No abstract

We compared the templae properties of a subgenomic RNA that onaed the authentic 5' and 3' ends of the vesicular stomat virus genome with those of RNAs in which the wild-tpe termini were engineered to extend their complementarity from 8 to 51 nucleotldes as seen in defective interfering RNAs. The RNA with authentic 5' and 3' ends dirted abundant t rpton but low replication. In contrast, RNAs with complement termini derived from either end of the genome replicated wellibut tra ibed poorly or not at all.These results have for undtandig the mechanisms of RNA replication and tra iption; they explain the replicative dominance of defective interfering RNAs and demonstrate that the extent ofterminal complementarity rather than its exact sequence is a major determinant of whether the template predominantiy directs nscription or replication.During infection the negative-stranded RNA genome of vesicular stomatitis virus (VSV) directs two distinct RNA synthetic processes: transcription offive discrete capped and polyadenylylated mRNAs and replication of a perfect fulllength copy of the genome. The same core RNA-dependent RNA polymerase is thought to execute both processes using as template a ribonucleoprotein complex ofthe viral genomic RNA. Transcription is the initial and predominant RNA synthetic event; synthesis of nucleocapsid (N) protein to encapsidate nascent genomic RNA is required for replication and is one trans-acting factor that modulates the balance between transcription and replication (1).To examine the role oftemplate sequences in the regulation of these two RNA synthetic processes, we established a system in which the entire replication cycle of a VSV defective interfering (DI) particle was reconstructed from cDNA clones (2). The DI RNA chosen for this work (DI-T) is a member ofthe copy-back family ofDI RNAs in which the 5' 2163 nt are derived from the wild-type (wt) VSV 5' terminus, but the 3' terminus is a 45-nt complement of the 5' terminus (3-6). DI genomes of this type have a replicative advantage over wt VSV and interfere with its replication (4). Deletion of internal regions of the DI-T genomic RNA and replacement with heterologous sequences have shown that the inverted repeats of the 5' end were sufficient to signal RNA replication (A. K. Pattnaik and G.W.W., unpublished data). Since the natural role of the 5' terminal sequence is to act exclusively as an origin of replication during synthesis of full-length'VSV negative strands, its presence at each end of the DI RNA has been proposed to account at least in part for the replicative advantage ofthis class ofDI particle and hence for its ability to interfere with the replication of wt VSV (4).In the work reported here, we took advantage of the ability to recover replicable RNAs from cDNA clones to compare the template properties ofRNA analogues that contained the authentic VSV 5' and 3' termini with those of RNAs engineered to have increasing extents of terminal complementarity. The results showed that RNAs that had the 3' end of the VSV geno...

Infectious defective interfering (DI) particles of the negative-stranded RNA virus vesicular stomatitis virus (VSV) have been recovered from negative-sense transcripts of a plasmid that contains a full-length cDNA derived from the DI-T particle genome. In order to determine the cis-acting sequences necessary for RNA replication, encapsidation, and budding and to approximate the minimal size of RNA that can be packaged into infectious particles, we constructed a series of internal deletions in the DI cDNA to generate plasmids that could be transcribed to yield RNAs which ranged in size from 2209 nucleotides down to 102 nucleotides. All the deletion plasmids retained at least 36 nucleotides from the 5'-terminus and 51 nucleotides from the 3'-terminus of the DI genome. In cells expressing the five VSV proteins, the deleted DI RNAs were examined for their ability to be encapsidated, to replicate, and to bud to produce infectious DI particles. An RNA as small as 191 nucleotides, which contained 46 nucleotides from the 5'-end and 145 nucleotides from the 3'-end of the DI genome was encapsidated, replicated, and budded at least as efficiently as the full-length wild-type DI RNA. In contrast, a 102-nucleotide RNA that contained only the 51 nucleotides from the 5'-end of the DI RNA and its perfect 51-nucleotide complement at the 3'-end replicated poorly and failed to bud infectious DI particles. However, an RNA with an insertion of 1499-nucleotide "stuffer" sequences of non-VSV origin between the two 51-nucleotide complementary termini not only replicated but also budded infectious particles. These data show that the signals necessary for RNA encapsidation, replication, and packaging into infectious DI particles are contained within the 5'-terminal 36 nucleotides and the 3'-terminal 51 nucleotides of the DI RNA genome. Furthermore, the results show that a heterologous sequence can be replicated and packaged into infectious particles if it is flanked by the DI RNA termini.

Interactions between cells from the ectoderm and mesoderm influence development of the endodermally-derived pancreas. While much is known about how mesoderm regulates pancreatic development, relatively little is understood about how and when the ectodermally-derived neural crest regulates pancreatic development and specifically, beta cell maturation. A previous study demonstrated that signals from the neural crest regulate beta cell proliferation and ultimately, beta cell mass. Here, we expand on that work to describe timing of neural crest arrival at the developing pancreatic bud and extend our knowledge of the non-cell autonomous role for neural crest derivatives in the process of beta cell maturation. We demonstrated that murine neural crest entered the pancreatic mesenchyme between the 26 and 27 somite stages (approximately 10.0 dpc) and became intermingled with pancreatic progenitors as the epithelium branched into the surrounding mesenchyme. Using a neural crest-specific deletion of the Forkhead transcription factor Foxd3, we ablated neural crest cells that migrate to the pancreatic primordium. Consistent with previous data, in the absence of Foxd3, and therefore the absence of neural crest cells, proliferation of Insulin-expressing cells and Insulin-positive area are increased. Analysis of endocrine cell gene expression in the absence of neural crest demonstrated that, although the number of Insulin-expressing cells was increased, beta cell maturation was significantly impaired. Decreased MafA and Pdx1 expression illustrated the defect in beta cell maturation; we discovered that without neural crest, there was a reduction in the percentage of Insulin-positive cells that co-expressed Glut2 and Pdx1 compared to controls. In addition, transmission electron microscopy analyses revealed decreased numbers of characteristic Insulin granules and the presence of abnormal granules in Insulin-expressing cells from mutant embryos. Together, these data demonstrate that the neural crest is a critical regulator of beta cell development on two levels: by negatively regulating beta cell proliferation and by promoting beta cell maturation.

A complete molecular understanding of ␤-cell mass expansion will be useful for the improvement of therapies to treat diabetic patients. During normal periods of metabolic challenges, such as pregnancy, ␤-cells proliferate, or self-renew, to meet the new physiological demands. The transcription factor Forkhead box D3 (Foxd3) is required for maintenance and self-renewal of several diverse progenitor cell lineages, and Foxd3 is expressed in the pancreatic primordium beginning at 10.5 d postcoitum, becoming localized predominantly to ␤-cells after birth. Here, we show that mice carrying a pancreas-specific deletion of Foxd3 have impaired glucose tolerance, decreased ␤-cell mass, decreased ␤-cell proliferation, and decreased ␤-cell size during pregnancy. In addition, several genes known to regulate proliferation, Foxm1, Skp2, Ezh2, Akt2, and Cdkn1a, are misregulated in islets isolated from these Foxd3 mutant mice. Together, these data place A pproximately 7% of pregnant women are affected by gestational diabetes mellitus (GDM), a disease resulting from the inability of the resident ␤-cell population to produce sufficient insulin during pregnancy (1). GDM increases the risk of complications to both mother and newborn child; the mother is more likely to develop type 2 diabetes later in life, and the child is more likely to be born with birth defects, macrosomia, and an increased risk of developing type 2 diabetes (2-6). During normal murine pregnancy, ␤-cells proliferate, or self-renew, thereby expanding the total ␤-cell mass to meet the mother's increasing demand for insulin (1,(7)(8)(9)(10)(11)(12). This mechanism of ␤-cell mass expansion during human pregnancy remains controversial. Analogous measurements are not ethically feasible in humans. However, morphological analyses of human pancreata indicate that ␤-cell mass is increased during pregnancy (13,14). Recent work analyzing pancreata from 38 cadaveric donors (18 pregnant, 20 controls) suggested that ␤-cells do not proliferate during human pregnancy. Instead, an increased number of smaller islets and insulin-positive cells was observed within the ductal epithelium (15). However, this work is not without caveats. It is well known that murine ␤-cells proliferate within a defined time window, and it is likely that the 18 pregnant human donors examined were not within an analogous gestational age (16,17). This study also included donors with inflammatory disease that may have adversely affected ␤-cell mass expansion (18). It is important to note that the pregnancy-associated hormones prolactin, placental lactogen (PL), and human growth hormone all stimulate ␤-cell proliferation in islets isolated from mice, rats, and humans, suggesting that cell proliferation is a conserved mechanism of Abbreviations: BrdU, 5-Bromo-2-deoxyuridine; Cdkn, cyclin-dependent kinase inhibitor; ES, embryonic stem; Ezh2, enhancer of zeste homolog 2; Foxa2, Forkhead box A2; Foxd3, Forkhead box D3; Foxm1, forkhead box M1; GDM, gestational diabetes mellitus; IPGTT, ip glucose tolerance testing;...

The enteric nervous system (ENS) arises from the coordinated migration, expansion and differentiation of vagal and sacral neural crest progenitor cells. During development, vagal neural crest cells enter the foregut and migrate in a rostro-to-caudal direction, colonizing the entire gastrointestinal tract and generating the majority of the ENS. Sacral neural crest contributes to a subset of enteric ganglia in the hindgut, colonizing the colon in a caudal-to-rostral wave. During this process, enteric neural crest-derived progenitors (ENPs) self-renew and begin expressing markers of neural and glial lineages as they populate the intestine. Our earlier work demonstrated that the transcription factor Foxd3 is required early in neural crest-derived progenitors for self-renewal, multipotency and establishment of multiple neural crest-derived cells and structures including the ENS. Here, we describe Foxd3 expression within the fetal and postnatal intestine: Foxd3 was strongly expressed in ENPs as they colonize the gastrointestinal tract and was progressively restricted to enteric glial cells. Using a novel Ednrb-iCre transgene to delete Foxd3 after vagal neural crest cells migrate into the midgut, we demonstrated a late temporal requirement for Foxd3 during ENS development. Lineage labeling of Ednrb-iCre expressing cells in Foxd3 mutant embryos revealed a reduction of ENPs throughout the gut and loss of Ednrb-iCre lineage cells in the distal colon. Although mutant mice were viable, defects in patterning and distribution of ENPs were associated with reduced proliferation and severe reduction of glial cells derived from the Ednrb-iCre lineage. Analyses of ENS-lineage and differentiation in mutant embryos suggested activation of a compensatory population of Foxd3-positive ENPs that did not express the Ednrb-iCre transgene. Our findings highlight the crucial roles played by Foxd3 during ENS development including progenitor proliferation, neural patterning, and glial differentiation and may help delineate distinct molecular programs controlling vagal versus sacral neural crest development.

Patients with mutations in the mitochondrial very-long-chain acyl-CoA dehydrogenase (VLCAD) gene are at risk for cardiomyopathy, myocardial dysfunction, ventricular tachycardia (VT), and sudden cardiac death. The mechanism is not known. Here we report a novel mechanism of VT in mice lacking VLCAD (VLCAD−/−). These mice exhibited polymorphic VT and increased incidence of VT after isoproterenol infusion. Polymorphic VT was induced in 10 out of 12 VLCAD−/− mice (83%) when isoproterenol was used. One out of 10 VLCAD−/− mice with polymorphic VT had VT with the typical bidirectional morphology. At the molecular level, VLCAD−/− cardiomyocytes showed increased levels of cardiac ryanodine receptor 2, phospholamban, and calsequestrin with increased [3H]ryanodine binding in heart microsomes. At the single cardiomyocyte level, VLCAD−/− cardiomyocytes showed significant increase in diastolic indo 1 and fura 2 fluorescence, with increased Ca2+ transient amplitude. These changes were associated with altered Ca2+ dynamics, to include: faster sarcomere contraction, larger time derivative of the upstroke, and shorter time-to-minimum sarcomere length compared with VLCAD+/+ control cells. The L-type Ca2+ current characteristics were not different under voltage-clamp conditions in the two VLCAD genotypes. Sarcoplasmic reticulum Ca2+ load measured as normalized integrated Na+/Ca2+ exchange current after rapid caffeine application was increased by 48% in VLCAD−/− cells. We conclude that intracellular Ca2+ handling represents a possible molecular mechanism of arrhythmias in mice and perhaps in VLCAD-deficient humans.