Inheritance of OCT4 predetermines fate choice in human embryonic stem cells.

It is well known that clonal cells can make different fate decisions, but it is unclear whether these decisions are determined during, or before, a cell's own lifetime. Here, we engineered an endogenous fluorescent reporter for the pluripotency factor OCT4 to study the timing of differentiation decisions in human embryonic stem cells. By tracking single-cell OCT4 levels over multiple cell cycle generations, we found that the decision to differentiate is largely determined before the differentiation stimulus is presented and can be predicted by a cell's preexisting OCT4 signaling patterns.

Primary Cilia Signaling Shapes the Development of Interneuronal Connectivity.

Appropriate growth and synaptic integration of GABAergic inhibitory interneurons are essential for functional neural circuits in the brain. Here, we demonstrate that disruption of primary cilia function following the selective loss of ciliary GTPase Arl13b in interneurons impairs interneuronal morphology and synaptic connectivity, leading to altered excitatory/inhibitory activity balance. The altered morphology and connectivity of cilia mutant interneurons and the functional deficits are rescued by either chemogenetic activation of ciliary G-protein-coupled receptor (GPCR) signaling or the selective induction of Sstr3, a ciliary GPCR, in Arl13b-deficient cilia.

Tissue-Specific Ablation of the LIF Receptor in the Murine Uterine Epithelium Results in Implantation Failure.

The cytokine leukemia inhibitory factor (LIF) is essential for rendering the uterus receptive for blastocyst implantation. In mice, LIF receptor expression (LIFR) is largely restricted to the uterine luminal epithelium (LE). LIF, secreted from the endometrial glands (GEs), binds to the LIFR, activating the Janus kinase-signal transducer and activation of transcription (STAT) 3 (Jak-Stat3) signaling pathway in the LE.

Gene expression analysis in the compartments of the murine uterus.

Embryo implantation, a key critical feature of mammalian pregnancy, involves co-ordinate interplay between an incoming blastocyst and a receptive uterus. Aberrations in signaling cascades during this process result in pregnancy loss in mammals, including women. Analysis of the complete uterus at any given point either during preparation for implantation or during and after embryo attachment and invasion makes it difficult to assign specific signaling mechanism to the individual cellular compartments of the uterus.

Disruption of SUMO-specific protease 2 induces mitochondria mediated neurodegeneration.

Post-translational modification of proteins by small ubiquitin-related modifier (SUMO) is reversible and highly evolutionarily conserved from yeasts to humans. Unlike ubiquitination with a well-established role in protein degradation, sumoylation may alter protein function, activity, stability and subcellular localization. Members of SUMO-specific protease (SENP) family, capable of SUMO removal, are involved in the reversed conjugation process.

Making BAC transgene constructs with lambda-red recombineering system for transgenic animals or cell lines.

The genomic DNA libraries based on Bacteria Artificial Chromosomes (BAC) are the foundation of whole genomic mapping, sequencing, and annotation for many species like mice and humans. With their large insert size, BACs harbor the gene-of-interest and nearby transcriptional regulatory elements necessary to direct the expression of the gene-of-interest in a temporal and cell-type specific manner. When replacing a gene-of-interest with a transgene in vivo, the transgene can be expressed with the same patterns and machinery as that of the endogenous gene.

Loss of Dishevelleds disrupts planar polarity in ependymal motile cilia and results in hydrocephalus.

Defects in ependymal (E) cells, which line the ventricle and generate cerebrospinal fluid flow through ciliary beating, can cause hydrocephalus. Dishevelled genes (Dvls) are essential for Wnt signaling, and Dvl2 has been shown to localize to the rootlet of motile cilia. Using the hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) mouse, we show that compound genetic ablation of Dvls causes hydrocephalus.

Six2 and Wnt regulate self-renewal and commitment of nephron progenitors through shared gene regulatory networks.

A balance between Six2-dependent self-renewal and canonical Wnt signaling-directed commitment regulates mammalian nephrogenesis. Intersectional studies using chromatin immunoprecipitation and transcriptional profiling identified direct target genes shared by each pathway within nephron progenitors. Wnt4 and Fgf8 are essential for progenitor commitment; cis-regulatory modules flanking each gene are cobound by Six2 and β-catenin and are dependent on conserved Lef/Tcf binding sites for activity.

Direct visualization of microtubules using a genetic tool to analyse radial progenitor-astrocyte continuum in brain.

Microtubule cytoskeletal dynamics of cortical progenitors and astroglial cells have critical roles in the emergence of normal functional organization of cerebral cortex and in disease processes such as tumorigenesis. However, tools to efficiently visualize these events are lacking. Here we describe a mouse genetic model to efficiently visualize and analyse radial progenitors, their astroglial progeny, and the microtubule cytoskeleton of these cells in the developing and adult brain.

Recombineering-based procedure for creating BAC transgene constructs for animals and cell lines.

The use of BAC/P1 as a vector for the generation of a transgene has gained popularity after the genomic annotation of many organisms was completed (often based on the respective BAC library). Large-scale generation of BAC transgenic mice has proven that BAC transgene approaches have less integration position effects and dosage artifacts when compared with traditional transgenic approaches. Also, a BAC can achieve the same tissue-specific expression as a knock-in of the same gene with less effort and shorter time of establishment.

The cytoskeletal adaptor protein band 4.1B is required for the maintenance of paranodal axoglial septate junctions in myelinated axons.

Precise targeting and maintenance of axonal domains in myelinated axons is essential for saltatory conduction. Caspr and Caspr2, which localize at paranodal and juxtaparanodal domains, contain binding sites for the cytoskeletal adaptor protein 4.1B.

Spatiotemporal ablation of myelinating glia-specific neurofascin (Nfasc NF155) in mice reveals gradual loss of paranodal axoglial junctions and concomitant disorganization of axonal domains.

The evolutionary demand for rapid nerve impulse conduction led to the process of myelination-dependent organization of axons into distinct molecular domains. These domains include the node of Ranvier flanked by highly specialized paranodal domains where myelin loops and axolemma orchestrate the axoglial septate junctions. These junctions are formed by interactions between a glial isoform of neurofascin (Nfasc(NF155)) and axonal Caspr and Cont.

Recombineering-based procedure for creating Cre/loxP conditional knockouts in the mouse.

Gene targeting in the mouse is an essential tool for studying gene function and creating models of human disease. The method described in this unit takes advantage of bacterial artificial chromosomes, Cre/loxP and FLPe/FRT systems, and recently evolved recombineering approaches to simplify the preparation of targeting constructs for generation of conditional knockout (CKO) animals. This method has been used to generate >30 CKO constructs, most of them successfully used to target mouse ES cells and establish mutant mice.

A BAC transgenic mouse model to analyze the function of astroglial SPARCL1 (SC1) in the central nervous system.

Extracellular matrix associated Sparc-like 1 (SC1/SPARCL1) can influence the function of astroglial cells in the developing and mature central nervous system (CNS). To examine SC1's significance in the CNS, we generated a BAC transgenic mouse model in which Sc1 is expressed in radial glia and their astrocyte derivatives using the astroglial-specific Blbp (Brain-lipid binding protein; [Feng et al., (1994) Neuron 12:895-908]) regulatory elements.

Mammary gland remodeling depends on gp130 signaling through Stat3 and MAPK.

The interleukin-6 (IL6) family of cytokines signals through the common receptor subunit gp130, and subsequently activates Stat3, MAPK, and PI3K. Stat3 controls cell death and tissue remodeling in the mouse mammary gland during involution, which is partially induced by IL6 and LIF. However, it is not clear whether Stat3 activation is mediated solely through the gp130 pathway or also through other receptors.

Cochlin, a secreted von Willebrand factor type a domain-containing factor, is regulated by leukemia inhibitory factor in the uterus at the time of embryo implantation.

Embryo implantation is a required step in the reproduction of all mammals. In mice, a transient rise in the uterine expression of leukemia inhibitory factor (LIF) occurs on d 4 of pregnancy and is essential for embryo implantation. However, which genes are regulated by LIF in the uterus at implantation has not been determined.

The lipid phosphatase LPP3 regulates extra-embryonic vasculogenesis and axis patterning.

Bioactive phospholipids, which include sphingosine-1-phosphate, lysophosphatidic acid, ceramide and their derivatives regulate a wide variety of cellular functions in culture such as proliferation, apoptosis and differentiation. The availability of these lipids and their products is regulated by the lipid phosphate phosphatases (LPPs). Here we show that mouse embryos deficient for LPP3 fail to form a chorio-allantoic placenta and yolk sac vasculature.

Loss of cyclooxygenase-2 retards decidual growth but does not inhibit embryo implantation or development to term.

Previous reports have described that female mice deficient in cyclooxygenase-2 (COX2) are largely infertile because of failure to ovulate, poor fertilization, and defective implantation and decidualization. In the present study, we reinvestigated reproduction in these mice and found they do show a reduction in the numbers of ovulated and fertilized eggs. However, we did not observe any substantial effect on embryo implantation frequencies or an inability of COX2-deficient females to support embryo development to weaning.