Linda Sutherland

An interspecies analysis reveals a key role for unmethylated CpG dinucleotides in vertebrate Polycomb complex recruitment.

The role of DNA sequence in determining chromatin state is incompletely understood. We have previously demonstrated that large chromosomal segments from human cells recapitulate their native chromatin state in mouse cells, but the relative contribution of local sequences versus their genomic context remains unknown. In this study, we compare orthologous chromosomal regions for which the human locus establishes prominent sites of Polycomb complex recruitment in pluripotent stem cells, whereas the corresponding mouse locus does not. Using recombination‐mediated cassette exchange at the mouse locus, we establish the primacy of local sequences in the encoding of chromatin state. We show that the signal for chromatin bivalency is redundantly encoded across a bivalent domain and that this reflects competition between Polycomb complex recruitment and transcriptional activation. Furthermore, our results suggest that a high density of unmethylated CpG dinucleotides is sufficient for vertebrate Polycomb recruitment. This model is supported by analysis of DNA methyltransferase‐deficient embryonic stem cells.

Efficient Gene Targeting by Homologous Recombination in Rat Embryonic Stem Cells

The rat is the preferred experimental animal in many biological studies. With the recent derivation of authentic rat embryonic stem (ES) cells it is now feasible to apply state-of-the art genetic engineering in this species using homologous recombination. To establish whether rat ES cells are amenable to in vivo recombination, we tested targeted disruption of the hypoxanthine phosphoribosyltransferase (hprt) locus in ES cells derived from both inbred and outbred strains of rats. Targeting vectors that replace exons 7 and 8 of the hprt gene with neomycinR/thymidine kinase selection cassettes were electroporated into male Fisher F344 and Sprague Dawley rat ES cells. Approximately 2% of the G418 resistant colonies also tolerated selection with 6-thioguanine, indicating inactivation of the hprt gene. PCR and Southern blot analysis confirmed correct site-specific targeting of the hprt locus in these clones. Embryoid body and monolayer differentiation of targeted cell lines established that they retained differentiation potential following targeting and selection. This report demonstrates that gene modification via homologous recombination in rat ES cells is efficient, and should facilitate implementation of targeted, genetic manipulation in the rat.

Tuning of β‐catenin activity is required to stabilize self‐renewal of rat embryonic stem cells

Stabilization of β‐catenin, through inhibition of glycogen synthase kinase 3 (GSK3) activity, in conjunction with inhibition of mitogen‐activated protein kinase kinase 1/2 (MEK) promotes self‐renewal of naïve‐type mouse embryonic stem cells (ESC). In developmentally more advanced, primed‐type, epiblast stem cells, however, β‐catenin activity induces differentiation. We investigated the response of rat ESCs to β‐catenin signaling and found that when maintained on feeder‐support cells in the presence of a MEK inhibitor alone (1i culture), the derivation efficiency, growth, karyotypic stability, transcriptional profile, and differentiation potential of rat ESC cultures was similar to that of cell lines established using both MEK and GSK3 inhibitors (2i culture). Equivalent mouse ESCs, by comparison, differentiated in identical 1i conditions, consistent with insufficient β‐catenin activity. This interspecies difference in reliance on GSK3 inhibition corresponded with higher overall levels of β‐catenin activity in rat ESCs. Indeed, rat ESCs displayed widespread expression of the mesendoderm‐associated β‐catenin targets, Brachyury and Cdx2 in 2i medium, and overt differentiation upon further increases in β‐catenin activity. In contrast, mouse ESCs were resistant to differentiation at similarly elevated doses of GSK3 inhibitor. Interestingly, without feeder support, moderate levels of GSK3 inhibition were necessary to support effective growth of rat ESC, confirming the conserved role for β‐catenin in ESC self‐renewal. This work identifies β‐catenin signaling as a molecular rheostat in rat ESC, regulating self‐renewal in a dose‐dependent manner, and highlights the potential importance of controlling flux in this signaling pathway to achieve effective stabilization of naïve pluripotency. Stem Cells 2013;31:2104–2115

Reprogramming Pig Fetal Fibroblasts Reveals a Functional LIF Signaling Pathway

Distinct signaling pathways are reported to maintain pluripotency in embryo-derived stem cells. Mouse embryonic stem cells (ESCs) respond to leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP)-mediated activity, whereas human ESCs depend upon Fibroblast growth factor (FGF) and activin signaling. In the majority of mammals investigated, however, the signals that support stem cell pluripotency are not well defined, as is evident by the persistent difficulties in maintaining authentic stable ESC lines. Induction of pluripotency by transcription factor-mediated reprogramming could provide an alternative way to produce ESC-like cells from nonpermissive species, and facilitate identification of core ESC signaling requirements. To evaluate the effectiveness of this approach in pigs, we transduced porcine foetal fibroblasts with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc, and maintained the resulting cultures in medium containing either LIF or FGF2. Alkaline phosphatase positive colonies with compact, mouse ESC-like morphology were preferentially recovered using serum-free medium supplemented with LIF. These cell lines expressed the endogenous stem cell transcription factors, OCT4, NANOG, and SOX2, and the cell surface marker SSEA-4, consistent with acquisition of an undifferentiated state. However, restricted differentiation potential, and persistent expression of retroviral transgenes indicated that reprogramming was incomplete. Interestingly, LIF activated both the transcription factor STAT3 and its target gene SOCS3, and stimulated cell growth, indicating functional coupling of the signaling pathway in these cells. This demonstration of LIF-dependence in reprogrammed pig cells supports the notion that the connection between LIF/STAT3 signaling and the core regulatory network of pluripotent stem cells is a conserved pathway in mammals.

Reduced levels of dopamine and altered metabolism in brains of HPRT knock-out rats: a new rodent model of Lesch-Nyhan Disease

Lesch-Nyhan disease (LND) is a severe neurological disorder caused by loss-of-function mutations in the gene encoding hypoxanthine phosphoribosyltransferase (HPRT), an enzyme required for efficient recycling of purine nucleotides. Although this biochemical defect reconfigures purine metabolism and leads to elevated levels of the breakdown product urea, it remains unclear exactly how loss of HPRT activity disrupts brain function. As the rat is the preferred rodent experimental model for studying neurobiology and diseases of the brain, we used genetically-modified embryonic stem cells to generate an HPRT knock-out rat. Male HPRT-deficient rats were viable, fertile and displayed normal caged behaviour. However, metabolomic analysis revealed changes in brain biochemistry consistent with disruption of purine recycling and nucleotide metabolism. Broader changes in brain biochemistry were also indicated by increased levels of the core metabolite citrate and reduced levels of lipids and fatty acids. Targeted MS/MS analysis identified reduced levels of dopamine in the brains of HPRT-deficient animals, consistent with deficits noted previously in human LND patients and HPRT knock-out mice. The HPRT-deficient rat therefore provides a new experimental platform for future investigation of how HPRT activity and disruption of purine metabolism affects neural function and behaviour.

Tuning differentiation signals for efficient propagation and in vitro validation of rat embryonic stem cell cultures.

The rat is one of the most commonly used laboratory animals in biomedical research and the recent isolation of genuine pluripotent rat embryonic stem (ES) cell lines has provided new opportunities for applying contemporary genetic engineering techniques to the rat and enhancing the use of this rodent in scientific research. Technical refinements that improve the stability of the rat ES cell cultures will undoubtedly further strengthen and broaden the use of these stem cells in biomedical research. Here, we describe a relatively simple and robust protocol that supports the propagation of germ line competent rat ES cells, and outline how tuning stem cell signaling using small molecule inhibitors can be used to both stabilize self-renewal of rat ES cell cultures and aid evaluation of their differentiation potential in vitro.

Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus

We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r−/− rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.

LIF-dependent survival of embryonic stem cells is regulated by a novel palmitoylated Gab1 signalling protein

ABSTRACT The cytokine leukaemia inhibitory factor (LIF) promotes self-renewal of mouse embryonic stem cells (ESCs) through activation of the transcription factor Stat3. However, the contribution of other ancillary pathways stimulated by LIF in ESCs, such as the MAPK and PI3K pathways, is less well understood. We show here that naive-type mouse ESCs express high levels of a novel effector of the MAPK and PI3K pathways. This effector is an isoform of the Gab1 (Grb2-associated binder protein 1) adaptor protein that lacks the N-terminal pleckstrin homology (PH) membrane-binding domain. Although not essential for rapid unrestricted growth of ESCs under optimal conditions, the novel Gab1 variant (Gab1β) is required for LIF-mediated cell survival under conditions of limited nutrient availability. This enhanced survival is absolutely dependent upon a latent palmitoylation site that targets Gab1β directly to ESC membranes. These results show that constitutive association of Gab1 with membranes through a novel mechanism promotes LIF-dependent survival of murine ESCs in nutrient-poor conditions. Highlighted Article: Embryonic stem cells rely upon expression of a novel lipid-modified Gab1 adaptor protein to promote survival under nutrient-poor conditions.

Gene targeting of the rat Csf1r locus using homologous recombination in ES cells

Program and Abstracts of the 10th Transgenic Technology Meeting (TT2011) TradeWinds Island Grand Resort, St Pete Beach, Florida, USA, October 24–26, 2011 The TT2011 Meeting is organized by the International Society for Transgenic Technologies (ISTT) (www.trans techsociety.org) TT2011 Organizing Committee LIuı́s Montoliu (National Center of Biotechnology—CNB, CSIC, Madrid, Spain), President Thom Saunders (University of Michigan, Ann Arbor, MI, USA), Vice-President Peter Sobieszczuk (University of Miami, Miami, FL, USA) Boris Jerchow (Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Germany) Carlisle Landel (Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA) Shirley Pease (Caltech, Pasadena, CA, USA) Johannes Wilbertz (KCTT, Karolinska Institute, Stockholm, Sweden) Elizabeth Williams (TASQ, University of Queensland, Brisbane, QLD, Australia) Tom Fielder (University of California-Irvine, CA, USA) Jan Parker-Thornburg (M. D. Anderson Cancer Center, Houston, TX, USA). TT2011 Registration and Administration (Technical

01-P020 MicroRNA regulation of ES cell differentiation

Hox genes are essential transcription factors that establish segmental identity on the head–tail axis of all animals. Proper regulation of Hox gene expression and function is in part accomplished through the actions of a multitude of noncoding RNAs. One important class of noncoding RNA, the micro-RNAs (miRNAs), regulates gene activity through translational repression. The miR-10 family is highly conserved in sequence and genomic position among the anterior Hox genes of all bilaterians. Nevertheless, no study has examined miR-10s role in modulating Hox gene function in protostomes. Here, we examined the expression and function of the miR-10 family mainly in the fruit fly Drosophila melanogaster and the flour beetle Tribolium castaneum. We observed changes in the miR-10 expression and in the precursor arm from which the mature miRNA sequence derives. Our results suggest the function of miR-10 during the establishment of the head–tail axis among insects has diverged.