SummaryNeurons in the mammalian neocortex arise from asymmetric divisions of progenitors residing in the ventricular zone. While in most progenitor divisions, the mitotic spindle is parallel to the ventricular surface, some progenitors reorient the spindle and divide in oblique orientations. Here, we use conditional deletion and overexpression of mouse Inscuteable (mInsc) to analyze the relevance of spindle reorientation in cortical progenitors. Mutating mInsc almost abolishes oblique and vertical mitotic spindles, while mInsc overexpression has the opposite effect. Our data suggest that oblique divisions are essential for generating the correct numbers of neurons in all cortical layers. Using clonal analysis, we demonstrate that spindle orientation affects the rate of indirect neurogenesis, a process where progenitors give rise to basal progenitors, which in turn divide symmetrically into two differentiating neurons. Our results indicate that the orientation of progenitor cell divisions is important for correct lineage specification in the developing mammalian brain.

During mammalian neurogenesis, progenitor cells can divide with the mitotic spindle oriented parallel or perpendicular to the surface of the neuroepithelium. Perpendicular divisions are more likely to be asymmetric and generate one progenitor and one neuronal precursor. Whether the orientation of the mitotic spindle actually determines their asymmetric outcome is unclear. Here, we characterize a mammalian homolog of Inscuteable (mInsc), a key regulator of spindle orientation in Drosophila. mInsc is expressed temporally and spatially in a manner that suggests a role in orienting the mitotic spindle in the developing nervous system. Using retroviral RNAi in rat retinal explants, we show that downregulation of mInsc inhibits vertical divisions. This results in enhanced proliferation, consistent with a higher frequency of symmetric divisions generating two proliferating cells. Our results suggest that the orientation of neural progenitor divisions is important for cell fate specification in the retina and determines their symmetric or asymmetric outcome.

The developmental potential of hematopoietic progenitors is restricted early on to either the erythromyeloid or lymphoid lineages. The broad developmental potential of Pax5−/− pro‐B cells is in apparent conflict with such a strict separation, although these progenitors realize the myeloid and erythroid potential with lower efficiency compared to the lymphoid cell fates. Here we demonstrate that ectopic expression of the transcription factors C/EBPα, GATA1, GATA2 and GATA3 strongly promoted in vitro macrophage differentiation and myeloid colony formation of Pax5−/− pro‐B cells. GATA2 and GATA3 expression also resulted in efficient engraftment and myeloid development of Pax5−/− pro‐B cells in vivo. The myeloid transdifferentiation of Pax5−/− pro‐B cells was accompanied by the rapid activation of myeloid genes and concomitant repression of B‐lymphoid genes by C/EBPα and GATA factors. These data identify the Pax5−/− pro‐B cells as lymphoid progenitors with a latent myeloid potential that can be efficiently activated by myeloid transcription factors. The same regulators were unable to induce a myeloid lineage switch in Pax5+/+ pro‐B cells, indicating that Pax5 dominates over myeloid transcription factors in B‐lymphocytes.

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The A 2A -adenosine receptor is a prototypical G s protein-coupled receptor but stimulates MAPK/ERK in a G s -independent way. The A 2A receptor has long been known to undergo restricted collision coupling with G s ; the mechanistic basis for this mode of coupling has remained elusive. Here we visualized agonist-induced changes in mobility of the yellow fluorescent protein-tagged receptor by fluorescence recovery after photobleaching microscopy. Stimulation with a specific A 2A receptor agonist did not affect receptor mobility. In contrast, stimulation with dopamine decreased the mobility of the D 2 receptor. When coexpressed in the same cell, the A 2A receptor precluded the agonist-induced change in D 2 receptor mobility. Thus, the A 2A receptor did not only undergo restricted collision coupling, but it also restricted the mobility of the D 2 receptor. Restricted mobility was not due to tethering to the actin cytoskeleton but was, in part, related to the cholesterol content of the membrane. Depletion of cholesterol increased receptor mobility but blunted activation of adenylyl cyclase, which was accounted for by impaired formation of the ternary complex of agonist, receptor, and G protein. These observations support the conclusion that the A 2A receptor engages G s and thus signals to adenylyl cyclase in cholesterol-rich domains of the membrane. In contrast, stimulation of MAPK by the A 2A receptor was not impaired. These findings are consistent with a model where the recruitment of these two pathways occurs in physically segregated membrane microdomains. Thus, the A 2A receptor is the first example of a G protein-coupled receptor documented to select signaling pathways in a manner dependent on the lipid microenvironment of the membrane.In the fluid mosaic model, the lipid bilayer is an isotropic milieu, in which membrane-embedded proteins diffuse in two dimensions and thus collide at random with each other (1). When applied to G protein-coupled receptors, the model predicts that G protein-coupled receptors move in a random walk, and, upon activation, this allows them to engage their cognate G proteins. This "collision coupling" mode of activation was validated in studies using the ␤-adrenergic receptor and its coupling to the effector enzyme adenylyl cyclase in turkey erythrocytes (2). However, experiments with the A 2 -adenosine receptor in turkey erythrocytes revealed kinetic properties of adenylyl cyclase activation that were incompatible with the collision coupling model. The results indicated a tight coupling of the adenosine receptor to G␣ s (3, 4); the term "restricted collision coupling" was coined to account for this altered mode of coupling. Restricted collision is not a feature unique to the avian A 2 -adenosine receptor, because it was also documented for the human A 2A receptor in platelet membranes (5). In addition, the A 2A -adenosine receptor has the unusual feature of forming a tight complex with G s , which persists in detergent solution, which is resistant to guanine nucleotides and which re...

The A 2A adenosine receptor is a prototypical G s -coupled receptor, but it also signals, e.g. to mitogen-activated protein (MAP) kinase, via a pathway that is independent of heterotrimeric G proteins. Truncation of the carboxyl terminus affects the strength of the signal through these alternative pathways. In a yeast two-hybrid interaction hunt, we screened a human brain library for proteins that bound to the juxtamembrane portion of the carboxyl terminus of the A 2A receptor. This approach identified ARNO/cytohesin-2, a nucleotide exchange factor for the small (monomeric) G proteins of the Arf (ADP-ribosylation factor) family, as a potential interaction partner. We confirmed a direct interaction by mutual pull down (of fusion proteins expressed in bacteria) and by immunoprecipitation of the proteins expressed in mammalian cells. To circumvent the long term toxicity associated with overexpression of ARNO/cytohesin-2, we created stable cell lines that stably expressed the A 2A receptor and where ARNO/cytohesin-2 or the dominant negative version E156K-ARNO/ cytohesin-2 was inducible by mifepristone. Cyclic AMP accumulation induced by an A 2A -specific agonist was neither altered by ARNO/cytohesin-2 nor by the dominant negative version. This was also true for agonistinduced desensitization. In contrast, expression of dominant negative E156K-ARNO/cytohesin-2 and of dominant negative T27N-Arf6 abrogated the sustained phase of MAP kinase stimulation induced by the A 2A receptor. We therefore conclude that ARNO/cytohesin-2 is required to support the alternative, heterotrimeric G protein-independent, signaling pathway of A 2A receptor, which is stimulation of MAP kinase.Over the last decade, it has been increasingly accepted that G protein-coupled receptors also bind regulatory proteins other than G proteins, arrestins, and G protein-coupled kinases, which are involved in effector regulation and desensitization, respectively (1). These accessory proteins include components of signaling cascades and bind to the carboxyl termini of various G protein-coupled receptors (2). The A 2A adenosine receptor has an unusually long intracellular carboxyl-terminal tail, 122 amino acids in man, when compared, for instance, to 34 residues in the carboxyl terminus of the A 1 adenosine receptor. Circumstantial evidence suggests that accessory proteins bind to the carboxyl terminus of the A 2A receptor (3). A 2A receptors can activate mitogen-activated protein (MAP) 1 kinase by a G␣ s -independent signaling pathway; this can be seen both in endothelial cells where the receptor is endogenously expressed (4) and upon heterologous expression in HEK293 cells (5). Truncation of the carboxyl terminus does not impair the ability of the A 2A adenosine receptor to stimulate MAP kinase but blunts stimulation of cAMP accumulation. In addition, fulllength and truncated receptors differ in their constitutive (agonist-independent) activity; this difference is only seen in intact cells and is lost upon membrane preparation, suggesting the loss of one o...

In this paper we propose a data augmentation methodology for training machine/deep learning gait recognition algorithms. While previously published methods generated synthetic data to augment training and/or testing sets [1] or to learn features invariant to certain conditions [2], they have incorporated a very limited number of covariate factors. To our knowledge, this is the first attempt to provide the ability to simultaneously generate synthetic data with so many controllable conditions for gait recognition.The combination of real motion capture, data preparation, avatar construction and scripted rendering allows synthetic frames to be generated, with almost arbitrary degrees of variation. Figure 1 shows a small sample of the controllable confounding factors that can be generated with the proposed methodology. Using the Gait Energy Image (GEI) [3] as gait features, extracted directly from the synthetically generated frames, we performed experiments to assess the level to which identity is preserved within the synthetic data sets. Results from our experiments -presented in Figure 2 -suggest that information about the identity of subjects is retained within the synthetically generated data. The experiments using GEIs as features within our dataset showed that augmenting a limited amount of real data with the synthetically generated data can yield identification of subjects with an accuracy of more than 95%. The results of this study suggest that synthetic data can be used to augment the training of gait recognition algorithms provided that the feature set (e.g. principal components) is expanded to include both real and synthetic examples of data.

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