Line Tg Research Articles

Identification and characterization of alternative promoters of zebrafish Rtn-4/Nogo genes in cultured cells and zebrafish embryos

In mammals, the Nogo family consists of Nogo-A, Nogo-B and Nogo-C. However, there are three Rtn-4/Nogo-related transcripts were identified in zebrafish. In addition to the common C-terminal region, the N-terminal regions of Rtn4-n/Nogo-C1, Rtn4-m/Nogo-C2 and Rtn4-l/Nogo-B, respectively, contain 9, 25 and 132 amino acid residues. In this study, we isolated the 5′-upstream region of each gene from a BAC clone and demonstrated that the putative promoter regions, P1-P3, are functional in cultured cells and zebrafish embryos. A transgenic zebrafish Tg(Nogo-B:GFP) line was generated using P1 promoter region to drive green fluorescent protein (GFP) expression through Tol2-mediated transgenesis. This line recapitulates the endogenous expression pattern of Rtn4-l/Nogo-B mRNA in the brain, brachial arches, eyes, muscle, liver and intestines. In contrast, GFP expressions by P2 and P3 promoters were localized to skeletal muscles of zebrafish embryos. Several GATA and E-box motifs are found in these promoter regions. Using morpholino knockdown experiments, GATA4 and GATA6 were involved in the control of P1 promoter activity in the liver and intestine, while Myf5 and MyoD for the control of P1 and P3 promoter activities in muscles. These data demonstrate that zebrafish Rtn4/Nogo transcripts might be generated by coupling mechanisms of alternative first exons and alternative promoter usage.

In Vivo Imaging of an Inducible Oncogenic Tumor Antigen Visualizes Tumor Progression and Predicts CTL Tolerance

Visualizing oncogene/tumor Ag expression by noninvasive imaging is of great interest for understanding processes of tumor development and therapy. We established transgenic (Tg) mice conditionally expressing a fusion protein of the SV40 large T Ag and luciferase (TagLuc) that allows monitoring of oncogene/tumor Ag expression by bioluminescent imaging upon Cre recombinase-mediated activation. Independent of Cre-mediated recombination, the TagLuc gene was expressed at low levels in different tissues, probably due to the leakiness of the stop cassette. The level of spontaneous TagLuc expression, detected by bioluminescent imaging, varied between the different Tg lines, depended on the nature of the Tg expression cassette, and correlated with Tag-specific CTL tolerance. Following liver-specific Cre-loxP site-mediated excision of the stop cassette that separated the promoter from the TagLuc fusion gene, hepatocellular carcinoma development was visualized. The ubiquitous low level TagLuc expression caused the failure of transferred effector T cells to reject Tag-expressing tumors rather than causing graft-versus-host disease. This model may be useful to study different levels of tolerance, monitor tumor development at an early stage, and rapidly visualize the efficacy of therapeutic intervention versus potential side effects of low-level Ag expression in normal tissues.

Behavioral analyses of transgenic mice harboring bipolar disorder candidate genes, IMPA1 and IMPA2

The inositol depletion hypothesis proposes the inhibition of IMPase ( myo-inositol monophosphatase) by lithium, a mood stabilizer, as a mechanism of lithium's efficacy. This hypothesis predicts that the upregulation of this biochemical pathway may underlie the pathophysiology of bipolar disorder. In favor of this idea, IMPA2 encoding IMPase is a candidate susceptibility gene for bipolar disorder and that the risk-conferring single nucleotide polymorphisms enhance the promoter activity of IMPA2. However, it is yet unknown whether such upregulation has a biological role in bipolar disorder. To address this issue, we generated transgenic mice for the two IMPase genes ( IMPA1 and IMPA2). The expression levels of the transgene were robust in IMPA2 Tg lines, but moderate in IMPA1 Tg lines, when compared to those of endogenous proteins. The transgenic mice behaved normally under durg-naïve conditions, and did not exhibit signs for manic change when an antidepressant amitriptyline was administrated. Interestingly, the male transgenic mice for IMPA2 exhibited a lithium-resistant phenotype in the forced swim test. The current study, as a whole, did not support a substantial role of the upregulation of IMPase in bipolar disorder, although the lithium-insensitivity trait seen in IMPA2 transgenic mice might represent some aspect relevant to the inositol depletion hypothesis.

Accelerated Liver Regeneration and Hepatocarcinogenesis in Mice Overexpressing Serine-45 Mutant β-Catenin

The Wnt/beta-catenin pathway is implicated in the pathogenesis of hepatocellular cancer (HCC). We developed a transgenic mouse (TG) in the FVB strain that overexpresses Ser45-mutated-beta-catenin in hepatocytes to study the effects on liver regeneration and cancer. In the two independent TG lines adult mice show elevated beta-catenin at hepatocyte membrane with no increase in the Wnt pathway targets cyclin-D1 or glutamine synthetase. However, TG hepatocytes upon culture exhibit a 2-fold increase in thymidine incorporation at day 5 (D5) when compared to hepatocytes from wildtype FVB mice (WT). When subjected to partial hepatectomy (PH), dramatic increases in the number of hepatocytes in S-phase are evident in TG at 40 and WT at 72 hours. Coincident with the earlier onset of proliferation, we observed nuclear translocation of beta-catenin along with an increase in total and nuclear cyclin-D1 protein at 40 hours in TG livers. To test if stimulation of beta-catenin induces regeneration, we used hydrodynamic delivery of Wnt-1 naked DNA to control mice, which prompted an increase in Wnt-1, beta-catenin, and known targets, glutamine synthetase (GS) and cyclin-D1, along with a concomitant increase in cell proliferation. beta-Catenin-overexpressing TG mice, when followed up to 12 months, showed no signs of spontaneous tumorigenesis. However, intraperitoneal delivery of diethylnitrosamine (DEN), a known carcinogen, induced HCC at 6 months in TG mice only. Tumors in TG livers showed up-regulation of beta-catenin, cyclin-D1, and unique genetic aberrations, whereas other canonical targets were unremarkable. beta-Catenin overexpression offers growth advantage during liver regeneration. Also, whereas no spontaneous HCC is evident, beta-catenin overexpression makes TG mice susceptible to DEN-induced HCC.

Calcium sensitivity, force frequency relationship and cardiac troponin I: Critical role of PKA and PKC phosphorylation sites

Transgenic models with pseudo phosphorylation mutants of troponin I, PKA sites at Ser 22 and 23 (cTnIDD22,23 mice) or PKC sites at Ser 42 and 44 (cTnIAD22,23DD42,44) displayed differential force–frequency relationships and afterload relaxation delay in vivo. We hypothesized that cTnI PKA and PKC phosphomimics impact cardiac muscle rate-related developed twitch force and relaxation kinetics in opposite directions. cTnIDD22,23 transgenic mice produce a force frequency relationship (FFR) equivalent to control NTG albeit at lower peak [Ca2+]i, while cTnIAD22,23DD42,44 TG mice had a flat FFR with normal peak systolic [Ca2+]i, thus suggestive of diminished responsiveness to [Ca2+]i at higher frequencies. Force–[Ca2+]i hysteresis analysis revealed that cTnIDD22,23 mice have a combined enhanced myofilament calcium peak response with an enhanced slope of force development and decline per unit of [Ca2+]i, whereas cTnIAD22,23DD42,44 transgenic mice showed the opposite. The computational ECME model predicts that the TG lines may be distinct from each other due to different rate constants for association/dissociation of Ca2+ at the regulatory site of cTnC. Our data indicate that cTnI phosphorylation at PKA sites plays a critical role in the FFR by increasing relative myofilament responsiveness, and results in a distinctive transition between activation and relaxation, as displayed by force–[Ca2+]i hysteresis loops. These findings may have important implications for understanding the specific contribution of cTnI to β-adrenergic inotropy and lusitropy and to adverse contractile effects of PKC activation, which is relevant during heart failure development.

Transgenic expression of prothymosin alpha on zebrafish epidermal cells promotes proliferation and attenuates UVB-induced apoptosis

This study generated a transgenic zebrafish line Tg(k18:Ptmaa-RFP) with overexpression of Prothymosin alpha type a (Ptmaa) in the skin epidermis. Red fluorescence first appears very weakly in the early stage, become stronger and mainly restricted in the nuclei of the epithelial cells from 3 dpf-larvae to adult fish. However, no evident morphological abnormalities were observed. Thus, overexpression of Ptmaa alone is not sufficient to cause disorganized growths or even cancer in zebrafish skin. Molecular and histological evidences showed that Tg(k18:Ptmaa-RFP) embryos have more proliferating cells in the pelvic fins [WT: 3.92 +/- 7.15; Tg(k18:Ptmaa-RFP): 38.00 +/- 10.87] and thicker skin [WT: 10.98 +/- 1.41 mum; Tg(k18:Ptmaa-RFP): 14.02 +/- 1.32 mum], indicating that overexpression of Ptmaa can promote proliferation. On the other hand, fewer apoptotic signals were found when Tg(k18:Ptmaa-RFP) embryos were exposed to UVB. Together with quantitative RT-PCR data, we suggest that UVB-induced epidermal cell apoptosis of zebrafish larvae can be attenuated by overexpression of Ptmaa through the enhancement of transcriptions of bcl2 mRNAs. Taken together, we conclude that overexpression of Ptmaa in zebrafish epidermal cells promotes proliferation and attenuates UVB-induced apoptosis but does not cause skin cancer.

Chronic stress-mutated presenilin 1 gene interaction perturbs neurogenesis and accelerates neurodegeneration

Recent evidence suggests that supplemental factors coincident with aging and genetic determinants might be involved in the initial progression of Alzheimer's disease (AD). Early studies also indicate that chronic stress decreases hippocampal neurogenesis. Here, we investigate the effect of chronic stress on hippocampal neurogenesis using a transgenic mouse line (Tg) that overexpresses human presenilin 1 (PS1) with a familial AD (FAD)-related mutation in order to elucidate how the combination of chronic stress and mutated genes affects the cytoarchitecture in the hippocampal granule cell layer (GCL), which contributes to spatial learning and memory. Using an original chronic intermittent restraint stress (CIRS) protocol, we examined the effect of stress on hippocampal neurogenesis and neurodegeneration by immunohistochemical analysis. After short-term CIRS, neurodegeneration in Tg mice was significantly increased in the hippocampus with an earlier onset and progression than in the non-stressed Tg mice. Moreover, after long-term CIRS, transitional neurodegeneration appeared to proceed along the neuronal circuit involved in cognitive function in stressed Tg mice. Although the number of Pax6-positive (+) cells (mostly granule neuron precursors) did not significantly decrease during CIRS in both non-Tg and Tg mice, doublecortin (DCX) + neuronal progenitor cells in the GCL were markedly influenced in Tg mice; they were significantly reduced without stress compared with non-stressed non-Tg mice and significantly increased by CIRS compared with non-stressed Tg mice. We conclude from these results that diverse responses against stressful experiences among genetically predisposed individuals could lead to cognitive dysfunction through retardation of neuronal maturation and neurodegeneration.

Effects of transgenic Pit-1 overexpression on calcium phosphate and bone metabolism

The type III inorganic phosphate (Pi) transporter Pit-1 was previously found to be preferentially expressed in developing long bones. Several studies also described a regulation of its expression in cultured bone cells by osteotropic factors, suggesting a role of this transporter in bone metabolism. In the present study, we investigated the effects of the transgenic overexpression of Pit-1 in Wistar male rats on calcium phosphate and bone metabolism. A threefold increase and doubling of Pi transport activity were recorded in primary cultured osteoblastic cells derived from calvaria of two transgenic (Tg) lines compared with wild-type littermates (WT), respectively. Skeletal development was not affected by the transgene, and bone mass, analyzed by DXA, was slightly decreased in Tg compared with WT. Enhanced Pi uptake in calvaria-derived osteoblasts from Pit-1 Tg was associated with a significantly decreased expression of alkaline phosphatase activity and a normal deposition and calcification of the collagenous matrix. In 4-month-old adult Tg rats, serum Pi and renal Pi transport were increased compared with WT. The decrease of serum Ca concentration was associated with increased serum parathyroid hormone levels. Variations in serum Pi in Pit-1 Tg rats were negatively correlated with serum fibroblast growth factor-23, whereas 1,25-dihydroxyvitamin D(3) was not affected by Pit-1 overexpression. In conclusion, transgenic Pit-1 overexpression in rats affected bone and calcium phosphate metabolism. It also decreased alkaline phosphatase activity in osteoblasts without influencing bone matrix mineralization as well as skeletal development.

Defects in the cerebella of conditional Neurod1 null mice correlate with effective Tg(Atoh1-cre) recombination and granule cell requirements for Neurod1 for differentiation

Neurod1 is a crucial basic helix-loop-helix gene for most cerebellar granule cells and mediates the differentiation of these cells downstream of Atoh1-mediated proliferation of the precursors. In Neurod1 null mice, granule cells die throughout the posterior two thirds of the cerebellar cortex during development. However, Neurod1 is also necessary for pancreatic beta-cell development, and therefore Neurod1 null mice are diabetic, which potentially influences cerebellar defects. Here, we report a new Neurod1 conditional knock-out mouse model created by using a Tg(Atoh1-cre) line to eliminate Neurod1 in the cerebellar granule cell precursors. Our data confirm and extend previous work on systemic Neurod1 null mice and show that, in the central lobules, granule cells can be eradicated in the absence of Neurod1. Granule cells in the anterior lobules are partially viable and depend on as yet unknown genes, but the Purkinje cells show defects not previously recognized. Interestingly, delayed and incomplete Tg(Atoh1-cre) upregulation occurs in the most posterior lobules; this leads to near normal expression of Neurod1 with a concomitant normal differentiation of granule cells, Purkinje cells, and unipolar brush cells in lobules IX and X. Our analysis suggests that Neurod1 negatively regulates Atoh1 to ensure a rapid transition from proliferative precursors to differentiating neurons. Our data have implications for research on medulloblastoma, one of the most frequent brain tumors of children, as the results suggest that targeted overexpression of Neurod1 under Atoh1 promoter control may initiate the differentiation of these tumors.

Delayed and Restricted Expression of UAS-Regulated GFP Gene in Early Transgenic Zebrafish Embryos by Using the GAL4/UAS System

A stable Tg(UAS:GFP) zebrafish line was generated and crossed with Tg(hsp70:GAL4) line, in which the GAL4 gene is under the control of an inducible zebrafish promoter derived from the heat shock 70 protein gene (hsp70). The dynamic green fluorescent protein (GFP) expression in early zebrafish embryos in the GAL4/UAS binary system was then investigated. We found that, at early developmental stages, expression of GFP effector gene was restricted and required a long recovery time to reach a detectable level. At later developmental stage (after 2 days postfertilization), GFP could be activated in multiple tissues in a shorter time, apparently due to a higher level of GAL4 messenger RNA induction. It appears that the type of tissues expressing GFP was dependent on whether they had been developed at the time of heat shock. Therefore, the delayed and restricted transgene expression should be taken into consideration when GAL4/UAS system is used to study transgene expression in early developmental stages.

DTA-mediated targeted ablation revealed differential interdependence of endocrine cell lineages in early development of zebrafish pancreas

Cell lineage analysis is critical in understanding the relationship between progenitors and differentiated cells as well as the mechanism underlying the process of differentiation. In order to study the zebrafish endocrine pancreas cell lineage, transgenic expression of diphtheria toxin gene A chain (DTA) under two cell type-specific promoters derived from the insulin ( ins) and somatostatin2 ( sst2) genes was used to ablate the two types of endocrine cells: insulin-producing β-cells and somatostatin-producing δ-cells, respectively. We found that ablation of β-cells resulted in a reduction of not only β-cells but also glucagon-producing α-cells; in contrast, δ-cells were largely unaffected. Ablation of δ-cells led to reduction of all three types of endocrine cells: α-, β-, and δ. Interestingly, α-cells were more profoundly affected in both β- and δ-cell ablations and were frequently reduced together with β- and δ-cells. By taking advantage of Tg( ins: gfp) and Tg( sst2: gfp) lines, we also monitored the changes of different types of endocrine cells in vivo after ablation and found that both β- and δ-cell populations significantly recovered by 3 dpf after their ablation and it seemed that δ-cells had a better capability of recovery than β-cells. Thus, our current observations indicated differential interdependence of these three cell lineages. The development of zebrafish α-cells, but not δ-cells, is dependent on β-cells, while the development of both α- and β-cells is dependent on δ-cells. In contrast, the development of δ-cells was independent of β-cells.

Analysis of prepulse inhibition in mouse lines overexpressing 22q11.2 orthologues

Animal models have been useful in elucidating the genetic basis of the cognitive and behavioural phenotypes associated with the 22q11.2 microdeletions. Loss-of-function models have implicated a number of genes as playing a role in prepulse inhibition (PPI) of the startle response. Here, we report the generation and initial analysis of bacterial artificial chromosome (BAC) transgenic (Tg) mice, overexpressing genes from within the 22q11.2 locus. We used engineered BAC constructs to generate Tg lines and quantitative RT-PCR to assess levels of gene expression in each line. We assessed PPI and open-field activity in mice from two low copy number lines. In Tg-1, a line overexpressing Prodh and Vpreb2, PPI was significantly increased at prepulse levels of 78 dB and 82 dB while no differences were found in activity measures. By contrast, no significant differences were found in PPI testing of the Tg-2 line overexpressing Zdhhc8, Ranbp1, Htf9c, T10, Arvcf and Comt. Taken together with previous loss-of-function reports, these findings suggest that Prodh has a key role in modulating the degree of sensorimotor gating in mice and possibly in humans and provide additional support for an important role of this pathway in modulating behavioural deficits associated with genomic gains or losses at 22q11.2.

10th Annual Congress of the German Association of Clinical Pharmacology

10th Annual Congress of the German Association of Clinical Pharmacology

Transgenic zebrafish line with over-expression of Hedgehog on the skin: a useful tool to screen Hedgehog-inhibiting compounds

We generated a transgenic line Tg(k18:shh:RFP) with overexpression of Sonic hedgehog in the skin epidermis. By 5 day-post-fertilization (dpf), many epidermal lesions were clearly observed, including a swollen yolk sac, epidermis growth malformation around the eyes and at the basement of the pectoral fins. Skin histology revealed embryos derived from Tg(k18:shh:RFP) displayed an elevated Nuclear/Cytoplasmic ratio and pleomorphic nuclei compared to their wild type littermates, suggesting the abnormal growth pattern on the epidermis of Tg(k18:shh:RFP) embryos were dysplasia. Later (by 7 dpf), Tg(k18:shh:RFP) embryos displayed broader pectoral fins which are similar to the polydactyly phenotypes of Nevoid basal cell carcinoma syndrome (NBCCS)/Gorlin patients and polydactylous mice. In addition, treatment with cyclopamine is able to enhance and prolong the survival rates and survival durations of Tg(k18:shh:RFP) embryos. In conclusion, this unique Tg(k18:shh:RFP) fish line, should be an excellent experimental animal for screening for a lower toxicity level of the new Hh-inhibitor and can even be used as a new anti-cancer drug-screening platform.

The molecular basis for transmission of human prion strains

Human prion diseases, such as Creutzfeldt‐Jakob disease (CJD), can occur in different "strains" which are characterized by their clinical onset, and by biochemical and neuropathological hallmarks of the post mortem brain. Human prion strain type is dramatically influenced by the M/V polymorphism at residue 129, with homozygous individuals much more likely to develop disease.We report a series of novel transgenic (Tg) mouse lines expressing various chimeras of mouse and human prion protein (PrP), which were inoculated with either of the two most common strains of sporadic (s) CJD, termed MM1 and VV2, or with the variant (v) CJD strain. The differential susceptibility for each strain allows a molecular dissection of the amino acid residues that influence strain propagation.One Tg line was susceptibility to sCJD(MM1) prions in less than 80 days, and defined six human PrP residues in the central region of the molecule that are essential for strain transmission. Conversely, for the sCJD(VV2) strain, transmission is regulated by residues in the terminal regions of the PrP sequence. Modification of strain characteristics on transmission of sCJD(VV2) and vCJD strains, also allowed us to formulate a general set of rules governing strain transformation.

A Membrane Associated mCherry Fluorescent Reporter Line for Studying Vascular Remodeling and Cardiac Function During Murine Embryonic Development

The development of the cardiovascular system is a highly dynamic process dependent on multiple signaling pathways regulating proliferation, differentiation, migration, cell-cell and cell-matrix interactions. To characterize cell and tissue dynamics during the formation of the cardiovascular system in mice, we generated a novel transgenic mouse line, Tg(Flk1::myr-mCherry), in which endothelial cell membranes are brightly labeled with mCherry, a red fluorescent protein. Tg(Flk1::myr-mCherry) mice are viable, fertile, and do not exhibit any developmental abnormalities. High levels of mCherry are expressed in the embryonic endothelium and endocardium, and expression is also observed in capillaries in adult animals. Targeting of the fluorescent protein to the cell membrane allows for subcellular imaging and cell tracking. By acquiring confocal time lapses of live embryos cultured on the microscope stage, we demonstrate that the newly generated transgenic model beautifully highlights the sprouting behaviors of endothelial cells during vascular plexus formation. We have also used embryos from this line to imaging the endocardium in the beating embryonic mouse heart, showing that Tg(Flk1::myr-mCherry) mice are suitable for the characterization of cardio dynamics. Furthermore, when combined with the previously described Tg(Flk1::H2B-EYFP) line, cell number in addition to cell architecture is revealed, making it possible to determine how individual endothelial cells contribute to the structure of the vessel.

Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors

AbstractThe transcription factor Runx1 is essential for the development of definitive hematopoietic stem cells (HSCs) during vertebrate embryogenesis and is transcribed from 2 promoters, P1 and P2, generating 2 major Runx1 isoforms. We have created 2 stable runx1 promoter zebrafish-transgenic lines that provide insight into the roles of the P1 and P2 isoforms during the establishment of definitive hematopoiesis. The Tg(runx1P1:EGFP) line displays fluorescence in the posterior blood island, where definitive erythromyeloid progenitors develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the aorta-gonad-mesonephros, with enhanced green fluorescent protein–labeled cells later populating the pronephros and thymus. This suggests that a function of runx1 promoter switching is associated with the establishment of discrete definitive blood progenitor compartments. These runx1 promoter–transgenic lines are novel tools for the study of Runx1 regulation and function in normal and malignant hematopoiesis. The ability to visualize and isolate fluorescently labeled HSCs should contribute to further elucidating the complex regulation of HSC development.

Availability of autoantigenic epitopes controls phenotype, severity, and penetrance in TCR Tg autoimmune gastritis

We examined TCR:MHC/peptide interactions and in vivo epitope availability to explore the Th1- or Th2-like phenotype of autoimmune disease in two TCR Tg mouse models of autoimmune gastritis (AIG). The TCR of strains A23 and A51 recognize distinct IA(d)-restricted peptides from the gastric parietal cell H/K-ATPase. Both peptides form extremely stable MHC/peptide (MHC/p) complexes. All A23 animals develop a Th1-like aggressive, inflammatory AIG early in life, while A51 mice develop indolent Th2-like AIG at 6-8 wk with incomplete penetrance. A51 T cells were more sensitive than A23 to low doses of soluble antigen and to MHC/p complexes. Staining with IA(d)/peptide tetramers was only detectable on previously activated T cells from A51. Thus, despite inducing a milder AIG, the A51 TCR displays a higher avidity for its cognate IA(d)/peptide. Nonetheless, in vivo proliferation of adoptively transferred A51 CFSE-labeled T cells in the gastric lymph node was relatively poor compared with A23 T cells. Also, DC from WT gastric lymph node, presenting processed antigen available in vivo, stimulated proliferation of A23 T cells better than A51. Thus, the autoimmune potential of these TCR in their respective Tg lines is strongly influenced by the availability of the peptide epitope, rather than by differential avidity for their respective MHC/p complexes.

Secondary motoneurons in juvenile and adult zebrafish: Axonal pathfinding errors caused by embryonic nicotine exposure

AbstractNicotine is a drug of abuse that has been reported to have many adverse effects on the developing nervous system. We previously demonstrated that embryonic exposure to nicotine alters axonal pathfinding of spinal secondary motoneurons in zebrafish. We hypothesize that these changes will persist into adulthood. The Tg(isl1:GFP) line of zebrafish, which expresses green fluorescent protein (GFP) in a subtype of spinal secondary motoneurons, was used to investigate potential long‐term consequences of nicotine exposure on motoneuron development. Anatomical characterization of Tg(isl1:GFP) zebrafish ranging between 3 and 30 days postfertilization (dpf) was initially performed in fixed tissue to characterize axonal trajectories in larval and juvenile fish. Tg(isl1:GFP) embryos were transiently exposed to 5–30 μM nicotine. They were then rescued from nicotine and raised into later stages of life (3–30 dpf) and fixed for microscopic examination. Morphological analysis revealed that nicotine‐induced abnormalities in secondary motoneuron anatomy were still evident in juvenile fish. Live imaging of Tg(isl1:GFP) zebrafish using fluorescent stereomicroscopy revealed that the nicotine‐induced changes in motoneuron axonal pathfinding persisted into adulthood. We detected abnormalities in 37‐dpf fish that were transiently exposed to nicotine as embryos. These fish were subsequently imaged over a 7‐week period of time until they were ≈3 months of age. These pathfinding errors of spinal secondary motoneuron axons detected at 37 dpf persisted within the same fish until 86 dpf, the latest age analyzed. These findings indicate that exposure to nicotine during embryonic development can have permanent consequences for motoneuron anatomy in zebrafish. J. Comp. Neurol. 512:305–322, 2009. © 2008 Wiley‐Liss, Inc.

Generation of living color transgenic zebrafish to trace somatostatin-expressing cells and endocrine pancreas organization

In the present study, both gfp and rfp transgenic zebrafish lines using a 2.5-kb zebrafish somatostain2 ( sst2) promoter were generated. During embryonic development, expression of GFP/RFP in the endocrine pancreas of transgenic embryos was initiated at ∼20 hpf and the number of GFP/RFP positive cells in the pancreas increased in subsequent stages; thus, our newly generated Tg( sst2:gfp) and Tg( sst2:rfp) lines faithfully recapitulated sst2 expression in endocrine pancreatic cells and provided a useful tool in analyzing the development of Sst2-producing δ-cells in the pancreas. By crossing these new transgenic lines with previously available transgenic lines targeted in insulin (Ins)-producing β-cells, Tg( ins:gfp) and Tg( ins:rfp), in combination with immunodetection of glucagon (Gcg)-producing α-cells and pancreatic polypeptide (PP)-producing PP-cells, the organization and composition of endocrine islets were investigated in both embryonic and adult pancreas. We found that there was always a big cluster of endocrine cells (principal islet) in the anterior-dorsal pancreas, followed by numerous smaller clusters (variable in size) of endocrine cells (secondary islets) along the anterior–posterior axis of the pancreas. All four types of endocrine cells were found in the principal islet, but secondary islets may or may not contain PP-cells. In addition, there were also discrete endocrine cells throughout the pancreas. In all co-localization experiments, we did not find any endocrine cells positive for more than one hormone markers, suggesting that these endocrine cells produce only a single hormone. In both principal and secondary islets, we found that β-cells were generally located in the center and non- β cells in the periphery; reminiscent of the “mantel–core” organization of islets of Langerhans in mammals where β-cells form the core and non- β-cells the mantel. In zebrafish primary islet, β-cells constitute most of the mass (∼50%), followed by δ-cells and α-cells (20–25% each), and PP-cells (1–2%); this is also similar to the composition of mammalian islets.