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. We further quantified how maternal OCT4 levels were transmitted to, and distributed between, daughter cells. As mother cells underwent division, newly established OCT4 levels in daughter cells rapidly became more predictive of final OCT4 expression status. These results imply that the choice between developmental cell fates can be largely predetermined at the time of cell birth through inheritance of a pluripotency factor.

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. Our results thus define a specific requirement for primary cilia-mediated GPCR signaling in interneuronal connectivity and inhibitory circuit formation.

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. JAK-STAT activation converts the LE to a receptive state so that juxtaposed blastocysts begin to implant. To specifically delete the LIFR in the LE, we derived a line of mice in which Cre recombinase was inserted into the endogenous lactoferrin gene (Ltf-Cre). Lactoferrin expression in the LE is induced by E2, and we demonstrate that Cre recombinase activity is restricted to the LE and GE. To determine the requirement of the LIFR in implantation, we derived an additional mouse line carrying a conditional (floxed) Lifrflx/flx gene. Crossing Ltf-Cre mice with Lifrflx/flx mice generated Lifrflx/Δ:LtfCre/+ females that were overtly normal but infertile. Many of these females, despite repeated matings, did not become pregnant. Unimplanted blastocysts were recovered from the Lifrflx/Δ:LtfCre/+ uteri and, when transferred to wild-type recipients, implanted normally, indicating that uterine receptivity rather than the embryo's competency is compromised. The loss of Lifr results in both the failure for STAT3 to translocate to the LE nuclei and a reduction in the expression of the LIF regulated gene Msx1 that regulates uterine receptivity. These results reveal that uterine expression of the LIFR is essential for embryo implantation and further define the components of the LIF signaling pathway necessary for effective implantation.

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. Here, we describe methods for the specific isolation of the luminal epithelium (LE) and subsequent analysis of gene expression/signaling pathways during embryo attachment. We further describe the analysis of RNA and proteins by specific techniques of quantitative PCR (qPCR), immunostaining and Western blotting of uterine tissues. These methods can be applied to the other cellular compartments of the uterus and embryo invasion and endometrial development. These techniques will be beneficial to investigators for delineating the mechanisms involved during embryo attachment and female reproduction as well as providing a means to studying highly dynamic changes in gene expression in tissues.

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. This chapter describes in detail a method of using lambda-red recombineering to make BAC transgene constructs with the integration of a transgene into a designated location within a BAC. As the final BAC construct will be used for transfection in cell lines or making transgenic animals, specific considerations with BAC transgenes such as genotyping, BAC coverage and integrity as well as quality of BAC DNA will be addressed. Not only does this approach provide a practical and effective way to modify large DNA constructs, the same recombineering principles can apply to smaller high copy plasmids as well as to chromosome engineering.

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. Although SUMO-specific proteases are known to reverse sumoylation in many well-defined systems, their importance in mammalian development and pathogenesis remains largely elusive. In patients with neurodegenerative diseases, aberrant accumulation of SUMO-conjugated proteins has been widely described. Several aggregation-prone proteins modulated by SUMO have been implicated in neurodegeneration, but there is no evidence supporting a direct involvement of SUMO modification enzymes in human diseases. Here we show that mice with neural-specific disruption of SENP2 develop movement difficulties which ultimately results in paralysis. The disruption induces neurodegeneration where mitochondrial dynamics is dysregulated. SENP2 regulates Drp1 sumoylation and stability critical for mitochondrial morphogenesis in an isoform-specific manner. Although dispensable for development of neural cell types, this regulatory mechanism is necessary for their survival. Our findings provide a causal link of SUMO modification enzymes to apoptosis of neural cells, suggesting a new pathogenic mechanism for neurodegeneration. Exploring the protective effect of SENP2 on neuronal cell death may uncover important preventive and therapeutic strategies for neurodegenerative diseases.

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. In hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) mutants, E cells differentiated normally, but the intracellular and intercellular rotational alignments of ependymal motile cilia were disrupted. As a consequence, the fluid flow generated by the hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) E cells was significantly slower than that observed in control mice. Dvls were also required for the proper positioning of motile cilia on the apical surface. Tamoxifen-induced conditional removal of Dvls in adult mice also resulted in defects in intracellular rotational alignment and positioning of ependymal motile cilia. These results suggest that Dvls are continuously required for E cell planar polarity and may prevent 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 ß-catenin and are dependent on conserved Lef/Tcf binding sites for activity. In vitro and in vivo analyses suggest that Six2 and Lef/Tcf factors form a regulatory complex that promotes progenitor maintenance while entry of ß-catenin into this complex promotes nephrogenesis. Alternative transcriptional responses associated with Six2 and ß-catenin cobinding events occur through non-Lef/Tcf DNA binding mechanisms, highlighting the regulatory complexity downstream of Wnt signaling in the developing mammalian kidney.

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. Using this tool, we demonstrate altered microtubule organization and capture dynamics in adenomatous polyposis coli-deficient radial progenitors. Further, using multiphoton microscopy, we show the utility of this tool in real-time imaging of astrocytes in living mouse brain and the short-term stable nature of astrocytes in cerebral cortex. Thus, this model will help explore the dynamics of radial progenitor/astrocyte development or dysfunction and the influence of microtubule functions during these events.

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 λ-RED recombinogenic system has been used to manipulate DNA constructs with site-directed mutagenesis, truncation, and tagging with an epitope tag or as a fusion protein by homologous recombination, as well as used here to modify many BACs with various transgenes. The recombineering plasmid, pKD46, is used to fabricate BAC transgenic constructs that can be used in generating transgenic organisms as well as used in mammalian cell culture.

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. The exact role of 4.1B in the organization and maintenance of axonal domains is still not clear. Here, we report the generation and characterization of 4.1B-null mice. We show that loss of 4.1B in the PNS results in mislocalization of Caspr at paranodes and destabilization of paranodal axoglial septate junctions (AGSJs) as early as postnatal day 30. In the CNS, Caspr localization is progressively disrupted and ultrastructural analysis showed paranodal regions that were completely devoid of AGSJs, with axolemma separated from the myelin loops, and loops coming off the axolemma. Most importantly, our phenotypic analysis of previously generated 4.1B mutants, used in the study by Horresh et al. (2010), showed that Caspr localization was not affected in the PNS, even after 1 year; and 4.1R was neither expressed, nor enriched at the paranodes. Furthermore, ultrastructural analysis of these 4.1B mutants showed destabilization of CNS AGSJs at ∼ 1 year. We also discovered that the 4.1B locus is differentially expressed in the PNS and CNS, and generates multiple splice isoforms in the PNS, suggesting 4.1B may function differently in the PNS versus CNS. Together, our studies provide direct evidence that 4.1B plays a pivotal role in interactions between the paranodal AGSJs and axonal cytoskeleton, and that 4.1B is critically required for long-term maintenance of axonal domains in myelinated axons.

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. Here we report the generation of myelinating glia-specific Nfasc(NF155) null mouse mutants. These mice exhibit severe ataxia, motor paresis, and death before the third postnatal week. In the absence of glial Nfasc(NF155), paranodal axoglial junctions fail to form, axonal domains fail to segregate, and myelinated axons undergo degeneration. Electrophysiological measurements of peripheral nerves from Nfasc(NF155) mutants revealed dramatic reductions in nerve conduction velocities. By using inducible PLP-CreER recombinase to ablate Nfasc(NF155) in adult myelinating glia, we demonstrate that paranodal axoglial junctions disorganize gradually as the levels of Nfasc(NF155) protein at the paranodes begin to drop. This coincides with the loss of the paranodal region and concomitant disorganization of the axonal domains. Our results provide the first direct evidence that the maintenance of axonal domains requires the fence function of the paranodal axoglial junctions. Together, our studies establish a central role for paranodal axoglial junctions in both the organization and the maintenance of axonal domains in myelinated axons.

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. Design and preparation of the CKO construct, as well as step-wise troubleshooting guidelines, are described in detail.

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. Characterization of these Blbf-Sc1 transgenic mice show elevated Sc1 transcript and protein in an astroglial selective pattern throughout the CNS. This model provides a novel in vivo system for evaluating the role of SC1 in brain development and function, in general, and for understanding SC1's significance in the fate and function of astroglial cells, in particular.

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. This question was explored in mice carrying two distinct mutations in the gp130 gene; one that resulted in the complete ablation of gp130 and one that led to the loss of Stat3 binding sites (gp130Delta/Delta). Deletion of gp130 specifically from mammary epithelium resulted in a complete loss of Stat3 activity and resistance to tissue remodeling comparable to that seen in the absence of Stat3. A less profound delay of mammary tissue remodeling was observed in gp130Delta/Delta mice. Stat3 tyrosine and serine phosphorylation was still detected in these mice suggesting that Stat3 activation could be the result of gp130 interfacing with other receptors. Experiments in primary mammary epithelial cells and transfected COS-7 cells revealed a p44/42 MAPK and EGFR-dependent Stat3 activation. Moreover, the gp130-dependent EGFR activation was independent of EGF ligands, suggesting a cytoplasmic interaction and cross-talk between these two receptors. These experiments establish that two distinct Stat3 signaling pathways emanating from gp130 are utilized in mammary tissue.

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. We performed a subtractive hybridization assay between luminal epithelial (LE) mRNAs from d 3 and 4 of pregnancy to find genes up-regulated on d 4 and which would be potentially regulated by LIF. One candidate, Coch-5b2, was up-regulated on the day of implantation. Coch mRNA localized to the LE of wild-type mice and was not detected in uteri from Lif-deficient mice. Treatment of LE with LIF, both in vitro and in vivo, resulted in the up-regulation of Coch. Coch is also highly expressed in other tissues, including the spleen and inner ear, but only in the uterus is Coch expression regulated by LIF. Mice were derived in which Coch was either deleted or tagged with a LacZ reporter. In mice carrying the tagged Coch gene, expression of Coch was detected in the LE and also at the site of embryo implantation. However, mice in which the Coch gene was deleted were normal, showing no overt defects in their reproduction. Although loss of Coch expression is not essential to reproduction in mice, it may serve as a useful marker for assessing the state of uterine receptivity in response to LIF at the onset of implantation.

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. Pseudopregnant COX2-null recipients do not show any alteration in the timing of implantation following blastocyst transfer, but they do show a delay in the initial rate of decidual growth after implantation that lags by approximately 24 h compared to that in heterozygous or wild-type recipients. These results support previous findings that COX2 has a role in mediating the initial uterine decidual response but is not essential to sustaining decidual growth and embryo development throughout the remainder of pregnancy.