p120 Catenin is required for normal tubulogenesis but not epithelial integrity in developing mouse pancreas.

The intracellular protein p120 catenin aids in maintenance of cell-cell adhesion by regulating E-cadherin stability in epithelial cells. In an effort to understand the biology of p120 catenin in pancreas development, we ablated p120 catenin in mouse pancreatic progenitor cells, which resulted in deletion of p120 catenin in all epithelial lineages of the developing mouse pancreas: islet, acinar, centroacinar, and ductal. Loss of p120 catenin resulted in formation of dilated epithelial tubules, expansion of ductal epithelia, loss of acinar cells, and the induction of pancreatic inflammation. Aberrant branching morphogenesis and tubulogenesis were also observed. Throughout development, the phenotype became more severe, ultimately resulting in an abnormal pancreas comprised primarily of duct-like epithelium expressing early progenitor markers. In pancreatic tissue lacking p120 catenin, overall epithelial architecture remained intact; however, actin cytoskeleton organization was disrupted, an observation associated with increased cytoplasmic PKCζ. Although we observed reduced expression of adherens junction proteins E-cadherin, ß-catenin, and α-catenin, p120 catenin family members p0071, ARVCF, and δ-catenin remained present at cell membranes in homozygous p120(f/f) pancreases, potentially providing stability for maintenance of epithelial integrity during development. Adult mice homozygous for deletion of p120 catenin displayed dilated main pancreatic ducts, chronic pancreatitis, acinar to ductal metaplasia (ADM), and mucinous metaplasia that resembles PanIN1a. Taken together, our data demonstrate an essential role for p120 catenin in pancreas development.

The tumor suppressor rpl36 restrains KRAS(G12V)-induced pancreatic cancer.

Ribosomal proteins are known to be required for proper assembly of mature ribosomes. Recent studies indicate an additional role for ribosomal proteins as candidate tumor suppressor genes. Pancreatic acinar cells, recently identified as effective cells of origin for pancreatic adenocarcinoma, display especially high-level expression of multiple ribosomal proteins. We, therefore, functionally interrogated the ability of two ribosomal proteins, rpl36 and rpl23a, to alter the response to oncogenic Kras in pancreatic acinar cells using a newly established model of zebrafish pancreatic cancer. These studies reveal that rpl36, but not rpl23a, acts as a haploinsufficient tumor suppressor, as manifested by more rapid tumor progression and decreased survival in rpl36(hi1807/+);ptf1a:gal4VP16(Tg);UAS:GFP-KRAS(G12V) fish compared with their rpl36(+/+);ptf1a:gal4VP16;UAS:GFP-KRAS(G12V) siblings. These results suggest that rpl36 may function as an effective tumor suppressor during pancreatic tumorigenesis.

Annotation of the zebrafish genome through an integrated transcriptomic and proteomic analysis.

Accurate annotation of protein-coding genes is one of the primary tasks upon the completion of whole genome sequencing of any organism. In this study, we used an integrated transcriptomic and proteomic strategy to validate and improve the existing zebrafish genome annotation. We undertook high-resolution mass-spectrometry-based proteomic profiling of 10 adult organs, whole adult fish body, and two developmental stages of zebrafish (SAT line), in addition to transcriptomic profiling of six organs. More than 7,000 proteins were identified from proteomic analyses, and ∼ 69,000 high-confidence transcripts were assembled from the RNA sequencing data. Approximately 15% of the transcripts mapped to intergenic regions, the majority of which are likely long non-coding RNAs. These high-quality transcriptomic and proteomic data were used to manually reannotate the zebrafish genome. We report the identification of 157 novel protein-coding genes. In addition, our data led to modification of existing gene structures including novel exons, changes in exon coordinates, changes in frame of translation, translation in annotated UTRs, and joining of genes. Finally, we discovered four instances of genome assembly errors that were supported by both proteomic and transcriptomic data. Our study shows how an integrative analysis of the transcriptome and the proteome can extend our understanding of even well-annotated genomes.

C1q/TNF-related protein 4 (CTRP4) is a unique secreted protein with two tandem C1q domains that functions in the hypothalamus to modulate food intake and body weight.

CTRP4 is a unique member of the C1q family, possessing two tandem globular C1q domains. Its physiological function is poorly defined. Here, we show that CTRP4 is an evolutionarily conserved, ∼34-kDa secretory protein expressed in the brain. In human, mouse, and zebrafish brain, CTRP4 expression begins early in development and is widespread in the central nervous system. Neurons, but not astrocytes, express and secrete CTRP4, and secreted proteins form higher-order oligomeric complexes. CTRP4 is also produced by peripheral tissues and circulates in blood. Its serum levels are increased in leptin-deficient obese (ob/ob) mice. Functional studies suggest that CTRP4 acts centrally to modulate energy metabolism. Refeeding following an overnight fast induced the expression of CTRP4 in the hypothalamus. Central administration of recombinant protein suppressed food intake and altered the whole-body energy balance in both chow-fed and high-fat diet-fed mice. Suppression of food intake by CTRP4 is correlated with a decreased expression of orexigenic neuropeptide (Npy and Agrp) genes in the hypothalamus. These results establish CTRP4 as a novel nutrient-responsive central regulator of food intake and energy balance.

Multiple ribosomal proteins are expressed at high levels in developing zebrafish endoderm and are required for normal exocrine pancreas development.

Ribosomal protein L (rpl) genes are essential for assembly of the 60S subunit of the eukaryotic ribosome and may also carry out additional extra-ribosomal functions. We have identified a common expression pattern for rpl genes in developing zebrafish larvae. After initially widespread expression in early embryos, the expression of multiple rpl genes becomes increasingly restricted to the endoderm. With respect to the pancreas, rpl genes are highly expressed in ptf1a-expressing pancreatic progenitors at 48 hpf, suggesting possible functional roles in pancreatic morphogenesis and/or differentiation. Utilizing two available mutant lines, rpl23a(hi2582) and rpl6(hi3655b), we found that ptf1a-expressing pancreatic progenitors fail to properly expand in embryos homozygous for either of these genes. In addition to these durable homozygous phenotypes, we also demonstrated recoverable delays in ptf1a-expressing pancreatic progenitor expansion in rpl23a(hi2582) and rpl6(hi3655b) heterozygotes. Disruptions in ribosome assembly are generally understood to initiate a p53-dependent cellular stress response. However, concomitant p53 knockdown was unable to rescue normal pancreatic progenitor expansion in either rpl23a(hi2582) or rpl6(hi3655b) mutant embryos, suggesting required and p53-independent roles for rpl23a and rpl6 in pancreas development.

Ribosomal biogenesis genes play an essential and p53-independent role in zebrafish pancreas development.

Mutations in the human Shwachman-Bodian-Diamond syndrome (SBDS) gene cause defective ribosome assembly and are associated with exocrine pancreatic insufficiency, chronic neutropenia and skeletal defects. However, the mechanism underlying these phenotypes remains unclear. Here we show that knockdown of the zebrafish sbds ortholog fully recapitulates the spectrum of developmental abnormalities observed in the human syndrome, and further implicate impaired proliferation of ptf1a-expressing pancreatic progenitor cells as the basis for the observed pancreatic phenotype. It is thought that diseases of ribosome assembly share a p53-dependent mechanism. However, loss of p53 did not rescue the developmental defects associated with loss of zebrafish sbds. To clarify the molecular mechanisms underlying the observed organogenesis defects, we performed transcriptional profiling to identify candidate downstream mediators of the sbds phenotype. Among transcripts displaying differential expression, functional group analysis revealed marked enrichment of genes related to ribosome biogenesis, rRNA processing and translational initiation. Among these, ribosomal protein L3 (rpl3) and pescadillo (pes) were selected for additional analysis. Similar to knockdown of sbds, knockdown or mutation of either rpl3 or pes resulted in impaired expansion of pancreatic progenitor cells. The pancreatic phenotypes observed in rpl3- and pes-deficient embryos were also independent of p53. Together, these data suggest novel p53-independent roles for ribosomal biogenesis genes in zebrafish pancreas development.

Cyclic caged morpholinos: conformationally gated probes of embryonic gene function.

Feeling a bit cagey: morpholino-based antisense reagents have been caged through oligonucleotide cyclization, enabling photocontrol of gene expression in zebrafish embryos and larvae. Using these reagents, the timing of exocrine cell fate commitment in the developing pancreas has been examined.

In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters.

Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae.

Graded levels of Ptf1a differentially regulate endocrine and exocrine fates in the developing pancreas.

The mechanisms regulating pancreatic endocrine versus exocrine fate are not well defined. By analyzing the effects of Ptf1a partial loss of function, we uncovered novel roles for this transcription factor in determining pancreatic fates. In a newly identified hypomorphic ptf1a mutant, pancreatic cells that would normally express ptf1a and become exocrine cells, express the endocrine marker Isl1, indicating a cell fate switch. Surprisingly, a milder reduction of Ptf1a leads to an even greater increase of ectopic endocrine cells, suggesting that Ptf1a also plays a role in promoting endocrine development. We propose that low levels of Ptf1a promote endocrine fate, whereas high levels repress endocrine fate and promote exocrine fate.

Nonapical symmetric divisions underlie horizontal cell layer formation in the developing retina in vivo.

Symmetric cell divisions have been proposed to rapidly increase neuronal number late in neurogenesis, but how critical this mode of division is to establishing a specific neuronal layer is unknown. Using in vivo time-lapse imaging methods, we discovered that in the laminated zebrafish retina, the horizontal cell (HC) layer forms quickly during embryonic development upon division of a precursor cell population. The precursor cells morphologically resemble immature, postmitotic HCs and express HC markers such as ptf1a and Prox1 prior to division. These precursors undergo nonapical symmetric division at the laminar location where mature HCs contact photoreceptors. Strikingly, the precursor cell type we observed generates exclusively HCs. We have thus identified a dedicated HC precursor, and our findings suggest a mechanism of neuronal layer formation whereby the location of mitosis could facilitate rapid contact between synaptic partners.

Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos.

We have adapted a novel multicistronic gene expression system involving viral peptides to the zebrafish. The viral 2A peptide allows production of multiple protein products from a single transgene. Based on highly inefficient peptide bond formation between glycine and proline residues within the 2A peptide, placement of 2A peptide sequence as a linker region between tandem cDNA's allows the stoichiometric translation of multiple unfused protein products. To test this system in zebrafish, we generated two different tandem reporter constructs employing eGFP and mCherry, separated by 2A peptide sequence. Using this system, we produced transgenic zebrafish in which fluorophores were produced as independent proteins from a single transcript. The successful application of this technology in zebrafish will be valuable for visually marking transgenic embryos and transgene-expressing cells, or in any situation where reliable expression of multiple transgenes is desired.

The Suppressor of Killer of prune, a unique glutathione S-transferase.

The prune-Killer of prune conditional dominant, lethal interaction in Drosophila was identified in the 1950s, but its mechanism remains unknown. We undertook a genetic screen for suppressors of this lethal interaction and identified a gene we named, Suppressor of Killer of prune Su(Kpn). Su(Kpn) is a unique protein with four N-terminal FLYWCH zinc-finger domains, an acidic domain and a C-terminal glutathione S-transferase (GST) domain. The GST domain of Su(Kpn) is of particular interest because GSTs are usually independent of other protein domains. While GSTs are generally thought of as detoxifying enzymes, they are also associated with cellular toxicity. We predict that the GST domain of the Su(Kpn) creates a toxic product in prune-Killer of prune flies that is lethal. The substrate of the Su(Kpn) remains unknown.

Loss-of-function mutations in a glutathione S-transferase suppress the prune-Killer of prune lethal interaction.

The prune gene of Drosophila melanogaster is predicted to encode a phosphodiesterase. Null alleles of prune are viable but cause an eye-color phenotype. The abnormal wing discs gene encodes a nucleoside diphosphate kinase. Killer of prune is a missense mutation in the abnormal wing discs gene. Although it has no phenotype by itself even when homozygous, Killer of prune when heterozygous causes lethality in the absence of prune gene function. A screen for suppressors of transgenic Killer of prune led to the recovery of three mutations, all of which are in the same gene. As heterozygotes these mutations are dominant suppressors of the prune-Killer of prune lethal interaction; as homozygotes these mutations cause early larval lethality and the absence of imaginal discs. These alleles are loss-of-function mutations in CG10065, a gene that is predicted to encode a protein with several zinc finger domains and glutathione S-transferase activity.

Functional correlates of mutation of the Asp32 and Gly34 residues of beta-catenin.

Beta-catenin is a multifunctional protein involved in both cadherin-mediated adhesion and the wnt signaling cascade. Mutations in exon 3 of beta-catenin have been identified in many cancers. In addition to disruption of key serine and threonine residues, mutations are frequently reported in other residues in exon 3 that are not kinase substrates. The most frequently mutated nonserine/threonine residues are D32 and G34. Since D32 and G34 are part of the ubiquitination destruction motif, DSGPhiXS, we hypothesize that this motif may contribute to disruption of beta-catenin homeostasis and lead to cellular transformation. We demonstrate that the mutants D32A and G34A exhibit no change in phosphorylation by GSK3beta, but display reduced ubiquitination compared to wild-type and S33A mutant beta-catenin. To assess the functional implications of these mutations, we created stable MDCK cell lines expressing these constructs. We found that stable cell lines harboring D32A-mutated beta-catenin were highly transformed, while S33A and G34 demonstrated only weak transforming properties in our assays. Despite altered ubiquitination status and increased transformation, the D32A mutant cell line does not display transcriptional activation of standard target genes. Therefore, D32A mutation may mediate transformation by an alternative beta-catenin-mediated signaling pathway.

beta-Catenin functions mainly as an adhesion molecule in patients with squamous cell cancer of the head and neck.

BACKGROUND: beta-catenin, depending on subcellular localization, plays a dual role in carcinogenesis: as a signaling factor (in the nucleus) and as an adhesion molecule (in cell membrane). In this study, we sought to determine the role of beta-catenin in head and neck carcinogenesis. METHODS: First, we studied the incidence of mutations of beta-catenin in a cohort of 60 head and neck squamous cell cancers (HNSCC). We subsequently evaluated the protein expression levels of beta-catenin in a cohort of oropharyngeal squamous cell cancer tissue microarray using a novel in situ method of quantitative protein analysis and correlated those with cyclin D1 levels and clinical and pathologic data. RESULTS: The mean follow-up time for survivors was 45 months and for all patients was 35 months. We found no mutations in the cohort of 60 HNSCC. beta-catenin displayed primarily membranous expression pattern. Patients with high tumor-node-metastasis stage were more likely to have high expression of beta-catenin (P = 0.040). Patients with low beta-catenin expression had a local recurrence rate of 79% compared with 29% for patients with high beta-catenin tumors (P = 0.0021). Univariate Cox regression revealed a hazard ratio for low beta-catenin tumors of 3.6 (P = 0.004). Kaplan-Meier analysis showed that patients with low beta-catenin expressing tumors trended toward worse 5-year disease-free survival (P = 0.06). In multivariate analysis, only beta-catenin expression status was an independent prognostic factor (P = 0.044) for local recurrence. Tumors with high beta-catenin had low cyclin D1 and vice versa (P = 0.007). CONCLUSIONS: The absence of activating beta-catenin mutations combined with the inverse correlation between beta-catenin levels with cyclin D1 levels and outcome suggest that beta-catenin mainly functions as an adhesion and not signaling molecule in HNSCC.

Functional correlates of mutations in beta-catenin exon 3 phosphorylation sites.

beta-Catenin-mediated signaling can be constitutively activated by truncation or mutation of serine and threonine residues in exon 3. Mutations in this region are observed in many human tumors. Examination of the locations of these mutations reveals interesting patterns; specifically, Ser45 and Thr41 appear more frequently in malignant tumors, and Ser37 and Ser33 are more common in benign entities. To test whether these patterns represent functional differences in beta-catenin signaling mechanisms, we generated mutations of each of these residues. Stable transformation of Madin-Darby canine kidney cells showed a transformed phenotype with each of the four mutations, as assessed by growth in soft agar and collagen. Functional assays including proliferation assays, cell shedding assays, and wounding assays demonstrated two groups. Ser45 and Thr41 represent a more transformed phenotype, whereas Ser37 and Ser33 behaved similarly to the vector in these assays. Assessment of downstream genes demonstrated increased activation of the beta-catenin target gene cyclin D1 by Ser45. Finally, we examined the kinase activity of I kappa B kinase-alpha and found that this kinase, unlike glycogen synthase kinase-3 beta, appears to preferentially phosphorylate Ser45 and Thr41, independent of priming by casein kinase-1. We conclude that these sites may represent an alternative (non-wnt) signaling pathway, which may be inappropriately activated in tumors with mutations of these residues.

Tissue microarray-based analysis shows phospho-beta-catenin expression in malignant melanoma is associated with poor outcome.

Beyond depth of invasion, there are very few prognostic markers to predict outcome in melanoma. It has been shown recently that the beta-catenin oncogene is mutated or shows altered subcellular localization suggesting that activation of beta-catenin mediated signaling plays a role in oncogenesis. We hypothesize that assessment of activated beta-catenin, as detected by a phospho-specific antibody, may be useful to predict outcome in melanoma. We use immuno-histochemical analysis of beta-catenin and phospho-beta-catenin, first to verify the specificity of the phospho-beta-catenin antibody and then to assay expression in a tissue microarray-based study. The subcellular localization of beta-catenin is membranous in some cases and cytoplasmic and nuclear in others. We validate the specificity of a ser33/37/thr41 phospho-beta-catenin antibody in transfected cells and show that the expression is almost exclusively localized to the nucleus in both cultured cells and human tissue. Evaluation of both total and phospho-beta-catenin antibodies showed that cytoplasmic/nuclear staining was more common in primary lesions, whereas nuclear phospho-beta-catenin was more common in metastatic lesions. High levels of nuclear phospho-beta-catenin are associated with significantly worse overall survival (51% vs. 25% overall survival at 5 years, p = 0.046). These results suggest that phospho-specific antibodies to beta-catenin define a unique subset of cases and that monitoring of phospho-beta-catenin expression may be useful for assessing prognosis in malignant melanoma.