Sergio Ottolenghi

Hippocampal development and neural stem cell maintenance require Sox2 -dependent regulation of Shh

Neural stem cells (NSCs) are controlled by diffusible factors. The transcription factor Sox2 is expressed by NSCs and Sox2 mutations in humans cause defects in the brain and, in particular, in the hippocampus. We deleted Sox2 in the mouse embryonic brain. At birth, the mice showed minor brain defects; shortly afterwards, however, NSCs and neurogenesis were completely lost in the hippocampus, leading to dentate gyrus hypoplasia. Deletion of Sox2 in adult mice also caused hippocampal neurogenesis loss. The hippocampal developmental defect resembles that caused by late sonic hedgehog (Shh) loss. In mutant mice, Shh and Wnt3a were absent from the hippocampal primordium. A SHH pharmacological agonist partially rescued the hippocampal defect. Chromatin immunoprecipitation identified Shh as a Sox2 target. Sox2-deleted NSCs did not express Shh in vitro and were rapidly lost. Their replication was partially rescued by the addition of SHH and was almost fully rescued by conditioned medium from normal cells. Thus, NSCs control their status, at least partly, through Sox2-dependent autocrine mechanisms.

Gene deletion as the cause of α thalassaemia: The severe form of α thalassaemia is caused by a haemoglobin gene deletion

Two independent groups show that the absence of all or part of the globin α-chain gene is the origin of the homozygous α thalassaemia.

Repression of kit Expression by Plzf in Germ Cells

ABSTRACT Male mice lacking expression of Plzf, a DNA sequence-specific transcriptional repressor, show progressive germ cell depletion due to exhaustion of the spermatogonial stem cell population. This is likely due to the deregulated expression of genes controlling the switch between spermatogonial self-renewal and differentiation. Here we show that Plzf directly represses the transcription of kit, a hallmark of spermatogonial differentiation. Plzf represses both endogenous kit expression and expression of a reporter gene under the control of the kit promoter region. A discrete sequence of the kit promoter, required for Plzf-mediated kit transcriptional repression, is bound by Plzf both in vivo and in vitro. A 3-bp mutation in this Plzf binding site abolishes the responsiveness of the kit promoter to Plzf repression. A significant increase in kit expression is also found in the undifferentiated spermatogonia isolated from Plzf−/− mice. Thus, we suggest that one mechanism by which Plzf maintains the pool of spermatogonial stem cells is through a direct repression of kit expression.

The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the Down critical region

We applied cDNA selection methods to a genomic clone (YAC 761B5) from chromosome 21 located in the so‐called ‘Down critical region’ in 21q22.3. Starting from human fetal heart and brain mRNAs we obtained and sequenced several cDNA clones. One of these clones (Down region aspartic protease (DRAP), named also BACE2 according to the gene nomenclature) revealed a striking nucleotide and amino acid sequence identity with several motifs present in members of the aspartic protease family. In particular the amino acid sequences comprising the two catalytic sites found in all mammalian aspartic proteases are perfectly conserved. Interestingly, the predicted protein shows a typical membrane spanning region; this is at variance with most other known aspartic proteases, which are soluble molecules. We present preliminary evidence, on the basis of in vitro translation studies and cell transfection, that this gene encodes a glycosylated protein which localizes mainly intracellularly but to some extent also to the plasma membrane. Furthermore DRAP/BACE2 shares a high homology with a newly described β‐secretase enzyme (BACE‐1) which is a transmembrane aspartic protease. The implications of this finding for Down syndrome are discussed.

Cardiomyogenesis in the Developing Heart Is Regulated by C-Kit–Positive Cardiac Stem Cells

Rationale: Embryonic and fetal myocardial growth is characterized by a dramatic increase in myocyte number, but whether the expansion of the myocyte compartment is dictated by activation and commitment of resident cardiac stem cells (CSCs), division of immature myocytes or both is currently unknown. Objective: In this study, we tested whether prenatal cardiac development is controlled by activation and differentiation of CSCs and whether division of c-kit–positive CSCs in the mouse heart is triggered by spontaneous Ca2+ oscillations. Methods and Results: We report that embryonic-fetal c-kit–positive CSCs are self-renewing, clonogenic and multipotent in vitro and in vivo. The growth and commitment of c-kit–positive CSCs is responsible for the generation of the myocyte progeny of the developing heart. The close correspondence between values computed by mathematical modeling and direct measurements of myocyte number at E9, E14, E19 and 1 day after birth strongly suggests that the organogenesis of the embryonic heart is dependent on a hierarchical model of cell differentiation regulated by resident CSCs. The growth promoting effects of c-kit–positive CSCs are triggered by spontaneous oscillations in intracellular Ca2+, mediated by IP3 receptor activation, which condition asymmetrical stem cell division and myocyte lineage specification. Conclusions: Myocyte formation derived from CSC differentiation is the major determinant of cardiac growth during development. Division of c-kit–positive CSCs in the mouse is promoted by spontaneous Ca2+ spikes, which dictate the pattern of stem cell replication and the generation of a myocyte progeny at all phases of prenatal life and up to one day after birth.

Spontaneous Calcium Oscillations Regulate Human Cardiac Progenitor Cell Growth

Rationale: The adult heart possesses a pool of progenitor cells stored in myocardial niches, but the mechanisms involved in the activation of this cell compartment are currently unknown. Objective: Ca2+ promotes cell growth raising the possibility that changes in intracellular Ca2+ initiate division of c-kit–positive human cardiac progenitor cells (hCPCs) and determine their fate. Methods and Results: Ca2+ oscillations were identified in hCPCs and these events occurred independently from coupling with cardiomyocytes or the presence of extracellular Ca2+. These findings were confirmed in the heart of transgenic mice in which enhanced green fluorescent protein was under the control of the c-kit promoter. Ca2+ oscillations in hCPCs were regulated by the release of Ca2+ from the endoplasmic reticulum through activation of inositol 1,4,5-triphosphate receptors (IP3Rs) and the reuptake of Ca2+ by the sarco-/endoplasmic reticulum Ca2+ pump (SERCA). IP3Rs and SERCA were highly expressed in hCPCs, whereas ryanodine receptors were not detected. Although Na+-Ca2+ exchanger, store-operated Ca2+ channels and plasma membrane Ca2+ pump were present and functional in hCPCs, they had no direct effects on Ca2+ oscillations. Conversely, Ca2+ oscillations and their frequency markedly increased with ATP and histamine which activated purinoceptors and histamine-1 receptors highly expressed in hCPCs. Importantly, Ca2+ oscillations in hCPCs were coupled with the entry of cells into the cell cycle and 5-bromodeoxyuridine incorporation. Induction of Ca2+ oscillations in hCPCs before their intramyocardial delivery to infarcted hearts was associated with enhanced engraftment and expansion of these cells promoting the generation of a large myocyte progeny. Conclusion: IP3R-mediated Ca2+ mobilization control hCPC growth and their regenerative potential.

Acetylation and MAPK phosphorylation cooperate to regulate the degradation of active GATA-1.

Regulation of transcription requires mechanisms to both activate and terminate transcription factor activity. GATA‐1 is a key haemopoietic transcription factor whose activity is increased by acetylation. We show here that acetylated GATA‐1 is targeted for degradation via the ubiquitin/proteasome pathway. Acetylation positively signals ubiquitination, suggesting that activation by acetylation simultaneously marks GATA‐1 for degradation. Promoter‐specific MAPK phosphorylation then cooperates with acetylation to execute protein loss. The requirement for both modifications is novel and suggests a way by which degradation of the active protein can be specifically regulated in response to external phosphorylation‐mediated signalling. As many transcription factors are activated by acetylation, we suggest that this might be a general mechanism to control transcription factor activity.

δβ-Thalassemia is due to a gene deletion

Abstract DNA has been prepared from peripheral blood or cultured skin fibroblasts obtained from three Sicilian and one Greek δβ-thalassemia homozygotes. Globin-gene analysis was carried out using a cDNA β probe, and the results indicate that δβ-thalassemia has arisen from a deletion of the β-globin genes. A similar result was obtained using DNA prepared from cultured skin fibroblasts from an individual homozygous for the Negro form of hereditary persistence of fetal hemoglobin (HPFH). In both cases, the deletion has spared the Gγ and Aγ loci directing the γ chains of hemoglobin F, but it has not been possible to demonstrate any difference between the size of the deletion involved in the production of δβ-thalassemia and that which gave rise to HPFH. These experiments provide further direct evidence that deletions of critical areas of the γ-δ-β gene cluster result in persistent γ chain synthesis in adult life.

The Ephrin A1–EphA2 System Promotes Cardiac Stem Cell Migration After Infarction

Rationale: Understanding the mechanisms that regulate trafficking of human cardiac stem cells (hCSCs) may lead to development of new therapeutic approaches for the failing heart. Objective: We tested whether the motility of hCSCs in immunosuppressed infarcted animals is controlled by the guidance system that involves the interaction of Eph receptors with ephrin ligands. Methods and Results: Within the cardiac niches, cardiomyocytes expressed preferentially the ephrin A1 ligand, whereas hCSCs possessed the EphA2 receptor. Treatment of hCSCs with ephrin A1 resulted in the rapid internalization of the ephrin A1–EphA2 complex, posttranslational modifications of Src kinases, and morphological changes consistent with the acquisition of a motile cell phenotype. Ephrin A1 enhanced the motility of hCSCs in vitro, and their migration in vivo following acute myocardial infarction. At 2 weeks after infarction, the volume of the regenerated myocardium was 2-fold larger in animals injected with ephrin A1–activated hCSCs than in animals receiving control hCSCs; this difference was dictated by a greater number of newly formed cardiomyocytes and coronary vessels. The increased recovery in myocardial mass with ephrin A1–treated hCSCs was characterized by further restoration of cardiac function and by a reduction in arrhythmic events. Conclusions: Ephrin A1 promotes the motility of EphA2-positive hCSCs, facilitates their migration to the area of damage, and enhances cardiac repair. Thus, in situ stimulation of resident hCSCs with ephrin A1 or their ex vivo activation before myocardial delivery improves cell targeting to sites of injury, possibly providing a novel strategy for the management of the diseased heart.

SOHLH1 and SOHLH2 control Kit expression during postnatal male germ cell development

How Kit expression is regulated in the germline remains unknown. SOHLH1 and SOHLH2, two bHLH transcription factors specifically expressed in germ cells, are involved in spermatogonia and oocyte differentiation. In the male, deletion of each factor causes loss of Kit-expressing spermatogonia in the prepuberal testis. In the female, SOHLH1 and SOHLH2 ablations cause oocyte loss in the neonatal ovary. To investigate whether Kit expression is regulated by these two factors in male germ cells, we examined SOHLH1 and SOHLH2 expression during fetal and postnatal mouse development. We found a strong positive correlation between Kit and the two transcription factors only in postnatal spermatogonia. SOHLH2 was enriched in undifferentiated spermatogonia, whereas SOHLH1 expression was maximal at Kit-dependent stages. Expression of SOHLH1, but not SOHLH2, was increased in postnatal mitotic germ cells by treatment with all-trans retinoic acid. We found that E-box sequences within the Kit promoter and its first intron can be transactivated in transfection experiments overexpressing Sohlh1 or Sohlh2. Co-transfection of both factors showed a cooperative effect. EMSA experiments showed that SOHLH1 and SOHLH2 can independently and cooperatively bind an E-box-containing probe. In vivo co-immunoprecipitations indicated that the two proteins interact and overexpression of both factors increases endogenous Kit expression in embryonic stem cells. SOHLH1 was found by ChIP analysis to occupy an E-box-containing region within the Kit promoter in spermatogonia chromatin. Our results suggest that SOHLH1 and SOHLH2 directly stimulate Kit transcription in postnatal spermatogonia, thus activating the signaling involved in spermatogonia differentiation and spermatogenetic progression.