Periportal hepatocyte proliferation at midgestation governs maternal glucose homeostasis in mice.

The maternal liver is challenged by metabolic demands throughout pregnancy. However, hepatocyte dynamics and their physiological significance in pregnancy remain unclear. Here, we show in mice that hepatocyte proliferation is spatiotemporally regulated in each liver lobular zone during pregnancy, with transient proliferation of periportal and pericentral hepatocytes during mid and late gestation, respectively. Using adeno-associated virus (AAV)-8-mediated expression of the cell cycle inhibitor p21 in hepatocytes, we show that inhibition of hepatocyte proliferation during mid, but not late, gestation impairs liver growth. Transcriptionally, genes involved in glucose/glycogen metabolism are downregulated in late pregnancy when midgestational hepatocyte proliferation is attenuated. In addition, hepatic glycogen storage is abolished, with concomitant elevated blood glucose concentrations, glucose intolerance, placental glycogen deposition, and fetal overgrowth. Laser capture microdissection and RNA-seq analysis of each liver lobular zone show zone-specific changes in the transcriptome during pregnancy and identify genes that are periportally expressed at midgestation, including the hyaluronan-mediated motility receptor (Hmmr). Knockdown of Hmmr in hepatocytes by AAV8-shHmmr suppresses periportal hepatocyte proliferation at midgestation and induces impaired hepatic glycogen storage, glucose intolerance, placental glycogen deposition and fetal overgrowth. Our results suggest that periportal hepatocyte proliferation during midgestation is critical for maternal glycogen metabolism and fetal size.

Delineation of biliary epithelial cell dynamics in maternal liver during pregnancy.

In pregnant mice, the maternal liver expands drastically during gestation, which is believed to be essential to accommodate various metabolic demands caused by physiological changes and fetal growth. Although hepatocyte proliferation and hypertrophy have been reported, little is known about the dynamics of biliary epithelial cells (BECs), which comprise the bile duct epithelium in the liver. Here, we show that BECs transiently proliferate during the early stage of gestation. Lineage tracing revealed that BEC progeny were retained in the bile duct epithelium and did not differentiate into hepatocytes, indicating BEC self-replication during pregnancy. RNA-sequencing analysis of BECs identified their early pregnancy-signature transcriptomes, which highlighted Yes-associated protein (YAP) signaling-related genes. Nuclear accumulation of YAP was enhanced in BECs during pregnancy but was barely detectable in hepatocytes. In addition, the pharmacological inhibition of YAP attenuated BEC proliferation and liver weight gain during pregnancy. Our results delineate the proliferation and transcriptomic dynamics of BECs during pregnancy and suggest the relevance of YAP-mediated signals.

Tbx3-dependent amplifying stem cell progeny drives interfollicular epidermal expansion during pregnancy and regeneration.

The skin surface area varies flexibly in response to body shape changes. Skin homeostasis is maintained by stem cells residing in the basal layer of the interfollicular epidermis. However, how the interfollicular epidermal stem cells response to physiological body shape changes remains elusive. Here, we identify a highly proliferative interfollicular epidermal basal cell population in the rapidly expanding abdominal skin of pregnant mice. These cells express Tbx3 that is necessary for their propagation to drive skin expansion. The Tbx3+ basal cells are generated from Axin2+ interfollicular epidermal stem cells through planar-oriented asymmetric or symmetric cell divisions, and express transit-amplifying cell marker CD71. This biased division of Axin2+ interfollicular epidermal stem cells is induced by Sfrp1 and Igfbp2 proteins secreted from dermal cells. The Tbx3+ basal cells promote wound repair, which is enhanced by Sfrp1 and Igfbp2. This study elucidates the interfollicular epidermal stem cell/progeny organisation during pregnancy and suggests its application in regenerative medicine.The abdominal skin expands rapidly during pregnancy. Here the authors show that a population of highly proliferative stem cell progenies expressing the transcription factor Tbx3 is required for abdominal skin expansion in pregnant mice.

A G-quadruplex structure at the 5' end of the H19 coding region regulates H19 transcription.

The H19 gene, one of the best known imprinted genes, encodes a long non-coding RNA that regulates cell proliferation and differentiation. H19 RNA is widely expressed in embryonic tissues, but its expression is restricted in only a few tissues after birth. However, regulation of H19 gene expression remains poorly understood outside the context of genomic imprinting. Here we identified evolutionarily conserved guanine (G)-rich repeated motifs at the 5' end of the H19 coding region that are consistent with theoretically deduced G-quadruplex sequences. Circular dichroism spectroscopy and electrophoretic mobility shift assays with G-quadruplex-specific ligands revealed that the G-rich motif, located immediately downstream of the transcription start site (TSS), forms a G-quadruplex structure in vitro. By using a series of mutant forms of H19 harboring deletion or G-to-A substitutions, we found that the H19-G-quadruplex regulates H19 gene expression. We further showed that transcription factors Sp1 and E2F1 were associated with the H19-G-quadruplex to either suppress or promote the H19 transcription, respectively. Moreover, H19 expression during differentiation of mouse embryonic stem cells appears to be regulated by a genomic H19 G-quadruplex. These results demonstrate that the G-quadruplex structure immediately downstream of the TSS functions as a novel regulatory element for H19 gene expression.

Dormant Pluripotent Cells Emerge during Neural Differentiation of Embryonic Stem Cells in a FoxO3-Dependent Manner.

One major concern over the clinical application of embryonic stem cell (ESC)-derived cells is the potentiation of latent tumorigenicity by residual undifferentiated cells. Despite the use of intensive methodological approaches to eliminate residual undifferentiated cells, the properties of these cells remain elusive. Here, we show that under a serum-free neural differentiation condition, residual undifferentiated cells markedly delay progression of their cell cycle without compromising their pluripotency. This dormant pluripotency was maintained during reculture of the cells under a serum-free condition, whereas upon serum stimulation, the cells exited the dormant state and restarted proliferation and differentiation into all three germ layers. Microarray analysis revealed a set of genes that is significantly upregulated in the dormant ESCs compared with their levels of regulation in proliferating ESCs. Among them, we identified the transcription factor Forkhead box O3 (FoxO3) to be an essential regulator of the maintenance of pluripotency in dormant ESCs. Our study demonstrates that the transition into the dormant state endows residual undifferentiated cells with FoxO3-dependent and leukemia inhibitory factor/serum-independent pluripotency.

c-Rel Regulates Inscuteable Gene Expression during Mouse Embryonic Stem Cell Differentiation.

Inscuteable (Insc) regulates cell fate decisions in several types of stem cells. Although it is recognized that the expression levels of mouse INSC govern the balance between symmetric and asymmetric stem cell division, regulation of mouse Insc gene expression remains poorly understood. Here, we showed that mouse Insc expression transiently increases at an early stage of differentiation, when mouse embryonic stem (mES) cells differentiate into bipotent mesendoderm capable of producing both endoderm and mesoderm in defined culture conditions. We identified the minimum transcriptional regulatory element (354 bases) that drives mouse Insc transcription in mES cells within a region >5 kb upstream of the mouse Insc transcription start site. We found that the transcription factor reticuloendotheliosis oncogene (c-Rel) bound to the minimum element and promoted mouse Insc expression in mES cells. In addition, short interfering RNA-mediated knockdown of either mouse INSC or c-Rel protein decreased mesodermal cell populations without affecting differentiation into the mesendoderm or endoderm. Furthermore, overexpression of mouse INSC rescued the mesoderm-reduced phenotype induced by knockdown of c-Rel. We propose that regulation of mouse Insc expression by c-Rel modulates cell fate decisions during mES cell differentiation.

PLK1-dependent activation of LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2.

Correct formation of the cell division axis requires the initial precise orientation of the mitotic spindle. Proper spindle orientation depends on centrosome maturation, and Polo-like kinase 1 (PLK1) is known to play a crucial role in this process. However, the molecular mechanisms that function downstream of PLK1 are not well understood. Here we show that LRRK1 is a PLK1 substrate that is phosphorylated on Ser 1790. PLK1 phosphorylation is required for CDK1-mediated activation of LRRK1 at the centrosomes, and this in turn regulates mitotic spindle orientation by nucleating the growth of astral microtubules from the centrosomes. Interestingly, LRRK1 in turn phosphorylates CDK5RAP2(Cep215), a human homologue of Drosophila Centrosomin (Cnn), in its γ-tubulin-binding motif, thus promoting the interaction of CDK5RAP2 with γ-tubulin. LRRK1 phosphorylation of CDK5RAP2 Ser 140 is necessary for CDK5RAP2-dependent microtubule nucleation. Thus, our findings provide evidence that LRRK1 regulates mitotic spindle orientation downstream of PLK1 through CDK5RAP2-dependent centrosome maturation.

PCTK1 regulates integrin-dependent spindle orientation via protein kinase A regulatory subunit KAP0 and myosin X.

Integrin-dependent cell-extracellular matrix (ECM) adhesion is a determinant of spindle orientation. However, the signaling pathways that couple integrins to spindle orientation remain elusive. Here, we show that PCTAIRE-1 kinase (PCTK1), a member of the cyclin-dependent kinases (CDKs) whose function is poorly characterized, plays an essential role in this process. PCTK1 regulates spindle orientation in a kinase-dependent manner. Phosphoproteomic analysis together with an RNA interference screen revealed that PCTK1 regulates spindle orientation through phosphorylation of Ser83 on KAP0, a regulatory subunit of protein kinase A (PKA). This phosphorylation is dispensable for KAP0 dimerization and for PKA binding but is necessary for its interaction with myosin X, a regulator of spindle orientation. KAP0 binds to the FERM domain of myosin X and enhances the association of myosin X-FERM with ß1 integrin. This interaction between myosin X-FERM and ß1 integrin appeared to be crucial for spindle orientation control. We propose that PCTK1-KAP0-myosin X-ß1 integrin is a functional module providing a link between ECM and the actin cytoskeleton in the ECM-dependent control of spindle orientation.

Pregnenolone functions in centriole cohesion during mitosis.

Cell division is controlled by a multitude of protein enzymes, but little is known about roles of metabolites in this mechanism. Here, we show that pregnenolone (P5), a steroid that is produced from cholesterol by the steroidogenic enzyme Cyp11a1, has an essential role in centriole cohesion during mitosis. During prometa-metaphase, P5 is accumulated around the spindle poles. Depletion of P5 induces multipolar spindles that result from premature centriole disengagement, which are rescued by ectopic introduction of P5, but not its downstream metabolites, into the cells. Premature centriole disengagement, induced by loss of P5, is not a result of precocious activation of separase, a key factor for the centriole disengagement in anaphase. Rather, P5 directly binds to the N-terminal coiled-coil domain of short-form of shugoshin 1 (sSgo1), a protector for centriole cohesion and recruits it to spindle poles in mitosis. Our results thus reveal a steroid-mediated centriole protection mechanism.

Inhibition of endocytic vesicle fusion by Plk1-mediated phosphorylation of vimentin during mitosis.

Endocytic vesicle fusion is inhibited during mitosis, but the molecular pathways that mediate the inhibition remain unclear. Here we uncovered an essential role of Polo-like kinase 1 (Plk1) in this mechanism. Phosphoproteomic analysis revealed that Plk1 phosphorylates the intermediate filament protein vimentin on Ser459, which is dispensable for its filament formation but is necessary for the inhibition of endocytic vesicle fusion in mitosis. Furthermore, this mechanism is required for integrin trafficking toward the cleavage furrow during cytokinesis. Our results thus identify a novel mechanism for fusion inhibition in mitosis and implicate its role in vesicle trafficking after anaphase onset.

ABL1 joins the cadre of spindle orientation machinery.

Directing the axis of cell division toward extrinsic and intrinsic cues plays a fundamental role in morphogenesis, asymmetric cell division, and stem cell self-renewal. Recent studies highlight the misorientation of the cell division axis as a cause of mammalian diseases, including polycystic kidney disease. Although the core regulators for oriented cell division have been identified in invertebrate model systems, we still have an imprecise picture of the relevant signaling networks in the mammalian system. The reasons for this include the lack of established approaches in mammalian cells to survey the molecules required for the spindle orientation. Here we summarize our recent study on a genome-scale RNA-mediated interference screen of human kinases to identify a new player for the oriented cell division in both culture cells and developing mammalian tissues.

ABL1 regulates spindle orientation in adherent cells and mammalian skin.

Despite the growing evidence for the regulated spindle orientation in mammals, a systematic approach for identifying the responsible genes in mammalian cells has not been established. Here we perform a kinase-targeting RNAi screen in HeLa cells and identify ABL1 as a novel regulator of spindle orientation. Knockdown of ABL1 causes the cortical accumulation of Leu-Gly-Asn repeat-enriched-protein (LGN), an evolutionarily conserved regulator of spindle orientation. This results in the LGN-dependent spindle rotation and spindle misorientation. In vivo inactivation of ABL1 by a pharmacological inhibitor or by ablation of the abl1 gene causes spindle misorientation and LGN mislocalization in mouse epidermis. Furthermore, ABL1 directly phosphorylates NuMA, a binding partner of LGN, on tyrosine 1774. This phosphorylation maintains the cortical localization of NuMA during metaphase, and ensures the LGN/NuMA-dependent spindle orientation control. This study provides a novel approach to identify genes regulating spindle orientation in mammals and uncovers new signalling pathways for this mechanism.

Dual role of Cdc42 in spindle orientation control of adherent cells.

The spindle orientation is regulated by the interaction of astral microtubules with the cell cortex. We have previously shown that spindles in nonpolarized adherent cells are oriented parallel to the substratum by an actin cytoskeleton- and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3]-dependent mechanism. Here, we show that Cdc42, a Rho family of small GTPases, has an essential role in this mechanism of spindle orientation by regulating both the actin cytoskeleton and PtdIns(3,4,5)P3. Knockdown of Cdc42 suppresses PI(3)K activity in M phase and induces spindle misorientation. Moreover, knockdown of Cdc42 disrupts the cortical actin structures in metaphase cells. Our results show that p21-activated kinase 2 (PAK2), a target of Cdc42 and/or Rac1, plays a key role in regulating actin reorganization and spindle orientation downstream from Cdc42. Surprisingly, PAK2 regulates spindle orientation in a kinase activity-independent manner. BetaPix, a guanine nucleotide exchange factor for Rac1 and Cdc42, is shown to mediate this kinase-independent function of PAK2. This study thus demonstrates that spindle orientation in adherent cells is regulated by two distinct pathways downstream from Cdc42 and uncovers a novel role of the Cdc42-PAK2-betaPix-actin pathway for this mechanism.

PtdIns(3,4,5)P3 regulates spindle orientation in adherent cells.

Cultured adherent cells divide on the substratum, leading to formation of the cell monolayer. However, how the orientation of this anchorage-dependent cell division is regulated remains unknown. We have previously shown that integrin-dependent adhesion orients the spindle parallel to the substratum, which ensures this anchorage-dependent cell division. Here, we show that phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is essential for this spindle orientation control. In metaphase, PtdIns(3,4,5)P3 is accumulated in the midcortex in an integrin-dependent manner. Inhibition of phosphatidylinositol-3-OH kinase (PI(3)K) reduces the accumulation of PtdIns(3,4,5)P3 and induces spindle misorientation. Introduction of PtdIns(3,4,5)P3 to these cells restores the midcortical accumulation of PtdIns(3,4,5)P3 and proper spindle orientation. PI(3)K inhibition causes dynein-dependent spindle rotations along the z-axis, resulting in spindle misorientation. Moreover, dynactin, a dynein-binding partner, is accumulated in the midcortex in a PtdIns(3,4,5)P3-dependent manner. We propose that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on spindles to the midcortex, and thereby orients the spindle parallel to the substratum.

Polo-like kinase 1 facilitates chromosome alignment during prometaphase through BubR1.

Plk1, an evolutionarily conserved M phase kinase, associates with not only spindle poles but also kinetochores during prometaphase. However, the role of Plk1 at kinetochores has been poorly understood. Here we show that BubR1 mediates the action of Plk1 at kinetochores for proper chromosome alignment. Our results show that BubR1 colocalizes with Plk1 at kinetochores of unaligned chromosomes and physically interacts with Plk1 in prometaphase cells. Down-regulation of Plk1 by small interfering RNA abolished the mobility-shifted, hyperphosphorylated form of BubR1 in the prometaphase-arrested cells. In addition, BubR1 was phosphorylated by Plk1 in vitro at two Plk1 consensus sites in the kinase domain of BubR1. The add-back of either wild-type BubR1 or BubR1 2E, in which the two Plk1 phosphorylation sites were replaced by glutamic acids, but not that of BubR1 2A, an unphosphorylatable mutant, rescued the chromosome alignment defects in BubR1-deficient cells. Moreover, when both Plk1 and BubR1 were down-regulated, the add-back of BubR1 2E, but not that of wild-type BubR1, rescued the chromosome alignment defects. These results taken together suggest that Plk1 facilitates chromosome alignment during prometaphase through BubR1.

Integrin-mediated adhesion orients the spindle parallel to the substratum in an EB1- and myosin X-dependent manner.

The orientation of mitotic spindles is tightly regulated in polarized cells, but it has been unclear whether there is a mechanism regulating spindle orientation in nonpolarized cells. Here we show that integrin-dependent cell adhesion to the substrate orients the mitotic spindle of nonpolarized cultured cells parallel to the substrate plane. The spindle is properly oriented in cells plated on fibronectin or collagen, but misoriented in cells on poly-L-lysine or treated with the RGD peptide or anti-beta1-integrin antibody, indicating requirement of integrin-mediated cell adhesion for this mechanism. Remarkably, this mechanism is independent of gravitation or cell-cell adhesion, but requires actin cytoskeleton and astral microtubules. Furthermore, myosin X and the microtubule plus-end-tracking protein EB1 are shown to play a role in this mechanism through remodeling of actin cytoskeleton and stabilization of astral microtubules, respectively. Our results thus uncover the existence of a mechanism that orients the spindle parallel to the cell-substrate adhesion plane, and identify crucial factors involved in this novel mechanism.