06-570 Sigma-AldrichAnti-phospho-Histone H3 (Ser10) Antibody, Mitosis Marker

Quiescent stem cells in adult tissues can be activated for homeostasis or repair. Neural stem cells (NSCs) in Drosophila are reactivated from quiescence in response to nutrition by the insulin signaling pathway. It is widely accepted that quiescent stem cells are arrested in G0 In this study, however, we demonstrate that quiescent NSCs (qNSCs) are arrested in either G2 or G0 G2-G0 heterogeneity directs NSC behavior: G2 qNSCs reactivate before G0 qNSCs. In addition, we show that the evolutionarily conserved pseudokinase Tribbles (Trbl) induces G2 NSCs to enter quiescence by promoting degradation of Cdc25String and that it subsequently maintains quiescence by inhibiting Akt activation. Insulin signaling overrides repression of Akt and silences trbl transcription, allowing NSCs to exit quiescence. Our results have implications for identifying and manipulating quiescent stem cells for regenerative purposes.

Limb skeletal elements originate from the limb progenitor cells, which undergo expansion and patterning to develop each skeletal element. Posterior-distal skeletal elements, such as the ulna/fibula and posterior digits develop in a Sonic hedgehog (Shh)-dependent manner. However, it is poorly understood how anterior-proximal elements, such as the humerus/femur, the radius/tibia and the anterior digits, are developed. Here we show that the zinc finger factors Sall4 and Gli3 cooperate for proper development of the anterior-proximal skeletal elements and also function upstream of Shh-dependent posterior skeletal element development. Conditional inactivation of Sall4 in the mesoderm before limb outgrowth caused severe defects in the anterior-proximal skeletal elements in the hindlimb. We found that Gli3 expression is reduced in Sall4 mutant hindlimbs, but not in forelimbs. This reduction caused posteriorization of nascent hindlimb buds, which is correlated with a loss of anterior digits. In proximal development, Sall4 integrates Gli3 and the Plzf-Hox system, in addition to proliferative expansion of cells in the mesenchymal core of nascent hindlimb buds. Whereas forelimbs developed normally in Sall4 mutants, further genetic analysis identified that the Sall4-Gli3 system is a common regulator of the early limb progenitor cells in both forelimbs and hindlimbs. The Sall4-Gli3 system also functions upstream of the Shh-expressing ZPA and the Fgf8-expressing AER in fore- and hindlimbs. Therefore, our study identified a critical role of the Sall4-Gli3 system at the early steps of limb development for proper development of the appendicular skeletal elements.

Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3(+) or BrdU(+) cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3(+)), astroglia (GFAP(+)), and microglia (Iba1(+) cells) were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day) at one dose/4-days resting or 5 doses (1 dose/day). Repeated URB597 treatment increased the plasma levels of the N-acylethanolamines oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3(+) and BrdU(+) subgranular cells as well as GFAP(+), Iba1(+) and cleaved caspase-3(+) cells, which was accompanied with decreased hippocampal expression of the cannabinoid CB1 receptor gene Cnr1 and Faah. In the hypothalami of these rats, the number of phospho-H3(+), GFAP(+) and 3-weeks-old BrdU(+) cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in Faah and/or Cnr1 expression and a negative energy context.

GABAergic neurons are the primary inhibitory cell type in the mature brain and their dysfunction is associated with important neurological conditions like schizophrenia and anxiety. We aimed to discover the underlying mechanisms for dorsal/ventral midbrain GABAergic neurogenesis. Previous work by us and others has provided crucial insights into the key function of Mgn and Mash1 genes in determining GABAergic neurotransmitter fate. Induction of dorsal midbrain GABAergic neurons does not take place at any time during development in either of the single mutant mice. However, GABAergic neurons in the ventral midbrain remained unchanged. Thus, the similarities in MB-GABAergic phenotype observed in the Mgn and Mash1 single mutants suggest the existence of other factors that take over the function of MGN and MASH1 in the ventral midbrain or the existence of different molecular mechanisms. We show that this process essentially depends on heterodimers and homodimers formed by MGN and MASH1 and deciphered the in vivo relevance of the interaction by phenotypic analysis of Mgn/Mash1 double knockout and compound mice. Furthermore, the combination of gain- and loss-of-function experiments in the developing midbrain showed co-operative roles for Mgn and Mash1 genes in determining GABAergic identity. Transcription factors belonging to the Enhancer-of-split-related and proneural families have long been believed to counterpart each other's function. This work uncovers a synergistic cooperation between these two families, and provides a novel paradigm for how these two families cooperate for the acquisition of MB-GABAergic neuronal identity. Understanding their molecular mechanisms is essential for cell therapy strategies to amend GABAergic deficits.

LKB1 plays important roles in governing energy homeostasis by regulating AMP-activated protein kinase (AMPK) and other AMPK-related kinases, including the salt-inducible kinases (SIKs). However, the roles and regulation of LKB1 in lipid metabolism are poorly understood. Here we show that Drosophila LKB1 mutants display decreased lipid storage and increased gene expression of brummer, the Drosophila homolog of adipose triglyceride lipase (ATGL). These phenotypes are consistent with those of SIK3 mutants and are rescued by expression of constitutively active SIK3 in the fat body, suggesting that SIK3 is a key downstream kinase of LKB1. Using genetic and biochemical analyses, we identify HDAC4, a class IIa histone deacetylase, as a lipolytic target of the LKB1-SIK3 pathway. Interestingly, we found that the LKB1-SIK3-HDAC4 signaling axis is modulated by dietary conditions. In short-term fasting, the adipokinetic hormone (AKH) pathway, related to the mammalian glucagon pathway, inhibits the kinase activity of LKB1 as shown by decreased SIK3 Thr196 phosphorylation, and consequently induces HDAC4 nuclear localization and brummer gene expression. However, under prolonged fasting conditions, AKH-independent signaling decreases the activity of the LKB1-SIK3 pathway to induce lipolytic responses. We also identify that the Drosophila insulin-like peptides (DILPs) pathway, related to mammalian insulin pathway, regulates SIK3 activity in feeding conditions independently of increasing LKB1 kinase activity. Overall, these data suggest that fasting stimuli specifically control the kinase activity of LKB1 and establish the LKB1-SIK3 pathway as a converging point between feeding and fasting signals to control lipid homeostasis in Drosophila.

During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G(1)-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G(2)- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. We bypass the 'mitotic mitochondrial fragmentation' checkpoint by inducing fragmented mitochondrial morphology and then measure the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophila S2R(+) cell and cells in the pouch region of wing imaginal disc of Drosophila larvae we show that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. Our experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. We provide evidence for the importance of mitochondrial superoxide in this process. Our results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors.

Mitotic repression of rRNA synthesis requires inactivation of the RNA polymerase I (Pol I)-specific transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of TAF(I)110 (TBP-associated factor 110) at a single threonine residue (T852). Upon exit from mitosis, T852 is dephosphorylated by Cdc14B, which is sequestered in nucleoli during interphase and is activated upon release from nucleoli at prometaphase. Mitotic repression of Pol I transcription correlates with transient nucleolar enrichment of the NAD(+)-dependent deacetylase SIRT1, which deacetylates another subunit of SL1, TAFI68. Hypoacetylation of TAFI68 destabilizes SL1 binding to the rDNA promoter, thereby impairing transcription complex assembly. Inhibition of SIRT1 activity alleviates mitotic repression of Pol I transcription if phosphorylation of TAF(I)110 is prevented. The results demonstrate that reversible phosphorylation of TAF(I)110 and acetylation of TAFI68 are key modifications that regulate SL1 activity and mediate fluctuations of pre-rRNA synthesis during cell cycle progression.

In vertebrates, the total number of vertebrae is precisely defined. Vertebrae derive from embryonic somites that are continuously produced posteriorly from the presomitic mesoderm (PSM) during body formation. We show that in the chicken embryo, activation of posterior Hox genes (paralogs 9-13) in the tail-bud correlates with the slowing down of axis elongation. Our data indicate that a subset of progressively more posterior Hox genes, which are collinearly activated in vertebral precursors, repress Wnt activity with increasing strength. This leads to a graded repression of the Brachyury/T transcription factor, reducing mesoderm ingression and slowing down the elongation process. Due to the continuation of somite formation, this mechanism leads to the progressive reduction of PSM size. This ultimately brings the retinoic acid (RA)-producing segmented region in close vicinity to the tail bud, potentially accounting for the termination of segmentation and axis elongation.

When cells enter mitosis, the anaphase-promoting complex/cyclosome (APC/C) is activated by phosphorylation and binding of Cdc20. The RXXL destruction box (D-box) of cyclin B1 only binds Cdc20 after release of the spindle checkpoint in metaphase, initiating cyclin B1 ubiquitination upon chromosome bi-orientation. However, we found that cyclin B1, through Cdk1 and Cks, is targeted to the phosphorylated APC/C(Cdc20) at the start of prometaphase, when the spindle checkpoint is still active. Here, we show that MASTL is essential for cyclin B1 recruitment to the mitotic APC/C and that this occurs entirely independently of Cdc20. Importantly, MASTL-directed binding of cyclin B1 to spindle checkpoint-inhibited APC/C(Cdc20) critically supports efficient cyclin B1 destruction after checkpoint release. A high incidence of anaphase bridges observed in response to MASTL RNAi may result from cyclin B1 remaining after securin destruction, which is insufficient to keep MASTL-depleted cells in mitosis but delays the activation of separase.

Aurora B regulates cytokinesis timing and plays a central role in the abscission checkpoint. Cellular events monitored by this checkpoint are beginning to be elucidated, yet signaling pathways upstream of Aurora B in this context remain poorly understood. Here we reveal a new connection between postmitotic genome surveillance and cytokinetic abscission. Underreplicated DNA lesions are known to be transmitted through mitosis and protected in newly formed nuclei by recruitment of 53BP1 and other proteins until repair takes place. We find that this genome surveillance initiates before completion of cytokinesis. Elevating replication stress increases this postmitotic process and delays cytokinetic abscission by keeping the abscission checkpoint active. We further find that ATR activity in midbody-stage cells links postmitotic genome surveillance to abscission timing and that Chk1 integrates this and other signals upstream of Aurora B to regulate when the final step in the physical separation of daughter cells occurs.