Pluripotency Factors Research Articles

Blastocoel expansion and AMOT degradation cooperatively promote YAP nuclear localization during epiblast formation

The epiblast is a pluripotent cell population formed in the late blastocyst stage of preimplantation embryos. During the process of epiblast formation from the inner cell mass (ICM) of the early blastocyst, activation of the Hippo pathway transcription factor TEAD by the nuclear translocation of the coactivator protein YAP is required for the robust expression of pluripotency factors. However, the mechanisms that alter YAP localization during epiblast formation remain unknown. Here, we reveal two such mechanisms. Expansion of the blastocoel promotes nuclear YAP localization by increasing cytoplasmic F-actin and reducing YAP phosphorylation. Additionally, cell differentiation regulates YAP. Expression of the junctional Hippo component, AMOT, gradually decreases during epiblast formation through a tankyrase-mediated degradation. SOX2 expression in the ICM is necessary for the reduction of AMOT and YAP phosphorylation. These two mechanisms function in parallel. Thus, the blastocoel–F-actin and SOX2–AMOT axes cooperatively suppress YAP phosphorylation and promote YAP nuclear localization during epiblast formation. The cooperation of these two distinct mechanisms likely contributes to the robustness of epiblast cell differentiation.

Overexpression of miR-192 Inhibits In Vitro Porcine Embryo Development by Inducing Oxidative Stress Damage and Impairing Mitochondrial Function.

Early embryonic development relies on intricately regulated gene expression, and miRNAs influence zygotic genome activation (ZGA), cleavage, and cell fate determination through post-transcriptional regulatory mechanisms. miR-192 is expressed in early pig embryos and participates in various reproductive processes. However, its role in pre-implantation pig embryo development remains poorly understood. In this study, we microinjected the miR-192 agonist (miR-192 agomir) into parthenogenetically activated pig embryos to evaluate its effects on early pig embryo development. Our findings indicate that compared to the control group (agomir NC), miR-192 agomir impairs the developmental capacity of parthenogenetic pig embryos to reach the 2-cell, 4-cell, and blastocyst stages. This impairment leads to imbalances in the oxidative-reductive system and abnormalities in mitochondrial function during the 4-cell stage, resulting in the significant accumulation of ROS, notable decreases in the expression of antioxidant enzymes CAT and SOD1 mRNA, reduction in mitochondrial membrane potential, and induction of apoptosis in pig blastocysts. Additionally, the overexpression of miR-192 inhibits the expression of its target genes YY1 and the pluripotency factor NANOG mRNA. In conclusion, this study reveals that the overexpression of miR-192 adversely affects early pig embryo development, providing new evidence for understanding the role miR-192 plays in reproduction.

Fate specification triggers a positive feedback loop of TEAD-YAP and NANOG to promote epiblast formation in preimplantation embryos.

In preimplantation embryos, epiblast (EPI) fate specification from the inner cell mass is controlled by the segregation of NANOG and GATA6 expression. TEAD-YAP interaction is activated during EPI formation, and is required for pluripotency factor expression. These events occur asynchronously with similar timing during EPI formation, and their relationship remains elusive. Here, we examined the relationship between NANOG-GATA6 and TEAD-YAP. The nuclear accumulation of YAP takes place only in EPI-specified cells, and a positive feedback loop operates between NANOG and TEAD-YAP. The effects of TEAD-YAP on SOX2 upregulation in EPI-specified cells are likely indirect. EPI fate specification also alters the response of Nanog, Sox2 and Cdx2 to TEAD-YAP. These results suggest that EPI-fate specification alters the transcriptional network from the morula-like to the EPI-specified state and activates TEAD-YAP to trigger a positive feedback loop with NANOG, which stabilizes the EPI fate. The coordinated occurrence of these processes in individual cells likely supports proper EPI formation under the condition of asynchronous EPI-fate specification.

LINE-1-Induced Retrotransposition Affects Early Preimplantation Embryo DNA Integrity and Pluripotency.

Retrotransposable elements are implicated in genome rearrangements and gene expression alterations that result in various human disorders. In the current study, we sought to investigate the potential effects of long interspersed elements-1 (LINE-1) overexpression on the integrity and methylation of DNA and on the expression of three major pluripotency factors (OCT4, SOX2, NANOG) during the preimplantation stages of human embryo development. Human MI oocytes were matured in vitro to MII and transfected through intracytoplasmic sperm injection (ICSI) either with an EGFP vector carrying a cloned active human LINE-1 retroelement or with the same EGFP vector without insert as control. The occurrence of retrotransposition events was screened by fluorescent microscopy. The in vitro preimplantation development as well as the methylation, pluripotency, and DNA double-strand breaks (DSBs) of the transfected embryos were examined. LINE-1 retrotransposons gave rise to new retrotransposition events in the transfected embryos. LINE-1 injected embryos were characterized by accelerated asymmetrical cell division, multiple cellular fragments, cleavage arrest, and degeneration. Early OCT4 expression remained unaltered, but cleavage arrest and a high fragmentation rate hindered the expression of SOX2/NANOG at the morula stage. Increased DNA DSBs were observed in cleavage-stage blastomeres, while no methylation changes were detected before the cleavage arrest. Our data provide evidence that LINE-1 retrotransposition in human preimplantation embryos may induce DNA DSBs, while at the same time, it appears to interfere with the expression patterns of pluripotency factors. The morphological, structural, and cleavage abnormalities of the transfected embryos show that aberrant retroelement expression may negatively affect human embryo development.

A PERTURBATION CELL ATLAS OF HUMAN INDUCED PLURIPOTENT STEM CELLS.

Towards comprehensively investigating the genotype-phenotype relationships governing the human pluripotent stem cell state, we generated an expressed genome-scale CRISPRi Perturbation Cell Atlas in KOLF2.1J human induced pluripotent stem cells (hiPSCs) mapping transcriptional and fitness phenotypes associated with 11,739 targeted genes. Using the transcriptional phenotypes, we created a minimum distortion embedding map of the pluripotent state, demonstrating rich recapitulation of protein complexes, such as strong co-clustering of MRPL, BAF, SAGA, and Ragulator family members. Additionally, we uncovered transcriptional regulators that are uncoupled from cell fitness, discovering potential novel pluripotency (JOSD1, RNF7) and metabolic factors (ZBTB41). We validated these findings via phenotypic, protein-interaction, and metabolic tracing assays. Finally, we propose a contrastive human-cell engineering framework (CHEF), a machine learning architecture that learns from perturbation cell atlases to predict perturbation recipes that achieve desired transcriptional states. Taken together, our study presents a comprehensive resource for interrogating the regulatory networks governing pluripotency.

UBR-5 and UBE2D mediate timely exit from stem fate via destabilization of poly(A)-binding protein PABP-2 in cell state transition

UBR5 E3 ligase has been associated with cancer susceptibility and neuronal integrity, with functions in chromatin regulation and proteostasis. However, the functions of ubr5 within animals remain unclear due to lethality in both mammals and flies when disrupted. Using Caenorhabditis elegans, we show that UBR-5 E3 ligase is required for timely exit of stem fate and complete transition into multiple cell type descendants in an ectodermal blast lineage. Animals lacking intact UBR-5 function simultaneously exhibit both stem fate and differentiated fate in the same descendant cells. A functional screen of UBR-5 physical interactors allowed us to identify the UBE2D2/3 E2 conjugase LET-70 working with UBR-5 to exit stem fate. Strikingly, we revealed that another UBR-5 physical interactor, namely the nuclear poly(A)-binding protein PABPN1 ortholog PABP-2, worked antagonistically to UBR-5 and LET-70. Lowering pabp-2 levels restored normal transition of cell state out of stemness and promoted normal cell fusion when either ubr-5 or let-70 UBE2D function was compromised. The UBR-5-LET-70 and PABP-2 switch works independently of the stem pool size determined by pluripotency factors like lin-28. UBR-5 limits PABP-2 protein and reverses the PABP-2-dependent gene expression program including developmental, proteostasis, and innate immunity genes. Loss of ubr-5 rescues the developmental stall when pabp-2 is compromised. Disruption of ubr-5 elevates PABP-2 levels and prolongs expression of ectodermal and muscle stem markers at the transition to adulthood. Additionally, ubr-5 mutants exhibit an extended period of motility during aging and suppress pabp-2-dependent early onset of immobility.

7704 Generating pituitary cells from iPSCs of patients with child onset congenital hypopituitarism harboring PROP1 pathogenic allelic variants

Abstract Disclosure: J.M. Marques: None. F. Fattahi: None. L.R. Carvalho: None. Introduction: Reduced or absent levels of pituitary hormones due to genetic or organic causes is known as combined pituitary hormone deficiency (CPHD) and PROP1 is the most frequent cause of CPHD, but more than 85% of the cases are without molecular diagnosis. Novel Techniques such as induced pluripotent stem cells (iPSC), can be used to generate patient’s specific pituitary cells for disease modeling. Aim: To generate iPSC from CO-CH patients’ peripheral blood mononuclear cells (PBMC) and differentiate them into pituitary cells. Methods: Peripheral blood was collected from 2 siblings (HC1 and HC2) harboring compound heterozygous PROP1 allelic variants and their parents (HC3 and HC4). PBMCs were isolated and expanded with MNC medium prior to nucleoporation and transfection with plasmids containing pluripotency genes (OCT4 and SOX2; cMYC and KLF). After 48h, transfected cells were transferred to Geltrex coated plates and fed with E8, NaB and βFGF until pluripotent colonies were formed. iPSCs were differentiated into cranial placode cells using pituitary placode conditioned medium (PPCM) until day 15. For pituitary cell maturation, pituitary conditioned medium (PPCM without SB431542) was used until day 30. qPCR and flow cytometry using the Kit BD Stemflow - Human Pluripotent Stem Cell Transcription Factor Analysis (BD Biosciences) for stem cells and the eBioscience™ Foxp3 / Transcription Factor Fixation/Permeabilization (Thermo Fisher Scientific) for the placode and pituitary markers were used. Pituitary hormonal levels from cell supernatant were measured at the Laboratory of Hormones and Molecular Genetics - HCFMUSP. Results: we generated iPSCs from patient and control blood cells. iPSCs were more than 90% positive for the pluripotency markers SOX2, NANOG, OCT3/4. Pituitary cells were generated with positivity for the placode marker SIX1 (58%) and pituitary markers, as LHX3 (56%) and PROP1 (59%), at day 15. Control cell lineages expressed higher levels of pituitary hormones at days 15 and 30 at RNA and protein levels while patient cells failed to induce hormone-producing cells properly. Pituitary hormonal release was impaired in patients pituitary cells. Conclusion: CO-CH patients’ PBMC were dedifferentiated to a pluripotent state, being able to generate pituitary hormone producing cells. Patient cells were able to mimic human phenotype. These cells can be used, in the future, to study CO-CH basis in patients specific background, to develop pituitary disease modeling as well as test possible new treatments. Presentation: 6/3/2024

Delayed Blastocyst Formation Reduces the Quality and Hatching Ability of Porcine Parthenogenetic Blastocysts by Increasing DNA Damage, Decreasing Cell Proliferation, and Altering Transcription Factor Expression Patterns.

The purpose of this study was to investigate the influence of blastocyst formation timing on the quality of porcine embryos derived from parthenogenetic activation. Newly formed blastocysts at days 6, 7, and 8 of culture [termed formation 6, 7, and 8 blastocysts (F6, F7, and F8 blastocysts)] were obtained, and a series of parameters related to the quality of blastocysts, including apoptosis incidents, DNA replication, pluripotent factors, and blastocyst hatching capacity, were assessed. Delayed blastocyst formation (F7 and/or F8 blastocysts) led to increased levels of ROS, DNA damage, and apoptosis while decreasing the mitochondrial membrane potential, DNA replication, Oct4 levels, and numbers of Sox2-positive cells. F7 blastocysts showed a significantly reduced hatching rate compared to F6 blastocysts; however, F8 blastocysts were unable to develop to the hatching stage. Collectively, our findings suggest a negative correlation between delayed blastocyst formation and blastocyst quality.

Modeling high-risk Wilms tumors enables the discovery of therapeutic vulnerability

Wilms tumor (WT) is the most common pediatric kidney cancer treated with standard chemotherapy. However, less-differentiated blastemal type of WT often relapses. To model the high-risk WT for therapeutic intervention, we introduce pluripotency factors into WiT49, a mixed-type WT cell line, to generate partially reprogrammed cells, namely WiT49-PRCs. When implanted into the kidney capsule in mice, WiT49-PRCs form kidney tumors and develop both liver and lung metastases, whereas WiT49 tumors do not metastasize. Histological characterization and gene expression signatures demonstrate that WiT49-PRCs recapitulate blastemal-predominant WTs. Moreover, drug screening in isogeneic WiT49 and WiT49-PRCs leads to the identification of epithelial- or blastemal-predominant WT-sensitive drugs, whose selectivity is validated in patient-derived xenografts (PDXs). Histone deacetylase (HDAC) inhibitors (e.g., panobinostat and romidepsin) are found universally effective across different WT and more potent than doxorubicin in PDXs. Taken together, WiT49-PRCs serve as a blastemal-predominant WT model for therapeutic intervention to treat patients with high-risk WT.

A-180 Pluripotency Factor DPPA4 in Cell lines of T-Cell Lymphoma

A-180 Pluripotency Factor DPPA4 in Cell lines of T-Cell Lymphoma

Resveratrol inhibits Lin28A expression and induces its degradation via the proteasomal pathway in NCCIT cells.

Lin28A is an oncoprotein overexpressed in several cancer types such as testicular, ovarian, colon, breast and lung cancers. As a pluripotency factor that promotes tumorigenesis, Lin28A is associated with more undifferentiated and aggressive tumors phenotypes. Moreover, Lin28A is a highly stable protein that is difficult to downregulate. The compound resveratrol (RSV) has anticancer effects. The present study aimed to elucidate the mechanisms underlying the downregulation of Lin28A protein expression by RSV in the NCCIT cell line. NCCIT cells were treated with different concentrations of RSV to investigate its effects on Lin28A expression. The mRNA expression levels of Lin28A and ubiquitin-specific protease 28 (USP28) were assessed using reverse transcription-quantitative PCR. Western blot analysis was employed to evaluate the protein levels of Lin28A, USP28 and phosphorylated Lin28A. In addition, in some experiments, cells were treated with a MAPK/ERK pathway inhibitor, and other experiments involved transfecting cells with small interfering RNAs targeting USP28. The results demonstrated that RSV significantly reduced Lin28A expression by destabilizing the protein; this effect was mediated by the ability of RSV to suppress the expression of USP28, a deubiquitinase that normally protects Lin28A from ubiquitination and degradation. Additionally, RSV inhibited phosphorylation of Lin28A via the MAPK/ERK pathway; this phosphorylation event has previously been shown to enhance the stability of Lin28A by increasing its half-life. This resulted in Lin28A degradation through the proteasomal pathway in NCCIT cells. The results provide further evidence of the anticancer activity of RSV, and identified Lin28A and USP28 as promising therapeutic targets. As a stable oncoprotein, downregulating Lin28A expression is challenging. However, the present study demonstrated that RSV can overcome this hurdle by inhibiting USP28 expression and MAPK/ERK signaling to promote Lin28A degradation. Furthermore, elucidating these mechanisms provides avenues for developing targeted cancer therapies.

A live single-cell reporter system reveals drug-induced plasticity of a cancer stem cell-like population in cholangiocarcinoma.

Cancer stem cells (CSC) play an important role in carcinogenesis and are acknowledged to be responsible for chemoresistance in cholangiocarcinoma (CCA). Studying CCA CSC has been challenging, due to lack of consensus CSC markers, and to their plastic nature. Since dual expression of the core pluripotent factors SOX2/OCT4 has been shown to correlate with poor outcome in CCA patients, we selected the SOX2/OCT4 activating short half-life GFP-based live reporter (SORE6-dsCopGFP) to study CSC dynamics at the single-cell level. Transduction of five human CCA cell lines resulted in the expression of 1.8-13.1% GFP-positive (SORE6POS) cells. By live imaging, we found that SORE6POS CCA cells possess self-renewal capacity and that they can be induced to differentiate. Significantly, the SORE6POS cells were highly tumorigenic, both in vitro and in vivo, thus implicating the characteristics of primary CSCs. When we then analyzed for selected CSC-related markers, we found that the majority of both CD133+/CD44+, and CD133+/LGR5+ CCA cells were SORE6POS cells. Exposing transduced cells to standard CCA chemotherapy revealed higher growth rate inhibition at 50% (GR50s) for SORE6POS cells compared to GFP-negative (SORE6NEG) ones indicating that these CSC-like cells were more resistant to the treatment. Moreover, the chemotherapy induced SORE6POS from SORE6NEG cells, while retaining the existing SORE6POS population. Finally, treatment of transduced cells with CDK4/6 inhibitors in vitro for 3 days resulted in a lowered CSC number in the culture. Thus, applying a live reporter system allowed us to elucidate the stem cell diversity and drug-induced plasticity of CCA CSCs. These findings have clear implications for future management of such patients.

Viral-mediated Oct4 overexpression and inhibition of Notch signaling synergistically induce neurogenic competence in mammalian Müller glia.

Retinal Müller glia in cold-blooded vertebrates can reprogram into neurogenic progenitors to replace neurons lost to injury, but mammals lack this ability. While recent studies have shown that transgenic overexpression of neurogenic bHLH factors and glial-specific disruption of NFI family transcription factors and Notch signaling induce neurogenic competence in mammalian Müller glia, induction of neurogenesis in wildtype glia has thus far proven elusive. Here we report that viral-mediated overexpression of the pluripotency factor Oct4 ( Pou5f1 ) induces transdifferentiation of wildtype mouse Müller glia into bipolar neurons and stimulates this process synergistically in parallel with Notch loss of function. Single cell multiomic analysis shows that Oct4 overexpression leads to widespread changes in gene expression and chromatin accessibility, inducing activity of both the neurogenic transcription factor Rfx4 and the Yamanaka factors Sox2 and Klf4. This study demonstrates that viral-mediated overexpression of Oct4 induces neurogenic competence in wildtype retinal Müller glia, identifying mechanisms that could be used in cell-based therapies for treating retinal dystrophies.

NCOA3 knockdown delays human embryo development

Embryonic development is a precisely controlled sequential process influenced by complex external and internal factors; therefore, this process holds paramount significance in the context of in vitro fertilization and embryo transfer (IVF-ET), with internal oocyte and embryo quality being pivotal in determining its success. Nuclear receptor coactivator 3 (NCOA3), a member of the p160 nuclear receptor coactivators family, has been extensively studied in tumorigenesis and reportedly plays a crucial role in maintaining pluripotency in mouse embryonic stem cells (ESCs). However, its functions in human embryo development remain largely unexplored. In this study, we collected human samples, including oocytes, zygotes, and embryos, from patients at the First Affiliated Hospital of Zhengzhou University to investigate whether NCOA3 regulates human embryonic development. To this end, we employed various assays, including immunofluorescence, quantitative real-time PCR (qPCR), microinjection, and RNA sequencing. Our findings suggested that NCOA3 expression level was low in inferior embryos (with >50 % fragmentation), and its presence is closely related to the expression of the pluripotency factor NANOG. Deletion of NCOA3 delays human embryonic development. Single-oocyte RNA sequencing revealed that NCOA3 primarily participates in metabolic alterations in oocytes. In sum, these findings elucidate the pivotal roles of NCOA3 in human embryonic development-NCOA3 deletion compromise the developmental potential of embryos. These mechanistic insights into the role of NCOA3 in human embryonic development not only advances our understanding of the intricate molecular mechanisms involved but also holds potential implications for improving assisted reproductive technologies (ART) and addressing developmental disorders in human embryos.

Genome access is transcription factor-specific and defined by nucleosome position

Mammalian gene expression is controlled by transcription factors (TFs) that engage sequence motifs in a chromatinized genome, where nucleosomes can restrict DNA access. Yet, how nucleosomes affect individual TFs remains unclear. Here, we measure the ability of over one hundred TF motifs to recruit TFs in a defined chromosomal locus in mouse embryonic stem cells. This identifies a set sufficient to enable the binding of TFs with diverse tissue specificities, functions, and DNA-binding domains. These chromatin-competent factors are further classified when challenged to engage motifs within a highly phased nucleosome. The pluripotency factors OCT4-SOX2 preferentially engage non-nucleosomal and entry-exit motifs, but not nucleosome-internal sites, a preference that also guides binding genome wide. By contrast, factors such as BANP, REST, or CTCF engage throughout, causing nucleosomal displacement. This supports that TFs vary widely in their sensitivity to nucleosomes and that genome access is TF specific and influenced by nucleosome position in the cell.

H3K4me2 distinguishes a distinct class of enhancers during the maternal-to-zygotic transition.

After egg fertilization, an initially silent embryonic genome is transcriptionally activated during the maternal-to-zygotic transition. In zebrafish, maternal vertebrate pluripotency factors Nanog, Pou5f3 (OCT4 homolog), and Sox19b (SOX2 homolog) (NPS) play essential roles in orchestrating embryonic genome activation, acting as "pioneers" that open condensed chromatin and mediate acquisition of activating histone modifications. However, some embryonic gene transcription still occurs in the absence of these factors, suggesting the existence of other mechanisms regulating genome activation. To identify chromatin signatures of these unknown pathways, we profiled the histone modification landscape of zebrafish embryos using CUT&RUN. Our regulatory map revealed two subclasses of enhancers distinguished by presence or absence of H3K4me2. Enhancers lacking H3K4me2 tend to require NPS factors for de novo activation, while enhancers bearing H3K4me2 are epigenetically bookmarked by DNA hypomethylation to recapitulate gamete activity in the embryo, independent of NPS pioneering. Thus, parallel enhancer activation pathways combine to induce transcriptional reprogramming to pluripotency in the early embryo.

Applications of Amniotic Membrane in Ophthalmology – New Perspectives in the Treatment of Eye Diseases

Amniotic membrane, a natural protective barrier surrounding the fetus, possesses unique biological properties, making it a promising tool in regenerative medicine. It contains pluripotent cells, collagen, anti-inflammatory cytokines, and growth factors. This article aims to present the current state of knowledge regarding the potential applications of amniotic membrane in ophthalmology, as well as the benefits and challenges associated with this promising therapy. Amniotic membrane, used in the form of a graft or extract, exhibits anti-inflammatory effects, accelerates tissue healing, and regenerates corneal epithelium. In ophthalmology, amniotic membrane is used in the treatment of corneal ulcers, limbal stem cell deficiency, burns, strabismus, glaucoma, and neoplastic changes. It can also be used in the reconstruction of the ocular surface and in vitreoretinal surgery. Studies demonstrate its effectiveness in improving symptoms of dry eye syndrome. Additionally, new methods of combining amniotic membrane with the ocular surface are being introduced. Amniotic membrane is also used as a carrier in stem cell culture. The development of technology and research on amniotic membrane lead to the discovery of new applications and the improvement of surgical techniques, opening up prospects for even broader utilization of its potential in the future.

Stemness and hybrid epithelial-mesenchymal profiles guide peritoneal dissemination of malignant mesothelioma and pseudomyxoma peritonei.

Intrabdominal dissemination of malignant mesothelioma (MM) and pseudomyxoma peritonei (PMP) is poorly characterized with respect to the stemness window which malignant cells activate during their reshaping on the epithelial-mesenchymal (E/M) axis. To gain insights into stemness properties and their prognostic significance in these rarer forms of peritoneal metastases (PM), primary tumor cultures from 55 patients selected for cytoreductive surgery with hyperthermic intraperitoneal chemotherapy were analyzed for cancer stem cells (CSC) by aldehyde dehydrogenase 1 (ALDH1) and spheroid formation assays, and for expression of a set of plasticity-related genes to measure E/M transition (EMT) score. Intratumor heterogeneity was also analyzed. Samples from PM of colorectal cancer were included for comparison. Molecular data were confirmed using principal component and cluster analyses. Associations with survival were evaluated using Kaplan-Meier and Cox regression models. The activity of acetylsalicylic acid (ASA), a stemness modifier, was tested in five cultures. Significantly increased amounts of ALDH1bright-cells identified high-grade PMP, and discriminated solid masses from ascitic/mucin-embedded tumor cells in both forms of PM. Epithelial/early hybrid EMT scores and an early hybrid expression pattern correlated with pluripotency factors were significantly associated with early peritoneal progression (p = .0343 and p = .0339, respectively, log-rank test) in multivariable models. ASA impaired spheroid formation and increased cisplatin sensitivity in all five cultures. These data suggest that CSC subpopulations and hybrid E/M states may guide peritoneal spread of MM and PMP. Stemness could be exploited as targetable vulnerability to increase chemosensitivity and improve patient outcomes. Additional research is needed to confirm these preliminary data.

The ribonucleoprotein complex factor Ybx1 stabilizes the maternal mRNA of the <i>ssx2ip</i> gene encoding the centrosome maturation protein in <i>Xenopus laevis</i> embryogenesis

Our study investigates the mechanisms that regulate early developmental gene expression in Xenopus laevis frog embryos. Our previous study demonstrated that maternal mRNAs of two developmentally significant genes, the nuclear retinoic acid receptor rxrg and the pluripotency factor pou5f3, form complexes with ribonucleoprotein complex Ybx1. Based on the results of the present study, we determined and demonstrated that the stability of the maternal mRNA ssx2ip, which encodes a conserved protein, also called Msd1 or ADIP, which is involved in centrosome maturation, is dependent on Ybx1. This research shows that Ybx1 forms a ribonucleoprotein complex with ssx2ip mRNA, which is mediated by its cold shock domain (CSD). This study confirms our hypothesis of Ybx1 selectively binding to maternal transcripts. It opens up new opportunities to study new mechanisms of gene expression regulation at the earliest stages of development by searching for possible cis-motifs for recognition by trans-regulators such as Ybx1.

Bone morphogenetic protein-3 is a negative regulator of transforming growth factor beta and fibrosis

Fibrosis results in one-third of all deaths globally and is a major healthcare challenge. Fibrosis is scarring caused by the excess deposition of extracellular matrix proteins by fibroblasts. Inhibition of pathways downstream of transforming growth factor β (TGF-β) a pluripotent growth factor, has potent antifibrotic effects in different organs. Here we show that loss of bone morphogenetic protein (BMP-3) is a feature of kidney fibrosis, independent of the initiating injury, suggesting loss of this cytokine is a core fibrotic mechanism. TGF-β decreased BMP3 expression in human fibroblasts is possibly a feed-forward loop that contributes to increased and sustained TGF-β activity. Recombinant human BMP-3 reduced TGF-β induced fibroblast contraction, migration and invasion, pathways that lead to scarring and tissue stiffening. BMP-3 reduced TGF-β stimulated collagen cross-linking, and Ox-LDL receptor 1, a regulator of collagen deposition. BMP-3 inhibited TGF-β stimulated lysyl oxidase activity. Lysyl oxidase mediated collagen cross-linking is a critical process in TGF-β induced fibrosis. We propose that BMP-3 alters fibroblast responses to TGF-β, shifting the balance from fibrosis to repair. Recombinant human BMP-3 shows promise for development as a novel therapeutic for fibrosis.