CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

Aims: Haploinsufficiency of the chromo-domain protein CHD7 underlies most cases of CHARGE syndrome, a multisystem birth defect including congenital heart malformation. Context specific roles for CHD7 in various stem, progenitor, and differentiated cell lineages have been reported. Previously, we showed severe defects when Chd7 is absent from cardiopharyngeal mesoderm (CPM). Here, we investigate altered gene expression in the CPM and identify specific CHD7-bound target genes with known roles in the morphogenesis of affected structures. / Methods and results: We generated conditional KO of Chd7 in CPM and analysed cardiac progenitor cells using transcriptomic and epigenomic analyses, in vivo expression analysis, and bioinformatic comparisons with existing datasets. We show CHD7 is required for correct expression of several genes established as major players in cardiac development, especially within the second heart field (SHF). We identified CHD7 binding sites in cardiac progenitor cells and found strong association with histone marks suggestive of dynamically regulated enhancers during the mesodermal to cardiac progenitor transition of mESC differentiation. Moreover, CHD7 shares a subset of its target sites with ISL1, a pioneer transcription factor in the cardiogenic gene regulatory network, including one enhancer modulating Fgf10 expression in SHF progenitor cells vs. differentiating cardiomyocytes. / Conclusion: We show that CHD7 interacts with ISL1, binds ISL1-regulated cardiac enhancers, and modulates gene expression across the mesodermal heart fields during cardiac morphogenesis

Caudal Fgfr1 disruption produces localised spinal mis-patterning and a terminal myelocystocele-like phenotype in mice

Closed spinal dysraphisms are poorly understood malformations classified as neural tube (NT) defects. Several, including terminal myelocystocele, affect the low spine. We previously identified a NT closure-initiating point, Closure 5, in the distal spine of mice. Here we document equivalent morphology of the caudal-most closing posterior neuropore (PNP) in mice and humans. Closure 5 forms in a region of active FGF signalling and pharmacological FGF receptor blockade impairs its formation in cultured mouse embryos. Conditional genetic deletion of Fgfr1 in caudal embryonic tissues with Cdx2Cre diminishes neuroepithelial proliferation, impairs Closure 5 formation and delays PNP closure. After closure, the distal NT of Fgfr1-disrupted embryos dilates to form a fluid-filled sac overlying ventrally flattened spinal cord. This phenotype resembles terminal myelocystocele. Histological analysis reveals regional and progressive loss of SHH and FOXA2-positive ventral NT domains, resulting in OLIG2-labelling of the ventral-most NT. The OLIG2-domain is also subsequently lost, eventually producing a NT entirely positive for the dorsal marker PAX3. Thus, a terminal myelocystocele-like phenotype can arise after completion of NT closure with localised spinal mis-patterning caused by disruption of FGFR1 signalling

Qualidade dos frutos de variedades de videira do Banco Ativo de Germoplasma da Embrapa Semiárido.

O objetivo deste trabalho foi caracterizar a qualidade de uvas tintas para consumo in natura e para processamento, conservadas no Banco Ativo de Germoplasma (BAG) de videira da Embrapa Semiárido

Investigating the role of Geminin in proliferation, migration and differentiation of pluripotent neural crest cells in transgenic mice

The neural crest is a multipotent cell population that is formed at the dorsal neural tube of vertebrate embryos during neurulation. After their formation, neural crest cells (NCCs) delaminate from the neural tube and migrate throughout the embryo following specific pathways, and give rise to a wide variety of structures, such as neural and glial cells of the peripheral nervous system (PNS), melanocytes, structures of the craniofacial skeleton, etc. Neural crest formation, self-renewal and differentiation require the coordination of proliferation and differentiation. Deregulation of these processes results in developmental diseases in humans, known as neurocristopathies. Geminin is a molecule that has the ability to regulate cell cycle progression and differentiation, through interactions with the licensing factor Cdt1, transcription factors and chromatin remodeling factors. Previous studies from our laboratory have shown that Geminin is an important regulator of self-renewal and differentiation of early cortical progenitors. In order to understand the mechanisms that control self-renewal and differentiation of multipotent neural crest cells (NCCs) and gain insight into the molecular mechanism of human diseases, we studied the role of Geminin in the formation, self-renewal and differentiation of NCCs. Towards this direction, we performed in vivo and in vitro experiments, using animal models that have been generated in our laboratory and allow the conditional inactivation of Geminin in neural crest cells. Our results showed that deletion of Geminin causes severe morphological malformations in embryos that are characterized by the absence of midbrain, branchial arches and severe craniofacial malformation. Mutant embryos are dying a few days before birth. Moreover, during early embryonic development, the neural crest-derived structures, such as cranial and dorsal root ganglia, the maxillary and the mandibular components, and enteric progenitor cells, were severely affected. The decrease of enteric neural crest cells resulted in the formation of aganglionic gut that resembles with the phenotype of Hirschsprung disease. The conditional inactivation of Geminin resulted in the decreased formation of naïve vagal neural crest cells, while enteric neural crest cells were dramatically reduced. Geminin deficient enteric neural crest sells show increased apoptosis at E9.5 and E10.5, and decreased cell proliferation at E9.5. These findings, combined with the decreased self-renewal capacity of enteric progenitor cells (EPCs) in vitro, suggest that Geminin is important for the self-renewal and the survival of ENS progenitor cells. In addition, deletion of Geminin resulted in decreased committed enteric neural crest cells that express enteric progenitor markers Phox2b, Ret and Mash1. In conclusion, our results highlight Geminin as an important molecule during the formation of multipotent neural crest cells. Geminin is required for the formation of vagal neural crest cells that colonize the gastrointestinal tract, and regulates survival and self-renewal of these cells, as well as their transition from a multipotent state to the committed enteric lineage of progenitor cells. Moreover, conditional inactivation of Geminin leads to Hirschsprung-like phenotype that could be used as model organisms to study disease pathogenesis and help in the discovery of new therapies.Τα κύτταρα της νευρικής ακρολοφίας είναι ένας πολυδύναμος πληθυσμός βλαστικών κυττάρων που δημιουργείται στη ραχιαία πλευρά του νευρικού σωλήνα των σπονδυλωτών κατά τη διάρκεια της νευριδίωσης. Μετά τη δημιουργία τους, τα κύτταρα της νευρικής ακρολοφίας μεταναστεύουν σε ολόκληρο το έμβρυο, ακολουθώντας συγκεκριμένα μονοπάτια, συνεισφέροντας στη δημιουργία μιας μεγάλης ποικιλίας δομών, όπως νευρικά και γλοιακά κύτταρα του περιφερικού νευρικού συστήματος (ΠΝΣ), μελανοκύτταρα, δομές που συμβάλλουν στο σκελετό του κρανίου και του προσώπου κλπ. Η δημιουργία, η αυτο- ανανέωση και η διαφοροποίηση των κυττάρων της νευρικής ακρολοφίας απαιτούν το συντονισμό των διεργασιών του κυτταρικού πολλαπλασιασμού και της κυτταρικής διαφοροποίησης. Η αδυναμία συντονισμού των παραπάνω διαδικασιών οδηγεί στην εμφάνιση ασθενειών στον άνθρωπο (neurocristopathies). Η Geminin είναι ένα μόριο που έχει την ικανότητα να ρυθμίζει την πρόοδο του κυτταρικού κύκλου, αλληλεπιδρώντας με τον παράγοντα αδειοδότησης της αντιγραφής Cdt1, και τη διαφοροποίηση, μέσω της αλληλεπίδρασής της με μεταγραφικούς παράγοντες και πρωτεΐνες αναδιαμόρφωσης της χρωματίνης. Προηγούμενες μελέτες του εργαστηρίου μας έχουν αναδείξει τη Geminin ως ένα σημαντικό ρυθμιστή των διαδικασιών της αυτο-ανανέωσης και διαφοροποίησης στα πρόδρομα νευρικά κύτταρα στον αναπτυσσόμενο φλοιό. Προκειμένου να κατανοήσουμε τους μηχανισμούς που ελέγχουν την αυτο-ανανέωση και τη διαφοροποίηση των πολυδύναμων κυττάρων της νευρικής ακρολοφίας και να κατανοήσουμε το μοριακό μηχανισμό ασθενειών στον άνθρωπο που σχετίζονται με την απορρύθμιση του ελέγχου της ικανότητας αυτο-ανανέωσης και διαφοροποίησης των πολυδύναμων κυττάρων της νευρικής ακρολοφίας μελετήσαμε το ρόλο της Geminin στη δημιουργία, την αυτο-ανανέωση, τον καθορισμό και τη διαφοροποίηση των κυττάρων της νευρικής ακρολοφίας. Προς αυτή την κατεύθυνση πραγματοποιήθηκαν τόσο in vivo όσο και in vitro πειράματα, χρησιμοποιώντας ζωικά μοντέλα τα οποία δημιουργήθηκαν από το εργαστήριο μας και στα οποία το γονίδιο της Geminin είχε αδρανοποιηθεί ειδικά στα κύτταρα της νευρικής ακρολοφίας. Τα αποτελέσματά μας έδειξαν ότι η απουσία της Geminin οδηγεί στη δημιουργία εμβρύων με σοβαρές μορφολογικές αλλοιώσεις, που κατά τα πρώιμα αναπτυξιακά στάδια χαρακτηρίζονται από την απουσία της δομής του μεσεγκεφάλου και των βραγχιακών τόξων και σε μεταγενέστερα αναπτυξιακά στάδια εμφανίζουν σοβαρή κρανιοπροσωπική δυσμορφία, με κατάληξη το θάνατο των εμβρύων, λίγες ημέρες πριν γεννηθούν. Επιπλέον, κατά τα πρώιμα αναπτυξιακά στάδια παρατηρήθηκαν σοβαρές αλλοιώσεις σε δομές που προέρχονται από τη νευρική ακρολοφία, όπως είναι τα κρανιακά και τα ραχιαία γάγγλια, οι γναθικές προεκβολές και τα πρόδρομα κύτταρα του εντερικού νευρικού συστήματος. Η μείωση του πληθυσμού των πρόδρομων κυττάρων του εντερικού νευρικού συστήματος (ΕΝΣ) οδήγησε στη δημιουργία ενός αγαγγλιονικού εντέρου, το οποίο παρομοιάζει με το φαινότυπο του ΕΝΣ στη νόσο Hirschsprung στον άνθρωπο. Η ιστοειδική αδρανοποίηση της Geminin οδήγησε στη μείωση των αδιαφοροποίητων κυττάρων νευρικής ακρολοφίας που δημιουργούνται στην αυχενική περιοχή του νευρικού σωλήνα και στην είσοδο μικρότερου αριθμού κυττάρων νευρικής ακρολοφίας στον γαστρεντερικό σωλήνα κατά τα πρώτα στάδια του αποικισμού του. Μελέτη των εντερικών κυττάρων νευρικής ακρολοφίας έδειξε ότι η αποσιώπηση της Geminin προκάλεσε την αύξηση της απόπτωσης κατά τις ηλικίες Ε9.5 και Ε10.5 και τη μείωση του κυτταρικού πολλαπλασιασμού τους κατά την ηλικία Ε9.5. Σε συνδυασμό με τη μειωμένη ικανότητα που δείχνουν τα πρόδρομα εντερικά κύτταρα να αυτο-ανανεώνονται, τα αποτελέσματά μας προτείνουν ότι η Geminin έχει σημαντικό ρόλο στην αυτο-ανανέωση και την επιβίωση των πρόδρομων κυττάρων του ΕΝΣ. Επιπλέον, η απουσία της Geminin οδηγεί στη μείωση των κυττάρων που έχουν καθορισμένη μοίρα και εκφράζουν τους δείκτες των πρόδρομων εντερικών κυττάρων Phox2b, Ret και Mash1, ενώ τα κύτταρα αυτά απουσία της Geminin παρουσιάζουν μειωμένη παραγωγή νευρικών κυττάρων, κατά την έναρξη της νευρωνικής διαφοροποίησης. Συμπερασματικά, τα αποτελέσματά μας αναδεικνύουν τη Geminin ως ένα σημαντικό μόριο κατά τη δημιουργία των πολυδύναμων κυττάρων της νευρικής ακρολοφίας. Επίσης η Geminin είναι απαραίτητη για τη δημιουργία των κυττάρων της νευρικής ακρολοφίας που αποικίζουν το γαστρεντερικό σωλήνα, ενώ ρυθμίζει την επιβίωση και την αυτο-ανανέωσή τους, καθώς και τη μετάβασή τους από την αρχικά αδιαφοροποίητη/πολυδύναμη κατάσταση στην εντερική αναπτυξιακή μοίρα. Επιπλέον η απουσία της Geminin δημιουργεί μύες οι οποίοι μιμούνται τη νόσο του Hirschsprung και αποτελούν ένα σημαντικό ζωικό μοντέλο για τη μελέτη των μηχανισμών της μοριακή παθογένειας της νόσου αλλά και στην εύρεση νέων θεραπειών

Analysis of IAP and <i>agouti</i> expression in <i>A^vy</i> ES cells.

<p>For qRT-PCR analyses, bar graphs represent mean+/− S.D. Results are representative of two independent experiments. <b>A.</b> qRT-PCR analysis of IAP expression. Expression levels are normalized to actin. RNA from <i>Dnmt1^c/c</i> ES cells was used as a positive control for IAP expression. <b>B.</b> qRT-PCR analysis of <i>IAP</i> expression in cells either untreated or treated with the DNA demethylating agent 5-azacytidine (5-aza, 7.5 µM). Expression levels are normalized to actin and expressed as fold change relative to the signal observed in untreated cells. <b>C.</b> qRT-PCR analysis of <i>agouti</i> expression in the various organs indicated. Expression levels are normalized to <i>GAPDH</i> and measured relative to expression level in heart. <b>D.</b> qRT-PCR analysis of <i>agouti</i> expression in cells either untreated or treated with the DNA demethylating agent 5-aza. Expression levels are normalized to GAPDH and measured relative to untreated signals. <b>E.</b> qRT-PCR analysis of <i>GFP</i> expression in the various cell lines indicated. RNA from <i>Oct4-GiP</i> ES cells was used as a positive control for GFP expression. Expression levels are normalized to actin. <b>F.</b> Western blot data using an anti-GFP antibody on whole cell lysates from <i>A^vy/A^GFP</i> ES cells either untreated or treated with 5-aza or the histone demethylating agent Tricostatin A (TSA, 40 nM). Whole cell lysate from <i>Oct4-GiP</i> ES cells was used as a positive control. Western blot using an anti-tubulin antibody was used as a loading control.</p

Methylation analysis of <i>A^vy</i> ES cell lines.

<p><b>A.</b><i>Sry</i> PCR genotyping result of the various cell lines used in this study. PCR on DNA from XY ES cells generate generates a 249 bp amplicon. Clone E1 is <i>Sry</i>⁻ and inferred to be XX. <b>B.</b> Methylation sensitive Southern blot data on DNA extracted from the various <i>A^vy</i> ES cell lines indicated. DNA extracted from <i>Dnmt1^−/−Dnmt3a^−/−Dnmt3b^−/−</i> (TKO) ES cells was included as a control for unmethylated signal. Asterisks indicate hypomethylated/unmethylated fragments. DNAs were digested with <i>Hpa</i>II restriction endonuclease. Probes used are as indicated. <b>C.</b> Bisulphite sequencing data using primers specific for the <i>A^vy</i> IAP LTR on DNA from the <i>A^vy</i> cell lines indicated. Passage numbers of <i>A^vy</i> ES cells are as follows: passage 6 (C12 and F8), passage 7 (B12, C11, D6, D11, E4, E7 and E9); passage 10 (C6), passage 25 (B5), passage 35 (B5_Neo), passage 45 (B5_GFP).</p

CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

CHARGE syndrome • CHD7 • Second heart field • Cardiopharyngeal mesoderm • Cardiac enhancer

Cdt1 Is Differentially Targeted for Degradation by Anticancer Chemotherapeutic Drugs

Background: Maintenance of genome integrity is crucial for the propagation of the genetic information. Cdt1 is a major component of the pre-replicative complex, which controls once per cell cycle DNA replication. Upon DNA damage, Cdt1 is rapidly targeted for degradation. This targeting has been suggested to safeguard genomic integrity and prevent rereplication while DNA repair is in progress. Cdt1 is deregulated in tumor specimens, while its aberrant expression is linked with aneuploidy and promotes tumorigenesis in animal models. The induction of lesions in DNA is a common mechanism by which many cytotoxic anticancer agents operate, leading to cell cycle arrest and apoptosis. Methodology/Principal Finding: In the present study we examine the ability of several anticancer drugs to target Cdt1 for degradation. We show that treatment of HeLa and HepG2 cells with MMS, Cisplatin and Doxorubicin lead to rapid proteolysis of Cdt1, whereas treatment with 5-Fluorouracil and Tamoxifen leave Cdt1 expression unaffected. Etoposide affects Cdt1 stability in HepG2 cells and not in HeLa cells. RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA. Conclusion/Significance: Our data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis. Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficac

Kidney Issues Associated with COVID-19 Disease

Infection with SARS-CoV-2 and the resulting COVID-19 can cause both lung and kidney damage. SARS-CoV-2 can directly infect renal cells expressing ACE2 receptors, resulting in kidney damage, and acute kidney injury (AKI) has been reported in COVID-19 hospitalized patients. The pathophysiology of COVID-19-associated AKI is multifactorial. Local and systemic inflammation, immune system dysregulation, blood coagulation disorders, and activation of the renin-angiotensin-aldosterone system (RAAS) are factors that contribute to the development of AKI in COVID 19 disease. COVID-19 patients with kidney involvement have a poor prognosis, and patients with chronic kidney disease (CKD) infected with SARS-CoV-2 have an increased mortality risk. CKD patients with COVID-19 may develop end-stage renal disease (ESRD) requiring dialysis. In particular, patients infected with SARS-CoV-2 and requiring dialysis, as well as patients who have undergone kidney transplantation, have an increased risk of mortality and require special consideration. Nephrologists and infectious disease specialists face several clinical dilemmas in the prophylaxis and treatment of CKD patients with COVID-19. This entry presents recent data showing the effects of COVID-19 on the kidneys and CKD patients and the challenges in the management of CKD patients with COVID-19, and discusses treatment strategies for these patients

5-Fluoruracil (5-FU) does alter Cdt1 protein expression levels in HepG2 but not in HeLa cells.

<p>HeLa and HepG2 cells were incubated for 6 h with 5-FU (0.1, 10 and 100 µg/ml) in the absence (lanes 1–4 and 9–10) or in the presence (lanes 5–8 and 12–14) of MG-132 (20 µM). Protein extracts were analyzed by Western blotting using antibodies against Cdt1, PARP, Geminin and Tubulin.</p