 3.23 Ga. Whole-rock major and trace element analyses suggest that the samples belong to two distinct petrogenetic groups. The first group is akin to the tonalite-trondhjemite-granodiorite (TTG) suite, representing highly fractionated magmas initially formed by partial melting of a basaltic crust. The second group, here classified as granites, is best interpreted by the remelting of a basaltic crust and the addition of more evolved material, and it is striking that TTG-like and granitic magmas occurred coevally in time and space. Overall, both groups were formed through intense intracrustal differentiation processes that lead to the loss of significant geochemical information about their original sources. High-precision Lu-Hf analyses in zircon allow to obtain such information and to trace back the isotopic composition of the Palaeoarchaean mantle. A clear change from superchondritic to subchondritic Hf isotope compositions is observed between 3.47 and 3.23 Ga. The superchondritic Hf isotope composition of the 3.47 Ga old granitoids substantiates derivation from a depleted mantle source that separated from the chondritic mantle prior to 3.8 Ga. The presence of ca. 3.5 Ga old inherited zircons in younger magmas suggests that crustal remelting processes were involved in their generation. We propose that all granitoids investigated in this study had their crustal sources originated from a single mantle–crust differentiation event that occurred at 3.50 Ga. This event resulted in the differentiation, from the same original mantle, of two distinct crustal reservoirs, i.e., a mafic reservoir with a 176Lu/177Hf ratio of 0.023, and a reservoir of intermediate/felsic composition ( 176Lu/ 177Hf=0.013). 3.32-3.31 Ga-old granitoids were produced by remelting of the mafic reservoir, whereas 3.43 and 3.23 Ga granitoids derived from the intermediate/felsic reservoir. Overall, our data suggest that protracted intracrustal remelting processes and differentiation have played a key role in the formation, evolution, and maturation of the building blocks of continents during the Palaeoarchaean.

Abstract P2060: Comprehensive Analysis Of 3’ End Sequencing Reveals Novel Polyadenylation Signals In Human Left Ventricle Failure

Background: Alternative polyadenylation (APA) regulates the genes post-transcriptionally and generates mRNA isoforms with different lengths of 3’ untranslated regions (3’UTR). APA dictates diverse cellular processes such as development, differentiation, metabolism, and protein localization. The dysregulation of APA leading to global 3’UTR changes has been well studied in several human diseases including cancer. However, the role of APA in the progression of cardiac fibrosis and eventually the left ventricle (LV) heart failure in patients remains elusive. Objective: To identify whether APA regulates the 3’UTR length in the failing LV hearts compared to the healthy hearts. Methods and Results: We used Poly(A)-Click-Seq to sequence the 3’ end of the transcripts and the PolyA-miner algorithm to comprehensively quantify the APA events in healthy and failing human LV specimens. We used the Poly-A index, a metric that determines the magnitude and position of the 3’UTR changes, to determine the shortening versus lengthening of 3′UTRs. Based on these scores, we identified more than 1000 genes with a significant shift in cleavage site usage in failing LV hearts compared to healthy. Moreover, the enrichment of these differentially regulated APA candidates revealed pathways involved in the progression of heart failure and molecular functions related to mRNA binding. So, by examining the differential polyadenylation events in these hearts, we identified disease-specific APA signatures. In addition, we also found the dysregulation in the core APA machinery proteins such as cleavage factor Im (CFIm) 25, 59, and 68 in the LV failing hearts compared to the healthy. Conclusion: Hence, we demonstrated that the regulation of the 3’UTR length through APA provides an extra layer of gene expression in human LV failure. Our results provide genome-wide polyadenylation maps of the human heart and show that APA of mRNA is dynamic in the progression of LV failure.

Dynamic switching of lateral inhibition spatial patterns.

Hes genes are transcriptional repressors activated by Notch. In the developing mouse neural tissue, HES5 expression oscillates in neural progenitors (Manning et al. 2019 Nat. Commun. 10, 1–19 (doi:10.1038/s41467-019-10734-8)) and is spatially organized in small clusters of cells with synchronized expression (microclusters). Furthermore, these microclusters are arranged with a spatial periodicity of three–four cells in the dorso-ventral axis and show regular switching between HES5 high/low expression on a longer time scale and larger amplitude than individual temporal oscillators (Biga et al. 2021 Mol. Syst. Biol. 17, e9902 (doi:10.15252/msb.20209902)). However, our initial computational modelling of coupled HES5 could not explain these features of the experimental data. In this study, we provide theoretical results that address these issues with biologically pertinent additions. Here, we report that extending Notch signalling to non-neighbouring progenitor cells is sufficient to generate spatial periodicity of the correct size. In addition, introducing a regular perturbation of Notch signalling by the emerging differentiating cells induces a temporal switching in the spatial pattern, which is longer than an individual cell’s periodicity. Thus, with these two new mechanisms, a computational model delivers outputs that closely resemble the complex tissue-level HES5 dynamics. Finally, we predict that such dynamic patterning spreads out differentiation events in space, complementing our previous findings whereby the local synchronization controls the rate of differentiation.

Variability of cross-tissue X-chromosome inactivation characterizes timing of human embryonic lineage specification events.

X-chromosome inactivation (XCI) is a random, permanent, and developmentally early epigenetic event that occurs during mammalian embryogenesis. We harness these features to investigate characteristics of early lineage specification events during human development. We initially assess the consistency of X-inactivation and establish a robust set of XCI-escape genes. By analyzing variance in XCI ratios across tissues and individuals, we find that XCI is shared across all tissues, suggesting that XCI is completed in the epiblast (in at least 6-16 cells) prior to specification of the germ layers. Additionally, we exploit tissue-specific variability to characterize the number of cells present during tissue-lineage commitment, ranging from approximately 20 cells in liver and whole blood tissues to 80 cells in brain tissues. By investigating the variability of XCI ratios using adult tissue, we characterize embryonic features of human XCI and lineage specification that are otherwise difficult to ascertain experimentally.