Cortical Development and Brain Malformations: Insights From the Differential Regulation of Early Events of DNA Replication

During the development of the cortex distinct populations of Neural Stem Cells (NSCs) are defined by differences in their cell cycle duration, self-renewal capacity and transcriptional profile. A key difference across the distinct populations of NSCs is the length of G1 phase, where the licensing of the DNA replication origins takes place by the assembly of a pre-replicative complex. Licensing of DNA replication is a process that is adapted accordingly to the cell cycle length of NSCs to secure the timed duplication of the genome. Moreover, DNA replication should be efficiently coordinated with ongoing transcription for the prevention of conflicts that would impede the progression of both processes, compromising the normal course of development. In the present review we discuss how the differential regulation of the licensing and initiation of DNA replication in different cortical NSCs populations is integrated with the properties of these stem cells populations. Moreover, we examine the implication of the initial steps of DNA replication in the pathogenetic mechanisms of neurodevelopmental defects and Zika virus-related microcephaly, highlighting the significance of the differential regulation of DNA replication during brain development

The Limits of Lawyering: Legal Opinions in Structured Finance

Significant controversy surrounds the issuance of legal opinions in structured finance transactions, particularly where accountants separately use these opinions, beyond their traditional primary use, for determining whether to characterize the transactions as debt. Reflecting at its core the unresolved boundaries between public and private in financial transactions, this controversy raises important issues of first impression: To what extent, for example, should lawyers be able to issue legal opinions that create negative externalities? Furthermore, what should differentiate the roles of lawyers and accountants in disclosing information to investors? Resolution of these issues not only helps to demystify the mystique, and untangle the morass, of legal-opinion giving but also affects the very viability of the securitization industry, which dominates American, and increasingly global, financing

Investigating the molecular role of Geminin in early embryonic neural stem cells

The cerebral cortex consists the outer layer of the mammalian brain and is one of the most complicated regions, characterised by excessive cell diversity. The cortex derives from the neural tube through a strictly regulated process that involves the generation of diverse types of neural stem and progenitor cells organised in discrete proliferating zones. Through the coordinated action of signalling molecules and transcription factors, cortical neurons are generated in the proliferating zones and subsequently they migrate radially towards the cortical plate. Additional intrinsic factors, like cell cycle regulation and chromatin landscape, are involved in the cell fate choices of neural stem cells. Genetic or environmental factors that compromise the proliferation or the integrity of neural stem cells during early development can lead to defective brain development and neurodevelopmental disorders. The accumulation of genetic alterations that affect the physiological functions of cells and usually lead to increased DNA damage and uncontrolled proliferation, is known as genomic instability. One of the main sources of genomic instability is replication stress which is described as DNA synthesis slow down or stalling of replication fork. Interestingly, replication stress-induced genomic instability during early development has been associated with microcephaly in humans. Geminin is a known nuclear protein that is characterised as a DNA replication inhibitor and as a molecule that is involved in cell fate choices. Here, we show that Geminin is essential for brain development in order to secure genome integrity during the early stages of cortical formation. In vivo deletion of Geminin from the neuroepithelial cells of the developing cortex caused increased accumulation of double strand breaks due to persistent replication stress, leading to cell cycle and mitotic defects. These aberrations were sufficient to induce apoptosis leading to a significant decrease in the stem cells pool and eventually to microcephaly. On the other hand, deletion of Geminin from radial glia cells did not cause replication stress or brain malformations. In order to elucidate the differential response between neuroepithelial and radial glia cells to Geminin deletion, we investigated possible differences in the regulation of the early events of DNA replication between the two populations. We have provided evidence that neuroepithelial cells license and fire more origins in order to complete DNA replication compared to radial glia cells. Additionally, we examined whether the length of the G1 phase, a well-described difference between neuroepithelial and radial glia cells contributes to the observed differences the exhibit the two populations upon Geminin. Establishing an in vitro system that permits the manipulation of G1 length we showed that the genomic instability caused by Geminin deletion is amplified as the G1 length decreases. We suggest that Geminin is essential for the establishment of a robust population of neuroepithelial cells early brain development. Our results support the dynamic regulation of DNA replication during cortical development suggesting that the protective role of Geminin in the maintenance of genome integrity during early development is defined by the intrinsic features of neuroepithelial cells.Ο φλοιός αποτελεί την εξωτερική στιβάδα του εγκεφάλου των θηλαστικών και είναι μία δομή που χαρακτηρίζεται από υψηλή κυτταρική ετερογένεια και πλούσια συνδεσιμότητα. Η ανάπτυξη του φλοιού αποτελεί μία διαδικασία που αρχίζει κατά τα αρχικά εμβρυϊκά στάδια και βασίζεται στη δημιουργία διακριτών πληθυσμών νευρικών βλαστικών και προγονικών κυττάρων τα οποία οργανώνονται σε νευρογενετικές ζώνες. Οι φλοιικοί νευρώνες δημιουργούνται σταδιακά στις νευρογενετικές ζώνες μέσω της συντονισμένης δράσης σηματοδοτικών μορίων και μεταγραφικών παραγόντων. Επιπρόσθετα ενδογενή χαρακτηριστικά των νευρικών βλαστικών κυττάρων, όπως η οργάνωση της χρωματίνης και η ρύθμιση του κυτταρικού κύκλου συμβάλουν στις διαδικασίες αυτοανανέωσης και διαφοροποίησης των νευρικών βλαστικών κυττάρων. Γενετικοί ή περιβαλλοντικοί παράγοντες οι οποίοι επηρεάζουν τις βασικές λειτουργίες των νευρικών βλαστικών κυττάρων κατά την εμβρυϊκή ανάπτυξη οδηγούν στην εμφάνιση νευροαναπτυξιακών ανωμαλιών και νευρολογικών διαταραχών. Πρόσφατα, η εμφάνιση γονιδιωματικής αστάθειας η οποία προκαλείται από το αντιγραφικό στρες συνδέθηκε με την εμφάνιση μικροκεφαλίας. Η γονιδιωματική αστάθεια αποτελεί μία κατάσταση η οποία προκαλείται από τη συσσώρευση γενετικών τροποποιήσεων επηρεάζοντας τις βασικές κυτταρικές λειτουργίες. Το αντιγραφικό στρες, το οποίο ορίζεται ως η επιβράδυνση ή η πλήρης διακοπή της αντιγραφής του DNA αποτελεί μία από τις πηγές γονιδιωματικής αστάθειας. H Geminin αποτελεί μία πρωτεΐνη η οποία έχει χαρακτηρισθεί για τον διττό ρόλο που έχει στη ρύθμιση της έναρξης της αντιγραφής του DNA και στις αποφάσεις διαφοροποίησης των βλαστικών κυττάρων. Τα αποτελέσματα της συγκεκριμένης εργασίας καταδεικνύουν ότι η Geminin είναι απαραίτητη για τη διασφάλιση της γονιδιωματικής σταθερότητας των νευροεπιθηλιακών κυττάρων τα οποία αποτελούν τον πρωταρχικό πληθυσμό νευρικών βλαστικών κυττάρων του εγκεφαλικού φλοιού. In vivo απαλοιφή της Geminin από τα νευροεπιθηλιακά κύτταρα εμβρύων μυών προκάλεσε τη συσσώρευση βλαβών στο DNA λόγω αντιγραφικού στρες τα οποία οδήγησαν στη διακοπή του κυτταρικού κύκλου καθώς και σε ανωμαλίες κατά τον διαχωρισμό των χρωμοσωμάτων. Τα παραπάνω είχαν ως αποτέλεσμα τον μειωμένο πολλαπλασιασμό και τελικά την απόπτωση των νευροεπιθηλιακών κυττάρων. Η δραματική μείωση των νευρικών βλαστικών κυττάρων είχε ως αποτέλεσμα τη διακοπή της φυσιολογικής ανάπτυξης του φλοιού προκαλώντας στα έμβρυα μυών τον φαινότυπο της μικροκεφαλίας. Επιπρόσθετη ανάλυση κατέδειξε ότι ο προστατευτικός ρόλος της Geminin έναντι του αντιγραφικού στρες χαρακτηρίζει μόνο τα νευροεπιθηλιακά κύτταρα, καθώς η ανάλυση που πραγματοποιήθηκε στα κύτταρα ακτινωτής γλοίας του αναπτυσσόμενου φλοιού δεν είχε παρόμοια αποτελέσματα. Προκειμένου να αποσαφηνίσουμε τη διαφορετική απόκριση των νευροεπιθηλιακών και των κυττάρων ακτινωτής γλοίας στην αποσιώπηση της Geminin εξετάσαμε ενδογενείς διαφορές μεταξύ των δύο πληθυσμών στις διαδικασίες αδειοδότησης και έναρξης της αντιγραφής του DNA. Τα αποτελέσματά μας υποστηρίζουν ότι τα νευροεπιθηλιακά κύτταρα αδειοδοτούν και χρησιμοποιούν περισσότερες αφετηρίες προκειμένου να ολοκληρώσουν την αντιγραφή του DNA σε σχέση με τα κύτταρα ακτινωτής γλοίας. Στη συνέχεια εξετάσαμε εάν η αυξημένη ευαισθησία των νευροεπιθηλιακών κυττάρων στην αποσιώπηση της Geminin οφείλεται στη σύντομη G1 φάση που τα χαρακτηρίζει. Χρησιμοποιώντας ένα in vitro σύστημα το οποίο μας επιτρέπει να ελέγχουμε τη διάρκεια της G1 δείξαμε ότι η γονιδιωματική αστάθεια η οποία προκαλείται από την αποσιώπηση της Geminin ενισχύεται καθώς η διάρκεια της G1 φάσης μειώνεται. Συνοψίζοντας, προτείνεται ότι η Geminin είναι απαραίτητη κατά τα αρχικά στάδια ανάπτυξης του εγκεφαλικού φλοιού προκειμένου να διασφαλίσει την ακεραιότητα του πληθυσμού των νευροεπιθηλιακών κυττάρων. Τα αποτελέσματα μας υποστηρίζουν τη δυναμική ρύθμιση των διαδικασιών έναρξης της αντιγραφής κατά την ανάπτυξη του φλοιού προτείνοντας ότι ο ρόλος της Geminin στη διατήρηση της γονιδιωματικής σταθερότητας εξαρτάται από τα ενδογενή χαρακτηριστικά των διακριτών πληθυσμών νευρικών βλαστικών και προγονικών κυττάρων που σχηματίζονται κατά την ανάπτυξη του φλοιού

Obesity-linked homologues TfAP-2 and Twz establish meal frequency in Drosophila melanogaster

In all animals managing the size of individual meals and frequency of feeding is crucial for metabolic homeostasis. In the current study we demonstrate that the noradrenalin analogue octopamine and the cholecystokinin (CCK) homologue Drosulfakinin (Dsk) function downstream of TfAP-2 and Tiwaz (Twz) to control the number of meals in adult flies. Loss of TfAP-2 or Twz in octopaminergic neurons increased the size of individual meals, while overexpression of TfAP-2 significantly decreased meal size and increased feeding frequency. Of note, our study reveals that TfAP-2 and Twz regulate octopamine signaling to initiate feeding; then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior. Intriguingly, we found that the mouse TfAP-2 and Twz homologues, AP-2β and Kctd15, co-localize in areas of the brain known to regulate feeding behavior and reward, and a proximity ligation assay (PLA) demonstrated that AP-2β and Kctd15 interact directly in a mouse hypothalamus-derived cell line. Finally, we show that in this mouse hypothalamic cell line AP-2β and Kctd15 directly interact with Ube2i, a mouse sumoylation enzyme, and that AP-2β may itself be sumoylated. Our study reveals how two obesity-linked homologues regulate metabolic homeostasis by modulating consummatory behavior

GemC1 governs multiciliogenesis through direct interaction with and transcriptional regulation of p73

A distinct combination of transcription factors elicits the acquisition of a specific fate and the initiation of a differentiation program. Multiciliated cells (MCCs) are a specialized type of epithelial cells that possess dozens of motile cilia on their apical surface. Defects in cilia function have been associated with ciliopathies that affect many organs, including brain and airway epithelium. Here we show that the geminin coiled-coil domain-containing protein 1 GemC1 (also known as Lynkeas) regulates the transcriptional activation of p73, a transcription factor central to multiciliogenesis. Moreover, we show that GemC1 acts in a trimeric complex with transcription factor E2F5 and tumor protein p73 (officially known as TP73), and that this complex is important for the activation of the p73 promoter. We also provide in vivo evidence that GemC1 is necessary for p73 expression in different multiciliated epithelia. We further show that GemC1 regulates multiciliogenesis through the control of chromatin organization, and the epigenetic marks/tags of p73 and Foxj1. Our results highlight novel signaling cues involved in the commitment program of MCCs across species and tissues. This article has an associated First Person interview with the first author of the paper

Detection of AP-2β and Kctd15 interactions in mHypoE-N25/2 cells using PLA.

<p>The images were acquired in single z-plane. PLA signals are shown in red (increased intensity projection) and the nuclei in blue (as stained by DAPI). Pictures were taken with 20× magnification. (A) PLA interaction between AP2β and Kctd15, (A₁) negative control without primary antibodies. Detection of AP-2β, Kctd15 and Ube2i interactions in mHypoE-N25/2 cells using PLA. The images were acquired in single z-plane. PLA signals are shown in red (increased intensity projection) and the nuclei in blue (as stained by DAPI). Pictures were taken with 20× magnification. (B) PLA interaction between AP-2β and Ube2i, (B₁) negative control without primary antibodies. (C) PLA interaction between Kctd15 and Ube2i, (C₁) negative control without primary antibodies. (D) Western analysis of mouse proteins, recovered from the entire brain, was performed using an anti-AP-2β antibody. Two bands were visible on the gel, the lower band corresponded to the predicted size of AP-2β of 48 kDa, while the upper band corresponds to sumoylated AP-2β, which should be should be ∼60 kDa.</p

Diet regulates <i>TfAP-2</i> and <i>Twz</i> transcript levels.

<p>(A) Relative levels of <i>Tfap-2</i> or <i>Twz</i> transcript in the heads of flies starved either for 24 or 48 h. (B) Relative levels of <i>Tfap-2</i> or <i>Twz</i> transcript in the heads of flies kept on various diets for 5 days. (A–B) RNA was collected from the heads of 50, 5–7 day old, males for each genotype. qPCR was repeated at least 7 times for each transcript. (C) Relative levels of <i>Tfap-2</i> transcript in the heads of control and <i>Twz</i> knockdown flies starved either for 24 h or kept on a low calorie diet (2.5 g·dl⁻¹∶2.5 g·dl⁻¹ sucrose∶brewer's yeast) for 5 days. RNA was collected from the heads of 50, 5–7 day old, males for each genotype. qPCR was repeated at least 7 times for each transcript. (D) Relative levels of <i>Tbh</i> or <i>Vmat</i> transcript in the heads of flies starved either for 24 or 48 h. (E) Relative levels of <i>Tbh</i> or <i>Vmat</i> transcript in the heads of flies kept on various diets for 5 days. (For all assays n = 10 qPCR runs; Different letters indicate similar groups (i.e. ‘a’ is significantly different than ‘b’ or ‘c’ and so on, one-way ANOVA with Bonferroni post hoc test for multiple comparisons, P<0.05). A single asterisk indicates significant difference in two-way ANOVA, P<0.05, while a double asterisk indicates significant difference in two-way ANOVA, P<0.005). Error bars represent the SE (SEM).</p

Epithelioids: Self-sustaining 3D epithelial cultures to study long-term processes

ABSTRACT Studying long-term biological processes such as the colonization of aging epithelia by somatic mutant clones has been slowed by the lack of suitable culture systems. Here we describe epithelioids, a facile, cost-effective method of culturing multiple mouse and human epithelia. Esophageal epithelioids self-maintain without passaging for at least a year, recapitulating the 3D structure, cell dynamics, transcriptome, and genomic stability of the esophagus. Live imaging over 5 months showed epithelioids replicate in vivo cell dynamics. Epithelioids enable the study of cell competition and mutant selection in 3D epithelia, and how anti-cancer treatments modulate the competition between transformed and wild type cells. Epithelioids are a novel method with a wide range of applications in epithelial tissues, particularly the study of long term processes, that cannot be accessed using other culture models

<i>Dsk</i> overexpression does not affect feeding behavior.

<p>(A–C) A CAFE assay was used to assess the total food intake (B), the average number of feeding bouts (C) and meal size (D) over a 24 hour period in 5–7 days old adult males. Five males were used for each replicate and the assay was repeated at least 10 times for each genotype. (D) Relative levels of <i>Dsk</i> transcript in the heads of flies starved either for 24 or 48 h. (E) Relative levels of <i>Dsk</i> transcript in the heads of flies kept on various diets for 5 days. (D,E) RNA was collected from the heads of 50, 5–7 day old, males for each genotype. qPCR was repeated at least 7 times for each transcript. (A,B and D: A single asterisk indicates significant difference, a non-parametric Kruskal-Wallis ANOVA was performed with Dunn's post hoc test for multiple comparisons with Dunn's post hoc test for multiple comparisons, P<0.05, while a double asterisk indicates significant difference P<0.005). (C: Different letters indicate similar groups (i.e. ‘a’ is significantly different than ‘b’ or ‘c’ and so on, A non-parametric Kruskal-Wallis ANOVA was performed with Dunn's post hoc test for multiple comparisons, P<0.05). Error bars represent the SE (SEM).</p

Diet regulates mouse hypothalamic <i>Tfap2b</i> and <i>Kctd15</i> transcript levels.

<p>(A,B) Relative level of (A) <i>Tfap2B</i> and (B) <i>Kctd15</i> expression in the hypothalamus from starved or obese male mice (n = 10 qPCR runs; one-way ANOVA with Bonferroni post hoc test for multiple comparisons, * P<0.05, ** P<0.005). (C) Mice were either fed a normal or high fat diet for 8 weeks. By week six mice raised on a high fat diet were significantly higher in weight than control mice (Two-way ANOVA with P<0.05 was calculated to ensure that the obese mice were significantly heavier than the controls). Error bars represent SD.</p