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RESEARCH ARTICLE| 15 September 2002
Involvement of the Polycomb-group gene Ring1B in the specification of the anterior-posterior axis in mice
Maki Suzuki,
Maki Suzuki
1Department of Molecular Embryology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
2Department of Oral Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan
*These authors contributed equally to this work
Yoko Mizutani-Koseki,
Yoko Mizutani-Koseki
Yu-ichi Fujimura,
Yu-ichi Fujimura
Hiro Miyagishima,
Hiro Miyagishima
Tomomi Kaneko,
Tomomi Kaneko
Yuki Takada,
Yuki Takada
Takeshi Akasaka,
Takeshi Akasaka
Hideki Tanzawa,
Hideki Tanzawa
Yoshihiro Takihara,
Yoshihiro Takihara
3Department of Develomental Biology and Medicine, Osaka Medical Center for Cancer and Cardiovascular Diseases, Nakamiti 1-3-3, Tousei-ku, Osaka, 537-8511, Japan
Megumi Nakano,
Megumi Nakano
4Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan
Hiroshi Masumoto,
Hiroshi Masumoto
Miguel Vidal,
Miguel Vidal
5Centro de Investigaciones Biologicas, Department of Developmental and Cell Biology, Velazquez 144, 28006 Madrid, Spain
Kyo-ichi Isono,
Kyo-ichi Isono
Haruhiko Koseki
6RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro, Tsurumi-ku, Yokohama 230-0045, Japan
†Author for correspondence (e-mail: koseki@med.m.chiba-u.ac.jp)
Accepted: 11 Jun 2002
https://doi.org/10.1242/dev.129.18.4171
Maki Suzuki, Yoko Mizutani-Koseki, Yu-ichi Fujimura, Hiro Miyagishima, Tomomi Kaneko, Yuki Takada, Takeshi Akasaka, Hideki Tanzawa, Yoshihiro Takihara, Megumi Nakano, Hiroshi Masumoto, Miguel Vidal, Kyo-ichi Isono, Haruhiko Koseki; Involvement of the Polycomb-group gene Ring1B in the specification of the anterior-posterior axis in mice. Development 15 September 2002; 129 (18): 4171–4183. doi: https://doi.org/10.1242/dev.129.18.4171
The products of the Polycomb group of genes form complexes that maintain the state of transcriptional repression of several genes with relevance to development and in cell proliferation. We have identified Ring1B, the product of the Ring1B gene (Rnf2 – Mouse Genome Informatics), by means of its interaction with the Polycomb group protein Mel18. We describe biochemical and genetic studies directed to understand the biological role of Ring1B. Immunoprecipitation studies indicate that Ring1B form part of protein complexes containing the products of other Polycomb group genes, such as Rae28/Mph1 and M33, and that this complexes associate to chromosomal DNA. We have generated a mouse line bearing a hypomorphic Ring1B allele, which shows posterior homeotic transformations of the axial skeleton and a mild derepression of some Hox genes (Hoxb4, Hoxb6 and Hoxb8) in cells anterior to their normal boundaries of expression in the mesodermal compartment. By contrast, the overexpression of Ring1B in chick embryos results in the repression of Hoxb9 expression in the neural tube. These results, together with the genetic interactions observed in compound Ring1B/Mel18 mutant mice, are consistent with a role for Ring1B in the regulation of Hox gene expression by Polycomb group complexes.
Polycomb, Ring1B, Mel18, Hox, Mouse
The Polycomb group (PcG) of genes was first identified by their requirement for the maintenance of the stable repression of Hox genes during the development of Drosophila melanogaster (Paro, 1995; Pirotta, 1997). Biochemical and immunohistochemical analyses indicate that Drosophila PcG gene products function as large multimeric protein complexes that are thought to act by changing the local chromatin structure (Paro, 1995; Pirotta, 1997). Synergistic genetic interactions between mutant alleles of different Drosophila PcG genes indicate their ability to affect the expression of Hox genes in a gene dose-dependent manner and are in line with the action of PcG gene products in multimeric protein complexes (Jurgens, 1985; Franke et al., 1992; Shao et al., 1999).
Mammalian genes that are structurally and functionally related to Drosophila PcG genes have been identified. The PcG gene products form parts of two different complexes. One complex contains the product of Eed (the ortholog of the Drosophila extra sex combs gene), and the products of Enx1 (Ezh2 – Mouse Genome Informatics) and Enx2 (the orthologs of the Drosophila enhancer of zeste gene). Eed interacts with histone deacetylases (Schumacher et al., 1996; Laible et al., 1997; van Lohuizen et al., 1998; Sewalt et al., 1998; van der Vlag and Otte, 1999). The second complex include the products of the paralogs of a subset of PcG genes. These genes exist as gene groups like Mel18 (Zfp144 – Mouse Genome Informatics) and Bmi1; M33 (Cbx2 – Mouse Genome Informatics), Mpc2 (Cbx4 – Mouse Genome Informatics) and Pc3 (Cbx8 – Mouse Genome Informatics); Rae28 (Edr1 – Mouse Genome Informatics)/Mph1 and Mph2 (Edr2 – Mouse Genome Informatics); and Ring1A (Ring1 – Mouse Genome Informatics) and Ring1B (Rnf2 – Mouse Genome Informatics) (Tagawa et al., 1990; van Lohuizen et al., 1991; Pearce et al., 1992; Satijn et al., 1997a; Hemenway et al., 2000; Nomura et al., 1994; Gunster et al., 1997; Schoorlemmer et al., 1997; Satijn et al., 1997b; Hemenway et al., 1998). This complex is similar to the Polycomb repressive complex 1 (PRC1) recently identified in Drosophila (Shao et al., 1999; Gebuhr et al., 2000). Indeed, mice bearing mutations in Mel18, Bmi1, M33, Mph1 or Ring1A show homeotic transformations of the axial skeleton and alterations in the expression patterns of Hox genes (Akasaka et al., 1996; van der Lugt et al., 1994; Core et al., 1997; Takihara et al., 1997; del Mar Lorente et al., 2000). Interestingly, Ring1A mutants exhibit anterior transformations, while the others show posterior transformations (del Mar Lorente et al., 2000). In addition, mice with compound mutations in these genes show synergistic enhancement of the phenotypes associated with the individual genes evidencing genetic interactions between these mutations (Bel et al., 1998; Akasaka et al., 2001). This complex is required for the maintenance of the stable repression of Hox genes, as indicated by progressive derepressions of Hoxb3 expression in migrating facial neural crest cells in Mel18/Bmi1 compound and Rae28/Mph1 mutant embryos (Tomotsune et al., 2000; Akasaka et al., 2001).
To gain further insight in mammalian PcG function, we searched for Mel18 interactors and found Ring1B. The Ring1B protein is closely related to the product of PcG gene Ring1A, and was first identified by its interaction with the product of M33 (Schoorlemmer et al., 1997). Ring1B was also found to interact with Bmi1 and Mph2 in yeasts (Hemenway et al., 1998). Here we provide evidence for the presence of Ring1B in a complex containing M33, Mel18 and Rae28/Mph1 in extracts prepared from 11.5 days post coitus (dpc) mouse embryo, which were found associated to chromosomal DNA. These observations strongly suggest that Ring1B is a component of the mammalian counterpart of Drosophila PRC1 complex, as Drosophila Ring protein is included in PRC1 (Saurin et al., 2001). To test this possibility functionally, we attempted to generate a loss-of-function mouse model for Ring1B. Here, we describe a mouse line bearing a hypomorphic mutation for Ring1B and show that this mutation is accompanied by limited homeotic transformations of the axial skeleton and alterations in Hox gene expression. In addition, we show a genetic interaction between the Ring1B and Mel18 mutations. We also performed a gain-of-function experiment by in ovo electroporation. Overexpression of Ring1B protein in the developing neural tube results in the repression of chick Hoxb9 expression. These results demonstrate that Ring1B plays a role in the anteroposterior (AP) specification of the paraxial mesoderm and neural tube in vertebrate embryos.
Yeast two hybrid assay
A Mel18 full-length cDNA was fused in-frame to the lexA-coding sequence in vector pBTM116 (Fields and Bartel, 2001). The L40 yeast strain containing lexA-HIS3 and lexA-lacZ reporter genes was first transformed with pBTM116-Mel18, and then with the mouse 11.0 dpc embryo Matchmaker cDNA library in pGAD10 (Gal4 activation domain fusion vector, Clontech), using the lithium acetate method. Six hundred independent colonies were isolated from 7×106 transformants on selective media plate lacking leucine, tryptophan and histidine and supplemented with 5 mM 3-aminotriazole. All these clones were restreaked onto the selection media and assayed for β-galactosidase activity by a filter assay (Vojtek et al., 1993). cDNA fragments were amplified by direct colony PCR from the His+lacZ+ positive clones, identified by sequencing and searched for using NCBI similarity search programs (BLASTn). Twenty clones, which were possible interactors of Mel18, were cured of the LexA fusion plasmid by growth on synthetic medium lacking leucine. Plasmid loss was confirmed by plating on both Leu-, Trp+ and Leu-, trp- plates. The selected clones grew only on trp+ media. The plasmid DNA was then prepared from those positive clones of interest and transformed into E. coli DH5α.
To test for interaction specificity, each pGAD10 variant containing Mel18, Bmi1, Ring1A, Ring1B, Mph2 or lamin was transformed into L40 (MATa), and each pBTM116 variant containing Mel18, Bmi1, Ring1A, Ring1B or Mph2 was transformed into AMR70 (MATα). Then the transformed clones were mated to each other in all possible combinations on YPAD plates. The Leu+Trp+ diploids were selected on Leu-Trp- plate and assayed for β-galactosidase activity using a filter assay.
In vitro transcription/translation and GST pull-down assay
The full-length and deleted Mel18 and Ring1B cDNAs were subcloned in the pcDNA3 vector (Invitrogen Japan, Tokyo, Japan). RNA was synthesized with 500 ng of supercoiled plasmids and translated in the presence of 10 μCi of [35S] methionine (Amersham Biosciences, Tokyo, Japan) using the TNT T7 Quick Coupled Reticulocyte Lysate System (Promega, Tokyo, Japan). Ten micrograms of plasmids containing GST fused to either intact or truncated Mel18 or Ring1B cDNAs or GST alone (as a negative control) were pre-bound to 10 μl of a 1:1 suspension of Glutathion Sepharose 4 Fast Flow (Amersham Biosciences, Tokyo, Japan) and then incubated on a rotating wheel with the in vitro translation mixture in 500 μl of NETN buffer (0.5% NP-40, 20 mM Tris-Cl pH 8.0, 11 mM NaCl, 1 mM EDTA and 100 mM PMSF) for 1 hour at 4°C Bound proteins were eluted by boiling in SDS sample buffer and were analyzed on 11% polyacrylamide gels. Gel images were obtained by BAS-2000II Bio-Imaging Analyzer (FUJIFILM, Tokyo, Japan).
The following antibodies were used in this study:
mouse anti-mouse Ring1B monoclonal antibody (clone 3) (Atsuta et al., 2001);
rabbit anti-mouse Ring1B antiserum (Garcia et al., 1999);
rabbit anti-mouse Ring1A antiserum (Schoorlemmer et al., 1997);
mouse anti-mouse Rae28/Mph1 monoclonal antibody (K. I., H. M. and H. K., unpublished);
rabbit anti-mouse Rae28/Mph1 antiserum (Y. T., unpublished);
goat anti-mouse Mel18 purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA);
mouse anti-mouse Bmi1 monoclonal antibody (Alkema et al., 1997);
rabbit anti-mouse M33 antiserum (Schoorlemmer et al., 1997);
rabbit anti-demethyl-HistoneH3 antiserum (Upstate Biotechnology, Lake Placid, NY) (Nakayama et al., 2001);
mouse anti-Bip monoclonal antibody (StressGen Biotechnologies, Victoria, B.C., Canada);
goat anti-Lamin B antibody (Santa Cruz Biotechnology, Santa Cruz, CA);
anti-HA epitope monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA);
rabbit anti-CENP-B antibody (BN1) (Kitagawa et al., 1995); and
goat anti-centromeric antigen (ACA) antiserum (Yoda et al., 1996).
Immunoprecipitations and western blot analysis
Total cellular extracts were prepared from 11.5 dpc mouse embryos. A single embryo was solublized by sonication in 1 ml of TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) using a Handy Sonic model UR-20P (TOMY seiko, Tokyo, Japan) followed by centrifugation at 9100 g for 5 minutes at 4°C to remove insoluble material. For immunoprecipitation, lysates were incubated with 1/2 volume of hybridoma culture supernatants containing either anti-Ring1B or anti-Rae28/Mph1 monoclonal antibodies, or with 1/30 volume of anti-Mel18 antiserum for 1 hour at 4°C with rocking. Immunocomplexes were captured with Protein A Sepharose beads (Amersham). Western blotting was performed as described previously (Atsuta et al., 2001).
Immunofluorescence staining
U2-OS human osteosarcoma cells were used for immunofluorescence staining analyses as described previously (Atsuta et al., 2001). For the analysis of mitotic cells, cells were fixed with 70% acetone at –20°C for 30 minutes and air dried. Fixed cells were incubated with blocking solution for 1 hour at room temperature, and incubated with the mixture of rabbit anti-CENP-B antibody or ACA serum (1:200 dilution) and anti-Ring1B antibody for 2 hours at 37°C. Cells were washed with phosphate-buffered saline containing 0.1% Tween 20 (PBST) for 5 minutes at room temperature four times, and then incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:160 dilution) (MBL, Nagoya, Japan) or fluorescein isothiocyanate-conjugated anti-human IgG (1:160 dilution) (MBL, Nagoya, Japan) and rhodamine-conjugated anti-mouse IgG (1:200 dilution) (MBL, Nagoya, Japan) diluted with PBST supplemented with 10% fetal bovine serum at 37°C for 1 hour. Cells on coverslips were then washed four times with PBST for 5 minutes at room temperature. Chromosomal DNA was stained with DAPI (1 μg/ml). Images were obtained with a fluorescence microscope (Carl Zeiss Japan, Tokyo, Japan) equipped with a cooled-CCD camera (PXL, Photometrics, CA) and IPLab software (Signal Analytics).
Chromatin purification by cesium chloride isopyknic centrifugation
Mouse embryos at 12.5 dpc were minced with scissors and subjected to chemical crosslinking with 1% formaldehyde in phosphate-buffered saline (PBS) for 30 minutes. Isolation of chromatin fraction was performed as described elsewhere (Orlando et al., 1997).
Generation of a hypomorphic allele of the Ring1B gene
A modified Ring1B allele was generated by gene targeting in R1 embryonic stem (ES) cells. Genomic Ring1B sequences were isolated from a mouse 129/SVJ genomic library. The targeting vector contains as homology arms, an EcoRI-SacI fragment containing two exons encoding the RING finger of Ring1B and a flanking EcoRI-HindIII fragment. MC1Neo and HSVtk genes were used as selectable cassettes for positive and negative selections, respectively (Fig. 6A). Thirty micrograms of the linearized plasmid were electroporated into 2×107 R1 ES cells. Two hundred and forty double resistant R1 clones were analyzed by Southern blot. One of the clones showed an alteration in the Ring1B locus and was used to generate germline chimeras as described (Nagy et al., 1993).
Single and double mutant mice
We generated homozygous Ring1B mutant mice by interbreeding heterozygous animals that had been backcrossed eight times into C57BL/6J. Mel18-deficient mice (Akasaka et al., 1996) from both C57BL/6J genetic background were also used in this study. Ring1B and Mel18 heterozygous mice in C57BL/6J genetic background were interbred to generate compound homozygous mutant mice. Skeletal phenotypes of Ring1B mutants were also investigated on the compound genetic background between 129/SvJ derived from R1 ES cells and C57BL/6J. Ring1B and Mel18 mutations were backcrossed onto C57BL/6J twice (N2). N2 heterozygotes were intercrossed to generate homozygous mutants. This hybrid line was maintained by brother-sister mating for at least ten generations.
Ring1B mutant mice were genotyped by PCR using the following oligonucleotides (Fig. 5A): RB22, 5′-GTTGAAGACTTCATTGTACC-3′ and RB18, 5′-GGAAGTCACATCTTAGCAGG-3′ for the Ring1B wild-type allele; and Neo2, 5′-CTTCCATCCGAGTACGTGCT-3′ and RB11, 5′-TTATCTTCTGCTCCACTACC-3′ for the mutant allele. Oligonucleotides used for genotyping Mel18 mutant mice have been described previously (Akasaka et al., 2001).
Phenotypic analysis
Skeletal analysis of 18.5 dpc or new born mice and in situ hybridization to 11.5 dpc embryo sections were performed as described previously (Kessel and Gruss, 1991). Hoxb4 from Dr R. Krumlauf, Hoxb6 from Dr K. Schughart and Hoxb8 from Dr J. Deschampus were used (Akasaka et al., 1996). Whole-mount in situ hybridization was performed as described previously with slight modifications (Wilkinson and Nieto, 1993). Briefly, maleic acid buffer (0.5 M maleic acid, 0.15 M NaCl, pH 7.5) was used for washes after antibody reaction. Chicken Hoxb9 provided by Dr A. Kuroiwa, and mouse Mel18 and Ring1B probes were used.
In ovo electroporation
cDNA fragments encoding the entire ORF of mouse Mel18 and Ring1B were amplified by PCR using primers that provided XhoI sites on both ends of the fragments for subcloning into the XhoI site of pCXN2 (kindly provided by Dr Jun-ichi Miyazaki, Osaka University, Japan) (Niwa et al., 1991). Empty pCXN2 vector was used as a negative control. In ovo electroporation was performed as described previously (Sakamoto et al., 1998; Nakamura and Funahashi, 2001). Expression vectors were dissolved at 2 μg/μl in PBS and mixed with 1/10 volume of 2 μg/μl β-galactosidase expression vector, pCHO110 (Amersham Biosciences, Tokyo, Japan), or green fluorescence protein (GFP) expression vector, pcDNA-GFP, and 1/20 volume of saturated Nile Blue solution. Fertilized chick eggs were purchased from Shiroyama chicken farm (Kanagawa, Japan) and cultured at 37.2°C before and after electroporation. The stages of chicken embryos were determined by counting somite number. After electroporation, embryos were re-incubated for varying length of time to allow expression of the introduced foreign DNA into one side of the neural tube and subsequently dissected and fixed in 4% paraformaldehyde in PBS.
In vitro interaction of Ring1B and Mel18
Among 63 Mel18-binding proteins identified in the screening of cDNAs from an 11.0 dpc mouse embryo library, 22, 8 and two cDNA clones encoded parts of Ring1B, Mph2 and Rae28/Mph1 proteins, respectively (T. A., unpublished). In order to asses the specificity of the interactions, we performed yeast two hybrid mating assays (Fig. 1A). Bmi1, which is known to interact with Ring1B and Mph2, was used as positive control and Lamin as a negative control (Hemenway et al., 1998). Because Ring1A, a structurally related protein to Ring1B, was never identified in our screening of Mel18 interactors, we also examined the interaction of Mel18 and Ring1A. As visualized by β-galactosidase activity, Mel18 interacted strongly with Ring1B and Mph2 but hardly with itself, Bmi1 or Ring1A in our experimental conditions.
To determine whether the Mel18 protein can directly interact with Ring1B, we used an in vitro GST pull-down assay (Fig. 1B). Radiolabeled full-length or truncated Mel18 and Ring1B proteins were synthesized in vitro and tested for binding activity to respective GST fusion proteins with full-length Ring1B (GST-Ring1B), a C-terminal-truncated Ring1B (GST-Ring1BΔC) (amino acids 1-188), N-terminal-truncated Ring1B (GST-Ring1BΔN) (amino acids 189-365) and Mel18 lacking PEST region (GST-Mel18ΔPEST) (amino acids 1-233). Radiolabeled full-length Mel18 (Mel18fl) was able to bind to GST-Ring1B and GST-Ring1BΔC, and weakly to GST-Ring1BΔN but exhibited no appreciable binding to GST-Sepharose alone (Fig. 1B, part a). Because we could not obtain a full-length Mel18 protein fused to GST, we instead prepared a GST-Mel18ΔPEST protein. Full-length Ring1B (Ring1Bfl) bound to GST-Mel18ΔPEST but not to GST-Sepharose (Fig. 1B, part b). These observations indicated potential direct interaction of Mel18 and Ring1B. Ring1BΔC fragment was capable to bind to GST-Mel18ΔPEST more efficiently than Ring1BΔN fragment (Fig. 1B, part).
In vivo interaction of Ring1B with PcG proteins
To obtain evidence for the in vivo association of Ring1B with mammalian PcG proteins in mammalian cells, we performed immunoprecipitation analysis using extracts from 11.5 dpc mouse embryos. We used anti-Mel18, anti-Ring1B or anti-Mph1 antibodies and preimmune rabbit serum as a negative control. A band with a mobility corresponding to 40 kDa was recognized with anti-Ring1B antibodies in the material immunoprecipitated by anti-Mel18, anti-Ring1B and anti-Rae28/Mph1 antibodies (Fig. 2A). This shows that protein complexes isolated from murine embryos containing Mel18 or Rae28/Mph1 also contain Ring1B.
Additional evidence for the in vivo association of Ring1B with Mel18 and other PcG proteins was obtained from their subcellular localization in U2-OS cells by indirect immunofluorescence (Fig. 2B-F). Endogenous Ring1B colocalized extensively with Mel18, Bmi1, Ring1A and Rae28/Mph1 in subnuclear speckles, previously termed PcG bodies, in interphase nuclei of U2-OS cells (Fig. 2B-E) (Saurin et al., 1998). Because a specific antibody against Mph2 was not available, we used U2-OS cells stably expressing Myc-tagged Mph2. In these cells endogenous Ring1B colocalized with Myc-tagged Mph2 (Fig. 2F). Taken together, these observations indicate that the PcG complexes in PcG bodies of interphase cells contain Ring1B.
Binding of Ring1B-containing PcG complexes to chromosomal DNA
Evidence for the association of PcG complexes to chromosomal DNA is available only for D. melanogaster (Zink and Paro, 1989; Orlando and Paro, 1993). To see whether mouse PcG complexes are also associated with chromosomal DNA, we analyzed formaldehyde crosslinked embryonic tissues after fractionation by cesium chloride isopyknic centrifugation for the presence of PcG proteins. Coomassie Brilliant Blue G-250 (CBB) staining showed most of the protein in fractions 1 to 3 but also in fractions 4 to 8 (Fig. 3B), whereas most chromosomal DNA was in fractions 5 to 7 (Fig. 3A). As a control, we studied the distribution of Bip, a luminal protein in the endoplasmic reticulum (ER) and nuclear envelope, which is thought not to interact with chromosomal DNA (Gething, 1999). The results showed that Bip was found in fractions 1 to 4, but it was absent from fractions 5 to 7 (Fig. 3C). A similar observation was obtained for Grp78, another ER luminal protein (Y. F., unpublished) (Gething, 1999). From the reciprocal distribution of chromosomal DNA and ER luminal proteins, we conclude that fractions 5 to 7 contain most of the chromatin-associated proteins. Distribution of dimethyl-Histone H3 to fractions 5 to 7 but not to fractions 1 and 2 revealed its similar distribution to chromosomal DNA (Fig. 3D) (Nakayama et al., 2001). This suggests that proteins that are not associated with chromatin are mainly distributed into fractions 1 and 2. Western blot analysis of these fractions showed that Ring1B and M33 were present in fractions 1 to 6 and Rae28/Mph1 in fractions 1 to 7 (Fig. 3D-F). Therefore, specific amounts of Ring1B, M33 and Rae28/Mph1 are associated with chromosomal DNA. Indeed, we have found that a significant amount of chromosomal DNA from fraction 6 could be co-immunoprecipitated by antibodies against Ring1B and Rae28/Mph1 (Y. F., unpublished). Our results also indicate that significant amount of Ring1B, M33 and Rae28/Mph1 are not closely associated with chromatin.
Association of Ring1B to the chromosomal DNA is cell cycle-dependent
It is known that in tissue culture cells RING1 and BMI1-containing PcG bodies associate with pericentromeric heterochromatin in a cell cycle-dependent manner (Saurin et al., 1998). Therefore, we asked what the localization of Ring1B would be during the various phases of the cell cycle. In the interphase nuclei of U2-OS cells, Ring1B showed a speckled distribution associated to centromeric regions, just as it was found for Ring1, Bmi1 and M33 (Fig. 4A,B) (Saurin et al., 1998; Voncken et al., 1999; Wang et al., 1997). At mitosis, the chromosomal signal of Ring1B was very much reduced. However, upon chromosome condensation at prophase, Ring1B proteins were still associated with condensed chromosomes (Fig. 4C). At prometaphase, following disassembly of the nuclear envelop and nuclear matrix, Ring1B staining was significantly weaker to that in interphase nuclei and dispersed into the cytoplasm, although a weak signal was still seen associated with part of pericentromeric chromatin regions (broken line in Fig. 4D). At anaphase, a clearer Ring1B signal was found associated to chromosomal structures, but most of the signal was still found evenly distributed in a speckle-free cytoplasm (Fig. 4E). Thus, the localization of Ring1B at pericentromeric heterochromatin is cell cycle regulated in a similar manner to that of Ring1 or Bmi1 (Saurin et al., 1998; Voncken et al., 1999).
Generation of a hypomorphic Ring1B allele
To start analyzing functionally Ring1B, we tried to inactivate the Ring1B locus in mice. In a gene targeting experiment, we obtained one clone with a modified Ring1B locus. Southern blot and sequence analysis of this locus showed that the selectable Neo cassette, together with some 0.8 kb sequences in one of the homology arms present in the targeting construct, were unexpectedly inserted between the exons labeled 3 and 4 (Fig. 5A-C). Because no other alterations were observed, there was the possibility that this modified Ring1B locus would synthesize an intact protein. We analyzed Ring1B gene expression in mice homozygous for the mutation and found that a cDNA encoding the full Ring1B ORF could be identified by RT-PCR (data not shown). However, the levels of Ring1B protein in these mice were significantly reduced compared with those in wild-type animals (Fig. 5D). Quantification of Ring1B signals in a Western blot of both homozygous and heterozygous mutant embryos, using the signals of LaminB to normalize for loading differences, showed that the levels of Ring1B in homozygous Ring1B mutant embryos were about 30% of those in heterozygous embryos. The reduced levels of Ring1B in the mutant mice, therefore, make it likely that this Ring1B allele is a hypomorphic one and is therefore designated as Ring1Breduced (Ring1Bred hereafter). Ring1Bred homozygotes were healthy and fertile.
Axial skeletal abnormalities and alterations in Hox gene expression in Ring1Bred/red mutants
Perturbation of PcG function in mice characteristically results in homeotic transformations and other alterations of the axial skeleton (Akasaka et al., 1996; van der Lugt et al., 1994; Core et al., 1997; Takihara et al., 1997; del Mar Lorente et al., 2000). Therefore, if Ring1B participates in such a function, it is possible that similar defects could be seen in Ring1Bred mice. We found reproducible alterations in the cervicothoracic boundary of the axial skeleton of homozygous Ring1Bred mice, but not wild type. Thus, in 61% of the mice, we found ectopic ribs associated with the 7th cervical (C7) vertebra (Fig. 6A, parts b and f, Fig. 6B). At a lower penetrance, 40% of mice, we found that the prominent spinous process characteristic of the 2nd thoracic vertebrae (T2) was on the T1. These alterations were seen even in heterozygous Ring1Bred mice, although at low frequency (Fig. 6B). Formation of an additional ossification center in the cranial region of the sternum indicated anterior shift of the sternum in Ring1Bred homozygotes (Fig. 6A, part f). Holes in the ossification centers in the upper region of the xyphoid process were observed (Fig. 6A, part f). This is also seen in Hoxc4 mutants (Saegusa et al., 1996). Thus, these alterations in the axial skeleton and sternum seen in Ring1Bred mice can be interpreted as posteriorizations of the vertebral identities: C7→T1 and T1→T2.
As the anterior boundaries of Hoxb4, Hoxb6 and Hoxb8 expression in the paraxial mesoderm demarcate the prospective vertebrae C2, C7 and T1, respectively, we asked whether their expression patterns would be altered in Ring1Bred mice. The analysis of 11.5 dpc embryos homozygous for the Ring1Bred allele showed weak but reproducible signals for Hoxb4 and Hoxb8 in the C1 and C7 prevertebrae, respectively, whereas no signals were detected in the wild-type littermates (Fig. 7). Ectopic expression of Hoxb6 was weakly seen in the prospective neural arch of C6 prevertebra (Fig. 7). The expression of Hoxb4, Hoxb6 and Hoxb8 in the neural tube was not significantly altered.
Thus, the homeotic transformations of the axial skeleton and the anteriorization of the rostral boundaries of Hox gene expression in the paraxial mesoderm seen in this hypomorphic Ring1B mouse line suggest that Ring1B plays a role in PcG function in mice.
Genetic interactions between Ring1Bred and Mel18 null-mutations
To evaluate the involvement of Ring1B in PcG function further, we investigated a possible genetic interaction with Mel18 by analyzing the axial skeleton of the offspring of crosses of Ring1Bred/+Mel18+/– mice. Mel18–/– mice with C57BL/6J genetic background now show a more homogeneous phenotype than when in a hybrid genetic background that we described previously and do not survive birth (Akasaka et al., 1996). The formation of ectopic arches in the occipital bone caused by the ectopic segmentation of the occipital bone, which was not observed in the hybrid genetic background, was now seen in every mouse (Fig. 6A, part c). The C1 vertebra was thinner than that of mice of a mixed genetic background and anterior shifts around the thoracolumbar, lumbosacral and sacrocaudal transition regions were uniformly seen in all Mel18–/– mice. Interestingly, ectopic ribs on the seventh cervical vertebrae were less prominent and penetrant (Fig. 6B).
Animals double homozygous for mutations in the Ring1Bred and Mel18 loci do not survive birth and showed homogeneous phenotypes, very similar to that of Mel18-null mice, except for the cervicothoracic region, in which almost perfect ribs were bilaterally articulated with an anteriorly shifted sternum in 50% of the mice. A unilateral association was seen in 8.6% of Ring1Bred/red and was never seen in Mel18 null mice (Fig. 6A, part d, part h, Fig. 6B). In summary, the phenotypes at the cervicothoracic level are more severe than the phenotypes of both single mutants in the C57BL/6J background, which provides genetic evidence for an interaction between mutations at the genes encoding Ring1B and Mel18.
By contrast, the posterior transformations seen in the axial skeleton of Mel18–/– mice were variably restored in Ring1Bred/redMel18–/– mice on the compound background between 129/SvJ derived from R1 ES cells and C57BL/6J, while enhanced phenotypes were also seen with less penetrance (Fig. 8). Double homozygotes were healthy and fertile on the compound background. We found three groups of mice homozygous for both Ring1Bred and Mel18, depending on the alterations in their axial skeletons. One group, comprising about half of the mice (7/15) of the mice showed phenotypes almost identical to those of Mel18–/– mice, except for the unilateral association of the seventh rib to the sternum (Fig. 8A, parts f, l and r). A second group, made with the other half of the mice (8/15), showed partial restoration of the posterior homeotic transformations typically seen in Mel18–/– to the wild-type phenotype. A representative phenotype of three mice in the latter group is the one shown in Fig. 8A (parts d, j and p). They showed normal occipital bones and C1 vertebra, with seven sternovertebral ribs and normally positioned lumbosacral and sacrocaudal boundaries, and anterior shift the thoracolumabar boundary (as represented by lack of 13th rib) (Fig. 8A, parts d, j and p). Thus, this group exhibited a wild-type axial skeleton, except for the alterations in the thoracolumbar transitional zone. Five mice in the latter group, represented by the specimen shown in Fig. 8A (parts e, k and q) exhibited posterior transformations of the occipital, cervical and upper thoracic regions of the axial skeleton characteristics of Mel18–/– mice. However, the lower thoracic, lumbar, sacral and caudal regions had a wild-type appearance. Therefore, the hybrid background does not allow the Ring1B mutation to lead to a clearly penetrant patterning defect. It also weakens the penetrance of abnormal phenotypes because of the Mel18 mutation at specific AP levels, and it leads to a partial rescue of the Mel18 mutant defects at all AP levels in the compound mutants. Strong interference of strain specific modifiers(s) in the hybrid background probably does not allow for the verification of genetic interaction observed in the C57BL/6.
Repression of cHoxb9 expression by overexpression of Ring1B protein
To examine the effect of a gain of function of Ring1B on Hox gene expression, we unilaterally overexpressed it in the developing neural tube of chicken embryos by in ovo electroporation (Fig. 9A, part a). For 0-5 somites embryos, only 10% of transfected embryos survived in our experimental conditions and most of surviving embryos exhibited extensive morphological defects in the neural tube. Thus, we used embryos older than the 6-somite stage. By means of this method, mouse Ring1B and Mel18 were successfully expressed in the developing spinal cord 24 hours after electroporation, but less intensely in the hindbrain (Fig. 9B, part a, Fig. 9C, part a). We investigated the expression of chicken Hoxb9 in the neural tube because its expression extends up to the level of 9th or 10th somite where stable ectopic expression of mouse Ring1B and Mel18 was seen (Fig. 9A, parts b and c). We first studied the expression of chicken Hoxb9 gene 48 hours after electroporation of chick embryos of various stages of development. In 6-12 somite embryos, we observed a significant posteriorization of the anterior boundary of chicken Hoxb9 expression in 67% of efficiently transfected embryos (Fig. 9B,D). The downregulation of chicken Hoxb9 expression by Ring1B usually extended along several somites (parentheses in Fig. 9B, parts b-d). The introduction of empty vector occasionally resulted in a subtle posteriorization of chicken Hoxb9 of about half somite width (4%) (Fig. 9D) (M. S., unpublished). The Ring1B-mediated repression of chicken Hoxb9 expression was much weaker when 12-18 somites embryos were electroporated (only 25% showed a posterior shift) and no effect was seen on 18-24 somite embryos (Fig. 9D). By analyzing embryos at 24, 36 and 48 hours after electroporation, we found it took 36 hours for the repression of chicken Hoxb9 expression to occur (Fig. 9E).
Similar experiments with Mel18 showed a very mild posteriorization of the anterior boundary of chicken Hoxb9 expression that affected only a somite in only 25% of the efficiently transfected embryos (Fig. 9C,D).
In this study, we provide ample evidence to indicate that Ring1B is a functional component of mammalian PcG complexes. Thus, co-immunoprecipitation and subnuclear localization studies show that Ring1B is found in protein complexes together with other PcG proteins such as Mel18, M33 and Rae28/Mph1. In addition, a hypomorphic loss-of-function mouse model revealed alterations in the axial skeleton and in the expression pattern of some Hox genes, consistent with a PcG function. Conversely, transient gain-of-function experiments in chick embryos showed a role of Ring1B in the repression of Hox gene expression. Finally we showed a genetic interaction between Ring1B and Mel18 resulting in the enhancement of Mel18-deficient phenotypes by the reduction of Ring1B dose on the C57BL/6J background. Taken together, these observations strongly qualify Ring1B as a mammalian PcG gene product.
The phenotype of the Ring1B mutant mice described here show some differences with those of mice with mutations in other PcG genes. Thus, the axial skeletal alterations in Ring1Bred/red mice are restricted to the cervicothoracic transitional zone, while they are seen along the entire axis in other PcG mutant mice (Akasaka et al., 1996; van der Lugt et al., 1994; Core et al., 1997; Takihara et al., 1997; del Mar Lorente et al., 2000). We believe this apparent difference does not imply that Ring1B function is restricted to the prospective cervicothoracic transitional region. First, the mutant mice show a reduction of Ring1B levels, whereas all other PcG mutant mice bear null mutations, and it is possible that specification events in that region are the most sensitive to Ring1B dose. Second, Ring1Bred/red embryos show a de-repression of Hoxb4 mesodermal expression in C1 prevertebra. The fact that neither a transformation of cervical vertebrae C1 to C2 nor any other alterations that affect these vertebrae are observed could be explained if the deregulation of other Hox genes required for C1 specification are not significantly affected in Ring1Bred/red mice. Third, the effect that the low levels of Ring1B has on the phenotype of the axial skeleton of mice lacking Mel18 affects regions throughout the entire axis in hybrid genetic background.
Present and previous biochemical studies indicate that Ring1B is found in protein complexes containing other PcG proteins, such as M33, RYBP, Mel18 and Rae28/Mph1 (Schoorlemmeret al., 1997; Garcia et al., 1999). Furthermore, we have shown for the first time that in mammals these PcG proteins are closely associated to chromosomal DNA, as it had been observed before in D. melanogaster, while significant quantities of them are not closely associated with chromatin (Zink and Paro, 1989; Orlando and Paro, 1993). Ring1B associates with a PcG multimeric complex through multiple protein-protein interactions, as suggested for other components of PcG complexes (Garcia et al., 1999). We have shown here that Ring1B binds Mel18 and Bmi1 via its N-terminal half, which includes a RING finger motif. However, it has been shown that Ring1B binds M33 and RYBP via C-terminal half (Schoorlemmer et al., 1997; Garcia et al., 1999). The structural similarities of Ring1B-binding domains of Mel18 and Bmi1 (94% identity of amino acids), suggest that they may associate with the same interacting surface on Ring1B. As Mel18 and Bmi1 are known to function redundantly during AP specification and protection against apoptosis, biological impacts might be similar between Ring1B/Mel18 and Ring1B/Bmi1 interactions (Akasaka et al., 2001). However, Hox gene expression is uniquely affected in Mel18- and Bmi1-deficient mice (Akasaka et al., 1996; van der Lugt et al., 1996). This suggests that Ring1B might preferentially interact with either Mel18 or Bmi1 in Hox gene-dependent manner. This preference could also be influenced by the other components of the PcG complexes as different sets of Hox genes are also affected in M33- and Rae28/Mph1-deficient mice (Core et al., 1997; Takihara et al., 1997).
Novel genetic interactions between Ring1Bred and Mel18-null mutations upon the compound genetic background suggest the polygenic regulation of mammalian PcG complexes. Restorations of posterior transformations caused by Mel18 null-mutation by Ring1Bred mutation suggests presence of compensatory machinery. In particular, Bmi1 could function to compensate Mel18 mutation. As Bmi1 is known to interact with not only Ring1B but also Rae28/Mph1 and Mph2, multimerization of Bmi1 and its interactors could be modified by unknown polymorphic elements (Hemenway et al., 1998). Molecular basis for genetic modifiers for PcG is not known; however, genes encoding constituents of PcG complex or proteins required for post-translational modification of PcG proteins could be involved (Voncken et al., 1999).
The repression of chicken Hoxb9 expression by transient high levels of mouse Ring1B and Mel18 in chicken embryos is in good agreement with the posteriorization of the rostral boundaries of Hox gene expression seen in Bmi1 and Ring1A transgenic mice (Alkema et al., 1995; del Mar Lorente et al., 2000). These data are also consistent with previous observations that most PcG proteins, including Ring1 family proteins act as transcriptional repressors when recruited to reporter constructs (Satijn et al., 1997b; Schoorlemmer et al., 1997). The anterior boundary of chicken Hoxb9 expression in the neural tube at the level of prospective 9th somite is already established in seven-somite embryos (Fig. 9A, part b). Thus, the overexpression of mouse Ring1B and Mel18 results in the active silencing of a chicken Hoxb9 gene, which is already transcriptionally active. Such an effect may result from the recruiting of ectopic Ring1B and Mel18 to regulatory regions of chicken Hoxb9, which, in turn, may alter locally the structure of the chromatin. However, overexpression of Ring1B does not affect chicken Hoxb9 expression in later stage embryos (Fig. 8C). Biochemical studies using purified PcG complexes and chromatin remodeling factors of the SWI/SNF provide clues on how PcG and trxG proteins counteract (Shao et al., 1999). PcG complexes act as a molecular lock when tethered on nucleosomes prior to exposure to the remodeling factors. Conversely, once exposed to SWI/SNF factors, PcG-mediated repression does not occur. If this is the case, the inability of Ring1B to repress chicken Hoxb9 expression at later stages in development may be due to the establishment of a trxG-dependent activation of chicken Hoxb9. Thus, the accessibility to, or the activity of Ring1B-containing PcG complexes on Hox clusters might be regulated in a developmental stage-dependent manner, as suggested in D. melanogaster (Orlando et al., 1998).
Interactions between Ring1B and Mel18. (A) Interaction in yeast cells indicated by the β-galactosidase activity. Yeast cellswere transformed with the indicated plasmids. Lamin was a negative control. (B) GST pull-down assays. (Part a) Binding of 35S-labeled full-length Mel18 protein (Mel18fl) to GST or GST fused to either full-length or truncated Ring1B (GST-Ring1Bfl, GST-Ring1BΔC or GST-Ring1BΔN, respectively). (Part b) Binding of 35S-labeled full-length Ring1B protein (Ring1Bfl) to GST or GST fused to truncated Mel18 (GST-Mel18ΔPEST) (amino acids 1-233). (Part c) Binding of 35S-labeled truncated Ring1B proteins (Ring1BΔC and Ring1BΔN) to GST or GST-Mel18ΔPEST.
In vivo association of Ring1B with mammalian PcG proteins. (A) Specific co-immunoprecipitation of Ring1B from 11.5 dpc mouse embryos using antibodies against Mel18 and Rae28/Mph1. The signals corresponding to the immunoglobulin heavy chains are indicated by arrowheads (Ig). (B-F) Comparative analysis of the subnuclear distributions of endogenous RING1B and MEL18 (B), BMI1 (C), RING1A (D), RAE28/MPH1 (E) and exogenous Mph2 (F) in normal or transfected human osteosarcoma U2-OS cells using indirect immunofluorescence. (Parts a) The signal of RING1B (green). (Parts b) The signals of endogenous MEL18 (B), BMI1 (C), RING1A (D), RAE28/MPH1 (E) and exogenous Mph2 (F) (red). (Parts c) Merged images. (Parts d) Phase contrast views. Monoclonal and polyclonal antibodies are indicated as M and P, respectively, in parentheses.
Association of Ring1B, M33 and Rae28/Mph1 with chromosomal DNA. Protein/DNA complex was concentrated by in vivo formaldehyde crosslinking and subsequent cesium chloride isopyknic centrifugation. (A) Chromosomal DNA in each fraction was visualized by Ethidium Bromide staining. λDNA digested with StyI was used as molecular weight markers. (B) Protein in each fraction was visualized by Coomassie Brilliant Blue (CBB) staining. Localization by western blot analysis of an ER luminal protein, Bip (C), dimethyl-Histone H3 (D), Ring1B (E), M33 (F) and Rae28/Mph1 (G).
Subcellular localization of RING1B in interphase and mitotic nuclei. (A,B) Distribution of RING1B and CENPB (A) and centromeric antigens (B) in interphase nucleus of U2-OS cells. (Parts a) Localization of endogenous RING1B (green). (Parts b) Localization of endogenous CENPB (A) and centromeric antigens (B; ACA) (red). (Parts c) DAPI (blue). (Parts d) Merged images. (Parts e) Higher magnification views of the boxed regions in parts d. (C) Distribution of RING1B (part a, green) and CENPB (part b, red) in prophase nucleus of U2-OS cells. (part c) DAPI (blue). (Part d) Merged images. (D) Distribution of RING1B (part a; green) and centromeric antigens (part b; red) in prometaphase nucleus of U2-OS cells. (Part c) DAPI (blue). (Part d) Merged images. (Part E) Distribution of RING1B (part a; green) and CENPB (part b; red) in anaphase nucleus of U2-OS cells. (Part c) DAPI (blue). (Part d) Merged images.
Disruption of the Ring1B gene in mice. (A) Diagram of the Ring1B locus, the targeting vector and of the modified allele. The Ring1B-coding exons are indicated by black boxes. The PGKneo and pMC1-tk expression cassettes were used for positive and negative selection, respectively. Position of relevant restriction sites (EcoRI, E; BamHI, B; HindIII, H; SalI, S; XhoI, X), location of probe and PCR primers, and sizes of diagnostic fragments are indicated. (B) Southern analysis of genomic DNA isolated from offspring of heterozygote matings after digestion with BamHI + HindIII and probed with the indicated 3′ probe shown in A. (C) PCR analysis of tail genomic DNA of the offspring of a heterozygous intercross. Primers RB22 and RB18 amplify a 1.4 kb of the wild-type allele, whereas primers Neo2 and RB11 amplify a 3.0 kb of the mutated allele. (D) Semi-quantitative analysis of Ring1B by western blot analysis of total proteins extracted from 11.5 dpc mouse embryos of the indicated genotypes probed with antibodies against Ring1B. To correct for loading differences, anti-lamin B antibodies and Coomassie Brilliant Blue (CBB) staining of the same gel were used.
Skeletal alterations of Ring1Bred/red, Mel18–/– and Ring1Bred/redMel18–/– mice. (A) The genotypes are indicated at the top. (Parts a-d) Lateral views of the upper part of the vertebral column. Yellow stars in parts c and d indicate a ectopic arch of the occipital bone. Black arrows in parts b-d indicate ectopic ribs associated with the 7th cervical vertebra. Red arrows indicate the prominent spinous process. Blue arrows in parts b and d indicate the additional ossification center of the sternum implying the anterior shift of the sternum. (Parts e-h) Ventral views of the rib cages. Black arrows in parts f and h indicate ectopic ribs associated with 7th cervical vertebra. (B) Various posterior transformations of the axial skeleton and their penetrance (indicated in parenthesis): (1) Supraoccipital bone→C1, appearance of the ectopic bones seen in the craniodorsal region of the C1 vertebra or ectopic arch of the occipital bones; (2) C1→C2, presence of the odontoid process on the C1 vertebra; (3) C2→C3, lack of the odontoid process from the C2 vertebra; (4) C7→T1, appearance of cervical ribs on C7; (5) T1→T2, prominent spinous process on T1; (6) T7→T8, dissociation of 7th rib from the sternum; (7) T13→L1, loss of the rib in 20th vertebra; (8) L5 or 6→S1, formation of the sacro-iliac joint in 25th or 26th vertebra; (9) S4→Ca1, appearance of the first caudal vertebra in 29th or 30th vertebra; (10) anterior shift of the sternum as revealed by an ectopic ossification.
Expression of Hoxb genes in 11.5 dpc wild-type and Ring1Bred/red mutant embryos. Lateral view of sagittal sections showing the expression of Hoxb4 (A,B), Hoxb6 (C,D) and Hoxb8 (E,F) in Ring1Bred/red (A,C,E) and wild type embryos (B,D,F). The number of prevertebrae starting at the proatlas are shown and segment boundaries are indicated by bars.
Skeletal alterations seen in Ring1Bred/red, Mel18–/– and Ring1Bred/redMel18–/– mice on the compound genetic background. (A) Respective genotype of Ring1B and Mel18 loci are indicated at the top. For Ring1Bred/redMel18–/–, three specimens are shown. (Parts a-f) Lateral views of the upper part of the vertebral column are shown. Stars in parts c and e indicate ectopic floating bone or ectopic arch of the occipital bone, respectively. Arrows in parts b, c, e and f indicate ectopic ribs associated with C7. (Parts g-l) Ventral views of the rib cages are shown. In part k, ectopic ossification center of the sternum as a consequence of complete anterior shift of the sternum and complete ectopic ribs are indicated by a blue arrow and black arrows, respectively. Note 7th ribs are articulated with the sternum in Ring1Bred/redMel18–/– mice. (Parts m-r) Ventral views of the thoracolumbar region are shown. Prospective T12 and T13 are indicated by respective numbers. Prospective lumbar vertebrae are indicated by red stars individually. (B) The frequency of the posterior transformations of the axial skeleton is schematically represented. The following parameters are scored and frequency of each alteration is indicated in parentheses: (1) supraoccipital bone→C1, appearance of the ectopic bones seen in the craniodorsal region of the C1 vertebra or ectopic arch of the occipital bones; (2) C1→C2, presence of the odontoid process on the C1 vertebra; (3) C2→C3, lack of the odontoid process from the C2 vertebra; (4) C7→T1, appearance of cervical ribs on C7; (5) T1→T2, prominent spinous process on T1; (6) T7→T8, dissociation of 7th rib from the sternum; (7) T13→L1, loss of the rib on 20th vertebra; (8) L1→T13, appearance of the rib on 21st vertebra; (9) L5 or 6→S1, formation of the sacro-iliac joint in 25th or 26th vertebra; and (10) S4→Ca1, appearance of the first caudal vertebra in 29th or 30th vertebra.
Repression of chicken Hoxb9 expression by overexpression of mouse Ring1B and Mel18. (A, part a) In ovo electroporation. A pair of electrodes held by a manipulator is inserted through a window and placed over the vitelline membrane overlying the embryo. Plasmid solution colored by Nile Blue is injected into the developing spinal cord as indicated by a yellow bracket. (parts b,c) Hoxb9 expression in seven-somite (part b) and 15-somite (part c) stage embryos. Somite boundaries are shown. (B, part a) Expression of mRing1B 24 hours after electroporation. (parts b-d) Repression of Hoxb9 expression observed 48 hours after electroporation. The anterior boundaries of Hoxb9 expression in the control sides are indicated by arrows. Downregulation of Hoxb9 expression by exogenous Ring1B are indicated by brackets. (C, part a) Expression of mMel18 24 hours after electroporation. (part b) Repression of cHoxb9 expression 48 hours after electroporation. The anterior boundary of Hoxb9 expression in the control side is indicated by an arrow. Downregulation of Hoxb9 expression by mouse Mel18 is indicated by a bracket. (D) Ring1B- and Mel18-dependent repression of Hoxb9 expression is influenced by the developmental stage. Frequency of affected embryos by transfection of mouse Ring1B, Mel18 and empty vectors were represented by closed, shaded and open bars, respectively. Mel18-dependent repression is less efficient than that of Ring1B and is similarly influenced by developmental stage. (E) Ring1B-dependent repression of Hoxb9 expression becomes obvious at least 36 hours after the electroporation. Asterisks in D and E indicate results from the identical series of experiments.
We are grateful to Drs R. Krumlauf, K. Schughart, J. Deschampus, A. Kuroiwa, J. Miyazaki and T. Aoe for the mouse Hoxb4 cDNA clone, mouse Hoxb6 cDNA clone, mouse Hoxb8 cDNA clone, chicken Hoxb9, pCXN2 vector and anti-Bip monoclonal antibody, respectively; to Dr K. Katsube for initial instructions for in ovo electroporation; to Drs T. Oda and M. Muramatsu for initial instructions for yeast two hybrid screening; and to Dr M. van Lohuizen for communication of unpublished data and anti-Bmi1 monoclonal antibody. We also thank Ms Sanae Takeda, Misao Uchida and Mr Shozo Sugimori for their help. This project was supported by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government; and by grants from the Naito Foundation, Mochida Foundation, Kanae Foundation and Uehara Memorial Foundation. Work in M. V.'s laboratory is supported by the Ministry of Science and Technology of Spain (grant PB97-1238) and the CAM (grant 08.1/0050/2000).
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). Positional cloning of a global regulator of anterior-posterior patterning in mice.
Sewalt, R. G., van der Vlag, J., Gunster, M. J., Hamer, K. M., den Blaauwen, J. L., Satijn, D. P., Hendrix, T., van Driel, R. and Otte, A. P. (
). Characterization of interactions between the mammalian polycomb-group proteins Enx1/EZH2 and EED suggests the existence of different mammalian polycomb-group protein complexes.
Shao, Z., Raible, F., Mollaaghababa, R., Guyon, J. R., Wu, C. T., Bender, W. and Kingston, R. E. (
). Stabilization of chromatin structure by PRC1, a Polycomb complex.
Tagawa, M., Sakamoto, T., Shigemoto, K., Matsubara, H., Tamura, Y., Ito, T., Nakamura, I., Okitsu, A., Imai, K. and Taniguchi, M. (
). Expression of novel DNA-binding protein with zinc finger structure in various tumor cells.
J. Biol. Chem
-20026.
Takihara, Y., Tomotsune, D., Shirai, M., Katoh-Fukui, Y., Nishii, K., Motaleb, M. A., Nomura, M., Tsuchiya, R., Fujita, Y., Shibata, Y., Higashinakagawa, T. and Shimada, K. (
). Targeted disruption of the mouse homologue of the Drosophila polyhomeotic gene leads to altered anteroposterior patterning and neural crest defects.
Tomotsune, D., Shirai, M., Takihara, Y. and Shimada, K. (
). Regulation of Hoxb3 expression in the hindbrain and pharyngeal arches by rae28, a member of the mammalian Polycomb group of genes.
Mech. Dev
van der Lugt, N. M., Domen, J., Linders, K., van Roon, M., Robanus-Maandag, E., te Riele, H., van der Valk, M., Deschamps, J., Sofroniew, M., van Lohuizen, M. and Berns, A. (
). Posterior transformation, neurological abnormalities, and severe hematopoietic defects in mice with a targeted deletion of the bmi-1 proto-oncogene.
van der Lugt, N. M., Alkema, M., Berns, A. and Deschamps, J. (
). The Polycomb-group homolog Bmi-1 is a regulator of murine Hox gene expression.
van der Vlag, J. and Otte, A. P. (
). Transcriptional repression mediated by the human polycomb-group protein EED involves histone deacetylation.
Nat. Genet
van Lohuizen, M., Verbeek, S., Scheijen, B., Wientjens, E., van der Gulden, H. and Berns, A. (
). Identification of cooperating oncogenes in E mu-myc transgenic mice by provirus tagging.
van Lohuizen, M., Tijms, M., Voncken, J. W., Schumacher, A., Magnuson, T. and Wientjens, E. (
). Interaction of mouse polycomb-group (Pc-G) proteins Enx1 and Enx2 with Eed: indication for separate Pc-G complexes.
Vojtek, A. B., Hollenberg, S. M. and Cooper, J. A. (
). Mammalian Ras interacts directly with the serine/threonine kinase Raf.
Voncken, J. W., Schweizer, D., Aagaard, L., Sattler, L., Jantsch, M. F. and van Lohuizen, M. (
). Chromatin-association of the Polycomb group protein BMI1 is cell cycle-regulated and correlates with its phosphorylation status.
J. Cell. Sci
Wang, G., Horsley, D., Ma, A., Otte, A. P., Hutchings, A., Butcher, G. W. and Singh, P. B. (
). M33, a mammalian homologue of Drosophila Polycomb localises to euchromatin within interphase nuclei but is enriched within the centromeric heterochromatin of metaphase chromosomes.
Cytogenet. Cell Genet
Wilkinson, D. G. and Nieto, M. A. (
). Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts.
Methods Enzymol
Yoda, K., Nakamura, N., Masumoto, H., Suzuki, N., Kitagawa, K., Nakano, M., Shinjo, A. and Okazaki, T. (
). A human centromere protein, CENP-B, has a DNA binding domain containing four potential alpha helices at the NH2 terminus, which is separable from dimerizing activity.
Zink, B. and Paro, R. (
). In vivo binding pattern of a trans-regulator of homoeotic genes in Drosophila melanogaster.


In the concert hall of the Alte Oper Frankfurt an architectonic staging, themed with the headline "departure growth future", made the guest be the heart of the 50th anniversary of the financial business dit.
Starting from the stage, ribbons – serving as media projection surfaces – form a three-dimensional sculpture. The distance to the stage dissolves, the guest becomes part of the game – the stage setting unfolds into the entire space.
Stock exchange diagrams give the form for the ribbons onto which half a century's story of success is projected, most notably the sustainability and innovative strength of the company.


This is a Travel Adventure by Luca & Vittorio. We did 1580 km in 2 days, from Cape Town (Western Cape) till Port Nolloth (Northern Cape) in 2 days. Official excuse was "we don't have internet at home so we go on a photographic trip". That trip actually became a sandboarding, driving, exploring trip across the Northern Cape.
But are you maybe interested in some pictures?


text:
Waypoint Labs
waypointlabs.com
About Waypoint Labs
Waypoint Labs is developing augmented reality software to change the way front-line workers capture, distribute, and access expert knowledge on the job.
Missing: Waypoint Labs's Product Demo & Case Studies
Missing: Waypoint Labs's Product & Differentiators
Expert Collections containing Waypoint Labs
Waypoint Labs is included in 2 Expert Collections, including AR/VR.
This collection includes companies creating hardware and/or software for augmented reality, virtual reality, and mixed reality applications.
US-based companies
Latest Waypoint Labs News
PTC Makes 3rd Augmented Reality Acquisition As AR Revenue Reaches $20M
PTC has made its third acquisition in the augmented reality space to provide an interactive, real-time training environment for industrial settings. The Needham, Mass.-based industrial software company announced its acquisition of Waypoint Labs, a startup born out of MIT in 2016 that allows trainers to build a three-dimensional set of instructions for workers in real-time using an AR headset such as the Microsoft HoloLens. "It's a brand new way to author work instructions," PTC CEO Jim Heppelmann (pictured) said during his keynote at the company's LiveWorx conference Monday. "It's software that enables humans to easily get from how a process is actually performed when it's performed and then convert that back into instructions for everyone else to use later." Financial terms of the deal were not disclosed. PTC's acquisition of Waypoint comes after the company acquired the Reality Editor software, another technology born out of MIT, in November 2017 and Vuforia, which became the basis for PTC's AR capabilities within the company's ThingWorx IoT platform, in November 2015. All of the company's AR products are now under the Vuforia brand, another development that was announced Monday. In a Q&A session Monday afternoon, Heppelmann revealed that PTC's AR business represents about $20 million in revenue with a 100 percent growth rate. He said Vuforia Engine, the core technology PTC had acquired from Qualcomm, now represents a minority of the AR business while PTC's enterprise solutions, like Vuforia Studio and View, bring in the most sales. "I'm very pleased with the AR business. It's early, but I wouldn't trade places with anybody. Not for anything," he said. During Heppelmann's keynote, a demonstrator used Waypoint to create a set of instructions for starting a lab procedure by using a series of hand gestures and voice commands with an AR headset. Using Waypoint's knowledge processing engine, the software converted the gestures and commands into a series of interactive text and images that were superimposed over the view of a second demonstrator with a headset. The company said Waypoint supports a range of industrial use cases. That includes manufacturers that can use Waypoint to create and distribute service manuals and product documentation. Jordan Reynolds, a senior manager at Kalypso, a Cleveland-based PTC partner, told CRN that his firm has already gone to production with AR solutions for a number of different companies, though he said most firms are still stuck in the proof-of-concept stage. "Everybody is investing in pilots. Few people have brought those pilots into production implementations that are ready for scale. Very few," Reynolds said. He commended PTC for the direction it has taken in adding more IoT and AR capabilities, giving the company a unique position in the industrial technology space. "What they did really well and very uniquely is they started with product development capabilities and that laid the foundation for smart connected product capabilities that involved IoT," Reynolds said
Waypoint Labs Frequently Asked Questions (FAQ)
When was Waypoint Labs founded?
Waypoint Labs was founded in 2016.
What is Waypoint Labs's latest funding round?
Waypoint Labs's latest funding round is Acquired.
Who are the investors of Waypoint Labs?
Investors of Waypoint Labs include PTC and Harvard Venture Incubation Program.

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text:
March 21, 2012 Memo
You are here: American University President Announcements March 21, 2012 Memo
About the President
Sylvia M. Burwell's Biography
Neil Kerwin, President Emeritus
President's Reports & Announcements
president@american.edu
President's Office Building, Room 1
Office of the President 4400 Massachusetts Avenue NW Washington, DC 20016-8060 United States
Memorandum March 21, 2012
AU Community
Neil Kerwin, President
Campus Plan Approval by D.C. Zoning Commission
I am pleased to inform you that on Thursday, March 8, the D.C. Zoning Commission approved AU's 2011 Campus Plan-which provides a "green light" to begin construction on a number of facilities projects to support AU's academic aspirations and enhance campus ambiance, while also emphasizing our commitment to be a sensitive neighbor.
The approval includes the East Campus project, which will convert the Nebraska Avenue parking lot into an eight-acre mixed use development comprising housing (590 beds), academic space, and offices; Nebraska Hall addition (150 beds); North Hall (360 beds), behind the President's Office Building; Mary Graydon Center addition; and Washington College of Law relocation to the Tenley Campus.
On Monday, March 26, we return for the Zoning Commission's deliberations on the final part of the Campus Plan-to seek the approval of construction plans for the law school's new Tenley Campus location.
In addition to building a number of priority projects, the university will have an array of responsibilities to fulfill and obligations to uphold. AU will house 67 percent of our undergraduates on campus by 2016; maintain a student population cap of 13,600 which includes 2,000 law students; comply with off-campus parking provisions and further monitor our traffic impact; manage objectionable off-campus student behavior; mitigate sound on the Jacobs Field; and strengthen efforts to engage neighborhood organizations in ongoing dialogue and dispute resolution.
In the next few weeks, the Zoning Commission will issue a written order that outlines the project approvals and the conditions of compliance. The life of this plan is for the next 10 years.
This entire Campus Plan process has lasted almost three years-we began with a campus discussion to identify and prioritize AU's space needs for the coming decades; assessed potential project sites; sought Board of Trustees approval; involved the neighboring community in extensive discussion and review by three different Advisory Neighborhood Commissions; and presented proposed plans before the D.C. Zoning Commission in public hearings. Along the way, we made significant changes to the plan-to modify projects, add new sites or drop others, alter designs, mitigate impact, and reaffirm AU's commitment that changes will be managed appropriately.
From envisioning initial concepts to earning final approval, the Campus Plan was a total team effort by many AU units and individuals who deserve special thanks-including the Office of Chief Financial Officer, Vice President and Treasurer, under whom Facilities Development, Campus Architect, and Community Relations all operate; the Office of the Vice President of Campus Life and especially the Dean of Students Office; the Office of the General Counsel; University Communications and Marketing; the Office of the President; and Goulston & Storrs, our land use counsel.
Also vital was participation by numerous other departments and AU alumni, faculty, staff, and students-including our Faculty Senate leadership; Student Government Association and Graduate Leadership Council; student ANC 3D representative; Alumni Association; and everyone who wrote letters of support, testified at public hearings, or spoke at community meetings or in private conversations. Thanks to everyone.
In the near future, I will provide an update on the March 26 Zoning Commission deliberation, details about the construction timeline, and measures we must implement as a good neighbor and community member. Having gone through a public process of rigorous debate over the past two years, we reassure our neighbors in the surrounding communities that we renew our commitment to be a vibrant citizen of Washington, D.C., and an integral and supportive part of our neighborhood.
This outcome truly will enable American University to begin a new chapter in its history. Thank you to everyone for this remarkable achievement.


text:
Sleater-Kinney, Spoon's Britt Daniel, The Thermals cover David Bowie's "Rebel Rebel" on New Year's Eve — watch
Plus, a tribute to George Michael with a performance of "Faith"
Ben Kaye
January 2, 2017 | 9:45am ET
Sleater-Kinney rang in the New Year in style with a sold-out gig at San Francisco's Masonic. As happens at any good NYE bash, they brought along some friends in openers The Thermals and the evening's DJ, Britt Daniel of Spoon. During Sleater-Kinney's set, all of these musicians joined together to pay tribute to those lost in 2016 with a pair of cover songs.
Carrie Brownstein and the band first welcomed out The Thermals for a rendition of George Michael's classic "Faith". Then Daniel, The Thermals' Hutch Harris, and Corin Tucker traded off verses on David Bowie's "Rebel Rebel" to close out the evening. Both songs ended up being the perfect message with which to steel oneself for the New Year, as Brownstein closed the show saying, "We love you. Protect yourself. Protect each other. Resist!"
Watch the performance of "Rebel Rebel" above, with a different angle and "Faith" viewable below.
"Rebel Rebel":
"Faith":
Britt Daniel
Sleater-Kinney, Spoon's Britt Daniel, The Thermals cover David Bowie's "Rebel Rebel" on New Year's Eve -- watch


text:
Q: Partial Differential Equations: Fourier Transform in Space and Time? Consider the one-dimensional wave equation:
$$\frac{1}{c^2}\frac{\partial^2}{\partial t^2}\psi(x, t) = \frac{\partial^2}{\partial x^2}\psi(x, t).$$
We can take the Fourier transform of $\psi(x,t)$ with respect to $x$:
$$\mathcal{F}\{\psi(x, t)\}(k) = \hat{\psi}(k, t).$$
Therefore, the wave equation becomes:
$$\frac{1}{c^2}\frac{\partial^2}{\partial t^2}\hat{\psi}(k, t) = - k^2 \hat{\psi}(k, t).$$
Although the resulting equation doesn't make much sense, can we repeat this process and take the Fourier transform of $\hat{\psi}(k, t)$ with respect to $t$ to have
$$\frac{l^2}{c^2}\hat{\hat{\psi}}(k, l) = k^2 \hat{\hat{\psi}}(k, l)?$$
In general, can we use the Fourier transform to eliminate all derivatives from any PDE?

A: I think your impression here is generally correct, but let's be specific: if we assume that the function $\psi(x,t)$ is a tempered distribution as a "function" of two variables, then, yes, we can take Fourier transforms, and a constant-coefficient PDE becomes an equation involving no derivatives.
For (linear) wave equations, the hypothesis that $\psi$ is tempered is not unreasonable.
For heat equations, there is a problem, both in physical terms and in mathematical terms. Physically, lots of scenarios cannot be run backward beyond some time $t_o$. Mathematically, in parallel, there are lots of (physically interesting/meaningful) set-ups where, as a function of $t$, $\psi(x,t)$ cannot be reasonably extended backward. In those cases, as in comments, one-sided things that accommodate boundare/initial conditions (Laplace...) are more relevant.

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                Accueil

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Call for Papers

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Women Techmakers Nouakchott est marque et globale du programme de Google pour les femmes dans la technologie. Women Techmakers offre visibilité, la communauté et les ressources pour les femmes dans la technologie pour stimuler l'innovation et la participation dans le domaine de Technologie.

Presenters – who should speak at the DevFest Conference?

developers (freelance and hired)

companies developing software (apps, tools, frameworks etc.)

research institutions with Google relevant activities

companies, organisations and individuals using Google technologies

If you belong to one of those groups you are cordially invited to submit a talk proposal (or a number of proposals). An independent program committee assesses the proposals and selects the actual conference talks.

Themes and topics – what do we expect?

Our team prepared set of interesting topics in but if you have any cool idea you want to share not from this list, feel free to submit it as well.

Android

Google I/O 2014

Material Design

Migration to ART

New Android Camera API

New Job Scheduler

Storage Access Framework

Elevation & Clipping

General

Android Continuous Integration

Loaders, Cursor Adapters

Android Animations

Patterns in Android development

Web

AngularJS (1x and 2.0)

Dart (AngularDart, server-side)

Polymer and WebComponents

Material Design in Web

ChromeOS (apps development)

Web Performance (tools, techniques, approaches)

Cloud

Google Cloud Platform

Compute Engine

App Engine

Storages (SQL, NoSQL, BigData)

APIs

Go (as a language for a Cloud Ready Apps)

Please submit your proposals here.

Deadline is September 1, 2014

Please note: There is no guarantee that a submission will be put onto the conference agenda!

The program committee picks the most interesting talk proposals and informs the selected submitters until September 15, 2014.

Any questions?

Email organizers at devfest@gdg.org.ua

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text:
Published Data Library (PDL)
Physiological, biomineralisation and structural measurements of the cold-water coral (CWC) Lophelia pertusa in response to increases in CO2 and temperature.
Hennige S. (1); Wicks L. (1); Kamenos N.A. (2); Perna G. (2); Findlay H.S. (3); Roberts J.M. (1)
(1) Heriot Watt University School of Life Sciences; (2) University of Glasgow Department of Geographical and Earth Sciences; (3) Plymouth Marine Laboratory
This dataset consists of physiological (respiration, growth), biomineralisation (crystal orientation, growth form) and structural (breaking strength) measurements of the cold-water coral Lophelia pertusa in response to the single and combined stressors of elevated temperature and CO2 over 12 months. Corals were collected from the Mingulay Reef Complex (UK) on RRS Discovery cruise D366/7 in June/July 2011. Live and dead corals were subjected to projected future CO2 and temperature scenarios at Heriot-Watt University to determine whether they can acclimate to projected future climate change. Data consists of respiration measurements, growth rates (measured through alkalinity anomaly technique), Scanning Electron Microscope (SEM) images of corals in aragonite undersaturated conditions, skeletal molecular bonding measurements (RAMAN spectroscopy), orientation/organisation images of newly accreted aragonite (through Electron Back Scatter Diffraction, EBSD), skeletal breaking strength, and carbonate chemistry of the experimental systems. The collection of these data and the subsequent synthesis and intercomparison were funded as part of the UKOA programme by NERC, DEFRA and DECC.
July 2011 - January 2014
Inner Seas off the West Coast of Scotland
Delimited,	Image
Discovery metadata record
Link to the related European Directory of Marine Environmental Data (EDMED) record
British Oceanographic Data Centre - Natural Environment Research Council, UK
doi:10.5285/13d58735-4252-109d-e053-6c86abc0bae4
Short DOI
doi:10/5q7
Citation text
Hennige S.; Wicks L.; Kamenos N.A.; Perna G.; Findlay H.S.; Roberts J.M.(2015). Physiological, biomineralisation and structural measurements of the cold-water coral (CWC) Lophelia pertusa in response to increases in CO2 and temperature. British Oceanographic Data Centre - Natural Environment Research Council, UK. doi:10/5q7.


We are proud to sell the finest products available. If anything you purchase from us fails to live up to your expectations, we will replace or refund the undamaged product within 35 days of the original purchase date. It's that simple. Questions or returns please email us at Wirelessoem@yahoo.com Please allows us to answer all Emails within 30 hours of a working business day.
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I just got this Sentiment Flags set/die from Lil' Inker Designs and was anxious to try it out. I think I'll get a lot of use out of this one! I paired it up with some of my Penny Black ducks. :) The colors on my card are from the CR84FN Challenge 34.
Wow, are we ever having some freaky hot weather this week! They're calling for a high of 104 degrees on Friday....yeee-ikes! I took Kara to Build a Bear Workshop this afternoon...figured it'd be best to just stay in the air conditioning.
I had time to do a little crafting today too...yay! I think the heat is what made me pull these warm, sunny colors. I die cut the butterflies three times, and popped two up on foam tape.
Hubby is grilling burgers on the grill and they're about done so I'm off to eat. Have a great night....and stay cool!
The main matted image is up on foam tape and I added some Stickles to the watermelon slice, hat band, and knife.
Hello all! TGIF??....TGIF! My SUPER sweet friend Silke sent me this gorgeous Flourishes set for my birthday and I couldn't wait to ink it up. Thanks, gf, I really love it!
I thought I'd try my hand at winning the entire Flourishes release (hey, you never know unless you try, right?) and play along in their Sketchy Challenge. The main image is up on foam tape, Stickles were added to the pink bow, and Diamond Glaze was added to the tip of the pen. The punched sentiment is also up on foam tape.
I used Ocean Paradise from Sweet and Sassy Stamps. Such a gorgeous set, Korin! The main panel and sentiment are up on foam tape and the layout is Sweet Stop Sketch 116. I added Stickles to the water and sand.
Okay, now we're off to the pool for the rest of the afternoon. Have a WONDERFUL day...and stay cool! It's in the 90's here today....YIKES!


text:
Watch: ROGUE ONE first official teaser
Posted by Joey Inigo | Apr 7, 2016 | Movies, TV, and Disney+ | 0
While Universal Studios is amidst celebrations of its brand new Wizarding World of Hary Potter, Disney has other distractions in mind with the release of the first official public teaser for the upcoming ROGUE ONE feature film.
Lucasfilm's ROGUE ONE, which takes place before the events of Star Wars: A New Hope, tells the story of unlikely heroes who have united to steal plans to the dreaded Death Star.
The cast includes Felicity Jones, Diego Luna, Ben Mendelsohn, Donnie Yen, Jiang Wen, Mads Mikkelsen, Alan Tudyk, Riz Ahmed, and Forest Whitaker.
ROGUE ONE is directed by Gareth Edwards, produced by Kathleen Kennedy, Allison Shearmur, and Simon Emanuel, executive produced by John Knoll and Jason McGatlin, and co-produced by John Swartz and Kiri Hart.
ROGUE ONE flies into theaters December 16, 2016.
Previous'D23 Destination D: Amazing Adventures' coming to Walt Disney World from Nov. 19
NextGo behind the voices of THE JUNGLE BOOK with this new featurette
New BEAUTY AND THE BEAST stills offer stunning look at upcoming film
Disney Channel confirms March 10 launch for ANDI MACK on digital platforms
APR. 2022 SALE: Find your fandom with discounted Disney-related apparel, stickers, cases and much more!
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Cuddle Lake outpost is an adventurous persons dream. This long lake offers everything a fishermen looks for in a trophy Pike lake. The spring torrent of incoming and outgoing water from the Utik River breaks up the ice in the bay first, which is where the camp is positioned bringing with it thousands of baitfish and feed for the resident trophy pike and Walleye that have gathered for this once a year feeding frenzy. This lake is known for producing double header trophy northern pike between 41" to 52 " in Length.
I now have exclusive rights to Pritchard Lake, which is just west of Cuddle. Pritchard Lake flows into Cuddle via an impressive set of rapids at the far west end of Cuddle. There are three boats and a tent camp set up at Pritchard Lake. Cuddle guests will have access via a portage trail. Future guests will have endless fishing opportunities between these two lakes and rivers.
The cabins at Cuddle were treated to an extensive renovation this past fall. A new large deck was added to the large cabin. Repeat customers will note many other improvements to the cabins, such as a wall moved within the large cabin to make room for a living room/lounge area with couches, etc.
In the spring, the fishing in the Utik River is tremendous both up and downstream. When water levels are normal, you can head downstream to the East all the way to the scenic Annesley Lake. Heading west you will have the chance to investigate the remaining 20 miles of bays, islands, and creeks in the lake. Cuddle Lake has had virtually zero fishing pressure by sport fishermen in the last 20 years. There are sure to be records that come out of this lake in the future.