Expression Of Nkx2 Research Articles

Differential regulation of a placental SAM68 and sFLT1 gene pathway and the relevance to maternal vitamin D sufficiency.

The goal of this study was to determine if an axis of placental gene expression associated with early onset and severe preeclampsia (EOSPE) was operative in term pregnancy and correlated with vitamin D sufficiency. qPCR analysis of NKX2-5, SAM68, sFLT1 and membrane bound VEGFR1/FLT1 mRNA expression was conducted in placentas from 43 subjects enrolled in a vitamin D3 pregnancy supplementation trial. Pair-wise rank order correlations between patient-specific gene expression levels were calculated, and their relationship to maternal 25(OH)D status was assessed by a two-sample Wilcoxon test. Additionally, we probed the mechanistic link between SAM68 and sFLT1 using siRNA depletion in a human trophoblast cell line model. Positive and highly significant correlations were found between SAM68 vs. sFLT1 and SAM68 vs. FLT1 expression levels, as were significant and differential correlations between the expression of these genes and perinatal 25(OH)D status. The variability when stratified by race/ethnicity was qualitatively distinct from those previously observed in EOSPE. Mechanistic studies confirmed a functional role for SAM68 protein in the regulation of sFLT1 expression. NKX2-5 expression was not significantly correlated with sFLT1 or SAM68 expression in these samples, suggesting that its expression may be significant at earlier stages of pregnancy or be restricted to pathological settings. These data further support our overarching hypothesis that SAM68 expression is a key determinant of VEGFR1 isoform expression in the placenta, and provide additional insights into how this gene pathway may be differentially deployed or modified in normal and pathological pregnancies.

Axenfeld-Rieger syndrome-associated mutants of the transcription factor FOXC1 abnormally regulate NKX2-5 in model zebrafish embryos

FOXC1 is a member of the forkhead family of transcription factors, and whose function is poorly understood. A variety of FOXC1 mutants have been identified in patients diagnosed with the autosomal dominant disease Axenfeld-Rieger syndrome, which is mainly characterized by abnormal development of the eyes, particularly those who also have accompanying congenital heart defects (CHD). However, the role of FOXC1 in CHD, and how these mutations might impact FOXC1 function, remains elusive. Our previous work provided one clue to possible function, demonstrating that zebrafish foxc1a, an orthologue of human FOXC1 essential for heart development, directly regulates the expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells. Abnormal expression of Nkx2-5 leads to CHD in mice and is also associated with CHD patients. Whether this link extends to the human system, however, requires investigation. In this study, we demonstrate that FOXC1 does regulate human NKX2-5 expression in a dose-dependent manner via direct binding to its proximal promoter. A comparison of FOXC1 mutant function in the rat cardiac cell line H9c2 and zebrafish embryos suggested that the zebrafish embryos might serve as a more representative model system than the H9c2 cells. Finally, we noted that three of the Axenfeld-Rieger syndrome FOXC1 mutations tested increased, whereas a fourth repressed the expression of NKX2-5 These results imply that mutant FOXC1s might play etiological roles in CHD by abnormally regulating NKX2-5 in the patients. And zebrafish embryos can serve as a useful in vivo platform for rapidly evaluating disease-causing roles of mutated genes.

A missense mutation of ErbB2 produces a novel mouse model of stillbirth associated with a cardiac abnormality but lacking abnormalities of placental structure.

BackgroundIn humans, stillbirth describes the death of a fetus before birth after 28 weeks gestation, and accounts for approximately 2.6 million deaths worldwide annually. In high-income countries, up to half of stillbirths have an unknown cause and are described as “unexplained stillbirths”; this lack of understanding impairs efforts to prevent stillbirth. There are also few animal models of stillbirth, but those that have been described usually have significant placental abnormalities. This study describes a novel mutant murine model of fetal death with atrial conduction block due to an ErbB2 missense mutation which is not associated with abnormal placental morphology.MethodsPhenotypic characterisation and histological analysis of the mutant mouse model was conducted. The mRNA distribution of the early cardiomyocyte marker Nkx2-5 was assessed via in situ hybridisation. Cardiac structure was quantified and cellular morphology evaluated by electron microscopy. Immunostaining was employed to quantify placental structure and cell characteristics on matched heterozygous and homozygous mutant placental samples.ResultsThere were no structural abnormalities observed in hearts of mutant embryos. Comparable Nkx2-5 expression was observed in hearts of mutants and controls, suggesting normal cardiac specification. Additionally, there was no significant difference in the weight, placenta dimensions, giant cell characteristics, labyrinth tissue composition, levels of apoptosis, proliferation or vascularisation between placentas of homozygous mutant mice and controls.ConclusionEmbryonic lethality in the ErbB2 homozygous mutant mouse cannot be attributed to placental pathology. As such, we conclude the ErbB2M802R mutant is a model of stillbirth with a non-placental cause of death. The mechanism of the atrial block resulting from ErbB2 mutation and its role in embryonic death is still unclear. Studying this mutant mouse model could identify candidate genes involved in stillbirth associated with structural or functional cardiac defects.

Second heart field-specific expression of Nkx2-5 requires promoter proximal interaction with Srf

The cardiac homeobox transcription factor Nkx2-5 is a major determinant of cardiac identity and cardiac morphogenesis. Nkx2-5 operates as part of a complex and mutually reinforcing network of early transcription factors of the homeobox, GATA zinc finger and MADS domain families to initiate the program of cardiac development and differentiation, particularly in outflow tract precursor cells in the second heart field (SHF). We have now found evidence for another aspect of cardiac transcription factor cooperativity between Nkx2-5 and the cardiac enriched MADS domain transcription factor Srf. Specifically, Srf interaction with an evolutionarily conserved binding site in the Nkx2-5 CpG island-like proximal promoter is required for cardiac specific expression mediated by an SHF enhancer, and for combinatorial activation of these elements by cardiac transcription factors. These results provide further insight into cooperative gene regulation during cardiogenesis at the level of promoter-enhancer interactions.

P6011NKX2-5 contributes to EndoMT and endothelial dysfunction in pulmonary arterial hypertension

Abstract Background The onset of inflammation, hypoxia or shear stress within blood vessels can result in endothelial-to-mesenchymal transition (EndoMT), a disease-associated process where endothelial cells (ECs) downregulate endothelial markers and acquire mesenchymal features. EndoMT is observed in patients with scleroderma-associated pulmonary hypertension (SSc-PAH), which have the highest mortality amongst all the scleroderma patient subgroups. The homeobox transcriptional factor NKX2-5 is fundamental for cardiovascular development. However, NKX2-5 expression has not been reported yet in ECs of adult pulmonary blood vessels. Purpose To investigate the role of NKX2-5 in the pulmonary endothelium of SSc-PAH. Methods Human pulmonary artery endothelial cells (HPAECs) were treated with a cocktail of TGF-β (5 ng/mL), TNF-α (5 ng/mL), and IL-1β (0.1 ng/mL) for 5 days. Immunofluorescence was used to detect NKX2-5 and other markers in ECs. Western blotting and qPCR evaluated, respectively, protein and gene expression. Lentiviral transduction forced NKX2-5 expression in the cells. Transendothelial electrical resistance (TEER) measurements evaluated endothelial barrier function. Pharmacological inhibition was performed to determine the pathways that lead to NKX2-5 activation. Casein kinase 2 (CK2)-inhibition (CX4945) of a chronic hypoxia mouse model of PAH was used to assess right ventricular systolic pressure (RVSP). Results Immunofluorescence showed a strong expression of NKX2-5 in the endothelium of SSc-PAH human lungs (p<0.0001). Western blot analysis demonstrated a 5.3-fold downregulation of CD31 (p<0.001), and an increased production of NKX2-5 (5.6-fold, p<0.0001) and of Procollagen I (12-fold, p=0.0009) after 5 days of cytokine stimulation on HPAECs. Relative mRNA expression has shown a 3-fold gene downregulation of CD31 (p=0.0002) and a reduction of VE-Cadherin (2.3-fold, p=0.0008) and of vWF (10.4-fold, p=0.003) in EndoMT, whereas gene expression of COL1α2 (8.5-fold, p<0.0001) and of NKX2-5 (1.5-fold, p=0.003) were upregulated. Immunofluorescence of cells has revealed a decreased VE-Cadherin expression concomitant with upregulation of NKX2-5 in EndoMT cells. Forced expression of NKX2-5 downregulated endothelial markers and endothelial barrier function was impaired whereas proliferation rate of cells was increased. Inhibition of PI3K, ERK5, ALK5 and CK2 reduced NKX2-5 protein expression within cells. CK2-inhibited mice under hypoxia conditions resembled the normoxia mice group by normalising RVSP. Conclusion HPAECs undergoing EndoMT express NKX2-5 in vitro and in vivo, via mediation of CK2, TGF-β, ERK5 and PI3K signalling. NKX2-5 downregulates key adherence junctional proteins, disrupting endothelial barrier function. This study highlights the involvement of NKX2-5 in EndoMT and in endothelial dysfunction, leading to vascular disease progression in SSc-PAH. Acknowledgement/Funding British Heart Foundation, Arthritis Research UK, Scleroderma Research UK and Royal Free Hospital Charity

Effects of cigarette smoke components on myocardial differentiation of mouse embryonic stem cells.

The heart is the first organ formed in the developing fetus, and abnormal development of the heart is a major cause of fetal death. The adverse effects of cigarette smoke on the heart have been well established, but it is not well understood how cigarette smoke components regulate signaling molecules and cardiac specific functions during the early differentiation stage of the embryonic heart. In this study, we identified changes in the size of mouse embryoid bodies (mEBs) in response to treatment with cigarette smoke extract (CSE) via regulation of HDAC2, p53, p21, and cyclin D1 protein expression, which are cardiac differentiation and cell-cycle markers, respectively. In addition, exposure of mouse embryonic stem cells (mESCs) to cigarette smoke components inhibited myocardial differentiation and development through the expression of HDAC1, HDAC2, GATA4, NKX2-5, TBX5, HAND1, and Troponin I. Long-term exposure studies showed that CSE and nicotine may delay the development of mouse cardiomyocytes from mESCs and inhibit the contractibility, which is a fundamental function of the heart. Taken together, these findings suggest that cigarette smoke components, including nicotine, may affect abnormal myocardial differentiation and development.

Chitosan-PVA-CNT nanofibers as electrically conductive scaffolds for cardiovascular tissue engineering

Conductive scaffolds are suitable candidates for cardiovascular tissue engineering (CTE) due to their similarity to the extracellular matrix of native tissue. Here, nanofiber scaffolds based on polyvinyl alcohol (PVA), chitosan (CS), and different concentrations of carbon nanotube (CNT) were produced using electrospinning. Scanning electron microscopy (SEM) image, mechanical test (elastic modulus: 130 ± 3.605 MPa), electrical conductivity (3.4 × 10−6 S/Cm), water uptake, cell adhesion, and cell viability (>80%) results of the PVA-CS-CNT1 scaffold revealed that the nanofiber containing 1% of CNT has optimal properties for cardiac differentiation. Afterwards, the differentiation of rat mesenchymal stem cells (MSCs) to cardiomyocytes was performed on the optimal scaffold by electrical stimulation in the presence of 5-azacytidine, TGF-β and ascorbic acid. The real-time qPCR results indicated that the expression of Nkx2.5, Troponin I, and β–MHC cardiac marker was increased significantly (>3 folds) in comparison to control group. Based on the findings of this study, the incorporation of MSCs, conductive scaffolds, and electrical stimulation seem to be a promising approach in CTE.

Hsa-miR-335 regulates cardiac mesoderm and progenitor cell differentiation

BackgroundWNT and TGFβ signaling pathways play critical regulatory roles in cardiomyocyte fate determination and differentiation. MiRNAs are also known to regulate different biological processes and signaling pathways. Here, we intended to find candidate miRNAs that are involved in cardiac differentiation through regulation of WNT and TGFβ signaling pathways.MethodsBioinformatics analysis suggested hsa-miR-335-3p and hsa-miR-335-5p as regulators of cardiac differentiation. Then, RT-qPCR, dual luciferase, TOP/FOP flash, and western blot analyses were done to confirm the hypothesis.ResultsHuman embryonic stem cells (hESCs) were differentiated into beating cardiomyocytes, and these miRNAs showed significant expression during the differentiation process. Gain and loss of function of miR-335-3p and miR-335-5p resulted in BRACHYURY, GATA4, and NKX2-5 (cardiac differentiation markers) expression alteration during the course of hESC cardiac differentiation. The overexpression of miR-335-3p and miR-335-5p also led to upregulation of CNX43 and TNNT2 expression, respectively. Our results suggest that this might be mediated through enhancement of WNT and TGFβ signaling pathways.ConclusionOverall, we show that miR-335-3p/5p upregulates cardiac mesoderm (BRACHYURY) and cardiac progenitor cell (GATA4 and NKX2-5) markers, which are potentially mediated through activation of WNT and TGFβ signaling pathways. Our findings suggest miR-335-3p/5p to be considered as a regulator of the cardiac differentiation process.

NKX2-8 deletion-induced reprogramming of fatty acid metabolism confers chemoresistance in epithelial ovarian cancer.

BackgroundAberrant fatty acid (FA) metabolism is a unique vulnerability of cancer cells and may present a promising target for cancer therapy. Our study aims to elucidate the molecular mechanisms by which NKX2–8 deletion reprogrammed FA metabolism-induced chemoresistance in epithelial ovarian cancer (EOC).MethodsThe deletion frequency and expression of NKX2–8 in 144 EOC specimens were assayed using Fluorescence in situ hybridization and immunochemical assays. The effects of NKX2–8 deletion and the fatty acid oxidation (FAO) antagonist Perhexiline on chemoresistance were examined by Annexin V and colony formation in vitro, and via an intraperitoneal tumor model in vivo. The mechanisms of NKX2–8 deletion in reprogrammed FA metabolism was determined using Chip-seq, metabolomic analysis, FAO assays and immunoprecipitation assays.FindingsNKX2–8 deletion was correlated with the overall and relapse-free survival of EOC patients. NKX2–8 inhibited the FAO pathway by epigenetically suppressing multiple key components of the FAO cascade, including CPT1A and CPT2. Loss of NKX2–8 resulted in reprogramming of FA metabolism of EOC cells in an adipose microenvironment and leading to platinum resistance. Importantly, pharmacological inhibition of FAO pathway using Perhexiline significantly counteracted NKX2–8 deletion-induced chemoresistance and enhanced platinum's therapeutic efficacy in EOC.InterpretationOur results demonstrate that NKX2–8 deletion-reprogrammed FA metabolism contributes to chemoresistance and Perhexiline might serve as a potential tailored treatment for patients with NKX2–8-deleted EOC.FundThis work was supported by Natural Science Foundation of China; Guangzhou Science and Technology Plan Projects; Natural Science Foundation of Guangdong Province; The Fundamental Research Funds for the Central Universities.

Role of Cdx factors in early mesodermal fate decisions.

Murine cardiac and hematopoietic progenitors are derived from Mesp1+ mesoderm. Cdx function impacts both yolk sac hematopoiesis and cardiogenesis in zebrafish, suggesting that Cdx family members regulate early mesoderm cell fate decisions. We found that Cdx2 occupies a number of transcription factor loci during embryogenesis, including key regulators of both cardiac and blood development, and that Cdx function is required for normal expression of the cardiogenic transcription factors Nkx2-5 and Tbx5 Furthermore, Cdx and Brg1, an ATPase subunit of the SWI/SNF chromatin remodeling complex, co-occupy a number of loci, suggesting that Cdx family members regulate target gene expression through alterations in chromatin architecture. Consistent with this, we demonstrate loss of Brg1 occupancy and altered chromatin structure at several cardiogenic genes in Cdx-null mutants. Finally, we provide evidence for an onset of Cdx2 expression at E6.5 coinciding with egression of cardiac progenitors from the primitive streak. Together, these findings suggest that Cdx functions in multi-potential mesoderm to direct early cell fate decisions through transcriptional regulation of several novel target genes, and provide further insight into a potential epigenetic mechanism by which Cdx influences target gene expression.

A Human iPSC Double-Reporter System Enables Purification of Cardiac Lineage Subpopulations with Distinct Function and Drug Response Profiles.

The diversity of cardiac lineages contributes to the heterogeneity of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs). Here, we report the generation of a hiPSC TBX5Clover2 and NKX2-5TagRFP double reporter to delineate cardiaclineages and isolate lineage-specific subpopulations. Molecular analyses reveal that four different subpopulations can be isolated based on the differential expression of TBX5 and NKX2-5, TBX5+NKX2-5+, TBX5+NKX2-5-, TBX5-NKX2-5+, and TBX5-NKX2-5-, mimicking the first heart field, epicardial, second heart field, and endothelial lineages, respectively. Genetic and functional characterization indicates that each subpopulation differentiates into specific cardiac cells. We further identify CORIN as a cell-surface marker for isolating the TBX5+NKX2-5+ subpopulation and demonstrate the use of lineage-specific CMs for precise drug testing. We anticipate that this tool will facilitate theinvestigation of cardiac lineage specification and isolation of specific cardiac subpopulations for drug screening, tissue engineering, and disease modeling.

Gene-environment interaction impacts on heart development and embryo survival.

Congenital heart disease (CHD) is the most common type of birth defect. In recent years, research has focussed on identifying the genetic causes of CHD. However, only a minority of CHD cases can be attributed to single gene mutations. In addition, studies have identified different environmental stressors that promote CHD, but the additive effect of genetic susceptibility and environmental factors is poorly understood. In this context, we have investigated the effects of short-term gestational hypoxia on mouse embryos genetically predisposed to heart defects. Exposure of mouse embryos heterozygous for Tbx1 or Fgfr1/Fgfr2 to hypoxia in utero increased the incidence and severity of heart defects while Nkx2-5+/- embryos died within 2 days of hypoxic exposure. We identified the molecular consequences of the interaction between Nkx2-5 and short-term gestational hypoxia, which suggest that reduced Nkx2-5 expression and a prolonged hypoxia-inducible factor 1α response together precipitate embryo death. Our study provides insight into the causes of embryo loss and variable penetrance of monogenic CHD, and raises the possibility that cases of foetal death and CHD in humans could be caused by similar gene-environment interactions.

Encapsulated miR-200c and Nkx2.1 in a nuclear/mitochondria transcriptional regulatory network of non-metastatic and metastatic lung cancer cells

BackgroundMicroRNAs are noncoding RNA molecules of ~ 22 nucleotides with diagnostic and therapeutic action [Curr Drug Targets, 2015. 16(12): p. 1381-403], affecting the expression of mRNAs involved in invasion, migration, and development [Oncotarget, 2015. 6(9): p. 6472-98, Cancer Manag Res, 2014. 6: p. 205-16]. miR-200c is part of the miR-200c/141 cluster on chromosome 12p13. Its mechanism of action when encapsulated is critical in lung cancer when patients express changes in miRNAs. miR-200c be a potential biomarkers for various lung diseases. As a potential therapy, miR-200c can impacts lives as target lung cancer is a leading cause of death with about 234,000 cases annually, high heterogeneity, complex screening, and a 5-year survival rate of 16% [CA Cancer J Clin, 2016.66(1): p. 7-30]. Encapsulated miR-200c efficiently enhances bioavailability, pharmacokinetics of therapeutics and targeting to cells, improves efficacy and provides potential cure.MethodsThe functions of miR-200c were determined in non-metastatic KW-634 and metastatic 821-T4 and 821-LN mouse lung cancer cell lines after various Nano vehicle treatments. Viability and cytotoxicity were determined by cell cycle and quantitative real-time PCR analyses were used to quantify levels of miR-200c and its target genes. In situ hybridization was used to visualize patterns of expression of miR-200c and others in the lung and many organs. Next-generation sequencing accession number GSE125000, invasion and migration assays using transwell chambers, and ActivSignal were used to elucidate the activation and inhibition profiles and perform direct expression measurements and modification of cellular components.ResultsDue to their effectiveness as intracellular vesicles transporting miR-200c into, out, and between parts of the cells, miR-200c is encapsulated with cholesterol, an integral part of the biological membranes with very important physical properties of the vehicle. Nano miR-200c showed efficient cellular uptake in KW-634, 821-T4, and 821-LN cells with important changes in gene expression and new isoforms. In KW-634, when treated with encapsulated miR-200c and compare to the non-encapsulated control; miR-29b increased by 5261-fold, and in 821-T4/LN, miR-1247 increased by 150-fold. Conversely, miR-1247 and miR-675 decreased by 348 and 1029.5-fold, respectively. miR-189 decreased by 34-fold in treated 821-T4 cells. A reduction of growth was observed only after 48 h of treatment with Nano miR-200c. Moreover, labeling the vehicle with carboxy-fluorescein showed that the encapsulated particles enter the nucleus and mitochondria. Encapsulated miR-200c by entering the cells, the nucleus and mitochondria, trigger changes in cell cycle phases with 4 up to 12 fold percentage in G2 and S phase respectively compare to miR-200c. Endogenous expression of Nkx2.1, miR-200c, and their targets Myb, Nfib, Six4 and Six1 showed an inverse correlation, as observed in development.ConclusionsLittle is known about miR-200c involvement in regulatory processes. Nano miR-200c affects invasion and migration mechanisms. The expression of encapsulated miR-200c contributes to the inhibition/activation of Kras, EMT, Hippo, regulatory pathways and blockers of metastasis. Delivery of miR-200c increases the expression of miR-29b, an EMY regulator, and miR-1247, an inhibitor of cancer genes, both tumor suppressors involved in lung metastasis. Encapsulated miR-200c act on different proteins that regulates cell cycle pathways. These findings represent a part of a regulatory network providing new insights towards improvement of therapy.

Bone morphogenetic protein 4 promotes the differentiation of Tbx18-positive epicardial progenitor cells to pacemaker-like cells.

Clarifying the mechanisms via which pacemaker- like cells are generated is critical for identifying novel targets for arrhythmia-associated disorders and constructing pacemakers with the ability to adapt to physiological requirements. T-box 18 (Tbx18)+ epicardial progenitor cells (EPCs) have the potential to differentiate into pacemaker cells. Although bone morphogenetic protein 4 (Bmp4) is likely to contribute, its role and regulatory mechanisms in the differentiation of Tbx18+ EPCs into pacemaker-like cells have remained to be fully elucidated. In the present study, the association between Bmp4, GATA binding protein 4 (Gata4) and hyperpolarization- activated cyclic nucleotide gated potassium channel 4 (Hcn4) to regulate NK2 homeobox 5 (Nkx2.5), which is known to be required for the differentiation of Tbx18+ EPCs into pacemaker-like cells, was assessed. Tbx18+ EPCs were isolated from Tbx18:Cre/Rosa26Renhanced yellow fluorescence protein (EYFP) murine embryos at embryonic day 11.5 and divided into the following four treatment groups: Control, Bmp4, Bmp4+LDN193189 (a Bmp inhibitor) and LDN193189. In vitro Bmp4 promoted the expression of Hcn4 in Tbx18+ EPCs via lineage tracing of Tbx18:Cre/Rosa26REYFP mice, which was likely due to upregulation of Gata4 expression. Gata4 knockdown experiments were then performed using the following five treatment groups: Control, control small interfering RNA (siRNA), Bmp4, Bmp4+siRNA targeting Gata4 (siGata4) and siGata4 group. Knockdown of Gata4 caused a downregulation of Hcn4 and an upregulation of Nkx2.5, but had no effect on Bmp4 expression. In conclusion, it was indicated that in Tbx18+ EPCs, the expression of Nkx2.5 was regulated by Bmp4 via Gata4. Taken together, these results provide important information on regulatory networks of pacemaker cell differentiation and may serve as a basis for further studies.

Single cell RNA-seq and ATAC-seq analysis of cardiac progenitor cell transition states and lineage settlement

Formation and segregation of cell lineages forming the heart have been studied extensively but the underlying gene regulatory networks and epigenetic changes driving cell fate transitions during early cardiogenesis are still only partially understood. Here, we comprehensively characterize mouse cardiac progenitor cells (CPCs) marked by Nkx2-5 and Isl1 expression from E7.5 to E9.5 using single-cell RNA sequencing and transposase-accessible chromatin profiling (ATAC-seq). By leveraging on cell-to-cell transcriptome and chromatin accessibility heterogeneity, we identify different previously unknown cardiac subpopulations. Reconstruction of developmental trajectories reveal that multipotent Isl1+ CPC pass through an attractor state before separating into different developmental branches, whereas extended expression of Nkx2-5 commits CPC to an unidirectional cardiomyocyte fate. Furthermore, we show that CPC fate transitions are associated with distinct open chromatin states critically depending on Isl1 and Nkx2-5. Our data provide a model of transcriptional and epigenetic regulations during cardiac progenitor cell fate decisions at single-cell resolution.

RPSA, a candidate gene for isolated congenital asplenia, is required for pre-rRNA processing and spleen formation in Xenopus.

A growing number of tissue-specific inherited disorders are associated with impaired ribosome production, despite the universal requirement for ribosome function. Recently, mutations in RPSA, a protein component of the small ribosomal subunit, were discovered to underlie approximately half of all isolated congenital asplenia cases. However, the mechanisms by which mutations in this ribosome biogenesis factor lead specifically to spleen agenesis remain unknown, in part due to the lack of a suitable animal model for study. Here we reveal that RPSA is required for normal spleen development in the frog, Xenopus tropicalis Depletion of Rpsa in early embryonic development disrupts pre-rRNA processing and ribosome biogenesis, and impairs expression of the key spleen patterning genes nkx2-5, bapx1 and pod1 in the spleen anlage. Importantly, we also show that whereas injection of human RPSA mRNA can rescue both pre-rRNA processing and spleen patterning, injection of human mRNA bearing a common disease-associated mutation cannot. Together, we present the first animal model of RPSA-mediated asplenia and reveal a crucial requirement for RPSA in pre-rRNA processing and molecular patterning during early Xenopus development.

Abstract 215: Induced Pluripotent Stem Cell-Derived Cardiomyocyte Proliferation is Enhanced by Co-culture With Female Mesenchymal Stem Cells

Background: Myocardial ischemia remains a leading cause of morbidity and mortality worldwide. Bone marrow-derived adult mesenchymal stem cells (MSCs) improve cardiac structure and function post-MI likely mediated in large part by paracrine factors. There is emerging evidence that female MSCs have greater therapeutic efficacy than male MSCs. Objective: We hypothesized that female MSCs promote cardiomyocyte differentiation, proliferation and contractility of induced pluripotent stem cells (iPSCs) better than male MSCs. Methods & Results: Human (h) iPSCs were co-cultured with male or female murine MSCs during or after differentiation into beating cardiomyocytes. Female MSCs were significantly more potent at stimulating ISL-1 and NKX2-5 expression in hiPSC-derived cardiac precursors and cardiomyocytes. Exosomes from female MSCs had lower levels of miR-17, a miR that directly represses Isl1 . hiPSCs co-cultured with female MSCs, exhibited a 2-fold greater increase in proliferation compared to male MSCs. In addition, ß 2 -adrenergic receptor expression was up regulated 3.5-fold in hiPSCs incubated with female MSCs compared to 2.5-fold for male MSCs. Conclusion: Our findings illustrate that there are significant differences between female and male MSCs in their ability to affect cardiomyogenesis of hiPSCs and suggest that this effect is mediated, at least in part, by reduced levels of miR-17. The observation that female MSCs have greater potential to improve cardiomyocyte proliferation and contractility has important implications for stem cell therapy.

Abstract LB-211: The cardiac transcription factor Nkx2-5 contributes to SRF-dependent AR action in prostate cancer

Abstract Ligand-activated androgen receptor (AR) drives prostate cancer (CaP) growth. Heterogeneity in AR action exists and AR often remains active when therapies targeting its ligand-activation have failed. Understanding the mechanisms by which AR mediates CaP progression may lead to new treatments. Previously, we identified a mechanism in which AR imparts androgen-responsiveness to the transcription factor Serum Response Factor (SRF). SRF-dependent AR action is enriched in CaP, correlates with CaP recurrence and is maintained in late-stage CaP. SRF binds constitutively at its target genes and becomes transcriptionally active via upstream signaling cascades or SRF cofactors. Our prior work showed that RhoA signaling transduces androgen regulation to a significant fraction, but not all, SRF target genes. Here, we isolate the homeodomain protein Nkx2-5 as a novel regulator of androgen-responsive SRF action. Nkx2-5 co-operates with SRF in transcriptional control of heart development. In CaP, Nkx2-5 promoter hypermethylation is a biomarker that distinguishes CaP from benign prostate, yet consequences of differential Nkx2-5 expression on CaP are unknown. In the CaP cell models LNCaP, C4-2 and VCaP, siRNA-mediated loss of Nkx2-5 increased basal and androgen-dependent expression of exogenously and endogenously expressed SRF target genes, and these effects were reversed after silencing of SRF. Conversely, overexpression of Nkx2-5 decreased androgen-responsiveness of SRF target genes. Co-IP and ChIP assays indicated androgen-stimulated SRF-Nkx2-5 interaction in CaP cell nuclei. Loss of Nkx2-5 resulted in distinct morphological changes in CaP cells, which depended entirely on SRF. In rhodamine phalloidin staining assays, loss of Nkx2-5 increased significantly (p<0.01) cell length, width and circumference, and the number of cell protrusions in cultures with and without androgen stimulation. Changes in cell shape were accompanied by modest decreases in CaP cell proliferation and migration, but significant (p<0.05) decreases in CaP cell-matrix adhesion. Nkx2-5 silencing increased the size of the cell nucleus and led to multilobed and grooved nuclei and enlarged nucleoli. These nuclear shape changes were not associated with alterations in DNA ploidy, but were associated with changed heterochromatin organization as reflected in a reduction in heterochromatin protein 1 immunostaining and nuclear content. Such Nkx2-5-dependent changes in nuclear shape and size and heterochromatin organization occurred also in non-malignant prostate epithelial RWPE2 cells. This work uncovered a novel role for Nkx2-5 in the transition from benign prostate to CaP, showing that Nkx2-5 regulates SRF transcriptional activity in CaP cells and demonstrating that differential Nkx2-5 expression alters cell-matrix adhesion and nuclear morphology, both processes that are associated with aggressive CaP progression. Citation Format: Salma Ben-Salem, Dhirodatta Senapati, Giridhar Mudduluru, Yixue Su, Varadha Balaji Venkadakrishnan, Hannelore V. Heemers. The cardiac transcription factor Nkx2-5 contributes to SRF-dependent AR action in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-211.

Gut microbiota-derived endotoxin enhanced the incidence of cardia bifida during cardiogenesis.

Cytotoxicity and inflammation-associated toxic responses could be induced by bacterial lipopolysaccharides (LPS) in vitro and in vivo, respectively. However, the mechanism involved in LPS-induced cardiac malformation in prenatal fetus is still unknown. In this study, we demonstrated that LPS was induced in gut microbiota imbalance mice, and next, LPS exposure during gastrulation in the chick embryo increased the incidence of cardia bifida. Gene transfection and tissue transplantation trajectory indicated that LPS exposure restricted the cell migration of cardiac progenitors to primary heart field in gastrula chick embryos. In vitro explant allograft of GFP-labeled anterior primitive streak demonstrated that LPS treatments could inhibit cell migration. A similar observation was also obtained from the cell migration assay of scratch wounds using primary culture of cardiomyocytes or H9c2 cells. In the embryos exposed to LPS, expressions of Nkx2.5 and GATA5 were disturbed. These genes are associated with cardiomyocyte differentiation when heart tube fusion occurs. Furthermore, pHIS3, C-caspase3 immunohistological staining indicated that cell proliferation decreased, cell apoptosis increased in the heart tube of chick embryo. Meanwhile, in vivo, pHIS3 immunohistological staining and Hochest/PI staining also draw the similar conclusions. The LPS exposure also caused the production of excess ROS, which might damage the cardiac precursor cells of developing embryos. At last, we showed that LPS-induced cardia bifida could be partially rescued through the addition of antioxidants. Together, these results reveal that excess ROS generation is involved in the LPS-induced defects in heart tube during chick embryo development.

Expression of Nkx2.5 and Isl1 in the cardiopharyngeal mesoderm of developing Xenopus embryos

The cardiopharyngeal field (CPF) is a mesodermal field that gives rise to the first (FHF) and second heart field (SHF)‐derived myocardium and the branchiomeric musculature. Isl1 and Nkx2.5 are transcription factors expressed in the CPF. Both genes are crucial for the specification of the myocardial cells derived from the SHF and for the further development of the heart in vertebrates. The importance of these genes is supported by the fact that perturbations in their expression lead to severe heart malformations and often result in embryonic death. In order to better understand the role of these two genes and their impact on the head and heart development, we analysed their expression in Xenopus laevis, which is potentially a good model to study the CPF and craniofacial defects in terms of evolutionary developmental medicine. In contrast to basal vertebrates, such as the urochordate Ciona, Xenopus' heart has an advanced morphology with several heart chambers and in‐ and outflow tracts. Concurrently, Xenopus is easy to rear in laboratory conditions. There are various genetic tools available to work with this species (e.g., TALENs, CRISPR/Cas9, ZFNs) and there are certain advantages for the study of embryology (e.g., external fertilization and large size of eggs) that favour this species over the mouse and chicken models.Whole‐mount in situ hybridization of embryos revealed that during early stages Isl1 and Nkx2.5 are co‐expressed in cardiac progenitor cells localized near the cement gland. Isl1 was also present in the stomodeum region and other cell populations; e.g., cephalic placodes and their associated ganglia or cranial paraxial mesoderm. At stage 25 (Nieuwkoop and Faber), Nkx2.5 was expressed in the FHF, SHF, and anterior endoderm. Later, it nearly separated from the Isl1 expression and became restricted to the heart tube, derived from the FHF. Isl1 was mainly expressed in the pharyngeal arch mesoderm adjacent to the heart and in the dorsolateral heart domain. At late stages, Nkx2.5 could be detected throughout the myocardium, while Isl1 was present in the foregut and cephalic ganglia. In spite of minor differences, our results conform to early studies in X. laevis. The comparison with expression patterns in mouse and chicken shows that Xenopus is indeed a good organism to study the CPF derivatives, like the myocardium and head musculature. Therefore, Xenopus can be further extensively used for developmental, evolutionary, and medical studies of the normal and abnormal head and heart myogenesis.Support or Funding InformationJMZ is supported by a Howard University College of Medicine pilot study award (BFPSAP: U100217).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.