Tight control of cell-cell communication is essential for the generation of a normally patterned embryo. A critical mediator of key cell-cell signaling events during embryogenesis is the highly conserved Wnt family of secreted proteins. Recent biochemical and genetic analyses have greatly enriched our understanding of how Wnts signal, and the list of canonical Wnt signaling components has exploded. The data reveal that multiple extracellular, cytoplasmic, and nuclear regulators intricately modulate Wnt signaling levels. In addition, receptor-ligand specificity and feedback loops help to determine Wnt signaling outputs. Wnts are required for adult tissue maintenance, and perturbations in Wnt signaling promote both human degenerative diseases and cancer. The next few years are likely to see novel therapeutic reagents aimed at controlling Wnt signaling in order to alleviate these conditions.

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The Wnt signaling pathway in development and disease.

The regulation of cell growth and survival can be subverted by a variety of genetic defects that alter transcriptional programs normally responsible for controlling cell number. High throughput analysis of these gene expression patterns should ultimately lead to the identification of minimal expression profiles that will serve as common denominators in assigning a cancer to a given category. In the course of defining the common denominators, though, we should not be too surprised to find that cancers within a single category may nevertheless exhibit seemingly disparate genetic defects. The wnt pathway has already provided an outstanding example of this. We now know of three regulatory genes in this pathway that are mutated in primary human cancers and several others that promote experimental cancers in rodents (Fig. 1). In all of these cases the common denominator is the activation of gene transcription by -catenin. The resulting gene expression profile should provide us with a signature common to those cancers carrying defects in the wnt pathway. In this review, the wnt pathway will be covered from the perspective of cancer, with emphasis placed on molecular defects known to promote neoplastic transformation in humans and in animal models.

/pdf/wnt-signaling-and-cancer-2fdsemer2h.pdf

Wnt signaling and cancer

The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling.

To resolve cellular heterogeneity, we developed a combinatorial indexing strategy to profile the transcriptomes of single cells or nuclei, termed sci-RNA-seq (single-cell combinatorial indexing RNA sequencing). We applied sci-RNA-seq to profile nearly 50,000 cells from the nematode Caenorhabditis elegans at the L2 larval stage, which provided >50-fold “shotgun” cellular coverage of its somatic cell composition. From these data, we defined consensus expression profiles for 27 cell types and recovered rare neuronal cell types corresponding to as few as one or two cells in the L2 worm. We integrated these profiles with whole-animal chromatin immunoprecipitation sequencing data to deconvolve the cell type–specific effects of transcription factors. The data generated by sci-RNA-seq constitute a powerful resource for nematode biology and foreshadow similar atlases for other organisms.

/pdf/comprehensive-single-cell-transcriptional-profiling-of-a-4eahlwn5oc.pdf

Comprehensive single-cell transcriptional profiling of a multicellular organism

Since its discovery as a protein associated with the cytoplasmic region of E-cadherin, beta-catenin has been shown to perform two apparently unrelated functions: it has a crucial role in cell-cell adhesion in addition to a signaling role as a component of the Wnt/wg pathway. Wnt/wg signaling results in beta-catenin accumulation and transcriptional activation of specific target genes during development. It is now apparent that deregulation of beta-catenin signaling is an important event in the genesis of a number of malignancies, such as colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma pilomatricomas, and prostate cancer. beta-catenin mutations appear to be a crucial step in the progression of a subset of these cancers, suggesting an important role in the control of cellular proliferation or cell death. The APC/beta-catenin pathway is highly regulated and includes players such as GSK3-beta, CBP, Groucho, Axin, Conductin, and TCF. c-MYC and cyclin D1 were recently identified as a key transcriptional targets of this pathway and additional targets are likely to emerge. Published 1999 John Wiley & Sons, Inc.

beta-catenin signaling and cancer.

Acceleration of lymphomagenesis in oncogene-bearing transgenic mice by slow-transforming retroviruses has proven a valuable tool in identifying cooperating oncogenes. We have modified this protocol to search for genes that can collaborate effectively with the transgene in later stages of tumor development. Propagation of tumors induced by Moloney murine leukemia virus (M-MuLV) in E mu-Pim1 or H2-K-myc transgenic mice by transplantation to syngeneic hosts permitted proviral tagging of 'progression' genes. Molecular cloning of common proviral insertion sites that were detected preferentially in transplanted tumors led to the identification of a novel gene, designated Frat1. The initial selection for integrations near Frat1 occurs in primary tumor cells that have already acquired proviruses in other common insertion sites, yielding primary lymphomas that contain only a minor fraction of tumor cells with an activated Frat1 allele. Transplantation of such primary lymphomas allows for a further expansion of tumor cell clones carrying a proviral insertion near Frat1, resulting in detectable Frat1 rearrangements in 17% of the transplanted E mu-Pim1 tumors and 30% of the transplanted H2-K-myc tumors, respectively. We have cloned and sequenced both the mouse Frat1 gene and its human counterpart. The proteins encoded by Frat1 and FRAT1 are highly homologous and their functions are thus far unknown. Tumor cell lines with high expression of Myc and Pim1 acquired an additional selective advantage in vivo upon infection with a Frat1-IRES-lacZ retrovirus, thus underscoring the role of Frat1 in tumor progression, and the ability of Frat1 to collaborate with Pim1 and Myc in lymphomagenesis.

/pdf/activation-of-a-novel-proto-oncogene-frat1-contributes-to-30tjm76vtt.pdf

Activation of a novel proto-oncogene, Frat1, contributes to progression of mouse T-cell lymphomas

Amplification of Tc1 flanking genomic sequence using an anchored PCR based method. (A) Schematic representation of the anchored PCR based approach. Genomic DNA was digested with Sau3A, resulting in small fragments consisting of Tc1 sequence and flanking genomic sequence. To amplify these fragments, a vectorette oligonucleotide anchor was ligated to the digested DNA. The Tc1 flanking fragments were amplified using primers that anneal to Tc1 and the vectorette anchor. Note that vectorette anchors can ligate at both ends of the fragments. The vectorette is, however, constructed in such a way that the vectorette PCR primers can only anneal after a complementary strand has been generated in the first round of PCR by synthesis from the Tc1-specific primer. Therefore, fragments containing only vectorette anchors, but no Tc1 sequence are not amplified. ∗, Internal Tc1 and vectorette primers containing restriction sites for EcoRI and SphI, respectively. (B) Southern blot analysis of the total vectorette PCR product using genomic DNA of a strain with a Tc1 insertion in the gene prk-2, compared with a similar strain without this insertion. Hybridization with a 32P-labeled prk-2 genomic probe shows specific amplification of a prk-2 fragment in the prk-2::Tc1 strain but not in the strain without this insertion.

https://www.pnas.org/content/pnas/93/25/14680.full.pdf

Transposon Tc1-derived, sequence-tagged sites in Caenorhabditis elegans as markers for gene mapping

Expression of a constitutively activated version of the heterotrimeric G protein alpha-subunit Galphas results in the swelling and vacuolization of a specific subset of ventral nerve cord motoneurons of Caenorhabditis elegans. A second site modifier (sgs-1) that completely suppresses this neuronal degeneration has been isolated. sgs-1 was cloned and was shown to encode an adenylyl cyclase which is most similar to mammalian adenylyl cyclase type IX. Mutations in sgs-1 change residues that are conserved among different adenylyl cyclases. These mutations are located in the two catalytic domains and in the first multiple transmembrane spanning region of the predicted protein. An sgs-1 reporter construct shows a general neuronal expression pattern, demonstrating that sgs-1 is expressed in the neurons that are susceptible to activated Galphas-induced cell death. A second C.elegans adenylyl cyclase gene (acy-2) was analyzed as well. In contrast to sgs-1, acy-2 shows a restricted expression pattern and loss of acy-2 function results in early larval lethality. These results suggest that SGS-1 is a target of Galphas signaling in motoneurons, whereas an interaction of Galphas with ACY-2, probably in the canal-associated neurons, is required for viability.

/pdf/g-protein-hyperactivation-of-the-caenorhabditis-elegans-3t9ewartnz.pdf

G protein hyperactivation of the Caenorhabditis elegans adenylyl cyclase SGS‐1 induces neuronal degeneration

https://www.cell.com/current-biology/pdf/S0960-9822(03)00164-7.pdf

Hendrik C. Korswagen

Papers

Activation of a novel proto-oncogene, Frat1, contributes to progression of mouse T-cell lymphomas

Transposon Tc1-derived, sequence-tagged sites in Caenorhabditis elegans as markers for gene mapping

G protein hyperactivation of the Caenorhabditis elegans adenylyl cyclase SGS‐1 induces neuronal degeneration

Hyperactivation of the G12-Mediated Signaling Pathway in Caenorhabditis elegans Induces a Developmental Growth Arrest via Protein Kinase C