Richard Alexander

Detection of functional haematopoietic stem cell niche using real-time imaging

Haematopoietic stem cell (HSC) niches, although proposed decades ago, have only recently been identified as separate osteoblastic and vascular microenvironments. Their interrelationships and interactions with HSCs in vivo remain largely unknown. Here we report the use of a newly developed ex vivo real-time imaging technology and immunoassaying to trace the homing of purified green-fluorescent-protein-expressing (GFP+) HSCs. We found that transplanted HSCs tended to home to the endosteum (an inner bone surface) in irradiated mice, but were randomly distributed and unstable in non-irradiated mice. Moreover, GFP+ HSCs were more frequently detected in the trabecular bone area compared with compact bone area, and this was validated by live imaging bioluminescence driven by the stem-cell-leukaemia (Scl) promoter–enhancer. HSCs home to bone marrow through the vascular system. We found that the endosteum is well vascularized and that vasculature is frequently localized near N-cadherin+ pre-osteoblastic cells, a known niche component. By monitoring individual HSC behaviour using real-time imaging, we found that a portion of the homed HSCs underwent active division in the irradiated mice, coinciding with their expansion as measured by flow assay. Thus, in contrast to central marrow, the endosteum formed a special zone, which normally maintains HSCs but promotes their expansion in response to bone marrow damage.

Flippase-mediated phospholipid asymmetry promotes fast Cdc42 recycling in dynamic maintenance of cell polarity

Lipid asymmetry at the plasma membrane is essential for such processes as cell polarity, cytokinesis and phagocytosis. Here we find that a lipid flippase complex, composed of Lem3, Dnf1 or Dnf2 (ref.xa0), has a role in the dynamic recycling of the Cdc42 GTPase, a key regulator of cell polarity, in yeast. By using quantitative microscopy methods, we show that the flippase complex is required for fast dissociation of Cdc42 from the polar cortex by the guanine nucleotide dissociation inhibitor. A loss of flippase activity, or pharmacological blockage of the inward flipping of phosphatidylethanolamine, a phospholipid with a neutral head group, disrupts Cdc42 polarity maintained by guanine nucleotide dissociation inhibitor-mediated recycling. Phosphatidylethanolamine flipping may reduce the charge interaction between a Cdc42 carboxy-terminal cationic region with the plasma membrane inner leaflet, enriched for the negatively charged lipid phosphatidylserine. Using a reconstituted system with supported lipid bilayers, we show that the relative composition of phosphatidylethanolamine versus phosphatidylserine directly modulates Cdc42 extraction from the membrane by guanine nucleotide dissociation inhibitor.

Distributed representation of chemical features and tunotopic organization of glomeruli in the mouse olfactory bulb

In the mammalian brain, similar features of the sensory stimuli are often represented in proximity in the sensory areas. However, how chemical features are represented in the olfactory bulb has been controversial. Questions have been raised as to whether specific chemical features of the odor molecules are represented by spatially clustered olfactory glomeruli. Using a sensitive probe, we have analyzed the glomerular response to large numbers of odorants at single glomerulus resolution. Contrary to the general view, we find that the representation of chemical features is spatially distributed in the olfactory bulb with no discernible chemotopy. Moreover, odor-evoked pattern of activity does not correlate directly with odor structure in general. Despite the lack of spatial clustering or preference with respect to chemical features, some structurally related odors can be similarly represented by ensembles of spatially distributed glomeruli, providing an explanation of their perceptual similarity. Whereas there is no chemotopic organization, and the glomeruli are tuned to odors from multiple classes, we find that the glomeruli are hierarchically arranged into clusters according to their odor-tuning similarity. This tunotopic arrangement provides a framework to understand the spatial organization of the glomeruli that conforms to the organizational principle found in other sensory systems.

Targeting of the SUN protein Mps3 to the inner nuclear membrane by the histone variant H2A.Z

Binding of histone H2A.Z to the SUN family member Mps3 is chromatin independent.

Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ

Cystic kidney diseases (CKDs) affect millions of people worldwide. The defining pathological features are fluid-filled cysts developing from nephric tubules due to defective flow sensing, cell proliferation and differentiation. The underlying molecular mechanisms, however, remain poorly understood, and the derived excretory systems of established invertebrate models (Caenorhabditis elegans and Drosophila melanogaster) are unsuitable to model CKDs. Systematic structure/function comparisons revealed that the combination of ultrafiltration and flow-associated filtrate modification that is central to CKD etiology is remarkably conserved between the planarian excretory system and the vertebrate nephron. Consistently, both RNA-mediated genetic interference (RNAi) of planarian orthologues of human CKD genes and inhibition of tubule flow led to tubular cystogenesis that share many features with vertebrate CKDs, suggesting deep mechanistic conservation. Our results demonstrate a common evolutionary origin of animal excretory systems and establish planarians as a novel and experimentally accessible invertebrate model for the study of human kidney pathologies. DOI: http://dx.doi.org/10.7554/eLife.07405.001

Regeneration of Sensory Hair Cells Requires Localized Interactions between the Notch and Wnt Pathways

In vertebrates, mechano-electrical transduction of sound is accomplished by sensory hair cells. Whereas mammalian hair cells are not replaced when lost, in fish they constantly renew and regenerate after injury. Inxa0vivo tracking and cell fate analyses of all dividing cells during lateral line hair cell regeneration revealed that support and hair cell progenitors localize to distinct tissue compartments. Importantly, we find that the balance between self-renewal and differentiation in these compartments is controlled by spatially restricted Notch signaling and its inhibition of Wnt-induced proliferation. The ability to simultaneously study and manipulate individual cell behaviors and multiple pathways inxa0vivo transforms the lateral line into a powerful paradigm to mechanistically dissect sensory organ regeneration. The striking similaritiesxa0toxa0other vertebrate stem cell compartments uniquely place zebrafish to help elucidate why mammals possess such low capacity to regenerate hair cells.

Acetylation of the SUN protein Mps3 by Eco1 regulates its function in nuclear organization

Acetylation of the SUN protein Mps3 by Eco1 regulates nuclear organization, including chromosome cohesion, telomere tethering, and gene silencing, without affecting Mps3 distribution in the nuclear membrane. It is one of the regulatory pathways known to affect chromosome positioning within the nucleus.

Egf Signaling Directs Neoblast Repopulation by Regulating Asymmetric Cell Division in Planarians.

A large population of proliferative stem cells (neoblasts) is required for physiological tissue homeostasis and post-injury regeneration in planarians. Recent studies indicate that survival of a few neoblasts after sublethal irradiation results in the clonalxa0expansion of the surviving stem cells and the eventual restoration of tissue homeostasis and regenerative capacity. However, the precise mechanisms regulating the population dynamics of neoblasts remain largely unknown. Here, we uncovered a central role for epidermal growth factor (EGF) signaling during inxa0vivo neoblast expansion mediated by Smed-egfr-3 (egfr-3) and its putative ligand Smed-neuregulin-7 (nrg-7). Furthermore, the EGF receptor-3 protein localizes asymmetrically on thexa0cytoplasmic membrane of neoblasts, and the ratioxa0of asymmetric to symmetric cell divisions decreases significantly in egfr-3(RNAi) worms. Our results not only provide the first molecular evidence of asymmetric stem cell divisions in planarians, but also demonstrate that EGF signaling likely functionsxa0as an essential regulator of neoblast clonal expansion.

Multispectral fingerprinting for improved in vivo cell dynamics analysis

BackgroundTracing cell dynamics in the embryo becomes tremendously difficult when cell trajectories cross in space and time and tissue density obscure individual cell borders. Here, we used the chick neural crest (NC) as a model to test multicolor cell labeling and multispectral confocal imaging strategies to overcome these roadblocks.ResultsWe found that multicolor nuclear cell labeling and multispectral imaging led to improved resolution of in vivo NC cell identification by providing a unique spectral identity for each cell. NC cell spectral identity allowed for more accurate cell tracking and was consistent during short term time-lapse imaging sessions. Computer model simulations predicted significantly better object counting for increasing cell densities in 3-color compared to 1-color nuclear cell labeling. To better resolve cell contacts, we show that a combination of 2-color membrane and 1-color nuclear cell labeling dramatically improved the semi-automated analysis of NC cell interactions, yet preserved the ability to track cell movements. We also found channel versus lambda scanning of multicolor labeled embryos significantly reduced the time and effort of image acquisition and analysis of large 3D volume data sets.ConclusionsOur results reveal that multicolor cell labeling and multispectral imaging provide a cellular fingerprint that may uniquely determine a cells position within the embryo. Together, these methods offer a spectral toolbox to resolve in vivo cell dynamics in unprecedented detail.

Proliferation-independent regulation of organ size by Fgf/Notch signaling

Organ morphogenesis depends on the precise orchestration of cell migration, cell shape changes and cell adhesion. We demonstrate that Notch signaling is an integral part of the Wnt and Fgf signaling feedback loop coordinating cell migration and the self-organization of rosette-shaped sensory organs in the zebrafish lateral line system. We show that Notch signaling acts downstream of Fgf signaling to not only inhibit hair cell differentiation but also to induce and maintain stable epithelial rosettes. Ectopic Notch expression causes a significant increase in organ size independently of proliferation and the Hippo pathway. Transplantation and RNASeq analyses revealed that Notch signaling induces apical junctional complex genes that regulate cell adhesion and apical constriction. Our analysis also demonstrates that in the absence of patterning cues normally provided by a Wnt/Fgf signaling system, rosettes still self-organize in the presence of Notch signaling. DOI: http://dx.doi.org/10.7554/eLife.21049.001