Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration.

Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation.

RNA-mediated interaction of Cajal bodies and U2 snRNA genes.

Cajal bodies (CBs) are nuclear structures involved in RNA metabolism that accumulate high concentrations of small nuclear ribonucleoproteins (snRNPs). Notably, CBs preferentially associate with specific genomic loci in interphase human cells, including several snRNA and histone gene clusters. To uncover functional elements involved in the interaction of genes and CBs, we analyzed the expression and subcellular localization of stably transfected artificial arrays of U2 snRNA genes. Although promoter substitution arrays colocalized with CBs, constructs containing intragenic deletions did not. Additional experiments identified factors within CBs that are important for association with the native U2 genes. Inhibition of nuclear export or targeted degradation of U2 snRNPs caused a marked decrease in the levels of U2 snRNA in CBs and strongly disrupted the interaction with U2 genes. Together, the results illustrate a specific requirement for both the snRNA transcripts as well as the presence of snRNPs (or snRNP proteins) within CBs. Our data thus provide significant insight into the mechanism of CB interaction with snRNA loci, strengthening the putative role for this nuclear suborganelle in snRNP biogenesis.

Stimulation of protein kinase C-dependent and -independent signaling pathways by bistratene A in intestinal epithelial cells.

The marine toxin bistratene A (BisA) potently induces cytostasis and differentiation in a variety of systems. Evidence that BisA is a selective activator of protein kinase C (PKC) delta implicates PKC delta signaling in the negative growth-regulatory effects of this agent. The current study further investigates the signaling pathways activated by BisA by comparing its effects with those of the PKC agonist phorbol 12-myristate 13-acetate (PMA) in the IEC-18 intestinal crypt cell line. Both BisA and PMA induced cell cycle arrest in these cells, albeit with different kinetics. While BisA produced sustained cell cycle arrest in G(0)/G(1) and G(2)/M, the effects of PMA were transient and involved mainly a G(0)/G(1) blockade. BisA also produced apoptosis in a proportion of the population, an effect not seen with PMA. Both agents induced membrane translocation/activation of PKC, with BisA translocating only PKC delta and PMA translocating PKC alpha, delta, and epsilon in these cells. Notably, while depletion of PKC alpha, delta, and epsilon abrogated the cell cycle-specific effects of PMA in IEC-18 cells, the absence of these PKC isozymes failed to inhibit BisA-induced G(0)/G(1) and G(2)/M arrest or apoptosis. The cell cycle inhibitory and apoptotic effects of BisA, therefore, appear to be PKC-independent in IEC-18 cells. On the other hand, BisA and PMA both promoted PKC-dependent activation of Erk 1 and 2 in this system. Thus, intestinal epithelial cells respond to BisA through activation of at least two signaling pathways: a PKC delta-dependent pathway, which leads to activation of mitogen-activated protein kinase and possibly cytostasis in the appropriate context, and a PKC-independent pathway, which induces both cell cycle arrest in G(0)/G(1) and G(2)/M and apoptosis through as yet unknown mechanisms.

Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium.

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

Coiled bodies preferentially associate with U4, U11, and U12 small nuclear RNA genes in interphase HeLa cells but not with U6 and U7 genes.

Coiled bodies (CBs) are nuclear organelles involved in the metabolism of small nuclear RNAs (snRNAs) and histone messages. Their structural morphology and molecular composition have been conserved from plants to animals. CBs preferentially and specifically associate with genes that encode U1, U2, and U3 snRNAs as well as the cell cycle-regulated histone loci. A common link among these previously identified CB-associated genes is that they are either clustered or tandemly repeated in the human genome. In an effort to identify additional loci that associate with CBs, we have isolated and mapped the chromosomal locations of genomic clones corresponding to bona fide U4, U6, U7, U11, and U12 snRNA loci. Unlike the clustered U1 and U2 genes, each of these loci encode a single gene, with the exception of the U4 clone, which contains two genes. We next examined the association of these snRNA genes with CBs and found that they colocalized less frequently than their multicopy counterparts. To differentiate a lower level of preferential association from random colocalization, we developed a theoretical model of random colocalization, which yielded expected values for chi2 tests against the experimental data. Certain single-copy snRNA genes (U4, U11, and U12) but not controls were found to significantly (p < 0.000001) associate with CBs. Recent evidence indicates that the interactions between CBs and genes are mediated by nascent transcripts. Taken together, these new results suggest that CB association may be substantially augmented by the increased transcriptional capacity of clustered genes. Possible functional roles for the observed interactions of CBs with snRNA genes are discussed.

Association of snRNA genes with coiled bodies is mediated by nascent snRNA transcripts.

BACKGROUND: Coiled bodies are nuclear organelles that are highly enriched in small nuclear ribonucleoproteins (snRNPs) and certain basal transcription factors. Surprisingly, coiled bodies not only contain mature U snRNPs but also associate with specific chromosomal loci, including gene clusters that encode U snRNAs and histone messenger RNAs. The mechanism(s) by which coiled bodies associate with these genes is completely unknown. RESULTS: Using stable cell lines, we show that artificial tandem arrays of human U1 and U2 snRNA genes colocalize with coiled bodies and that the frequency of the colocalization depends directly on the transcriptional activity of the array. Association of the genes with coiled bodies was abolished when the artificial U2 arrays contained promoter mutations that prevent transcription or when RNA polymerase II transcription was globally inhibited by alpha-amanitin. Remarkably, the association was also abolished when the U2 snRNA coding regions were replaced by heterologous sequences. CONCLUSIONS: The requirement for the U2 snRNA coding region indicates that association of snRNA genes with coiled bodies is mediated by the nascent U2 RNA itself, not by DNA or DNA-bound proteins. Our data provide the first evidence that association of genes with a nuclear organelle can be directed by an RNA and suggest an autogenous feedback regulation model.

Colonocyte differentiation is associated with increased expression and altered distribution of protein kinase C isozymes.

BACKGROUND & AIMS: Colon cancer cells express reduced levels of protein kinase C (PKC). This study examines the regulation of PKC isozymes in normal colonic epithelium, as a basis for understanding the significance of alterations in this enzyme system in colon carcinogenesis. METHODS: The expression and localization of PKC isozymes in mouse and rat colonocytes at different developmental stages were determined using a combined morphological and biochemical approach. PKC alpha expression was compared in colonic adenocarcinomas and adjacent normal mucosa by immunoblot analysis. RESULTS: Mouse and rat colonocytes express PKC alpha, beta II, delta, epsilon, and zeta. Relatively low levels of these isozymes were detected in proliferating cells of the crypt base, predominantly in the cytosolic compartment. Coincident with colonocyte growth arrest/differentiation, PKC isozyme expression markedly increased in both the cytosolic and, more significantly, in the membrane/cytoskeletal fraction. Colonic tumors express reduced levels of PKC alpha, an isozyme that has been implicated in negative control of intestinal cell growth. CONCLUSIONS: These findings are supportive of a role for certain PKC isozyme(s) in signaling pathways mediating postmitotic events in colonocytes in situ, and suggest that diminished activity of these pathway(s) may contribute to the alterations in growth control/differentiation associated with colonic neoplasia.

The U22 host gene (UHG): chromosomal localization of UHG and distribution of U22 small nucleolar RNA.

The U22 host gene (UHG) is very unusual because it encodes a spliced, polyadenylated RNA that has little apparent coding capacity and is rapidly degraded. The stable RNA products from this locus are actually encoded within eight different introns of the UHG pre-RNA. These small nucleolar RNAs (snoRNAs) assemble into ribonucleoproteins, some of which have been shown to function in rRNA processing and modification. In order to more fully characterize the locus, we have mapped UHG to chromosome 11q13 by fluorescence in situ hybridization (FISH). Radiation hybrid mapping placed this sequence-tagged site with very high probability (lod >19) to chromosome 11, approximately 10.1 cR distal to framework marker WI-8652. We also investigated the possibility that the expression of UHG was subject to genomic imprinting. Several laboratories have shown that non-protein-coding mRNAs are frequently associated with imprinted domains in mammalian cells. We used a novel somatic cell hybrid method to assay parent-of-origin effects in the expression of UHG alleles and found that, unlike XIST, IPW, and H19, this RNA is expressed biparentally. Additional FISH experiments using anti-U22 oligonucleotides revealed that, as with U3, this sno-RNA is localized throughout the nucleolus.

Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro.

The human parvovirus adeno-associated virus (AAV) is unique in its ability to target viral integration to a specific site on chromosome 19 (ch-19). Recombinant AAV (rAAV) vectors retain the ability to integrate but have apparently lost this ability to target. In this report, we characterize the terminal-repeat-mediated integration for wild-type (wt), rAAV, and in vitro systems to gain a better understanding of these differences. Cell lines latent for either wt or rAAV were characterized by a variety of techniques, including PCR, Southern hybridization, and fluorescence in situ hybridization analysis. More than 40 AAV-rAAV integration junctions were cloned, sequenced, and then subjected to comparison and analysis. In both immortalized and normal diploid human cells, wt AAV targeted integration to ch-19. Integrated provirus structures consisted of head-to-tail tandem arrays with the majority of the junction sequences involving the AAV inverted terminal repeats (ITRs). No complete viral ITRs were directly observed. In some examples, the AAV p5 promoter sequence was found to be fused at the virus-cell junction. Data from dot blot analysis of PCR products were consistent with the occurrence of inversions of genomic and/or viral DNA sequences at the wt integration site. Unlike wt provirus junctions, rAAV provirus junctions mapped to a subset of non-ch-19 sequences. Southern analysis supported the integration of proviruses from two independent cell lines at the same locus on ch-2. In addition, provirus terminal repeat sequences existed in both the flip and flop orientations, with microhomology evident at the junctions. In all cases with the exception of the ITRs, the vector integrated intact. rAAV junction sequence data were consistent with the occurrence of genomic rearrangement by deletion and/or rearrangement-translocation at the integration locus. Finally, junctions formed in an in vitro system between several AAV substrates and the ch-19 target site were isolated and characterized. Linear AAV substrates typically utilized the end of the virus DNA substrate as the point of integration, whereas products derived from AAV terminal repeat hairpin structures in the presence or absence of Rep protein resembled AAV-ch-19 junctions generated in vivo. These results describing wt AAV, rAAV, and in vitro integration junctions suggest that the viral integration event itself is mediated by terminal repeat hairpin structures via nonviral cellular recombination pathways, with specificity for ch-19 in vivo requiring additional viral components. These studies should have an important impact on the use of rAAV vectors in human gene therapy.

Protein kinase C isozyme-mediated cell cycle arrest involves induction of p21(waf1/cip1) and p27(kip1) and hypophosphorylation of the retinoblastoma protein in intestinal epithelial cells.

The molecular mechanisms underlying protein kinase C (PKC) isozyme-mediated control of cell growth and cell cycle progression are poorly understood. Our previous analysis of PKC isozyme regulation in the intestinal epithelium in situ revealed that multiple members of the PKC family undergo changes in expression and subcellular distribution precisely as the cells cease proliferating in the mid-crypt region, suggesting that activation of one or more of these molecules is involved in negative regulation of cell growth in this system (Saxon, M. L., Zhao, X., and Black, J. D. (1994) J. Cell Biol. 126, 747-763). In the present study, the role of PKC isozyme(s) in control of intestinal epithelial cell growth and cell cycle progression was examined directly using the IEC-18 immature crypt cell line as a model system. Treatment of IEC-18 cells with PKC agonists resulted in translocation of PKC alpha, delta, and epsilon from the soluble to the particulate subcellular fraction, cell cycle arrest in G1 phase, and delayed transit through S and/or G2/M phases. PKC-mediated cell cycle arrest in G1 was accompanied by accumulation of the hypophosphorylated, growth-suppressive form of the retinoblastoma protein and induction of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). Reversal of these cell cycle regulatory effects was coincident with activator-induced down-regulation of PKC alpha, delta, and epsilon. Differential down-regulation of individual PKC isozymes revealed that PKC alpha in particular is sufficient to mediate cell cycle arrest by PKC agonists in this system. Taken together, the data implicate PKC alpha in negative regulation of intestinal epithelial cell growth both in vitro and in situ via pathways which involve modulation of Cip/Kip family cyclin-dependent kinase inhibitors and the retinoblastoma growth suppressor protein.

Human genes encoding U3 snRNA associate with coiled bodies in interphase cells and are clustered on chromosome 17p11.2 in a complex inverted repeat structure.

Coiled bodies (CBs) are nuclear organelles whose morphological structure and molecular composition have been conserved from plants to animals. Furthermore, CBs are often found to co-localize with specific DNA loci in both mammalian somatic nuclei and amphibian oocytes. Much as rDNA sequences are called nucleolus organizers, we term these coiled body-associated sequences 'coiled body organizers' (CBORs). The only sequences that have been shown to be CBORs in human cells are the U1, U2 and histone gene loci. We wanted to determine whether other snRNA genes might also act as CBORs. In this paper we show that human U3 genes (the RNU3 locus) preferentially associate with CBs in interphase cells. In addition, we have analyzed the genomic organization of the RNU3 locus by constructing a BAC and P1 clone contig. We found that, unlike the RNU1 and RNU2 loci, U3 genes are not tandemly repeated. Rather, U3 genes are clustered on human chromosome 17p11.2, with evidence for large inverted duplications within the cluster. Thus all of the CBORs identified to date are composed of either tandemly repeated or tightly clustered genes. The evolutionary and cell biological consequences of this type of organization are discussed.

Characterization of the mouse histone gene cluster on chromosome 13: 45 histone genes in three patches spread over 1Mb.

The histone gene cluster on mouse chromosome 13 has been isolated and characterized. Using overlapping YAC clones containing histone genes from chromosome 13, a contig of approximately 2 Mb has been defined. It contains 45 histone genes, organized in three patches containing tightly clustered genes. An 80-kb patch (patch III) containing 12 histone genes is near one end of the contig, and a similar-sized patch (patch I) containing 15 histone genes is near the other end of the contig, located at least 500 kb from the central patch (patch II) of histone genes. The entire cluster contains six histone H1 genes, including the testis-specific histone H1t gene that maps to the middle of the cluster. All nine histone H3 genes in this cluster have been sequenced, and their level of expression determined. Each histone H3 gene is distinct, with five genes encoding the H3.2 protein subtype and four genes encoding the H3.1 protein. They are all expressed, with each histone H3 gene accounting for a small proportion of the total histone H3 mRNA.

Characterization of the 55-kb mouse histone gene cluster on chromosome 3.

The histone gene cluster on mouse chromosome 3 has been isolated as a series of overlapping P1 clones, covering 110-120 kb, by probing with the histone H3-614 gene that had been mapped previously to mouse chromosome 3. There are genes for 10 core histone proteins present in a 55-kb cluster within this contig. There are three histone H3 genes, two of which are identical; four histone H2a genes, two of which are identical, one histone H4 gene; and two histone H2b genes. These histone H3 and H2a genes encode approximately 40% of the total H3 and H2a mRNA, whereas the histone H4 and histone H2b genes encode < 10% of the total H4 and H2b mRNA. There are no histone H1 genes present in this cluster. All of the histone H2a genes encode histone H2a.2 proteins (or variants of H2a.2), and account for all the H2a.2 genes in the mouse genome. All three histone H3 genes encode the histone H3.2 protein. A 21-kb region containing the adjacent H3-614 and H2a-614 genes has been duplicated and is present in an inverted repeat separated by 4.5 kb. The other two H2a genes are adjacent, with the 3' ends of their mRNAs separated by only 49 nucleotides in the DNA and the U7 snRNP binding sites separated by only 20 nucleotides. One of the histone H2b genes has lost the stem-loop sequence characteristic of the replication-dependent histone mRNAs and encodes only polyadenylated mRNAs.

Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells.

Coiled bodies (CBs) are nuclear organelles whose structures appear to be highly conserved in evolution. In rapidly cycling cells, they are typically located in the nucleoplasm but are often found in contact with the nucleolus. The CBs in human cells contain a unique protein, called p80-coilin. Studies on amphibian oocyte nuclei have revealed a protein within the "sphere" organelle that shares significant structural similarity to p80-coilin. Spheres and CBs are also highly enriched in small nuclear ribonucleoproteins and other RNA-processing components. We present evidence that, like spheres, CBs contain U7 small nuclear RNA (snRNA) and associate with specific chromosomal loci. Using biotinylated 2'-O-methyl oligonucleotides complementary to the 5' end of U7 snRNA and fluorescence in situ hybridization, we show that U7 is distributed throughout the nucleoplasm, excluding nucleoli, and is concentrated in CBs. Interestingly, we found that CBs often associate with subsets of the histone, U1, and U2 snRNA gene loci in interphase HeLa-ATCC and HEp-2 monolayer cells. However, in a strain of suspension-grown HeLa cells, called HeLa-JS1000, we found a much lower rate of association between CBs and snRNA genes. Possible roles for CBs in the metabolism of these various histone and snRNAs are discussed.

A perinucleolar compartment contains several RNA polymerase III transcripts as well as the polypyrimidine tract-binding protein, hnRNP I.

We have investigated the subcellular organization of the four human Y RNAs. These RNAs, which are transcribed by RNA polymerase III, are usually found complexed with the Ro autoantigen, a 60-kD protein. We designed 2'-OMe oligoribonucleotides that were complementary to accessible single-stranded regions of Y RNAs within Ro RNPs and used them in fluorescence in situ hybridization. Although all four Y RNAs were primarily cytoplasmic, oligonucleotides directed against three of the RNAs hybridized to discrete structures near the nucleolar rim. We have termed these structures "perinucleolar compartments" (PNCs). Double labeling experiments with appropriate antisera revealed that PNCs are distinct from coiled bodies and fibrillar centers. Co-hybridization with a genomic DNA clone spanning the human Y1 and Y3 genes showed that PNCs are not stably associated with the transcription site for these Y RNAs. Although 5S rDNA was often located near the nucleolar periphery, PNCs are not associated with 5S gene loci. Two additional pol III transcripts, the RNA components of RNase P and RNase MRP, did colocalize within PNCs. Most interestingly, the polypyrimidine tract-binding protein hnRNP I/PTB was also concentrated in this compartment. Possible roles for this novel nuclear subdomain in macromolecular assembly and/or nucleocytoplasmic shuttling of these five pol III transcripts, along with hnRNP I/PTB, are discussed.