Wac: a new Augmin subunit required for chromosome alignment but not for acentrosomal microtubule assembly in female meiosis

The bipolar spindle forms without centrosomes naturally in female meiosis and by experimental manipulation in mitosis. Augmin is a recently discovered protein complex required for centrosome-independent microtubule generation within the spindle in Drosophila melanogaster cultured cells. Five subunits of Augmin have been identified so far, but neither their organization within the complex nor their role in developing organisms is known. In this study, we report a new Augmin subunit, wee Augmin component (Wac). Wac directly interacts with another Augmin subunit, Dgt2, via its coiled-coil domain. Wac depletion in cultured cells, especially without functional centrosomes, causes severe defects in spindle assembly. We found that a wac deletion mutant is viable but female sterile and shows only a mild impact on somatic mitosis. Unexpectedly, mutant female meiosis showed robust microtubule assembly of the acentrosomal spindle but frequent chromosome misalignment. For the first time, this study establishes the role of an Augmin subunit in developing organisms and provides an insight into the architecture of the complex

Haemogenic Gastruloids Recapitulate Developmental Haematopoiesis and Provide an Ontogeny-Relevant Context to Dissect the Origins of Infant Leukemia

Meeting abstract presented at the 64th ASH Annual Meeting and Exposition, New Orleans, LA, USA, 10-13 Dec 2022..Modelling of developmental hematopoiesis has historically been challenging due to the inability to produce hematopoietic stem cells (HSC) and recapitulate microenvironment interactions ex vivo. Gastruloids are 3D aggregates of embryonic stem (ES) cells which display developmentally-specific spatial and temporal organization that recapitulate gastrulation. We adapted the gastruloid protocol to introduce hematopoietic signalling cues, and generated an in vitro model of embryonic hematopoiesis that sequentially recapitulates the formation of hemogenic endothelium, hematopoietic progenitors, and pre-HSC, over a culture period of 216 hours. Flow cytometry analysis detected the presence of c-Kit+ endothelium at 120h, followed by emergence of CD41+ hematopoietic progenitors at 144h, and the appearance of CD45+ cells from 192h. CD45+ cells were observed in small clusters adjoining endothelium-lined structures, reminiscent of developmental hemogenic-to-endothelial transition and intra-aortic clusters. Single-cell RNA sequencing revealed specification of pre-definitive and definitive waves of embryonic hematopoiesis, aligning 144h-CD41+ cells with erythro-myeloid progenitors (EMP), and late CD45+ with lympho-myeloid progenitors and pre-HSC, altogether supporting the hemogenic gastruloid as a model that is temporally and topographically congruous with the embryo. The close recapitulation of developmental ontogeny led us to explore hemogenic gastruloids to understand cell and stage-specific susceptibility to forms of Acute Myeloid Leukaemia exclusively observed in infants. The chromosomal translocation t(7;12)(q36;p13), characterized by the ectopic overexpression of the MNX1 gene, is found in up to one third of infant AML cases, but has been challenging to model using conventional strategies, largely due to the inability of MNX1 to transform adult hematopoietic cells. The age-selectivity of t(7;12) has been proposed to reflect a transient developmental window for a target cell of origin absent in adult life, but its nature is yet to be defined. In order to identify the context of MNX1-driven leukemogenesis, we produced hemogenic gastruloids using lentiviral-transduced mouse ES cells in which we overexpressed MNX1 as a proxy of t(7;12). Although MNX1 did not interfere with ES cell pluripotent cultures, it primed incipient hemogenic programmes and promoted hemogenic gastruloid formation. Critically, expression of MNX1 resulted in transformation of gastruloid-derived hematopoietic cells, as assessed by serial colony-forming cell replating, with expansion of a phenotypic myeloid cell, a phenomenon not observed in adult tissues. Detailed analysis of the cellular composition of MNX1-overexpressing hemogenic gastruloids revealed a significant effect in the output of CD41+ and c-Kit+ populations at 144h, but no effect in CD45+ cells at 192-216h, suggesting that the target of MNX1 lies within the EMP stage, an observation supported by single-cell RNA-seq analysis of MNX1 vs control gastruloids. Systematic comparison of the temporal transcriptional profiles of hemogenic gastruloids, MNX1-overexpressing gastruloids, and t(7;12) patients, pinpoints the target cell of MNX1 at the HE-to-EMP transition. In summary, we propose a novel model of embryonic hematopoiesis capable of capturing developmentally-relevant cellularity and topography of the early hematopoietic microenvironment, with the ability to mechanistically elucidate developmental associations of infant leukemia

Anastral spindle assembly and γ-tubulin in Drosophila oocytes

<p>Abstract</p> <p>Background</p> <p>Anastral spindles assemble by a mechanism that involves microtubule nucleation and growth from chromatin. It is still uncertain whether γ-tubulin, a microtubule nucleator essential for mitotic spindle assembly and maintenance, plays a role. Not only is the requirement for γ-tubulin to form anastral <it>Drosophila </it>oocyte meiosis I spindles controversial, but its presence in oocyte meiosis I spindles has not been demonstrated and is uncertain.</p> <p>Results</p> <p>We show, for the first time, using a bright GFP fusion protein and live imaging, that the <it>Drosophila </it>maternally-expressed γTub37C is present at low levels in oocyte meiosis I spindles. Despite this, we find that formation of bipolar meiosis I spindles does not require functional γTub37C, extending previous findings by others. Fluorescence photobleaching assays show rapid recovery of γTub37C in the meiosis I spindle, similar to the cytoplasm, indicating weak binding by γTub37C to spindles, and fits of a new, potentially more accurate model for fluorescence recovery yield kinetic parameters consistent with transient, diffusional binding.</p> <p>Conclusions</p> <p>The FRAP results, together with its mutant effects late in meiosis I, indicate that γTub37C may perform a role subsequent to metaphase I, rather than nucleating microtubules for meiosis I spindle formation. Weak binding to the meiosis I spindle could stabilize pre-existing microtubules or position γ-tubulin for function during meiosis II spindle assembly, which follows rapidly upon oocyte activation and completion of the meiosis I division.</p

Dendritic structural plasticity

Dendrites represent the compartment of neurons primarily devoted to collecting and computating input. Far from being static structures, dendrites are highly dynamic during development and appear to be capable of plastic changes during the adult life of animals. During development, it is a combination of intrinsic programs and external signals that shapes dendrite morphology; input activity is a conserved extrinsic factor involved in this process. In adult life, dendrites respond with more modest modifications of their structure to various types of extrinsic information, including alterations of input activity. Here, the author reviews classical and recent evidence of dendrite plasticity in invertebrates and vertebrates and current progress in the understanding of the molecular mechanisms that underlie this plasticity. Importantly, some fundamental questions such as the functional role of dendrite remodeling and the causal link between structural modifications of neurons and plastic processes, including learning, are still open. (c) 2011 Wiley Periodicals, Inc. Develop Neurobiol 72: 7386, 201

Centrosomes and microtubule organisation during Drosophila development

Are the microtubule-organising centers of the different cell types of a metazoan interchangeable? If not, what are the differences between them? Do they play any role in the differentiation processes to which these cells are subjected? Nearly one hundred years of centrosome research has established the essential role of this organelle as the main microtubule-organising center of animal cells. But only now are we starting to unveil the answers to the challenging questions which are raised when the centrosome is studied within the context of a pluricellular organism. In this review we present some of the many examples which illustrate how centrosomes and microtubule organisation changes through development in Drosophila and discuss some of its implications

Essential role for gamma-tubulin in the acentriolar female meiotic spindle of Drosophila.

Microtubule nucleation in vivo requires gamma-tubulin, a highly conserved component of microtubule-organizing centers. In Drosophila melanogaster there are two gamma-tubulin genes, gammaTUB23C and gammaTUB37C. Here we report the cytological and molecular characterization of the 37C isoform. By Western blotting, this protein can only be detected in ovaries and embryos. Antibodies against this isoform predominantly label the centrosomes in embryos from early cleavage divisions until cycle 15, but fail to reveal any particular localization of gamma-tubulin in the developing egg chambers. The loss of function of this gene results in female sterility and has no effect on viability or male fertility. Early stages of oogenesis are unaffected by mutations in this gene, as judged both by morphological criteria and by localization of reporter genes, but the female meiotic spindle is extremely disrupted. Nuclear proliferation within the eggs laid by mutant females is also impaired. We conclude that the expression of the 37C gamma-tubulin isoform of D. melanogaster is under strict developmental regulation and that the organization of the female meiotic spindle requires gamma-tubulin

Slit and Robo regulate dendrite branching and elongation of space-filling neurons in Drosophila

Space-filling neurons extensively sample their receptive fields with fine dendritic branches. In this study we show that a member of the conserved Robo receptor family, Robo, and its ligand Slit regulate the dendritic differentiation of space-filling neurons. Loss of Robo or Slit function leads to faster elongating and less branched dendrites of the complex and space-filling class IV multi-dendritic dendrite-arborization (md-da) neurons in the Drosophila embryonic peripheral nervous system, but not of the simpler class I neurons. The total dendrite length of Class IV neurons is not modified in robo or slit mutant embryos. Robo mediates this process cell-autonomously. Upon Robo over-expression in md-da neurons the dendritic tree is simplified and time-lapse analysis during larval stages indicates that this is clue to reduction in the number of newly formed branches. We propose that Slit, through Robo, provides an extrinsic signal to coordinate the growth rate and the branching level of space-filling neurons, thus allowing them to appropriately cover their target field. (C) 2008 Elsevier Inc. All rights reserved

Slit and Robo regulate dendrite branching and elongation of space-filling neurons in Drosophila

Space-filling neurons extensively sample their receptive fields with fine dendritic branches. In this study we show that a member of the conserved Robo receptor family, Robo, and its ligand Slit regulate the dendritic differentiation of space-filling neurons. Loss of Robo or Slit function leads to faster elongating and less branched dendrites of the complex and space-filling class IV multi-dendritic dendrite-arborization (md-da) neurons in the Drosophila embryonic peripheral nervous system, but not of the simpler class I neurons. The total dendrite length of Class IV neurons is not modified in robo or slit mutant embryos. Robo mediates this process cell-autonomously. Upon Robo over-expression in md-da neurons the dendritic tree is simplified and time-lapse analysis during larval stages indicates that this is clue to reduction in the number of newly formed branches. We propose that Slit, through Robo, provides an extrinsic signal to coordinate the growth rate and the branching level of space-filling neurons, thus allowing them to appropriately cover their target field. (C) 2008 Elsevier Inc. All rights reserved

The Drosophila myosin VI Jaguar is required for basal protein targeting and correct spindle orientation in mitotic neuroblasts

Asymmetric cell divisions generate cellular diversity. In Drosophila, embryonic neuroblasts target cell fate determinants basally, rotate their spindles by 90 degrees to align with the apical-basal axis, and divide asymmetrically in a stem cell-like fashion. In this process, apically localized Bazooka recruits Inscuteable and other proteins to form an apical complex, which then specifies spindle orientation and basal localization of the cell fate determinants and their adapter proteins such as Miranda. Here we report that Miranda localization requires the unconventional myosin VI Jaguar (Jar). In jar null mutant embryos, Miranda is delocalized and the spindle is misoriented, but the Inscuteable crescent remains apical. Miranda directly binds to Jar, raising the possibility that Miranda and its associated proteins are translocated basally by this actin-based motor. Our studies demonstrate that a class VI myosin is necessary for basal protein targeting and spindle orientation in neuroblasts