Id genes are essential for early heart formation.

Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

Retinoic acid control of pax8 during renal specification of Xenopus pronephros involves hox and meis3.

Development of the Xenopus pronephros relies on renal precursors grouped at neurula stage into a specific region of dorso-lateral mesoderm called the kidney field. Formation of the kidney field at early neurula stage is dependent on retinoic (RA) signaling acting upstream of renal master transcriptional regulators such as pax8 or lhx1. Although lhx1 might be a direct target of RA-mediated transcriptional activation in the kidney field, how RA controls the emergence of the kidney field remains poorly understood. In order to better understand RA control of renal specification of the kidney field, we have performed a transcriptomic profiling of genes affected by RA disruption in lateral mesoderm explants isolated prior to the emergence of the kidney field and cultured at different time points until early neurula stage. Besides genes directly involved in pronephric development (pax8, lhx1, osr2, mecom), hox (hoxa1, a3, b3, b4, c5 and d1) and the hox co-factor meis3 appear as a prominent group of genes encoding transcription factors (TFs) downstream of RA. Supporting the idea of a role of meis3 in the kidney field, we have observed that meis3 depletion results in a severe inhibition of pax8 expression in the kidney field. Meis3 depletion only marginally affects expression of lhx1 and aldh1a2 suggesting that meis3 principally acts upstream of pax8. Further arguing for a role of meis3 and hox in the control of pax8, expression of a combination of meis3, hoxb4 and pbx1 in animal caps induces pax8 expression, but not that of lhx1. The same combination of TFs is also able to transactivate a previously identified pax8 enhancer, Pax8-CNS1. Mutagenesis of potential PBX-Hox binding motifs present in Pax8-CNS1 further allows to identify two of them that are necessary for transactivation. Finally, we have tested deletions of regulatory sequences in reporter assays with a previously characterized transgene encompassing 36.5 â€‹kb of the X. tropicalis pax8 gene that allows expression of a truncated pax8-GFP fusion protein recapitulating endogenous pax8 expression. This transgene includes three conserved pax8 enhancers, Pax8-CNS1, Pax8-CNS2 and Pax8-CNS3. Deletion of Pax8-CNS1 alone does not affect reporter expression, but deletion of a 3.5 â€‹kb region encompassing Pax8-CNS1 and Pax8-CNS2 results in a severe inhibition of reporter expression both in the otic placode and kidney field domains.

Caged Dexamethasone to Photo-control the Development of Embryos through Activation of the Glucocorticoid Receptor.

Efficient tools for controlling molecular functions with exquisite spatiotemporal resolution are much in demand to investigate biological processes in living systems. Here we report an easily synthesized caged dexamethasone for photo-activating cytoplasmic proteins fused to the glucocorticoid receptor. In the dark, it is stable in vitro as well as in vivo in both zebrafish (Danio rerio) and Xenopus sp, two significant models of vertebrates. In contrast, it liberates dexamethasone upon UV illumination, which has been harnessed to interfere with developmental steps in embryos of these animals. Interestingly, this new system is biologically orthogonal to the one for photo-activating proteins fused to the estrogen ERT receptor, which brings great prospect for activating two distinct proteins down to the single cell level.

Multiple hPOT1-TPP1 cooperatively unfold contiguous telomeric G-quadruplexes proceeding from 3' toward 5', a feature due to a 3'-end binding preference and to structuring of telomeric DNA.

Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5'TTAGGG repeats and ends with a 3' single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1-TPP1 preferentially proceeds from 3' toward 5'. We explain this directionality in terms of two factors: (i) the preference of hPOT1-TPP1 for the binding site situated at the 3' end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1-TPP1 in potassium. By comparing binding in K+ and in Li+, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3'-end and proceeding toward 5'.

Energy-Dependent Three-Body Loss in 1D Bose Gases.

We study the loss of atoms in quantum Newton's cradles with a range of average energies and transverse confinements. We find that the three-body collision rate in one-dimension is strongly energy dependent, as predicted by a strictly 1D theory. We adapt the theory to atoms in waveguides, then, using detailed momentum measurements to infer all the collisions that occur, we compare the observed loss to the adapted theory and find that they agree well.

Repression of TERRA Expression by Subtelomeric DNA Methylation Is Dependent on NRF1 Binding.

Chromosome ends are transcribed into long noncoding telomeric repeat-containing RNA (TERRA) from subtelomeric promoters. A class of TERRA promoters are associated with CpG islands embedded in repetitive DNA tracts. Cytosines in these subtelomeric CpG islands are frequently methylated in telomerase-positive cancer cells, and demethylation induced by depletion of DNA methyltransferases is associated with increased TERRA levels. However, the direct evidence and the underlying mechanism regulating TERRA expression through subtelomeric CpG islands methylation are still to establish. To analyze TERRA regulation by subtelomeric DNA methylation in human cell line (HeLa), we used an epigenetic engineering tool based on CRISPR-dCas9 (clustered regularly interspaced short palindromic repeats - dead CRISPR associated protein 9) associated with TET1 (ten-eleven 1 hydroxylase) to specifically demethylate subtelomeric CpG islands. This targeted demethylation caused an up-regulation of TERRA, and the enhanced TERRA production depended on the methyl-sensitive transcription factor NRF1 (nuclear respiratory factor 1). Since AMPK (AMP-activated protein kinase) is a well-known activator of NRF1, we treated cells with an AMPK inhibitor (compound C). Surprisingly, compound C treatment increased TERRA levels but did not inhibit AMPK activity in these experimental conditions. Altogether, our results provide new insight in the fine-tuning of TERRA at specific subtelomeric promoters and could allow identifying new regulators of TERRA.

Investigating the Effect of Mono- and Dimeric 360A G-Quadruplex Ligands on Telomere Stability by Single Telomere Length Analysis (STELA).

Telomeres are nucleoprotein structures that cap and protect the natural ends of chromosomes. Telomeric DNA G-rich strands can form G-quadruplex (or G4) structures. Ligands that bind to and stabilize G4 structures can lead to telomere dysfunctions by displacing shelterin proteins and/or by interfering with the replication of telomeres. We previously reported that two pyridine dicarboxamide G4 ligands, 360A and its dimeric analogue (360A)2A, were able to displace in vitro hRPA (a single-stranded DNA-binding protein of the replication machinery) from telomeric DNA by stabilizing the G4 structures. In this paper, we perform for the first time single telomere length analysis (STELA) to investigate the effect of G4 ligands on telomere length and stability. We used the unique ability of STELA to reveal the full spectrum of telomere lengths at a chromosome terminus in cancer cells treated with 360A and (360A)2A. Upon treatment with these ligands, we readily detected an increase of ultrashort telomeres, whose lengths are significantly shorter than the mean telomere length, and that could not have been detected by other methods.

Characterization of potential TRPP2 regulating proteins in early Xenopus embryos.

Transient receptor potential cation channel-2 (TRPP2) is a nonspecific Ca2+ -dependent cation channel with versatile functions including control of extracellular calcium entry at the plasma membrane, release of intracellular calcium ([Ca2+ ]i) from internal stores of endoplasmic reticulum, and calcium-dependent mechanosensation in the primary cilium. In early Xenopus embryos, TRPP2 is expressed in cilia of the gastrocoel roof plate (GRP) involved in the establishment of left-right asymmetry, and in nonciliated kidney field (KF) cells, where it plays a central role in early specification of nephron tubule cells dependent on [Ca2+ ]i signaling. Identification of proteins binding to TRPP2 in embryo cells can provide interesting clues about the mechanisms involved in its regulation during these various processes. Using mass spectrometry, we have therefore characterized proteins from late gastrula/early neurula stage embryos coimmunoprecipitating with TRPP2. Binding of three of these proteins, golgin A2, protein kinase-D1, and disheveled-2 has been confirmed by immunoblotting analysis of TRPP2-coprecipitated proteins. Expression analysis of the genes, respectively, encoding these proteins, golga2, prkd1, and dvl2 indicates that they are likely to play a role in these two regions. Golga2 and prkd1 are expressed at later stage in the developing pronephric tubule where golgin A2 and protein kinase-D1 might also interact with TRPP2. Colocalization experiments using exogenously expressed fluorescent versions of TRPP2 and dvl2 in GRP and KF reveal that these two proteins are generally not coexpressed, and only colocalized in discrete region of cells. This was observed in KF cells, but does not appear to occur in the apical ciliated region of GRP cells.

G4 motifs affect origin positioning and efficiency in two vertebrate replicators.

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome-wide studies in vertebrates have recently identified a consensus G-rich motif potentially able to form G-quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.

Short in-Frame Insertions/Deletions in the Coding Sequence of the α-Globin Gene. Consequences of the 3D Structure and Resulting Phenotypes: Hb Choisy as an Example.

A small group of hemoglobin (Hb) variants result from 'in-frame' deletion/insertion (del/ins). We describe a new variant of this group (Hb Choisy), found on the α1 gene, which is the exact counterpart of a previously published deletional variant, Hb J-Biskra [codons 51-58 (or codons 52-59) (-24 bp) (-TCTGCCCAGGTTAAGGGCCACGGC); HBA1: c.157_180del (or HBA2)]. In Hb J-Biskra, the sequence Ser-Ala-Gln-Val-Lys-Gly-His-Gly located from positions α52(E1) to α59(E8) is deleted, while in Hb Choisy the same sequence (Ser-Ala-Gln-Val-Lys-Gly-His-Gly) is inserted at position α52(E1). The variant carrying the insertion appears to be less damaging than the one with the deletion. A possible explanation could be that the additional sequence is located in the C to E interhelical region, and is less disturbing to the general structure of the globin chain. This insertion/deletion (ins/del) is likely favored by the repetition, at an interval of 16 nucleotides, of an eight nucleotide sequence. Comparison of variants of this group, found in the HbVar database, shows that structural modifications resulting from insertions are frequently less damaging than that caused by deletions.

5' to 3' Unfolding Directionality of DNA Secondary Structures by Replication Protein A: G-QUADRUPLEXES AND DUPLEXES.

The replication protein A (RPA) is a single-stranded DNA-binding protein that plays an essential role in DNA metabolism. RPA is able to unfold G-quadruplex (G4) structures formed by telomeric DNA sequences, a function important for telomere maintenance. To elucidate the mechanism through which RPA unfolds telomeric G4s, we studied its interaction with oligonucleotides that adopt a G4 structure extended with a single-stranded tail on either side of the G4. Binding and unfolding was characterized using several biochemical and biophysical approaches and in the presence of specific G4 ligands, such as telomestatin and 360A. Our data show that RPA can bind on each side of the G4 but it unwinds the G4 only from 5' toward 3'. We explain the 5' to 3' unfolding directionality in terms of the 5' to 3' oriented laying out of hRPA subunits along single-stranded DNA. Furthermore, we demonstrate by kinetics experiments that RPA proceeds with the same directionality for duplex unfolding.

TRPP2-dependent Ca2+ signaling in dorso-lateral mesoderm is required for kidney field establishment in Xenopus.

In Xenopus laevis embryos, kidney field specification is dependent on retinoic acid (RA) and coincides with a dramatic increase of Ca(2+) transients, but the role of Ca(2+) signaling in the kidney field is unknown. Here, we identify TRPP2, a member of the transient receptor potential (TRP) superfamily of channel proteins encoded by the pkd2 gene, as a central component of Ca(2+) signaling in the kidney field. TRPP2 is strongly expressed at the plasma membrane where it might regulate extracellular Ca(2+) entry. Knockdown of pkd2 in the kidney field results in the downregulation of pax8, but not of other kidney field genes (lhx1, osr1 and osr2). We further show that inhibition of Ca(2+) signaling with an inducible Ca(2+) chelator also causes downregulation of pax8, and that pkd2 knockdown results in a severe inhibition of Ca(2+) transients in kidney field explants. Finally, we show that disruption of RA results both in an inhibition of intracellular Ca(2+) signaling and of TRPP2 incorporation into the plasma membrane of kidney field cells. We propose that TRPP2-dependent Ca(2+) signaling is a key component of pax8 regulation in the kidney field downstream of RA-mediated non-transcriptional control of TRPP2.

Human POT1 is required for efficient telomere C-rich strand replication in the absence of WRN.

Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.

Pax8 and Pax2 are specifically required at different steps of Xenopus pronephros development.

The respective role of Pax2 and Pax8 in early kidney development in vertebrates is poorly understood. In this report, we have studied the roles of Pax8 and Pax2 in Xenopus pronephros development using a loss-of-function approach. Our results highlight a differential requirement of these two transcription factors for proper pronephros formation. Pax8 is necessary for the earliest steps of pronephric development and its depletion leads to a complete absence of pronephric tubule. Pax2 is required after the establishment of the tubule pronephric anlage, for the expression of several terminal differentiation markers of the pronephric tubule. Neither Pax2 nor Pax8 is essential to glomus development. We further show that Pax8 controls hnf1b, but not lhx1 and Osr2, expression in the kidney field as soon as the mid-neurula stage. Pax8 is also required for cell proliferation of pronephric precursors in the kidney field. It may exert its action through the wnt/beta-catenin pathway since activation of this pathway can rescue MoPax8 induced proliferation defect and Pax8 regulates expression of the wnt pathway components, dvl1 and sfrp3. Finally, we observed that loss of pronephros in Pax8 morphants correlates with an expanded vascular/blood gene expression domain indicating that Pax8 function is important to delimit the blood/endothelial genes expression domain in the anterior part of the dorso-lateral plate.

Hb Savaria [α49(CE7)Ser→Arg; HBA2: c.150C > A]: A New Case and Complete Description.

Hb Savaria [α49(CE7)Ser→Arg; HBA2: c.150C > A] is a rare hemoglobin (Hb) variant, initially described in Eastern Europe but present worldwide. It belongs to that class of variants which can be confused with Hb S [ß6(A3)Glu→Val; HBB: c.20A > T] by automated protein analysis and thus needs special tests for proper identification. Because it could arise from different nucleotide substitutions and according to the rules of the Human Genome Variation Society (HGVS) nomenclature, three 'Hb Savaria' variants are possible. In the case reported here it resulted from the HBA2: c.148A > C change.

Hb Olivet (HBA1: C.40G > A; p.Ala14Thr), a Novel Silent Hemoglobin Variant in Two Families of Distinct Origin.

We report two families, members of which are carriers of a novel hemoglobin (Hb) variant that was named Hb Olivet [α13(A11)Ala→Thr (α1) (GCC > ACC); HBA1: c.40G > A; p.Ala14Thr]. The analysis of these cases allowed a clear description of this anomaly that behaves as a silent Hb. In the first family, of Portuguese ethnicity living in France, the proband, a 24-year-old male and his 57-year-old mother, both appeared to be carriers. The son presented with borderline mean corpuscular volume (MCV), while the mother was normocytic and normochromic. Hemoglobin separation on capillary electrophoresis (CE) was normal, while a slightly asymmetric peak was observed on high performance liquid chromatography (HPLC). In a second family, originally from Surinam but living in The Netherlands, the proband, a 6-year-old girl, showed a mild microcytosis at low ferritin levels. The abnormal Hb was inherited from the mother who was clearly iron depleted, was not present in the sister and brother of the proband. The microcytic hypochromic anemia was only shown in two out of a total of four carriers. It therefore seems likely that iron depletion is causative as two carriers are completely normal. Characterization and genotype/phenotype correlation are briefly described.

DNA damage signaling induced by the G-quadruplex ligand 12459 is modulated by PPM1D/WIP1 phosphatase.

The triazine derivative 12459 is a potent G-quadruplex ligand that triggers apoptosis or delayed growth arrest, telomere shortening and G-overhang degradation, as a function of its concentration and time exposure to the cells. We have investigated here the DNA damage response induced by 12459 in A549 cells. Submicromolar concentrations of 12459 triggers a delayed Chk1-ATR-mediated DNA damage response associated with a telomeric dysfunction and a G2/M arrest. Surprisingly, increasing concentrations of 12459 leading to cell apoptosis induced a mechanism that bypasses the DNA damage signaling and leads to the dephosphorylation of Chk1 and γ-H2AX. We identified the phosphatase Protein Phosphatase Magnesium dependent 1D/Wild-type P53-Induced Phosphatase (PPM1D/WIP1) as a factor responsible for this dephosphorylation. SiRNA-mediated depletion of PPM1D/WIP1 reactivates the DNA damage signaling by 12459. In addition, PPM1D/WIP1 is activated by reactive oxygen species (ROS) induced by 12459. ROS generated by 12459 are sufficient to trigger an early DNA damage in A549 cells when PPM1D/WIP1 is depleted. However, ROS inactivation by N-acetyl cysteine (NAC) treatment does not change the apoptotic response induced by 12459. Because PPM1D expression was recently reported to modulate the recruitment of DNA repair molecules, our data would suggest a cycle of futile protection against 12459, thus leading to a delayed mechanism of cell death.

Closure of Isolated Congenital Coronary Artery Fistula: Long-Term Outcomes and Rate of Re-intervention.

Long-term outcome after closure of isolated congenital coronary artery fistula (ICCAF) is poorly documented. To assess late outcome after ICCAF closure, a 1983-2013 retrospective study included all patients who attempted an ICCAF closure and whose follow-up was ≥1 year. ICCAF was diagnosed in 23 patients [median age 6.9 years (0.1-70.5 years), 13 children]. ICCAF was symptomatic in 12 patients (52.2 %). First intervention was either a transcatheter embolization (n = 19 patients, 82.6 %) or a surgical ligation (n = 4 patients, 17.4 %). After a follow-up of 9.0 years (2.8-33.5), neither death nor late ischemic event occurred but one patient was transplanted, because of postoperative myocardial infarction. Late ICCAF recanalization occurred in eight patients, leading to successful embolization of the shunt in all patients after a delay of 9.8 years (5.7-13.8 years) from the first intervention. Re-intervention occurred later in children (p = 0.0027), with a 50 and 37.5 % freedom from re-intervention in adults compared to a 100 and 89.0 % in children, respectively, at 1 and 6 years of follow-up. At last follow-up, coronary artery diameter had decreased from a mean z score of 12.0 ± 7.7 to a mean z score of 6.0 ± 6.0 (p = 0.002). Long-term outcome after ICCAF closure is excellent, with neither death nor late ischemic event, and a significant decrease in coronary artery diameter with time. Late follow-up is of paramount importance, as one-third of patients will require a re-intervention for late shunt recanalization.

Catheter-based interventions for modified Blalock-Taussig shunt obstruction: a 20-year experience.

Thrombotic occlusion of a modified Blalock-Taussig (BT) shunt is rare, leading to life-threatening hypoxemia. Rescue percutaneous interventions may allow recanalization of the systemic-to-pulmonary shunt but data on large patients' scales are lacking. We aimed to describe safety and effectiveness of catheter-based interventions to restore modified BT shunt patency. All patients who attempted transcatheter intervention for thrombotic occlusion of a modified BT shunt at our Institution from 1994 to 2014 were reviewed. Characteristics, management, and outcomes of the 28 identified patients were analyzed. Thirty-three procedures were performed at a median age of 0.6 years old (range 0.03-32.1 years) and a median weight of 5.8 kg (range 2.2-82 kg). Percutaneous intervention consisted in 33 balloon angioplasty (100 %) and 14 stent implantations (42.4 %). Thrombolytic agents were also used in 6.1 % cases. No peri-procedural death occurred but complications were observed in five patients (15.2 %), including one catheter-induced transient complete atrioventricular block, one cardiac tamponade, and one massive thrombo-embolic stroke. Early procedural success was obtained in 28 patients (84.8 %) and remained long-lasting in 26 patients (78.8 %). A young age and a low body-weight at the time of the procedure were significantly associated with procedural failure (p = 0.0364 and p = 0.0247, respectively). Although technically challenging and carrying potential major complications, transcatheter intervention can be considered as an efficient rescue strategy to restore patency in case of thrombotic obstruction of a modified BT shunt.

Cubilin, a high affinity receptor for fibroblast growth factor 8, is required for cell survival in the developing vertebrate head.

Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity.