PPARα regulates endothelial progenitor cell maturation and myeloid lineage differentiation through a NADPH oxidase-dependent mechanism in mice

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Epigenetic regulation in neural crest development : role of DNA methyltransferases 3A and 3B

The neural crest is a group of migratory, multipotent stem cells that play a crucial role in many aspects of embryonic development. This uniquely vertebrate cell population forms within the dorsal neural tube but then emigrates out and migrates long distances to different regions of the body. These cells contribute to formation of many structures such as the peripheral nervous system, craniofacial skeleton, and pigmentation of the skin. Why some neural tube cells undergo a change from neural to neural crest cell fate is unknown as is the timing of both onset and cessation of their emigration from the neural tube. In recent years, growing evidence supports an important role for epigenetic regulation as a new mechanism for controlling aspects of neural crest development. In this thesis, I dissect the roles of the de novo DNA methyltransferases (DNMTs) 3A and 3B in neural crest specification, migration and differentiation. First, I show that DNMT3A limits the spatial boundary between neural crest versus neural tube progenitors within the neuroepithelium. DNMT3A promotes neural crest specification by directly mediating repression of neural genes, like Sox2 and Sox3. Its knockdown causes ectopic Sox2 and Sox3 expression at the expense of neural crest territory. Thus, DNMT3A functions as a molecular switch, repressing neural to favor neural crest cell fate. Second, I find that DNMT3B restricts the temporal window during which the neural crest cells emigrate from the dorsal neural tube. Knockdown of DNMT3B causes an excess of neural crest emigration, by extending the time that the neural tube is competent to generate emigrating neural crest cells. In older embryos, this resulted in premature neuronal differentiation. Thus, DNMT3B regulates the duration of neural crest production by the neural tube and the timing of their differentiation. My results in avian embryos suggest that de novo DNA methylation, exerted by both DNMT3A and DNMT3B, plays a dual role in neural crest development, with each individual paralogue apparently functioning during a distinct temporal window. The results suggest that de novo DNA methylation is a critical epigenetic mark used for cell fate restriction of progenitor cells during neural crest cell fate specification. Our discovery provides important insights into the mechanisms that determine whether a cell becomes part of the central nervous system or peripheral cell lineages.

Osteoclast formation elicited by interleukin-33 stimulation is dependent upon the type of osteoclast progenitor

Osteoclasts are bone resorbing multinucleated cells (MNCs) derived from macrophage progenitors. IL-33 has been reported to drive osteoclastogenesis independently of receptor activator of NFκB ligand (RANKL) but this remains controversial as later studies did not confirm this. We found IL-33 clearly elicited functional dentine-resorbing osteoclast formation from human adult monocytes. However, monocytes from only 3 of 12 donors responded this way, while all responded to RANKL. Human cord blood-derived progenitors and murine bone marrow macrophages lacked an osteoclastogenic response to IL-33. In RAW264.7 cells, IL-33 elicited NFκB and p38 responses but not NFATc1 signals (suggesting poor osteoclastogenic responses) and formed only mononuclear tartrate-resistant acid phosphatase positive (TRAP(+)) cells. Since TGFβ boosts osteoclastogenesis in RAW264.7 cells we employed an IL-33/TGFβ co-treatment, which resulted in small numbers of MNCs expressing key osteoclast markers TRAP and calcitonin receptors. Thus, IL-33 possesses weak osteoclastogenic activity suggesting pathological significance and, perhaps, explaining previous conflicting reports.

La perturbation du locus Nr2f1-K12 entraine une différenciation gliale précoce dans un nouveau modèle murin de mégacôlon aganglionnaire

La maladie de Hirschsprung est une affection congénitale de la motilité intestinale caractérisée par un segment aganglionnaire dans le côlon terminal. Un criblage génétique par mutation insertionnelle aléatoire chez la souris nous a permis d’identifier la lignée transgénique Spot dont les homozygotes souffrent de mégacôlon aganglionnaire. L’analyse d’intestins d’embryons mutants a révélé une baisse de prolifération et un délai de migration des cellules de la crête neurale entériques (CCNe) progénitrices dus à leur différenciation gliale précoce, entrainant un défaut de colonisation de l’intestin et une aganglionose du côlon. Le séquençage du génome Spot indique que le transgène s’est inséré à l’intérieur du locus K12-Nr2f1 sur le chromosome 13, une région dépourvue de gènes préalablement associés à la maladie, perturbant également une séquence non-codante très conservée dans l’évolution. K12 est un gène d’ARN long non codant (ARNlnc) et antisens du gène Nr2f1, lui-même impliqué dans la gliogénèse du système nerveux central. Le séquençage du transcriptome des CCN a montré une surexpression de Nr2f1 et des formes courtes de K12 chez Spot et des essais luciférase ont révélé l’activité répressive de l’élément conservé. Nous avons observé l’expression de K12 dans les CCNe et sa localisation subcellulaire dans des zones transcriptionnellement actives du noyau. Avec l’émergence des ARNlnc régulateurs, ces données nous permettent de pointer deux nouveaux gènes candidats associés à une différenciation gliale prématurée du SNE menant au mégacôlon aganglionnaire, en supposant que la régulation de Nr2f1 se fait par son antisens, K12.

La perturbation du locus Nr2f1-K12 entraine une différenciation gliale précoce dans un nouveau modèle murin de mégacôlon aganglionnaire

La maladie de Hirschsprung est une affection congénitale de la motilité intestinale caractérisée par un segment aganglionnaire dans le côlon terminal. Un criblage génétique par mutation insertionnelle aléatoire chez la souris nous a permis d’identifier la lignée transgénique Spot dont les homozygotes souffrent de mégacôlon aganglionnaire. L’analyse d’intestins d’embryons mutants a révélé une baisse de prolifération et un délai de migration des cellules de la crête neurale entériques (CCNe) progénitrices dus à leur différenciation gliale précoce, entrainant un défaut de colonisation de l’intestin et une aganglionose du côlon. Le séquençage du génome Spot indique que le transgène s’est inséré à l’intérieur du locus K12-Nr2f1 sur le chromosome 13, une région dépourvue de gènes préalablement associés à la maladie, perturbant également une séquence non-codante très conservée dans l’évolution. K12 est un gène d’ARN long non codant (ARNlnc) et antisens du gène Nr2f1, lui-même impliqué dans la gliogénèse du système nerveux central. Le séquençage du transcriptome des CCN a montré une surexpression de Nr2f1 et des formes courtes de K12 chez Spot et des essais luciférase ont révélé l’activité répressive de l’élément conservé. Nous avons observé l’expression de K12 dans les CCNe et sa localisation subcellulaire dans des zones transcriptionnellement actives du noyau. Avec l’émergence des ARNlnc régulateurs, ces données nous permettent de pointer deux nouveaux gènes candidats associés à une différenciation gliale prématurée du SNE menant au mégacôlon aganglionnaire, en supposant que la régulation de Nr2f1 se fait par son antisens, K12.

Mass assembly and morphological transformations since z ∼ 3 from CANDELS

We quantify the evolution of the stellar mass functions (SMFs) of star-forming and quiescent galaxies as a function of morphology from z ∼ 3 to the present. Our sample consists of ∼50 000 galaxies in the CANDELS fields (∼880 arcmin^2), which we divide into four main morphological types, i.e. pure bulge-dominated systems, pure spiral disc-dominated, intermediate two-component bulge+disc systems and irregular disturbed galaxies. At z ∼ 2, 80 per cent of the stellar mass density of star-forming galaxies is in irregular systems. However, by z ∼ 0.5, irregular objects only dominate at stellar masses below 10^9 M_⊙. A majority of the star-forming irregulars present at z ∼ 2 undergo a gradual transformation from disturbed to normal spiral disc morphologies by z ∼ 1 without significant interruption to their star formation. Rejuvenation after a quenching event does not seem to be common except perhaps for the most massive objects, because the fraction of bulge-dominated star-forming galaxies with M^*/M_⊙ > 10^10.7 reaches 40 per cent at z < 1. Quenching implies the presence of a bulge: the abundance of massive red discs is negligible at all redshifts over 2 dex in stellar mass. However, the dominant quenching mechanism evolves. At z > 2, the SMF of quiescent galaxies above M^* is dominated by compact spheroids. Quenching at this early epoch destroys the disc and produces a compact remnant unless the star-forming progenitors at even higher redshifts are significantly more dense. At 1 < z < 2, the majority of newly quenched galaxies are discs with a significant central bulge. This suggests that mass quenching at this epoch starts from the inner parts and preserves the disc. At z < 1, the high-mass end of the passive SMF is globally in place and the evolution mostly happens at stellar masses below 10^10 M_⊙. These low-mass galaxies are compact, bulge-dominated systems, which were environmentally quenched: destruction of the disc through ram-pressure stripping is the likely process.

Book review. Made to matter: White fathers, stolen generations

The topic of white fathers of Aboriginal children has been a long time coming into focus. Aboriginal mothers and children and their loss by being taken into white custody—these matters have been thoroughly, often heart-rendingly explored in artefacts and documentaries in a variety of genres. The white male progenitors have been much less visible except as occasions of scandal. An Aboriginal child without a known father made the child very vulnerable and, conveniently (from an administrative point of view), able to be drawn into the white ‘care’ system where the state became by proxy the stern father in a process that obliterated Aboriginal kinship claims. Probyn-Rapsey notes perspicaciously that although they are often written about (by Aboriginal relatives, biographers, scholars, government officials, Protectors, white women activists, parliamentarians), the fathers of the children rarely speak in their own voices on the subject of their paternity of mixed race children. This study examines the next best thing: textual traces of them in reports, letters, diaries, novels and especially Aboriginal memoirs.

Muscle damage and inflammation during recovery from exercise

Unaccustomed exercise consisting of eccentric (i.e., lengthening) muscle contractions often results in muscle damage characterized by ultrastructural alterations in muscle tissue, clinical signs and symptoms (e.g., reduced muscle strength and range of motion, increased muscle soreness and swelling, efflux of myocellular proteins). The time course of recovery following exercise-induced muscle damage depends on the extent of initial muscle damage, which in turn is influenced by the intensity and duration of exercise, joint angle/muscle length and muscle groups used during exercise. The effects of these factors on muscle strength, soreness and swelling are well characterized. By contrast, much less is known about how they affect intramuscular inflammation and molecular aspects of muscle adaptation/remodeling. Although inflammation has historically been viewed as detrimental for recovery from exercise, it is now generally accepted that inflammatory responses-if tightly regulated-are integral to muscle repair and regeneration. Animal studies have revealed that other cell types including mast cells, eosinophils, CD8 and T regulatory lymphocytes, fibro-adipogenic progenitors and pericytes also help to facilitate muscle tissue regeneration. However, more research is required to determine whether these cells respond to exercise-induced muscle damage. A large body of research has investigated the efficacy of physicotherapeutic, pharmacological and nutritional interventions for reducing the signs and symptoms of exercise-induced muscle damage, with mixed results. More research is needed to examine if/how these treatments influence inflammation and muscle remodeling during recovery from exercise.

Rôle du gène de fusion MLL-ENL dans le développement et le maintien du phénotype leucémique

Les translocations chromosomiques du gène MLL sont connues pour mener au développement de leucémies aiguës. La translocation avec un de ses partenaires de fusion les plus communs, ENL, peut engendrer des leucémies aiguës de plusieurs types différents pour cette même translocation. Une fois la leucémogenèse initiée par la fusion MLL-ENL, son rôle quant au maintien du phénotype leucémique n’est pas encore bien connu à ce jour. Pour mieux comprendre l’importance de MLL-ENL après la leucémogenèse, des cellules souches/progénitrices de sang de cordon ombilical humain purifiées ont ainsi été transduites par un virus exprimant le gène de fusion MLL-ENL bordé par des sites LoxP ainsi que le marqueur eGFP. Ces cellules infectées ont ensuite été injectées dans notre modèle de souris immunodéficientes irradiées et placées sous observation pendant 24 semaines pour voir le développement de leucémies aiguës. Elles ont alors été sacrifiées et les cellules la moelle osseuse et de la rate ont ensuite été analysées par cytométrie en flux pour déterminer si la xénogreffe a engendré une leucémie dans notre modèle ainsi que le phénotype de celle-ci. Les souris injectées par les cellules infectées par le MLL-ENL ont généré uniquement des leucémies lymphoïdes aiguës de type B. Les cellules de ces leucémies primaires isolées ont été par la suite infectées par un lentivirus exprimant la cre-recombinase et le marqueur BFP afin d’exciser le gène MLL-ENL des cellules leucémiques grâce aux sites LoxP. Les cellules ont ensuite été triées pour le marqueur BFP et injectées dans des souris secondaires pour de voir si les cellules leucémiques souches pouvaient toujours régénérer la leucémie. Les conséquences de l’absence de MLL-ENL dans le maintien du phénotype leucémique n’ont cependant pas pu être vérifiées à cause d’une erreur dans la séquence de la cre-recombinase, mais nous avons observé la régénération des leucémies secondaires.

Using induced pluripotent stem cells (IPSCS) as a replacement for in vivo models to screen novel therapies in chronic myeloid leukaemia (CML)

Tyrpsine kinase inhibitors (TKIs) effectively target progenitors and mature leukaemic cells but prove less effective at eliminating leukaemic stem cells (LSCs) in patients with chronic myeloid leukaemia (CML). Several reports indicate that the TGFβ superfamily pathway is important for LSC survival and quiescence. We conducted extensive microarray analyses to compare expression patterns in normal haemopoietic stem cells (HSC) and progenitors with CML LSC and progenitor populations in chronic phase (CP), accelerated phase (AP) and blast crisis (BC) CML. The BMP/SMAD pathway and downstream signalling molecules were identified as significantly deregulated in all three phases of CML. The changes observed could potentiate altered autocrine signalling, as BMP2, BMP4 (p<0.05), and ACTIVIN A (p<0.001) were all down regulated, whereas BMP7, BMP10 and TGFβ (p<0.05) were up regulated in CP. This was accompanied by up regulation of BMPRI (p<0.05) and downstream SMADs (p<0.005). Interestingly, as CML progressed, the profile altered, with BC patients showing significant over-expression of ACTIVIN A and its receptor ACVR1C. To further characterise the BMP pathway and identify potential candidate biomarkers within a larger cohort, expression analysis of 42 genes in 60 newly diagnosed CP CML patient samples, enrolled on a phase III clinical trial (www.spirit-cml.org) with greater than 12 months follow-up data on their response to TKI was performed. Analysis revealed that the pathway was highly deregulated, with no clear distinction when patients were stratified into good, intermediate and poor response to treatment. One of the major issues in developing new treatments to target LSCs is the ability to test small molecule inhibitors effectively as it is difficult to obtain sufficient LSCs from primary patient material. Using reprogramming technologies, we generated induced pluripotent stem cells (iPSCs) from CP CML patients and normal donors. CML- and normal-derived iPSCs were differentiated along the mesodermal axis to generate haemopoietic and endothelial precursors (haemangioblasts). IPSC-derived haemangioblasts exhibited sensitivity to TKI treatment with increased apoptosis and reduction in the phosphorylation of downstream target proteins. 4 Dual inhibition studies were performed using BMP pathway inhibitors in combination with TKI on CML cell lines, primary cells and patient derived iPSCs. Results indicate that they act synergistically to target CML cells both in the presence and absence of BMP4 ligand. Inhibition resulted in decreased proliferation, irreversible cell cycle arrest, increased apoptosis, reduced haemopoietic colony formation, altered gene expression pattern, reduction in self-renewal and a significant reduction in the phosphorylation of downstream target proteins. These changes offer a therapeutic window in CML, with intervention using BMP inhibitors in combination with TKI having the potential to prevent LSC self-renewal and improve outcome for patients. By successfully developing and validating iPSCs for CML drug screening we hope to substantially reduce the reliance on animal models for early preclinical drug screening in leukaemia.

The locations of recent supernovae near the Sun from modelling 60Fe transport

The signature of 60Fe in deep-sea crusts indicates that one or more supernovae exploded in the solar neighbourhood about 2.2 million years ago1–4. Recent isotopic analysis is consistent with a core-collapse or electron-capture supernova that occurred 60 to 130 parsecs from the Sun5. Moreover, peculiarities in the cosmic ray spectrum point to a nearby supernova about two million years ago6. The Local Bubble of hot, diffuse plasma, in which the Solar System is embedded, originated from 14 to 20 supernovae within a moving group, whose surviving members are now in the Scorpius– Centaurus stellar association7,8. Here we report calculations of the most probable trajectories and masses of the supernova progenitors, and hence their explosion times and sites. The 60Fe signal arises from two supernovae at distances between 90 and 100 parsecs. The closest occurred 2.3 million years ago at present-day galactic coordinates l = 327°, b = 11°, and the second-closest exploded about 1.5 million years ago at l = 343°, b = 25°, with masses of 9.2 and 8.8 times the solar mass, respectively. The remaining supernovae, which formed the Local Bubble, contribute to a smaller extent because they happened at larger distances and longer ago (60Fe has a half- life of 2.6 million years9,10). There are uncertainties relating to the nucleosynthesis yields and the loss of 60Fe during transport, but they do not influence the relative distribution of 60Fe in the crust layers, and therefore our model reproduces the measured relative abundances very well.