Expression Of Nkx2 Research Articles

NKX2-5, a modifier of skeletal muscle pathology due to RNA toxicity.

RNA toxicity is implicated in a number of disorders; especially those associated with expanded repeat sequences, such as myotonic dystrophy (DM1). Previously, we have shown increased NKX2-5 expression in RNA toxicity associated with DM1. Here, we investigate the relationship between NKX2-5 expression and muscle pathology due to RNA toxicity. In skeletal muscle from mice with RNA toxicity and individuals with DM1, expression of Nkx2-5 or NKX2-5 and its downstream targets are significantly correlated with severity of histopathology. Using C2C12 myoblasts, we show that over-expression of NKX2-5 or mutant DMPK 3'UTR results in myogenic differentiation defects, which can be rescued by knockdown of Nkx2-5, despite continued toxic RNA expression. Furthermore, in a mouse model of NKX2-5 over-expression, we find defects in muscle regeneration after induced damage, similar to those seen in mice with RNA toxicity. Using mouse models of Nkx2-5 over-expression and depletion, we find that NKX2-5 levels modify disease phenotypes in mice with RNA toxicity.

Directed differentiation of patient-specific induced pluripotent stem cells identifies the transcriptional repression and epigenetic modification of NKX2-5, HAND1, and NOTCH1 in hypoplastic left heart syndrome.

The genetic basis of hypoplastic left heart syndrome (HLHS) remains unknown, and the lack of animal models to reconstitute the cardiac maldevelopment has hampered the study of this disease. This study investigated the altered control of transcriptional and epigenetic programs that may affect the development of HLHS by using disease-specific induced pluripotent stem (iPS) cells. Cardiac progenitor cells (CPCs) were isolated from patients with congenital heart diseases to generate patient-specific iPS cells. Comparative gene expression analysis of HLHS- and biventricle (BV) heart-derived iPS cells was performed to dissect the complex genetic circuits that may promote the disease phenotype. Both HLHS- and BV heart-derived CPCs were reprogrammed to generate disease-specific iPS cells, which showed characteristic human embryonic stem cell signatures, expressed pluripotency markers, and could give rise to cardiomyocytes. However, HLHS-iPS cells exhibited lower cardiomyogenic differentiation potential than BV-iPS cells. Quantitative gene expression analysis demonstrated that HLHS-derived iPS cells showed transcriptional repression of NKX2-5, reduced levels of TBX2 and NOTCH/HEY signaling, and inhibited HAND1/2 transcripts compared with control cells. Although both HLHS-derived CPCs and iPS cells showed reduced SRE and TNNT2 transcriptional activation compared with BV-derived cells, co-transfection of NKX2-5, HAND1, and NOTCH1 into HLHS-derived cells resulted in synergistic restoration of these promoters activation. Notably, gain- and loss-of-function studies revealed that NKX2-5 had a predominant impact on NPPA transcriptional activation. Moreover, differentiated HLHS-derived iPS cells showed reduced H3K4 dimethylation as well as histone H3 acetylation but increased H3K27 trimethylation to inhibit transcriptional activation on the NKX2-5 promoter. These findings suggest that patient-specific iPS cells may provide molecular insights into complex transcriptional and epigenetic mechanisms, at least in part, through combinatorial expression of NKX2-5, HAND1, and NOTCH1 that coordinately contribute to cardiac malformations in HLHS.

Insulin‐Like Growth Factor Binding Protein 4 Enhances Cardiomyocytes Induction in Murine‐Induced Pluripotent Stem Cells

Insulin-like growth factor binding protein 4 (IGFBP4) has been reported to play critical role in cardiomyocytes differentiation of embryonic stem cells (ESCs). But whether it promotes cardiomyocytes induction of iPSCs is unclear. In the present study, we aim to explore the role of IGFBP4 in the cardiogenesis of mouse iPSCs. We observed that IGFBP4 treatment at late stage during differentiation process of mouse iPSCs greatly enhanced the beating frequency of embryoid bodies (EBs). The expressions of Nkx2.5 (cardiac-specific transcription factor), α-MHC, α-actinin, and Troponin I (cardiac-specific protein) were significantly enhanced by IGFBP4 treatment. Immunostaining analysis showed that α-MHC, TNNT2 and connexin 43, typical cardiac markers, were obviously expressed in isolated cardiomyocytes from iPSCs with or without IGFBP4 treatment. Further study revealed that IGFBP4 had little effect on the apoptosis of EBs, but it significantly promoted the proliferation of cardiomyocytes from iPSCs characterized by higher ratio EdU positive cells in differentiated cardiomyocytes. We next observed that IGFBP4 inhibited β-catenin expression in cytosol of EBs at late stage during differentiation of iPSCs. Knockdown of β-catenin using a siRNA technique promoted the proliferation of differentiated cardiomyocytes and enhanced cardiomyocytes induction of iPSCs, suggesting that the effect of IGFBP4 on cardiomyocytes differentiation of iPSCs has relationship with β-catenin signaling pathway. In conclusion, IGFBP4 promotes cardiogenesis of iPSCs by enhancing the proliferation of differentiated cardiomyocytes through inhibiting β-catenin signaling.

Placental Nkx2-5 and target gene expression in early-onset and severe preeclampsia

Objective: Preeclampsia (PE) affects 2–8% of pregnancies worldwide and is a significant source of maternal and neonatal morbidity and mortality. However, the mechanisms underlying PE are poorly understood and major questions regarding etiology and risk factors remain to be addressed. Our objective was to examine whether abnormal expression of the cardiovascular developmental transcription factor, Nkx2-5, was associated with early onset and severe preeclampsia (EOSPE). Methods: Using qPCR and immunohistochemical assay, we examined expression of Nkx2-5 and target gene expression in EOSPE and control placental tissue. We tested resulting mechanistic hypotheses in cultured cells using shRNA knockdown, qPCR, and western blot. Results: Nkx2-5 is highly expressed in racially disparate fashion (Caucasians > African Americans) in a subset of early EOSPE placentae. Nkx2-5 mRNA expression is highly correlated (Caucasians > African Americans) to mRNA expression of the preeclampsia marker sFlt-1, and of the Nkx2-5 target and RNA splicing factor, Sam68. Knockdown of Sam68 expression in cultured cells significantly impacts sFlt-1 mRNA isoform generation in vitro, supporting a mechanistic hypothesis that Nkx2-5 impacts EOSPE severity in a subset of patients via upregulation of Sam68 to increase sFlt-1 expression. Expression of additional Nkx2-5 targets potentially regulating metabolic stress response is also elevated in a racially disparate fashion in EOSPE. Conclusions: Expression of Nkx2-5 and its target genes may directly influence the genesis and racially disparate severity, and define a mechanistically distinct subclass of EOSPE.

C-Abl inhibitor imatinib enhances insulin production by β cells: c-Abl negatively regulates insulin production via interfering with the expression of NKx2.2 and GLUT-2.

Chronic myelogenous leukemia patients treated with tyrosine kinase inhibitor, Imatinib, were shown to have increased serum levels of C-peptide. Imatinib specifically inhibits the tyrosine kinase, c-Abl. However, the mechanism of how Imatinib treatment can lead to increased insulin level is unclear. Specifically, there is little investigation into whether Imatinib directly affects β cells to promote insulin production. In this study, we showed that Imatinib significantly induced insulin expression in both glucose-stimulated and resting β cells. In line with this finding, c-Abl knockdown by siRNA and overexpression of c-Abl markedly enhanced and inhibited insulin expression in β cells, respectively. Unexpectedly, high concentrations of glucose significantly induced c-Abl expression, suggesting c-Abl may play a role in balancing insulin production during glucose stimulation. Further studies demonstrated that c-Abl inhibition did not affect the major insulin gene transcription factor, pancreatic and duodenal homeobox-1 (PDX-1) expression. Of interest, inhibition of c-Abl enhanced NKx2.2 and overexpression of c-Abl in β cells markedly down-regulated NKx2.2, which is a positive regulator for insulin gene expression. Additionally, we found that c-Abl inhibition significantly enhanced the expression of glucose transporter GLUT2 on β cells. Our study demonstrates a previously unrecognized mechanism that controls insulin expression through c-Abl-regulated NKx2.2 and GLUT2. Therapeutic targeting β cell c-Abl could be employed in the treatment of diabetes or β cell tumor, insulinoma.

Investigation of spinal cerebrospinal fluid-contacting neurons expressing PKD2L1: evidence for a conserved system from fish to primates.

Over 90 years ago, Kolmer and Agduhr identified spinal cerebrospinal fluid-contacting neurons (CSF-cNs) based on their morphology and location within the spinal cord. In more than 200 vertebrate species, they observed ciliated neurons around the central canal that extended a brush of microvilli into the cerebrospinal fluid (CSF). Although their morphology is suggestive of a primitive sensory cell, their function within the vertebrate spinal cord remains unknown. The identification of specific molecular markers for these neurons in vertebrates would benefit the investigation of their physiological roles. PKD2L1, a transient receptor potential channel that could play a role as a sensory receptor, has been found in cells contacting the central canal in mouse. In this study, we demonstrate that PKD2L1 is a specific marker for CSF-cNs in the spinal cord of mouse (Mus musculus), macaque (Macaca fascicularis) and zebrafish (Danio rerio). In these species, the somata of spinal PKD2L1+ CSF-cNs were located below or within the ependymal layer and extended an apical bulbous extension into the central canal. We found GABAergic PKD2L1-expressing CSF-cNs in all three species. We took advantage of the zebrafish embryo for its transparency and rapid development to identify the progenitor domains from which pkd2l1+ CSF-cNs originate. pkd2l1+ CSF-cNs were all GABAergic and organized in two rows—one ventral and one dorsal to the central canal. Their location and marker expression is consistent with previously described Kolmer–Agduhr cells. Accordingly, pkd2l1+ CSF-cNs were derived from the progenitor domains p3 and pMN defined by the expression of nkx2.2a and olig2 transcription factors, respectively. Altogether our results suggest that a system of CSF-cNs expressing the PKD2L1 channel is conserved in the spinal cord across bony vertebrate species.

SIRPA, VCAM1 and CD34 identify discrete lineages during early human cardiovascular development

The study of human cardiogenesis would benefit from a detailed cell lineage fate map akin to that established for the haematopoietic lineages. Here we sought to define cell lineage relationships based on the expression of NKX2-5 and the cell surface markers VCAM1, SIRPA and CD34 during human cardiovascular development. Expression of NKX2-5GFP was used to identify cardiac progenitors and cardiomyocytes generated during the differentiation of NKX2-5GFP/w human embryonic stem cells (hESCs). Cardiovascular cell lineages sub-fractionated on the basis of SIRPA, VCAM1 and CD34 expression were assayed for differentiation potential and gene expression. The NKX2-5posCD34pos population gave rise to endothelial cells that rapidly lost NKX2-5 expression in culture. Conversely, NKX2-5 expression was maintained in myocardial committed cells, which progressed from being NKX2-5posSIRPApos to NKX2-5posSIRPAposVCAM1pos. Up-regulation of VCAM1 was accompanied by the expression of myofilament markers and reduced clonal capacity, implying a restriction of cell fate potential. Combinatorial expression of NKX2-5, SIRPA, VCAM1 and CD34 can be used to define discrete stages of cardiovascular cell lineage differentiation. These markers identify specific stages of cardiomyocyte and endothelial lineage commitment and, thus provide a scaffold for establishing a fate map of early human cardiogenesis.

Effect of NRG-1/ErbB Signaling Intervention on the Differentiation of Bone Marrow Stromal Cells Into Sinus Node–like Cells

The neuregulin-1 (NRG-1)/ErbB signaling pathway is a crucial regulator of cardiac development and plays an important role in the formation of the cardiac special conduction system. To establish a rat bone marrow stromal cell (BMSC) cardiomyocyte (CM)-like differentiation model, BMSCs were treated with 5-azacytidine and fibroblast growth factor basic (FGF-basic) for 24 hours and then cocultured with neonatal rat CMs in a Transwell culture system. The feasibility of regulating the differentiation of BMSCs into sinoatrial node cells by manipulating the NRG-1/ErbB pathway was investigated. Three weeks after induction, reverse transcription-polymerase chain reaction analysis revealed that inhibition of NRG-1/ErbB signaling (using AG1478) greatly enhanced the expression of HCN4, Tbx3, and Tbx2. Additionally, Tbx3 protein levels were higher than in the control group and even produced distinct nodal-type action potentials. The expression of Nkx2.5 in the NRG-1 group (treated with exogenous NRG-1) was higher than the other 2 groups. The expression of phospho-Akt was also increased in the NRG-1 group but decreased in the AG1478 group. Together, these data demonstrate that inhibiting the endogenous NRG-1/ErbB signaling pathway when rat BMSCs differentiate into CMs can greatly enhance the pacemaker phenotype. Akt signaling may be one of the underlying molecular mechanisms responsible for these results.

PT356 VCP746, a novel A1/A2 adenosine receptor agonist, reduces hypertrophy in cardiac myocytes and collagen synthesis in cardiac fibroblasts and renal mesangial cells

Introduction: The establishment of induced pluripotent stem cells (iPSCs) potentially provides a unique resource for generating patient specific cardiomyocytes to for cardiac repair. However, the poor efficiency and yield of cardiomyocytes currently limits the application of this technology. Insulin-like growth factor binding protein 4 (IGFBP4) has been reported to play critical role in cardiomyocytes differentiation of embryonic stem cells (ESCs). But whether it promotes iPSCs into cardiomyocytes is unclear. Objectives: In the present study, we aim to explore the role of IGFBP4 in the cardiogenesis of mouse iPSCs. Methods: For differentiation, iPSCs were trypsinized and EBs were generated by the threedimensional hanging-drop method. EBs were grown in hanging drops for three days (day 0 to day 3), each drop initially consisted of 1000 cells in 30 mL of differentiation medium (growth medium without LIF). IGFBP4 (Sino Biological Inc.) was added at day 3-8,day 3-6 and day 6-8 period under 1 mg/ mL to determine the effective time-window. Results: We observed that IGFBP4 treatment at late-stage during differentiation process of mouse iPSCs greatly enhanced the beating frequency of embryoid bodies (EBs). The expressions of Nkx2.5 (cardiac specific transcription factor), a-MHC, a-actinin and Troponin I (cardiac specific protein) were significantly enhanced by IGFBP4 treatment. Immunostaining analysis showed that a-MHC, TNNT2 and connex43, typical cardiac markers, were obviously expressed in isolated cardiomyocytes from iPSCs with or without IGFBP4 treatment. Further study revealed that IGFBP4 had little effect on the apoptosis of EBs. But it significantly promoted the proliferation of cardiomycytes from iPSCs chracterized by higher ratio BrdU positive cells in differentiated cardiomyocytes. We next observed that IGFBP4 inhibted b-catenin expression in cytosol of EBs at late-stage during differentiation of iPSCs. Knockdown of b-catenin using a siRNA technique promoted the proliferation of differentiated cardiomyocytes and enhancing cardiomyocytes induction of iPSCs, suggesting that the effect of IGFBP4 on cardiomyocyte differentiation of iPSCs has relationship with b-catenin signaling pathway. Conclusion: In conclusion, IGFBP4 promotes cardiogenesis of iPSCs by enhancing the proliferation of differentiated cardiomyocytes through b-catenin signaling. Disclosure of Interest: None Declared

PT353 Identification of miRNA networks in pathological cardiac remodelling-New therapeutic targets

Introduction: The establishment of induced pluripotent stem cells (iPSCs) potentially provides a unique resource for generating patient specific cardiomyocytes to for cardiac repair. However, the poor efficiency and yield of cardiomyocytes currently limits the application of this technology. Insulin-like growth factor binding protein 4 (IGFBP4) has been reported to play critical role in cardiomyocytes differentiation of embryonic stem cells (ESCs). But whether it promotes iPSCs into cardiomyocytes is unclear. Objectives: In the present study, we aim to explore the role of IGFBP4 in the cardiogenesis of mouse iPSCs. Methods: For differentiation, iPSCs were trypsinized and EBs were generated by the threedimensional hanging-drop method. EBs were grown in hanging drops for three days (day 0 to day 3), each drop initially consisted of 1000 cells in 30 mL of differentiation medium (growth medium without LIF). IGFBP4 (Sino Biological Inc.) was added at day 3-8,day 3-6 and day 6-8 period under 1 mg/ mL to determine the effective time-window. Results: We observed that IGFBP4 treatment at late-stage during differentiation process of mouse iPSCs greatly enhanced the beating frequency of embryoid bodies (EBs). The expressions of Nkx2.5 (cardiac specific transcription factor), a-MHC, a-actinin and Troponin I (cardiac specific protein) were significantly enhanced by IGFBP4 treatment. Immunostaining analysis showed that a-MHC, TNNT2 and connex43, typical cardiac markers, were obviously expressed in isolated cardiomyocytes from iPSCs with or without IGFBP4 treatment. Further study revealed that IGFBP4 had little effect on the apoptosis of EBs. But it significantly promoted the proliferation of cardiomycytes from iPSCs chracterized by higher ratio BrdU positive cells in differentiated cardiomyocytes. We next observed that IGFBP4 inhibted b-catenin expression in cytosol of EBs at late-stage during differentiation of iPSCs. Knockdown of b-catenin using a siRNA technique promoted the proliferation of differentiated cardiomyocytes and enhancing cardiomyocytes induction of iPSCs, suggesting that the effect of IGFBP4 on cardiomyocyte differentiation of iPSCs has relationship with b-catenin signaling pathway. Conclusion: In conclusion, IGFBP4 promotes cardiogenesis of iPSCs by enhancing the proliferation of differentiated cardiomyocytes through b-catenin signaling. Disclosure of Interest: None Declared

Characterization of the hypothalamus of Xenopus laevis during development. II. The basal regions

The expression patterns of conserved developmental regulatory transcription factors and neuronal markers were analyzed in the basal hypothalamus of Xenopus laevis throughout development by means of combined immunohistochemical and in situ hybridization techniques. The connectivity of the main subdivisions was investigated by in vitro tracing techniques with dextran amines. The basal hypothalamic region is topologically rostral to the basal diencephalon and is composed of the tuberal (rostral) and mammillary (caudal) subdivisions, according to the prosomeric model. It is dorsally bounded by the optic chiasm and the alar hypothalamus, and caudally by the diencephalic prosomere p3. The tuberal hypothalamus is defined by the expression of Nkx2.1, xShh, and Isl1, and rostral and caudal portions can be distinguished by the distinct expression of Otp rostrally and Nkx2.2 caudally. In the mammillary region the xShh/Nkx2.1 combination defined the rostral mammillary area, expressing Nkx2.1, and the caudal retromammillary area, expressing xShh. The expression of xLhx1, xDll4, and Otp in the mammillary area and Isl1 in the tuberal region highlights the boundary between the two basal hypothalamic territories. Both regions are strongly connected with subpallial regions, especially those conveying olfactory/vomeronasal information, and also possess abundant intrahypothalamic connections. They show reciprocal connections with the diencephalon (mainly the thalamus), project to the midbrain tectum, and are bidirectionally related to the rhombencephalon. These results illustrate that the basal hypothalamus of anurans shares many features of specification, regionalization, and hodology with amniotes, reinforcing the idea of a basic bauplan in the organization of this prosencephalic region in all tetrapods.

Primary culture of avian embryonic heart forming region cells to study the regulation of vertebrate early heart morphogenesis by vitamin A

BackgroundImportant knowledge about the role of vitamin A in vertebrate heart development has been obtained using the vitamin A-deficient avian in ovo model which enables the in vivo examination of very early stages of vertebrate heart morphogenesis. These studies have revealed the critical role of the vitamin A-active form, retinoic acid (RA) in the regulation of several developmental genes, including the important growth regulatory factor, transforming growth factor-beta2 (TGFβ2), involved in early events of heart morphogenesis. However, this in ovo model is not readily available for elucidating details of molecular mechanisms determining RA activity, thus limiting further examination of RA-regulated early heart morphogenesis. In order to obtain insights into RA-regulated gene expression during these early events, a reliable in vitro model is needed. Here we describe a cell culture that closely reproduces the in ovo observed regulatory effects of RA on TGFβ2 and on several developmental genes linked to TGFβ signaling during heart morphogenesis.ResultsWe have developed an avian heart forming region (HFR) cell based in vitro model that displays the characteristics associated with vertebrate early heart morphogenesis, i.e. the expression of Nkx2.5 and GATA4, the cardiogenesis genes, of vascular endothelial growth factor (VEGF-A), the vasculogenesis gene and of fibronectin (FN1), an essential component in building the heart, and the expression of the multifunctional genes TGFβ2 and neogenin (NEO). Importantly, we established that the HFR cell culture is a valid model to study RA-regulated molecular events during heart morphogenesis and that the expression of TGFβ2 as well as the expression of several TGFβ2-linked developmental genes is regulated by RA.ConclusionsOur findings reported here offer a biologically relevant experimental in vitro system for the elucidation of RA-regulated expression of TGFβ2 and other genes involved in vertebrate early cardiovascular morphogenesis.

Effects of Fluid Dynamic Forces Created by Rotary Orbital Suspension Culture on Cardiomyogenic Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have the potential to become an effective resource for regenerative medicine in heart disease. The efficiency of cardiomyocyte differentiation changes drastically depending on the quality of hESC aggregates known as embryoid bodies (EBs). This study compares the production of EBs in rotary orbital suspension culture at various speeds with that in a simple static suspension culture. None of the outgrowths of EBs formed in static suspension culture developed beating. In contrast, outgrowths from EBs formed at 100 rpm had the highest rate of beating, 70%, and increased the expression of Nkx2.5, a master gene of cardiomyocyte differentiation. Outgrowths from EBs developed at slower rotational speeds showed more endoderm gene expression. At faster speeds, the expression of ectoderm markers increased. A computational hydrodynamic simulation showed that the liquid medium shear stress on the bottom was restricted to the periphery at 30 and 55 rpm and the center at 120 rpm. The analysis showed that at 100 rpm, the shear stress was uniformly distributed in the dish. Our results suggest that shear stress by fluid dynamic forces induces the differentiation of specific cell phenotypes from hESCs depending on rotational speed. For cardiomyocyte differentiation, 100 rpm was the most effective speed.

NKX2‐5, SIL/TAL and TLX3/HOX11L2 expression in Egyptian pediatric T‐cell acute lymphoblastic leukemia

Cohorts of T-cell acute lymphoblastic leukemia (T-ALL) patients show regional geographic differences in incidence, biological features and clinical outcome, implying that in different populations, cases may harbor different genetic lesions than those reported elsewhere. In this study, we prospectively evaluated the frequency and the clinical relevance of NKX2-5, TLX3/HOX11L2 and SIL/TAL expression in Egyptian childhood T-ALL. NKX2-5, TLX3/HOX11L2 and SIL/TAL expression were tested in peripheral blood and/or bone marrow of 83 newly diagnosed Egyptian childhood T-ALL patients. NKX2-5 expression was detected in 11/83 cases (13%), TLX3/HOX11L2 (5/83, 6%) and SIL/TAL (4/83, 5%). Initial central nervous system involvement was significantly higher in the NKX2-5 positive versus negative patients (P = 0.009). The follow-up period was a median of 65.5 months. The 5-year leukemia-free and event-free survival rates of the whole T-ALL population were 70 ± 6% and 58 ± 6%, respectively. The 5-year leukemia-free and event-free survival rates of NKX2-5 were 86 ± 13% and 60 ± 16%, respectively. There were no statistically significant differences in clinical presentation, biological features, initial response to chemotherapy, or subsequent treatments between the subgroups and the total population. Egyptian T-ALL cases seemed to have a different genetic pattern compared to other populations, with a lower incidence of TLX3/HOX11L2 and SIL/TAL but a higher incidence of NKX2-5 expression than recorded in Western countries.

Proper Development of the Outer Longitudinal Smooth Muscle of the Mouse Pylorus Requires Nkx2-5 and Gata3

Infantile hypertrophic pyloric stenosis is a common birth anomaly characterized by obstruction of the pyloric lumen. A genome-wide association study implicated NKX2-5, which encodes a transcription factor that is expressed in embryonic heart and pylorus, in the pathogenesis of infantile hypertrophic pyloric stenosis. However, the function of the NKX2-5 in pyloric smooth muscle development has not been examined directly. We investigated the pattern of Nkx2-5 during the course of murine pyloric sphincter development and examined coexpression of Nkx2-5 with Gata3 and Sox9-other transcription factors with pyloric-specific mesenchymal expression. We also assessed pyloric sphincter development in mice with disruption of Nkx2-5 or Gata3. We used immunofluorescence analysis to compare levels of NKX2-5, GATA3, and SOX9 in different regions of smooth muscle cells. Pyloric development was assessed in mice with conditional or germline deletion of Nkx2-5 or Gata3, respectively. Gata3, Nkx2-5, and Sox9 are coexpressed in differentiating smooth muscle cells of a distinct fascicle of the pyloric outer longitudinal muscle. Expansion of this fascicle coincides with development of the pyloric sphincter. Disruption of Nkx2-5 or Gata3 causes severe hypoplasia of this fascicle and alters pyloric muscle shape. Although expression of Sox9 requires Nkx2-5 and Gata3, there is no apparent hierarchical relationship between Nkx2-5 and Gata3 during pyloric outer longitudinal muscle development. Nkx2-5 and Gata3 are independently required for the development of a pyloric outer longitudinal muscle fascicle, which is required for pyloric sphincter morphogenesis in mice. These data indicate that regulatory changes that alter Nkx2-5 or Gata3 expression could contribute to pathogenesis of infantile hypertrophic pyloric stenosis.

Chicken β-globin insulators fail to shield the nkx2.5 promoter from integration site effects in zebrafish

Genetic lineage tracing and conditional mutagenesis are developmental genetics techniques reliant on precise tissue-specific expression of transgenes. In the mouse, high specificity is usually achieved by inserting the transgene into the locus of interest through homologous recombination in embryonic stem cells. In the zebrafish, DNA containing the transgenic construct is randomly integrated into the genome, usually through transposon-mediated transgenesis. Expression of such transgenes is affected by regulatory features surrounding the integration site from general accessibility of chromatin to tissue-specific enhancers. We tested if the 1.2kb cHS4 insulators derived from the chicken β-globin locus can shield a transgene from chromosomal position effects in the zebrafish genome. As our test promoters, we used two different-length versions of the zebrafish nkx2.5. We found that flanking a transgenic construct by cHS4 insulation sequences leads to overall increase in the expression of nkx2.5:mRFP. However, we also observed a very high degree of variability of mRFP expression, indicating that cHS4 insulators fail to protect nkx2.5:mRFP from falling under the control of enhancers in the vicinity of integration site.

Morphological and molecular bases of cardiac development

The heart is a mesoderm-derived organ, whose formation is regulated by various genes. Initially, the most important is expression of Nkx2.5, CR1, pitx2, anf and mhc2a, which are responsible for differentiation of cardiomyocytes. In a later phase activation of mhc2b, pitx2c, mesp1, pcmf1, vmhc, xin, mcl2v, mlc2a, mlc2a, mef2, hand1 and hand2 was revealed. Their expression is regulated by various molecules, including transcription (XIN, GATA, MEF, Tbx5, Baf60c, PECAM, tie-2, MEF2) and growth (VEGF, FGF, PDGF) factors, as well as proteins (i.e., dickkopf-1, cerberus, cytotactin, fibrillin, nodal, thrombomodulin, Wnt, bone morphometric ones - BMP2, BMP 4, BMP5, BMP7) and other substances, such as retinoid and folic acid. Crucial steps in cardiac organogenesis are development of the ventricle and atrial formation, as well as septation and valve formation. Any disturbances of such processes may lead to various congenital heart diseases and defects that could be initiated by various genetic, epigenetic or environmental factors. The most common heart malformations are: stenosis (coarctation) of the aorta and pulmonary trunk, bicuspid aortic valve, atrial and/or ventricular septal defect, persistent truncus arteriosus (Botallo duct), transposition of the great vessels, tricuspid atresia, hypoplastic left and right heart, as well as syndrome of Lutembachera, Cantrell, Ebstein, Eisenmenger and Shone and trilogy, tetralogy, pentalogy of Fallot.

Abstract 142: Modeling Drug-Induced Long QT Syndrome with Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Long QT syndrome (LQTS) is characterized by prolonged cardiac repolarization time and increased risk of ventricular arrhythmia. LQTS can be either inherited or induced notably after drugs intake. Mutations in genes encoding cardiac ion channels have been reported to underlie inherited LQTS. In contrast, drug-induced LQTS (diLQTS) most frequently arises from altered function of the hERG channel; the risk of developing diLQTS varies largely between subjects and most people who have life-threatening diLQTS have no known genetic risk factors. We investigated whether the susceptibility to develop diLQTS observed in vivo can be recapitulated in vitro using patient-specific induced pluripotent stem cell (iPSC) technology. We collected skin fibroblasts from ten subjects who developed significant diLQTS after administration of Sotalol and/or Erythromycin. Ten other individuals who displayed no changes in QT interval after administration of the same drugs, were selected. iPSC were generated by retroviral delivery of Oct4, Sox2, Nanog and Klf4 in 17 of the 20 individuals. We report preliminary results obtained from iPSC-derived cardiomyocytes (iPSC-CMs) of two subjects. All experiments were performed in a blinded fashion without knowledge of the associated clinical phenotype. Cardiac differentiation of iPSC resulted in the generation of spontaneously beating embryoid bodies. iPSC-CMs showed positive staining for TNNT2, ACTN2 and Cx43. Gene expression analysis confirmed the expression of NKX2.5, MLC2v, MYH6 and MYH7, and of the relevant KCNH2 gene. The two lines had similar basal electrophysiological properties as assessed by measurements of action potential (AP) by patch-clamp technique and extracellular field potentials (FP) using micro-electrode array (MEA). E4031, a classical HERG blocker, significantly prolonged the FP duration (FPD) in a dose-dependent manner in both lines (EC50: 30.19 and 51.57 respectively). When both Sotalol and Erythromicin were used, FPD was prolonged in one of the two samples in a dose-dependent manner (EC50Sotalol: 100; EC50Erythr: 9.64) while drug response was blunted in the other cell line. This study suggests that patient-specific iPSC can be used to model the functional abnormalities observed in acquired diLQTS.

Genetic Abnormalities inFOXP1Are Associated with Congenital Heart Defects

The etiology for the majority of congenital heart defects (CHD) is unknown. We identified a patient with unbalanced atrioventricular septal defect (AVSD) and hypoplastic left ventricle who harbored an ~0.3 Mb monoallelic deletion on chromosome 3p14.1. The deletion encompassed the first four exons of FOXP1, a gene critical for normal heart development that represses cardiomyocyte proliferation and expression of Nkx2.5. To determine whether FOXP1 mutations are found in patients with CHD, we sequenced FOXP1 in 82 patients with AVSD or hypoplastic left heart syndrome. We discovered two patients who harbored a heterozygous c.1702C>T variant in FOXP1 that predicted a potentially deleterious substitution of a highly conserved proline (p.Pro568Ser). This variant was not found in 287 controls but is present in dbSNP at a 0.2% frequency. The orthologous murine Foxp1 p.Pro596Ser mutant protein displayed deficits in luciferase reporter assays and resulted in increased proliferation and Nkx2.5 expression in cardiomyoblasts. Our data suggest that haploinsufficiency of FOXP1 is associated with human CHD.

Large particle multiphoton flow cytometry to purify intact embryoid bodies exhibiting enhanced potential for cardiomyocyte differentiation

Embryoid bodies (EBs) are large (>100 μm) 3D microtissues composed of stem cells, differentiating cells and extracellular matrix (ECM) proteins that roughly recapitulate early embryonic development. EBs are widely used as in vitro model systems to study stem cell differentiation and the complex physical and chemical interactions contributing to tissue development. Though much has been learned about differentiation from EBs, the practical and technical difficulties of effectively probing and properly analyzing these 3D microtissues has limited their utility and further application. We describe advancement of a technology platform developed in our laboratory, multiphoton flow cytometry (MPFC), to detect and sort large numbers of intact EBs based on size and fluorescent reporters. Real-time and simultaneous measurement of size and fluorescence intensity are now possible, through the implementation of image processing algorithms in the MPFC software. We applied this platform to purify populations of EBs generated from murine induced pluripotent stem (miPS) cells exhibiting enhanced potential for cardiomyocyte differentiation either as a consequence of size or expression of NKX2-5, a homeodomain protein indicative of precardiac cells. Large EBs (330-400 μm, diameter) purified soon after EB formation showed significantly higher potential to form cardiomyocytes at later time points than medium or small EBs. In addition, EBs expressing NKX2-5 soon after EB formation were more likely to form beating areas, indicative of cardiomyocyte differentiation, at later time points. Collectively, these studies highlight the ability of the MPFC to purify EBs and similar microtissues based on preferred features exhibited at the time of sorting or on features indicative of future characteristics or functional capacity.