Sept4/ARTS is required for stem cell apoptosis and tumor suppression.

Inhibitor of Apoptosis Proteins (IAPs) are frequently overexpressed in tumors and have become promising targets for developing anti-cancer drugs. IAPs can be inhibited by natural antagonists, but a physiological requirement of mammalian IAP antagonists remains to be established. Here we show that deletion of the mouse Sept4 gene, which encodes the IAP antagonist ARTS, promotes tumor development. Sept4-null mice have increased numbers of hematopoietic stem and progenitor cells, elevated XIAP protein, increased resistance to cell death, and accelerated tumor development in an Eµ-Myc background. These phenotypes are partially suppressed by inactivation of XIAP. Our results suggest that apoptosis plays an important role as a frontline defense against cancer by restricting the number of normal stem cells.

Spatially coordinated kinase signaling regulates local axon degeneration.

In addition to being a hallmark of neurodegenerative disease, axon degeneration is used during development of the nervous system to prune unwanted connections. In development, axon degeneration is tightly regulated both temporally and spatially. Here, we provide evidence that degeneration cues are transduced through various kinase pathways functioning in spatially distinct compartments to regulate axon degeneration. Intriguingly, glycogen synthase kinase-3 (GSK3) acts centrally, likely modulating gene expression in the cell body to regulate distally restricted axon degeneration. Through a combination of genetic and pharmacological manipulations, including the generation of an analog-sensitive kinase allele mutant mouse for GSK3ß, we show that the ß isoform of GSK3, not the α isoform, is essential for developmental axon pruning in vitro and in vivo. Additionally, we identify the dleu2/mir15a/16-1 cluster, previously characterized as a regulator of B-cell proliferation, and the transcription factor tbx6, as likely downstream effectors of GSK3ß in axon degeneration.

Critical role for Kit-mediated Src kinase but not PI 3-kinase signaling in pro T and pro B cell development.

The Kit receptor functions in hematopoiesis, lymphocyte development, gastrointestinal tract motility, melanogenesis, and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo, we have generated knock-in mice in which docking sites for PI 3-kinase (KitY719) or Src kinase (KitY567) have been mutated. Whereas steady-state hematopoiesis is normal in KitY719F/Y719F and KitY567F/Y567F mice, lymphopoiesis is affected differentially. The KitY567F mutation, but not the KitY719F mutation, blocks pro T cell and pro B cell development in an age-dependent manner. Thus, the Src family kinase, but not the PI 3-kinase docking site in Kit, mediates a critical signal for lymphocyte development. In agreement with these results, treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec) leads to deficits in pro T and pro B cell development, similar to those seen in KitY567F/Y567F and KitW/W mice. The two mutations do not affect embryonic gametogenesis but the KitY719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the KitY567F mutation does not affect this process. Therefore, Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo.

The Sept4 septin locus is required for sperm terminal differentiation in mice.

The murine septin4 gene (Sept4) has been implicated in diverse cellular functions, including cytokinesis, apoptosis, and tumor suppression. Here, we investigated the function of Sept4 proteins during mouse development by creating a targeted deletion of the Sept4 genomic locus. Sept4 mutant mice are viable but male sterile due to immotile and structurally defective sperm. During spermatogenesis, Sept4 proteins were essential for proper mitochondrial architecture and establishment of the annulus, a ring-like structure in the tail region of sperm. In addition, Sept4 mutant sperm showed defects in the elimination of residual cytoplasm during sperm maturation and had increased staining for the caspase inhibitor XIAP. This is consistent with a role of the proapoptotic Sept4 protein ARTS in promoting caspase-mediated removal of cytoplasm via inhibition of XIAP. Our results indicate that Sept4 proteins play distinct but evolutionarily conserved functions in different cellular compartments.

CSF1R mutations in hereditary diffuse leukoencephalopathy with spheroids are loss of function.

Hereditary diffuse leukoencephalopathy with spheroids (HDLS) in humans is a rare autosomal dominant disease characterized by giant neuroaxonal swellings (spheroids) within the CNS white matter. Symptoms are variable and can include personality and behavioural changes. Patients with this disease have mutations in the protein kinase domain of the colony-stimulating factor 1 receptor (CSF1R) which is a tyrosine kinase receptor essential for microglia development. We investigated the effects of these mutations on Csf1r signalling using a factor dependent cell line. Corresponding mutant forms of murine Csf1r were expressed on the cell surface at normal levels, and bound CSF1, but were not able to sustain cell proliferation. Since Csf1r signaling requires receptor dimerization initiated by CSF1 binding, the data suggest a mechanism for phenotypic dominance of the mutant allele in HDLS.

Glucose sensor O-GlcNAcylation coordinates with phosphorylation to regulate circadian clock.

Posttranslational modifications play central roles in myriad biological pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a critical factor in regulating complex GSK3ß-dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3ß. Interestingly, OGT activity is regulated by GSK3ß; hence, OGT and GSK3ß exhibit reciprocal regulation. Modulating O-GlcNAcylation levels alter circadian period length in both mice and Drosophila; conversely, protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addition, O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662-S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine-tune circadian clock.

Notch lineages and activity in intestinal stem cells determined by a new set of knock-in mice.

The conserved role of Notch signaling in controlling intestinal cell fate specification and homeostasis has been extensively studied. Nevertheless, the precise identity of the cells in which Notch signaling is active and the role of different Notch receptor paralogues in the intestine remain ambiguous, due to the lack of reliable tools to investigate Notch expression and function in vivo. We generated a new series of transgenic mice that allowed us, by lineage analysis, to formally prove that Notch1 and Notch2 are specifically expressed in crypt stem cells. In addition, a novel Notch reporter mouse, Hes1-EmGFP(SAT), demonstrated exclusive Notch activity in crypt stem cells and absorptive progenitors. This roster of knock-in and reporter mice represents a valuable resource to functionally explore the Notch pathway in vivo in virtually all tissues.

A role for kit receptor signaling in Leydig cell steroidogenesis.

Kit and its ligand, Kitl, function in hematopoiesis, melanogenesis, and gametogenesis. In the testis, Kitl is expressed by Sertoli cells and Kit is expressed by spermatogonia and Leydig cells. Kit functions are mediated by receptor autophosphorylation and subsequent association with signaling molecules, including phosphoinositide (PI) 3-kinase. We previously characterized the reproductive consequences of blocking Kit-mediated PI 3-kinase activation in KitY(719F)/Kit(Y719F) knockin mutant male mice. Only gametogenesis was affected in these mice, and males are sterile because of a block in spermatogenesis during the spermatogonial stages. In the present study, we investigated effects of the Kit(Y719F) mutation on Leydig cell development and steroidogenic function. Although the seminiferous tubules in testes of mutant animals are depleted of germ cells, the testes contain normal numbers of Leydig cells and the Leydig cells in these animals appear to have undergone normal differentiation. Evaluation of steroidogenesis in mutant animals indicates that testosterone levels are not significantly reduced in the periphery but that LH levels are increased 5-fold, implying an impairment of steroidogenesis in the mutant animals. Therefore, a role for Kit signaling in steroidogenesis in Leydig cells was sought in vitro. Purified Leydig cells from C57Bl6/J male mice were incubated with Kitl, and testosterone production was measured. Kitl-stimulated testosterone production was 2-fold higher than that in untreated controls. The Kitl-mediated testosterone biosynthesis in Leydig cells is PI 3-kinase dependent. In vitro, Leydig cells from mutant mice were steroidogenically more competent in response to LH than were normal Leydig cells. In contrast, Kitl-mediated testosterone production in these cells was comparable to that in normal cells. Because LH levels in mutant males are elevated and LH is known to stimulate testosterone biosynthesis, we proposed a model in which serum testosterone levels are controlled by elevated LH secretion. Leydig cells of mutant males, unable to respond effectively to Kitl stimulation, initially produce lower levels of testosterone, reducing testosterone negative feedback on the hypothalamic-pituitary axis. The consequent secretion of additional LH, under this hypothesis, causes a restoration of normal levels of serum testosterone. Kitl, acting via PI 3-kinase, is a paracrine regulator of Leydig cell steroidogenic function in vivo.