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Abstract

Transgene driven expression of Escherichia coli nitroreductase (NTR1.0) renders animal cells susceptible to the antibiotic metronidazole (MTZ). Many NTR1.0/MTZ ablation tools have been reported in zebrafish, which have significantly impacted regeneration studies. However, NTR1.0-based tools are not appropriate for modeling chronic cell loss as prolonged application of the required MTZ dose (10 mM) is deleterious to zebrafish health. We established that this dose corresponds to the median lethal dose (LD50) of MTZ in larval and adult zebrafish and that it induced intestinal pathology. NTR2.0 is a more active nitroreductase engineered from Vibrio vulnificus NfsB that requires substantially less MTZ to induce cell ablation. Here, we report on the generation of two new NTR2.0-based zebrafish lines in which acute β-cell ablation can be achieved without MTZ-associated intestinal pathology. For the first time, we were able to sustain β-cell loss and maintain elevated glucose levels (chronic hyperglycemia) in larvae and adults. Adult fish showed significant weight loss, consistent with the induction of a diabetic state, indicating that this paradigm will allow the modeling of diabetes and associated pathologies.

An AT motif-dependent axis, modulated by the transcription factor Zfhx3, influences the circadian clock in mice. In particular, gain of function of Zfhx3significantly shortens circadian rhythms and alters the transcriptional activity of an important class of neuropeptides that controls intercellular signaling in the suprachiasmatic nucleus (SCN) of the hypothalamus. The ZFHX3/AT axis revealed an important, largely cell-nonautonomous control of the circadian clock. Here, by studying the recently identified circadian mouse mutant Zfhx3Sci/+, we identify significant effects on sleep homeostasis, a phenomenon that is outside the canonical circadian clock system and that is modulated by the activity of those neuropeptides at a circuit level. We show that the Zfhx3Sci/+mutation accelerates the circadian clock at both the hourly scale (i.e., advancing circadian rhythms) and the seconds-to-minutes scale (i.e., anticipating behavioral responses) in mice. The in vivo results are accompanied by a significant presence of sleep targets among protein-protein interactions of the Zfhx3Sci/+-dependent network.

Numerous lines of evidence have highlighted the involvement of the dopamine system in the pathophysiology of schizophrenia. Association studies of dopaminergic genes such as the dopamine D2 receptor gene (DRD2), however, have produced contradictory results. To test the hypothesis that DRD2polymorphisms are associated with schizophrenia, we investigated two DRD2-related polymorphisms (TaqI A1A2 or rs1800497 and –141-C InsDelor rs1799732) in a Spanish population isolate from northern Spain consisting of 165 controls and 119 patients with schizophrenia. The TaqI A1 allele was less frequent in schizophrenic patients than in controls (P0.002). A similar association was found for the TaqI A2A2 genotype (P0.0003). No association was found for the DRD2−141-C InsDelpolymorphism. The strong association between a potentially functional polymorphism, downstream of the DRD2gene and schizophrenia, suggests that the direct or indirect functional effects of this polymorphism, acting on either the ANKK1or DRD2genes, may play a role in the pathophysiology of schizophrenia.

The T cell costimulatory molecule CD28 is important for T cell survival, yet both the signaling pathways downstream of CD28 and the apoptotic pathways they antagonize remain poorly understood. Here we demonstrate that CD4+ T cells from CD28-deficient mice show increased susceptibility to Fas-mediated apoptosis via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. Protein kinase B (PKBα/Akt1) is an important serine/threonine kinase that promotes survival downstream of PI3K signals. To understand how PI3K-mediated signals downstream of CD28 contribute to T cell survival, we examined Fas-mediated apoptosis in T cells expressing an active form of PKBα. Our data demonstrate that T cells expressing active PKB are resistant to Fas-mediated apoptosis in vivo and in vitro. PKB transgenic T cells show reduced activation of caspase-8, BID, and caspase-3 due to impaired recruitment of procaspase-8 to the death-inducing signaling complex (DISC). Similar alterations are seen in T cells from mice which are haploinsufficient for PTEN, a lipid phosphatase that regulates phosphatidylinositol-3,4,5-trisphosphate (PIP3) and influences PKBα activity. These findings provide a novel link between CD28 and an important apoptosis pathway in vivo, and demonstrate that PI3K/PKB signaling prevents apoptosis by inhibiting DISC assembly.

Muscular dystrophy is frequently caused by disruption of the dystrophin-glycoprotein complex (DGC), which links muscle cells to the extracellular matrix. Dystroglycan, a central component of the DGC, serves as a laminin receptor via its extracellular α subunit, and interacts with dystrophin (and thus the actin cytoskeleton) through its integral membrane β subunit. We have removed the function of dystroglycan in zebrafish embryos. In contrast to mouse, where dystroglycan mutations lead to peri-implantation lethality, dystroglycan is dispensable for basement membrane formation during early zebrafish development. At later stages, however, loss of dystroglycan leads to a disruption of the DGC, concurrent with loss of muscle integrity and necrosis. In addition, we find that loss of the DGC leads to loss of sarcomere and sarcoplasmic reticulum organisation. The DGC is required for long-term survival of muscle cells in zebrafish, but is dispensable for muscle formation. Dystroglycan or the DGC is also required for normal sarcomere and sarcoplasmic reticulum organisation. Because zebrafish embryos lacking dystroglycan share several characteristics with human muscular dystrophy, they should serve as a useful model for the disease. In addition, knowing the dystroglycan null phenotype in zebrafish will facilitate the isolation of other molecules involved in muscular dystrophy pathogenesis.

Basement membranes are thought to be essential for organ formation, providing the scaffold on which individual cells organize to form complex tissues. Laminins are integral components of basement membranes. To understand the development of a simple vertebrate organ, we have used positional cloning to characterize grumpy and sleepy, two zebrafish loci known to control notochord formation, and find that they encode laminin β1 and laminin γ1, respectively. Removal of either chain results in the dramatic loss of laminin 1 staining throughout the embryo and prevents formation of the basement membrane surrounding the notochord. Notochord cells fail to differentiate and many die by apoptosis. By transplantation, we demonstrate that, for both grumpy and sleepy, notochord differentiation can be rescued by exogenous sources of the missing laminin chain, although notochordal sources are also sufficient for rescue. These results demonstrate a clear in vivo requirement for laminin β1 and laminin γ1 in the formation of a specific vertebrate organ and show that laminin or the laminin-dependent basement membrane is essential for the differentiation of chordamesoderm to notochord.

The purpose of this study was to investigate whether aging alters serotonergic innervation of the superior colliculus and pretectum in rats. The superior colliculus has one of the highest concentrations of serotonin in the rat central nervous system. Young and old male F344 rats (<6 months, and >18 months, albino and pigmented) were used in all experiments. Coronal sections through the superior colliculus and pretectum were incubated with antibodies to serotonin, the serotonin 2A receptor, and the serotonin transporter. Immunocytochemical staining was analyzed semi-quantitatively. The results indicate that with age there is an increase in serotonin immunoreactivity throughout the entire superior colliculus and pretectum, a decrease in levels of serotonin 2A receptor staining in select layers of superior colliculus, and no change in serotonin transporter immunoreactivity. Albino rats differ from pigmented rats in that they have enhanced serotonergic immunoreactivity in the superficial layers of superior colliculus, a region that receives direct retinal input. These data suggest that the age-related changes in the serotonergic system in the superior colliculus and pretectum may account for some of the alterations in light-mediated behaviors with aging.

In preparation for a forthcoming revision of the family Aristolochiaceae in Australia and New Guinea, a total of 14 new species and two new subspecies are described. Due to the fact that Aristolochiaceae within the region fall into two distinctive, well-defined groups of species, it was considered necessary to examine the affinities of these within the family. One group belongs to Aristolochia, whereas the other is clearly accommodated in Pararistolochia, the latter previously regarded as being solely a tropical African genus. The distinction between Pararistolochiaand Aristolochiais discussed. It is concluded that the Australia and New Guinea taxa were found to substantiate circumscription of Pararistolochia. Two new species of Aristolochia( A. nauseifoliaand A. goliathiana) are described, as well as 12 species of Pararistolochia (P. alexandriana, P. australopithecurus, P. biakensis, P. kepara, P. manokwariensis, P. meridionaliana, P. paradisiana, P. peninsulensis, P. tithonusiana, P. sepikensis, P. sparusifolia, P. linearifolia). Two subspecies of P. meridionaliana(subsp. popondettensisand subsp. milnensis) are described. Eleven other taxa from Australia and New Guinea are newly combined under Pararistolochia: P. deltanthaF. Muell., 1868; P. dictyophlebiaMerr. & Perry, 1948; P. dielsianaO.C. Schmidt, 1923; P. englerianaO.C. Schmidt, 1927; P. gracilifoliaO.C. Schmidt, 1923; P. laheyanaBail., 1912; P. momandulK. Sch., 1889; P. praevenosaF. Muell., 1861; P. pithecurusRidl., 1914; P. schlechteriLaut., 1905; as well as P. decandraDing Hou, 1983, from Borneo.

The growing interest in integrating the arts across the curriculum represents an important change from previous concerns about teaching the structure of individual disciplines. It will require a change in the concept of cognition in the arts that is currently dominant in our psychology. This concept—the ‘symbol systems’ account—restricts thinking to one medium or symbol system at a time, whether the medium is a medium of perception—as in ‘visual thinking’—or of expression-as with drawing. The restriction delegitimizes the kind of cross-media connections in thought that integrated learning requires, including connections with culture and student purposes. The new interest will require of art educators a more equal balance between epistemological and psychological considerations and a more generous view of the role of language in learning.