Gene Expression Dynamics Research Articles

Rethinking eco‐evo studies of gene expression for non‐model organisms in the genomic era

AbstractRecent advances in genomic technology, including the rapid development of long‐read sequencing technology and single‐cell RNA‐sequencing methods, are poised to significantly expand the kinds of studies that are feasible in ecological genomics. In this perspective, we review these new technologies and discuss their potential impact on gene expression studies in non‐model organisms. Although traditional RNA‐sequencing methods have been an extraordinarily powerful tool to apply functional genomics in an ecological context, bulk RNA‐seq approaches often rely on de novo transcriptome assembly, and cannot capture expression changes in rare cell populations or distinguish shifts in cell type abundance. Advancements in genome assembly technology, particularly long‐read sequencing, and improvements in the scalability of single‐cell RNA‐sequencing (scRNA‐seq) offer unprecedented resolution in understanding cellular heterogeneity and gene regulation. We discuss the potential of these technologies to enable disentangling differential gene regulation from cell type composition differences and uncovering subtle expression patterns masked by bulk RNA‐seq. The integration of these approaches provides a more nuanced understanding of the ecological and evolutionary dynamics of gene expression, paving the way for refined models and deeper insights into the generation of biodiversity.

M2WISH: An easy and efficient protocol for whole‐mount mRNAin situ hybridization that allows 3D cell resolution of gene expression in Arabidopsis thaliana

SUMMARYGene expression analysis is essential for understanding the mechanisms involved in plant development. Here, we developed M2WISH, a protocol based on MicroWave treatment for Wholemount mRNA In Situ Hybridization in Arabidopsis. By permeabilizing tissues without damaging cellular organization this protocol results in high and homogeneous hybridization yields that enable systematic analysis of gene expression dynamics. Moreover, when combined with cellular histochemical staining, M2WISH successfully provides a cellular resolution of gene expression. Thus, we demonstrate the robustness of M2WISH with 10 genes on roots, aerial meristems, leaves, and embryos in the seed. We applied M2WISH to study the spatial dynamics of WUSCHEL (WUS) and CLAVATA3 (CLV3) expression during in vitro meristematic conversion of roots into shoot apical meristems. Thus, we showed that shoot apical meristems could arise from two different types of root structures that differed by their CLV3 gene expression patterns. We constructed 3D cellular representations of WUS and CLV3 gene co‐expression pattern and stressed the variability inherent to meristem conversion. Thus, this protocol generates a large amount of data on the localization of gene expression, which can be used to model complex systems.

Biclustering analysis on tree-shaped time-series single cell gene expression data of Caenorhabditis elegans

BackgroundIn recent years, gene clustering analysis has become a widely used tool for studying gene functions, efficiently categorizing genes with similar expression patterns to aid in identifying gene functions. Caenorhabditis elegans is commonly used in embryonic research due to its consistent cell lineage from fertilized egg to adulthood. Biologists use 4D confocal imaging to observe gene expression dynamics at the single-cell level. However, on one hand, the observed tree-shaped time-series datasets have characteristics such as non-pairwise data points between different individuals. On the other hand, the influence of cell type heterogeneity should also be considered during clustering, aiming to obtain more biologically significant clustering results.ResultsA biclustering model is proposed for tree-shaped single-cell gene expression data of Caenorhabditis elegans. Detailedly, a tree-shaped piecewise polynomial function is first employed to fit non-pairwise gene expression time series data. Then, four factors are considered in the objective function, including Pearson correlation coefficients capturing gene correlations, p-values from the Kolmogorov-Smirnov test measuring the similarity between cells, as well as gene expression size and bicluster overlapping size. After that, Genetic Algorithm is utilized to optimize the function.ConclusionThe results on the small-scale dataset analysis validate the feasibility and effectiveness of our model and are superior to existing classical biclustering models. Besides, gene enrichment analysis is employed to assess the results on the complete real dataset analysis, confirming that the discovered biclustering results hold significant biological relevance.

Dynamics of pax7 expression during development, muscle regeneration, and in vitro differentiation of satellite cells in rainbow trout (Oncorhynchus mykiss).

Essential for muscle fiber formation and hypertrophy, muscle stem cells, also called satellite cells, reside beneath the basal lamina of the muscle fiber. Satellite cells have been commonly identified by the expression of the Paired box 7 (Pax7) due to its specificity and the availability of antibodies in tetrapods. In fish, the identification of satellite cells remains difficult due to the lack of specific antibodies in most species. Based on the development of a highly sensitive in situ hybridization (RNAScope®) for pax7, we showed that pax7+ cells were detected in the undifferentiated myogenic epithelium corresponding to the dermomyotome at day 14 post-fertilization in rainbow trout. Then, from day 24, pax7+ cells gradually migrated into the deep myotome and were localized along the muscle fibers and reach their niche in satellite position of the fibres after hatching. Our results showed that 18 days after muscle injury, a large number of pax7+ cells accumulated at the wound site compared to the uninjured area. During the in vitro differentiation of satellite cells, the percentage of pax7+ cells decreased from 44% to 18% on day 7, and some differentiated cells still expressed pax7. Taken together, these results show the dynamic expression of pax7 genes and the follow-up of these muscle stem cells during the different situations of muscle fiber formation in trout.

Single-cell transcriptome profiling reveals the spatiotemporal distribution of triterpenoid saponin biosynthesis and transposable element activity in Gynostemma pentaphyllum shoot apexes and leaves

Gynostemma pentaphyllum (Thunb.) Makino is an important producer of dammarene-type triterpenoid saponins. These saponins (gypenosides) exhibit diverse pharmacological benefits such as anticancer, antidiabetic, and immunomodulatory effects, and have major potential in the pharmaceutical and health care industries. Here, we employed single-cell RNA sequencing (scRNA-seq) to profile the transcriptomes of more than 50,000 cells derived from G. pentaphyllum shoot apexes and leaves. Following cell clustering and annotation, we identified five major cell types in shoot apexes and four in leaves. Each cell type displayed substantial transcriptomic heterogeneity both within and between tissues. Examining gene expression patterns across various cell types revealed that gypenoside biosynthesis predominantly occurred in mesophyll cells, with heightened activity observed in shoot apexes compared to leaves. Furthermore, we explored the impact of transposable elements (TEs) on G. pentaphyllum transcriptomic landscapes. Our findings the highlighted the unbalanced expression of certain TE families across different cell types in shoot apexes and leaves, marking the first investigation of TE expression at the single-cell level in plants. Additionally, we observed dynamic expression of genes involved in gypenoside biosynthesis and specific TE families during epidermal and vascular cell development. The involvement of TE expression in regulating cell differentiation and gypenoside biosynthesis warrant further exploration. Overall, this study not only provides new insights into the spatiotemporal organization of gypenoside biosynthesis and TE activity in G. pentaphyllum shoot apexes and leaves but also offers valuable cellular and genetic resources for a deeper understanding of developmental and physiological processes at single-cell resolution in this species.

LSD2 Is an Epigenetic Player in Multiple Types of Cancer and Beyond

Histone demethylases, enzymes responsible for removing methyl groups from histone proteins, have emerged as critical players in regulating gene expression and chromatin dynamics, thereby influencing various cellular processes. LSD2 and LSD1 have attracted considerable interest among these demethylases because of their associations with cancer. However, while LSD1 has received significant attention, LSD2 has not been recognized to the same extent. In this study, we conduct a comprehensive comparison between LSD2 and LSD1, with a focus on exploring LSD2’s implications. While both share structural similarities, LSD2 possesses unique features as well. Functionally, LSD2 shows diverse roles, particularly in cancer, with tissue-dependent roles. Additionally, LSD2 extends beyond histone demethylation, impacting DNA methylation, cancer cell reprogramming, E3 ubiquitin ligase activity and DNA damage repair pathways. This study underscores the distinct roles of LSD2, providing insights into their contributions to cancer and other cellular processes.

USING FOETAL SKELETOGENESIS TO EVALUATE AND IMPROVE IN VITRO CHONDROGENESIS PROTOCOLS

The reliable production of _in vitro_ chondrocytes that faithfully recapitulate _in vivo_ development would be of great benefit for orthopaedic disease modelling and regenerative therapy(1,2). Current efforts are limited by off-target differentiation, resulting in a heterogeneous product, and by the lack of comparison to human tissue, which precludes detailed evaluation of _in vitro_ cells(3,4).We performed single-cell RNA-sequencing of long bones dissected from first-trimester fetal limbs to form a detailed ‘atlas’ of endochondral ossification. Through 100-gene in-situ sequencing, we placed each sequenced cell type into its anatomical context to spatially resolve the process of endochondral ossification. We then used this atlas to perform deconvolution on a series of previously published bulk transcriptomes generated from _in vitro_ chondrogenesis protocols to evaluate their ability to accurately produce chondrocytes.We then applied single-nuclear RNA-sequencing to cells from the best performing protocol collected at multiple time points to allow direct comparison between the differentiation of _in vitro_ and _in vivo_ cells.We captured 275,000 single fetal cells, profiling the development of chondrocytes from multipotent mesenchymal progenitors to hypertrophic cells at full transcriptomic breadth. Using this atlas as the ground truth for evaluating _in vitro_ cells, we found substantial variability in cell states produced by each protocol, with many showing little similarity to _in vivo_ cells, and all exhibiting off-target differentiation.Trajectory alignment between _in vivo_ and _in vitro_ single-cell data revealed key differences in gene expression dynamics between _in vitro_ and _in vivo cells,_ with several osteoblastic transcription factors erroneously unregulated _in vitro,_ including _FOXO1._Using this information, we inhibited _FOXO1_ in culture to successfully increase chondrocyte yield _in vitro._This study presents a new framework for evaluating tissue engineering protocols, using single-cell data to drive improvement and bring the prospect of true engineered cartilage closer to reality.

SlNAC3 suppresses cold tolerance in tomatoes by enhancing ethylene biosynthesis.

Low temperature stress poses a significant challenge to the productivity of horticultural crops. The dynamic expression of cold-responsive genes plays a crucial role in plant cold tolerance. While NAC transcription factors have been extensively studied in plant growth and development, their involvement in regulating plant cold tolerance remains poorly understood. In this study, we focused on the identification and characterisation of SlNAC3 as the most rapid and robust responsive gene in tomato under low temperature conditions. Manipulating SlNAC3 through overexpression or silencing resulted in reduced or enhanced cold tolerance, respectively. Surprisingly, we discovered a negative correlation between the expression of CBF and cold tolerance in the SlNAC3 transgenic lines. These findings suggest that SlNAC3 regulates tomato cold tolerance likely through a CBF-independent pathway. Furthermore, we conducted additional investigations to identify the molecular mechanisms underlying SINAC3-mediated cold tolerance in tomatoes. Our results revealed that SlNAC3 controls the transcription of ethylene biosynthetic genes, thereby bursting ethylene release in response to cold stress. Indeed, the silencing of these genes led to an augmentation in cold tolerance. This discovery provides valuable insights into the regulatory pathways involved in ethylene-mediated cold tolerance in tomatoes, offering potential strategies for developing innovative approaches to enhance cold stress resilience in this economically important crop species.

Unveiling the Dynamics behind Glioblastoma Multiforme Single-Cell Data Heterogeneity.

Glioblastoma Multiforme is a brain tumor distinguished by its aggressiveness. We suggested that this aggressiveness leads single-cell RNA-sequence data (scRNA-seq) to span a representative portion of the cancer attractors domain. This conjecture allowed us to interpret the scRNA-seq heterogeneity as reflecting a representative trajectory within the attractor's domain. We considered factors such as genomic instability to characterize the cancer dynamics through stochastic fixed points. The fixed points were derived from centroids obtained through various clustering methods to verify our method sensitivity. This methodological foundation is based upon sample and time average equivalence, assigning an interpretative value to the data cluster centroids and supporting parameters estimation. We used stochastic simulations to reproduce the dynamics, and our results showed an alignment between experimental and simulated dataset centroids. We also computed the Waddington landscape, which provided a visual framework for validating the centroids and standard deviations as characterizations of cancer attractors. Additionally, we examined the stability and transitions between attractors and revealed a potential interplay between subtypes. These transitions might be related to cancer recurrence and progression, connecting the molecular mechanisms of cancer heterogeneity with statistical properties of gene expression dynamics. Our work advances the modeling of gene expression dynamics and paves the way for personalized therapeutic interventions.

The effect of adipose-derived mesenchymal stem cell transplantation on ovarian mitochondrial dysfunction in letrozole-induced polycystic ovary syndrome in rats: the role of PI3K-AKT signaling pathway

ObjectiveThe present study aimed to elucidate how mesenchymal stem cells (MSCs) application could efficiently attenuate pathological changes of letrozole-induced poly cystic ovary syndrome (PCOS) by modulating mitochondrial dynamic via PI3K-AKT pathway.MethodsThirty-two female rats were randomly divided into four experimental groups: Sham, PCOS, PCOS + MSCs, and PCOS + MSCs + LY294002. The Sham group received 0.5% w/v carboxymethyl cellulose (CMC); the PCOS group received letrozole (1 mg/kg, daily) in 0.5% CMC for 21 days. Animals in the PCOS + MSCs group received 1 × 106 MSCs/rat (i.p,) on the 22th day of the study. In the PCOS + MSCs + LY294002 group, rats received LY294002 (PI3K-AKT inhibitor) 40 min before MSC transplantation. Mitochondrial dynamic gene expression, mitochondrial membrane potential (MMP), citrate synthase (CS) activity, oxidative stress, inflammation, ovarian histological parameters, serum hormone levels, homeostatic model assessment for insulin resistance (HOMA-IR), insulin and glucose concentrations, p-PI3K and p-AKT protein levels were evaluated at the end of the experiment.ResultsPCOS rats showed a significant disruption of mitochondrial dynamics and histological changes, lower MMP, CS, ovary super oxide dismutase (SOD) and estrogen level. They also had a notable rise in insulin and glucose concentrations, HOMA-IR, testosterone level, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels, ovarian malondialdehyde (MDA) content as well as a notable decrease in p-PI3K and p-AKT protein levels compared to the Sham group. In the PCOS + MSCs group, the transplantation of MSCs could improve the above parameters. Administration of LY294002 (PI3K-AKT pathway inhibitor) deteriorated mitochondrial dynamic markers, oxidative stress status, inflammation markers, hormonal levels, glucose, and insulin levels and follicular development compared to the PCOS + MSCs group.ConclusionsThis study demonstrated that the protective effects of MSC transplantation in regulating mitochondrial dynamics, promoting mitochondrial biogenesis, competing with redox status and inflammation response were mainly mediated through the PI3K-AKT pathway in the PCOS model.

Transcriptomic and lipidomic analysis of the differential pathway contribution to the incorporation of erucic acid to triacylglycerol during Pennycress seed maturation.

Thlaspi arvense (Pennycress) is an emerging feedstock for biofuel production because of its high seed oil content enriched in erucic acid. A transcriptomic and a lipidomic study were performed to analyze the dynamics of gene expression, glycerolipid content and acyl-group distribution during seed maturation. Genes involved in fatty acid biosynthesis were expressed at the early stages of seed maturation. Genes encoding enzymes of the Kennedy pathway like diacylglycerol acyltransferase1 (TaDGAT1), lysophosphatidic acid acyltransferase (TaLPAT) or glycerol 3-phosphate acyltransferase (TaGPAT) increased their expression with maturation, coinciding with the increase in triacylglycerol species containing 22:1. Positional analysis showed that the most abundant triacylglycerol species contained 18:2 at sn-2 position in all maturation stages, suggesting no specificity of the lysophosphatidic acid acyltransferase for very long chain fatty acids. Diacylglycerol acyltransferase2 (TaDGAT2) mRNA was more abundant at the initial maturation stages, coincident with the rapid incorporation of 22:1 to triacylglycerol, suggesting a coordination between Diacylglycerol acyltransferase enzymes for triacylglycerol biosynthesis. Genes encoding the phospholipid-diacylglycerol acyltransferase (TaPDAT1), lysophosphatidylcholine acyltransferase (TaLPCAT) or phosphatidylcholine diacylglycerolcholine phosphotransferase (TaPDCT), involved in acyl-editing or phosphatidyl-choline (PC)-derived diacylglycerol (DAG) biosynthesis showed also higher expression at the early maturation stages, coinciding with a higher proportion of triacylglycerol containing C18 fatty acids. These results suggested a higher contribution of these two pathways at the early stages of seed maturation. Lipidomic analysis of the content and acyl-group distribution of diacylglycerol and phosphatidyl-choline pools was compatible with the acyl content in triacylglycerol at the different maturation stages. Our data point to a model in which a strong temporal coordination between pathways and isoforms in each pathway, both at the expression and acyl-group incorporation, contribute to high erucic triacylglycerol accumulation in Pennycress.

H2O2-Induced Oxidative Stress Responses in Eriocheir sinensis: Antioxidant Defense and Immune Gene Expression Dynamics

Eriocheir sinensis, a key species in China’s freshwater aquaculture, is threatened by various diseases, which were verified to be closely associated with oxidative stress. This study aimed to investigate the response of E. sinensis to hydrogen peroxide (H2O2)-induced oxidative stress to understand the biological processes behind these diseases. Crabs were exposed to different concentrations of H2O2 and their antioxidant enzyme activities and gene expressions for defense and immunity were measured. Results showed that activities of antioxidant enzymes—specificallysuperoxide dismutase (SOD), catalase (CAT), total antioxidant capacity(T-AOC), glutathione (GSH), and glutathione peroxidase (GSH-Px)—varied with exposure concentration and duration, initially increasing then decreasing. Notably, SOD, GSH-Px, and T-AOC activities dropped below control levels at 96 h. Concurrently, oxidative damage markers, including malondialdehyde (MDA), H2O2, and 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels, increased with exposure duration. The mRNA expression of SOD, CAT, and GSH-Px also showed an initial increase followed by a decrease, peaking at 72 h. The upregulation of phenoloxidaseloxidase (proPO) and peroxinectin (PX) was also detected, but proPO was suppressed under high levels of H2O2. Heat shock protein 70 (HSP70) expression gradually increased with higher H2O2 concentrations, whereas induced nitrogen monoxide synthase (iNOS) was upregulated but decreased at 96 h. These findings emphasize H2O2’s significant impact on the crab’s oxidative and immune responses, highlighting the importance of understanding cellular stress responses for disease prevention and therapy development.

Cell coupling compensates for changes in single-cell Her6 dynamics and provides phenotypic robustness.

We investigate the effect of altering the protein expression dynamics of the bHLH transcription factor Her6 at the single-cell level in the embryonic zebrafish telencephalon. Using an homozygote endogenous Her6:Venus reporter and 4D single-cell tracking, we show that Her6 oscillates in neural telencephalic progenitors and that fusion of protein destabilisation domain (PEST) alters its expression dynamics, causing most cells to downregulate Her6 prematurely. However, counterintuitively, oscillatory cells increase with some expressing Her6 at high levels, resulting in increased heterogeneity of Her6 expression in the population. These tissue-levels changes appear to be an emergent property of coupling between single-cells as revealed by experimentally disrupting Notch signalling and by computationally modelling alterations in Her6 protein stability. Despite the profound differences in the single-cell Her6 dynamics, the size of the telencephalon is only transiently altered, and differentiation markers do not exhibit significant differences early on, while a small increase is observed at later developmental stages. Our study suggests that cell coupling provides a compensation strategy, whereby an almost normal phenotype is maintained even though single-cell gene expression dynamics are abnormal, granting phenotypic robustness.

Dynamic transcriptome profiling revealed a key gene ZmJMJ20 and pathways associated with cadmium stress in maize

Cadmium (Cd) pollution in soil poses a global concern due to its serious impacts on human health and ecological security. In plants, tremendous efforts have been made to identify some key genes and pathways in Cd stress responses. However, studies on the roles of epigenetic factors in response to Cd stress were still limited. In the study, we first gain insight into the gene expression dynamics for maize seedlings under 0 h, 12 h, and 72 h Cd stress. As a result, six distinct groups of genes were identified by hierarchical clustering and principal component analysis. The key pathways associated with 12 h Cd stress were protein modifications including protein ubiquitination, signal transduction by protein phosphorylation, and histone modification. Whereas, under 72 h stress, main pathways were involved in biological processes including phenylalanine metabolism, response to oxygen-containing compounds and metal ions. Then to be noted, one of the most highly expressed genes at 12 h under Cd treatment is annotated as histone demethylases (ZmJMJ20). The evolutionary tree analysis and domain analysis showed that ZmJMJ20 belonged to the JmjC-only subfamily of the Jumonji-C (JmjC) family, and ZmJMJ20 was conserved in rice and Arabidopsis. After 72 h of Cd treatment, the zmjmj20 mutant created by EMS treatment manifested less severe chlorosis/leaf yellowing symptoms compared with wild-type plants, and there was no significant difference in Fv/Fm and φPSII value before and after Cd treatment. Moreover, the expression levels of several photosynthesis-related down-regulated genes in EMS mutant plants were dramatically increased compared with those in wild-type plants at 12 h under Cd treatment. Our results suggested that ZmJMJ20 plays an important role in the Cd tolerance response pathway and will facilitate the development of cultivars with improved Cd stress tolerance.

Investigation of post-immunization immune response gene expression kinetics in lymphoid tissues of White Leghorn and Indian native chicken

Understanding the basal expression kinetics of genes related to innate immunity aids effective genetic selection for immune response and how host genetics behaves with tolerance to Newcastle disease (ND) is obscure. It was hence aimed to investigate post-immunization mRNA expression kinetics of some immune response genes in White Leghorn, Aseel and Kadaknath chicken. Day-old chicks were vaccinated with ND RDF-1 strain with booster dose at 28-days of age at the experimental layer farm of ICAR-Central Avian Research Institute, Izatnagar (India) and lymphoid tissues were collected in RNAlater at 42-days of age. Total RNA was extracted and first strand cDNA was synthesized. Relative quantification of mRNA expression of IL1-β, IFN-γ, iNOS and TLR15 gene was assessed by qRT-PCR. The mRNA expressions significantly varied among the genes in spleen and thymus, and moderate expression of IFN-γ in all chicken genotypes suggested good protection against Newcastle disease virus. IL1-β-mRNA expressed at the highest level (P˂ 0.05) in spleen followed by thymus and bursa, whereas IFN-γ and iNOS expressed highly (P˂ 0.05) in thymus followed by spleen and bursa. In case of TLR15, the expression was significantly the highest in spleen followed by bursa and thymus. Kadaknath had the highest (P˂ 0.05) expression of IL1-β in thymus and that of iNOS in spleen, whereas Aseel showed the least (P > 0.05) expression than Kadaknath and White Leghorn. The study revealed the existence of wide variation in basal expression levels of immune response genes among different lymphoid tissues and lack of discernible differences in the expression between the chicken genotypes.

Comprehensive genomic features indicative for Notch responsiveness.

Transcription factor RBPJ is the central component in Notch signal transduction and directly forms a coactivator complex together with the Notch intracellular domain (NICD). While RBPJ protein levels remain constant in most tissues, dynamic expression of Notch target genes varies depending on the given cell-type and the Notch activity state. To elucidate dynamic RBPJ binding genome-wide, we investigated RBPJ occupancy by ChIP-Seq. Surprisingly, only a small set of the total RBPJ sites show a dynamic binding behavior in response to Notch signaling. Compared to static RBPJ sites, dynamic sites differ in regard to their chromatin state, binding strength and enhancer positioning. Dynamic RBPJ sites are predominantly located distal to transcriptional start sites (TSSs), while most static sites are found in promoter-proximal regions. Importantly, gene responsiveness is preferentially associated with dynamic RBPJ binding sites and this static and dynamic binding behavior is repeatedly observed across different cell types and species. Based on the above findings we used a machine-learning algorithm to predict Notch responsiveness with high confidence in different cellular contexts. Our results strongly support the notion that the combination of binding strength and enhancer positioning are indicative of Notch responsiveness.

Dynamics of CD44+ bovine nucleus pulposus cells with inflammation

Intervertebral Disc (IVD) degeneration has been associated with a chronic inflammatory response, but knowledge on the contribution of distinct IVD cells, namely CD44, to the progression of IVD degeneration remains elusive. Here, bovine nucleus pulposus (NP) CD44 cells were sorted and compared by gene expression and proteomics with the negative counterpart. NP cells were then stimulated with IL-1b (10 ng/ml) and dynamics of CD44 gene and protein expression was analyzed upon pro-inflammatory treatment. The results emphasize that CD44 has a multidimensional functional role in IVD metabolism, ECM synthesis and production of neuropermissive factors. CD44 widespread expression in NP was partially associated with CD14 and CD45, resulting in the identification of distinct cell subsets. In conclusion, this study points out CD44 and CD44-based cell subsets as relevant targets in the modulation of the IVD pro-inflammatory/degenerative cascade.

Unveiling Gene Expression Dynamics during Early Embryogenesis in Cynoglossus semilaevis: A Transcriptomic Perspective.

Commencing with sperm-egg fusion, the early stages of metazoan development include the cleavage and formation of blastula and gastrula. These early embryonic events play a crucial role in ontogeny and are accompanied by a dramatic remodeling of the gene network, particularly encompassing the maternal-to-zygotic transition. Nonetheless, the gene expression dynamics governing early embryogenesis remain unclear in most metazoan lineages. We conducted transcriptomic profiling on two types of gametes (oocytes and sperms) and early embryos (ranging from the four-cell to the gastrula stage) of an economically valuable flatfish-the Chinese tongue sole Cynoglossus semilaevis (Pleuronectiformes: Cynoglossidae). Comparative transcriptome analysis revealed that large-scale zygotic genome activation (ZGA) occurs in the blastula stage, aligning with previous findings in zebrafish. Through the comparison of the most abundant transcripts identified in each sample and the functional analysis of co-expression modules, we unveiled distinct functional enrichments across different gametes/developmental stages: actin- and immune-related functions in sperms; mitosis, transcription inhibition, and mitochondrial function in oocytes and in pre-ZGA embryos (four- to 1000-cell stage); and organ development in post-ZGA embryos (blastula and gastrula). These results provide insights into the intricate transcriptional regulation of early embryonic development in Cynoglossidae fish and expand our knowledge of developmental constraints in vertebrates.

Longitudinal profiling of human androgenotes through single-cell analysis unveils paternal gene expression dynamics in early embryo development.

How do transcriptomics vary in haploid human androgenote embryos at single cell level in the first four cell cycles of embryo development? Gene expression peaks at the fourth cell cycle, however some androcytes exhibit unique transcriptional behaviors. The developmental potential of an embryo is determined by the competence of the oocyte and the sperm. However, studies of the contribution of the paternal genome using pure haploid androgenotes are very scarce. This study was performed analyzing the single-cell transcriptomic sequencing of 38 androcytes obtained from 10 androgenote bioconstructs previously produced in vitro (de Castro et al., 2023). These results were analyzed through different bioinformatics software such as g: Profiler, GSEA, Cytoscape, and Reactome. Single cell sequencing was used to obtain the transcriptomic profiles of the different androcytes. The results obtained were compared between the different cycles studied using the DESeq2 program and functional enrichment pathways using g: Profiler, Cytoscape, and Reactome. A wave of paternally driven transcriptomic activation was found during the third-cell cycle, with 1128 upregulated and 225 downregulated genes and the fourth-cell cycle, with 1373 upregulated and 286 downregulated genes, compared to first-cell cycle androcytes. Differentially expressed routes related to cell differentiation, DNA-binding transcription, RNA biosynthesis and RNA polymerase II transcription regulatory complex, and cell death were found in the third and fourth with respect to the first-cell cycle. Conversely, in the fourth cell cycle, 153 downregulated and 332 upregulated genes were found compared with third cell cycle, associated with differentially expressed processes related to E-box binding and zinc finger protein 652 (ZNF652) transcription factor. Further, significant overexpression of LEUTX, PRAMEF1, DUXA, RFPL4A, TRIM43, and ZNF675 found in androgenotes, compared to biparental embryos, highlights the paternal contributions to zygote genome activation. All raw sequencing data are available through the Gene Expression Omnibus (GEO) under accessions number: GSE216501. Extrapolation of biological events from uniparental constructs to biparental embryos should be done with caution. Maternal and paternal genomes do not act independently of each other in a natural condition. The absence of one genome may affect gene transcription of the other. In this sense, the haploid condition of the bioconstructs could mask the transcriptomic patterns of the single cells. The results obtained demonstrated the level of involvement of the human paternal haploid genome in the early stages of embryo development as well as its evolution at the transcriptomic level, laying the groundwork for the use of these bioconstructs as reliable models to dispel doubts about the genetic role played by the paternal genome in the early cycles of embryo development. This study was funded by Instituto de Salud Carlos III (ISCIII) through the project 'PI22/00924', co-funded by European Regional Development Fund (ERDF); 'A way to make Europe'. F.D. was supported by the Spanish Ministry of Economy and Competitiveness through the Miguel Servet program (CPII018/00002). M.J.E. was supported by Instituto de Salud Carlos III (PI19/00577 [M.J.E.]) and FI20/00086. P.dC. was supported by a predoctoral grant for training in research into health (PFIS PI19/00577) from the Instituto de Salud Carlos III. All authors declare having no conflict of interest with regard to this trial.

Activated and Naïve Allogenic Human Placental Mesenchymal Stromal Cells Exert an Immunomodulatory Effect on Hidradenitis Suppurativa Patient Peripheral Blood Mononuclear Cells.

This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from patients with hidradenitis suppurativa (HS). Blood samples were collected from 3 healthy and 3 patients with HS. Isolated PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin solution. The PBMCs of patients with HS were co-cultured with naïve MSCs (n-MSCs), activated with tumor necrosis factor (TNF)-α (10 ng/mL) and interferon (IFN)-γ (10 ng/mL) MSCs (a-MSCs), or adalimumab (30 μg/mL). The division index (proliferation inhibition) of PBMCs was analyzed by flow cytometry using the Proliferation Modeling tool after 5 days of coculture. The relative inflammatory gene expression dynamics and cytokine secretion were quantified in triplicate using real-time polymerase chain reaction (PCR) and Luminex assays. PBMCs from the HS control group showed statistically significant increases in interleukin (IL)-6 and IFN-γ cytokine concentrations and IL-17A gene expression when compared with healthy subjects. Statistically significant reduction of the division index was found in the a-MSCs group (P = 0.04). Also, the Luminex assay revealed significantly reduced proinflammatory cytokine concentrations of IL-9 (P = 0.022) and IL-17A (P = 0.022) in the a-MSCs group with the same trend of numerical lowering in n-MSCs group when compared to HS control. The results of real-time PCR revealed a numerical increase in the expression of the IL-1β, IL-36α, and TNF-α genes in both the a-MSCs and n-MSCs groups compared with the HS control. In conclusion, our findings suggest that MSCs can effectively curb PBMCs proliferation and suppress the production of inflammatory cytokines. Moreover, the preactivation of MSCs with IFN-γ and TNF-α before use can enhance their therapeutic effectiveness. Nevertheless, a larger sample size is imperative to validate these results.