High-resolution, three-dimensional mapping of gene expression using GeneExpressMap (GEM).

The analysis of temporal and spatial patterns of gene expression is critically important for many kinds of developmental studies, including the construction of gene regulatory networks. Recently, multiplex, fluorescent, whole mount in situ hybridization (multiplex F-WMISH), applied in combination with confocal microscopy, has emerged as the method of choice for high-resolution, three-dimensional (3D) mapping of gene expression patterns in developing tissues. We have developed an image analysis tool, GeneExpressMap (GEM), that facilitates the rapid, 3D analysis of multiplex F-WMISH data at single-cell resolution. GEM assigns F-WMISH signal to individual cells based upon the proximity of cytoplasmic hybridization signal to cell nuclei. Here, we describe the features of GEM and, as a test of its utility, we use GEM to analyze patterns of regulatory gene expression in the non-skeletogenic mesoderm of the early sea urchin embryo. GEM greatly extends the power of multiplex F-WMISH for analyzing patterns of gene expression and is a valuable tool for gene network analysis and many other kinds of developmental studies.

Leptin secretion in horses: effects of dexamethasone, gender, and testosterone.

Five experiments were performed to evaluate the effects of dexamethasone (DEX), gender, and testosterone on plasma leptin concentrations in horses. In experiment 1, plasma leptin, insulin, glucose, and IGF-1 concentrations were increased (P < 0.01) in stallions following five daily injections of DEX (125 microg/kg BW). In experiment 2, leptin concentrations increased (P < 0.01) in mares, geldings, and stallions following a single injection of DEX, and the response was greater (P < 0.01) in mares and geldings than in stallions. The gender effect was confounded by differences in body condition scores and diet; however, based on stepwise regression analysis, both BCS and gender were significant sources of variation in the best fit model for pre-DEX leptin concentrations (R(2) = 0.65) and for maximum leptin response to DEX (R(2) = 0.75). In experiment 3, in which mares and stallions were pair-matched based on age and body condition and fed similar diets, mares again had higher (P < 0.01) leptin concentrations than stallions after DEX treatment as used in experiment 2. In experiment 4, there was no difference (P > 0.1) in plasma leptin response in mares following four single-injection doses of DEX from 15.6 to 125 microg/kg BW. In experiment 5, treatment of mares with testosterone propionate every other day for 5 days did not alter (P > 0.1) plasma leptin concentrations or the leptin response to DEX. In conclusion, multiple injections of DEX increase leptin concentrations in stallions, as does a single injection in mares (as low as 15.6 microg/kg BW), geldings and stallions. The greater leptin levels observed in mares and geldings relative to stallions were due partially to their greater body condition and partially to the presence of hyperleptinemic individuals; however, even after accounting for body condition and diet, mares still had greater leptin concentrations than stallions after DEX administration. Elevation of testosterone levels in mares for approximately 10 days did not alter leptin concentrations in mares.

Effect of dexamethasone, feeding time, and insulin infusion on leptin concentrations in stallions.

Three experiments tested the hypotheses that daily cortisol rhythm, feeding time, and/or insulin infusion affect(s) leptin secretion in stallions. Ten mature stallions received ad libitum hay and water and were fed a grain concentrate once daily at 0700. In Exp. 1, stallions received either a single injection of dexamethasone (125 microg/kg BW i.m.; n = 5) or vehicle (controls; n = 5) at 0700 on d -1. Starting 24 h later, blood samples were collected every 2 h for 36 h via jugular venipuncture. Cortisol in control stallions varied (P < 0.01) with time, with a morning peak and evening nadir; dexamethasone suppressed (P < 0.01) cortisol concentrations. Leptin and insulin were greater (P < 0.01) in the treated stallions, as was the insulin response to feeding (P < 0.01). Leptin in control stallions varied (P < 0.01) in a diurnal pattern, peaking approximately 10 h after onset of eating. This pattern of leptin secretion was similar, although of greater magnitude (P < 0.01), in treated stallions. In Exp. 2, five stallions were fed the concentrate portion of their diet daily at 0700 and five were switched to feeding at 1900. After 14 d on these regimens, blood samples were collected every 4 h for 48 h and then twice daily for 5 d. Cortisol varied diurnally (P = 0.02) and was not altered (P = 0.21) by feeding time. Insulin and leptin increased (P < 0.01) after feeding, and the peaks in insulin and leptin were shifted 12 h by feeding at 1900. In Exp. 3, six stallions were used in two 3 x 3 Latin square experiments. Treatments were 1) normal daily meal at 0700; 2) no feed for 24 h; and 3) no feed and a bolus injection of insulin (0.4 mIU/kg BW i.v.) followed by infusion of insulin (1.2 mIU.kg BW(-1).min(-1)) for 180 min, which was gradually decreased to 0 by 240 min; sufficient glucose was infused to maintain euglycemia. Plasma insulin increased (P < 0.01) in stallions when they were meal-fed (to approximately 150 microIU/mL) or infused with insulin and glucose (to approximately 75 microIU/mL), but insulin remained low (10 microIU/mL or less) when they were not fed. The increases in insulin were paralleled by gradual increases (P < 0.01) in leptin concentrations 3 to 4 h later in stallions fed or infused with insulin and glucose. When stallions were not fed, leptin concentrations remained low. These results demonstrate that feeding time, and more specifically the insulin increase associated with a meal, not cortisol rhythm, drives the postprandial increase in plasma leptin concentrations in horses.

Early development of ocular dominance columns.

The segregation of lateral geniculate nucleus (LGN) axons into ocular dominance columns is believed to involve a prolonged, activity-dependent sorting process. However, visualization of early postnatal ferret LGN axons by direct LGN tracer injections revealed segregated ocular dominance columns <7 days after innervation of layer 4. These early columns were unaffected by experimentally induced imbalances in retinal activity, implying that different mechanisms govern initial column formation and their modification during the subsequent critical period. Instead of activity-dependent plasticity, we propose that ocular dominance column formation relies on the targeting of distinct axonal populations to defined locales in cortical layer 4.

Conservation of absolute foveal area in New World monkeys. A constraint on eye size and conformation.

The foveal specializations of five New World monkeys, the marmoset, Callithrix jacchus; the golden-handed tamarin, Saguinus midas niger; the squirrel monkey, Saimiri ustius; the capuchin monkey, Cebus apella; and the howler monkey, Alouatta caraya were compared. Although retinal area varies by over a factor of two in these monkeys, the area of the fovea does not covary with retinal area and remains approximately the same absolute size, as measured by the dimensions of the high density region of cones, or the rod-free region. This constancy in foveal size also holds for rhesus monkeys and humans, bringing the variation in retinal area to a factor of five. Alouatta caraya is unusual, distinguished by a very high central cone density and a small rod-free zone. Physiological constraints that might limit foveal area over a wide range of eye sizes are considered.

Development of ocular dominance columns in the absence of retinal input.

The initial establishment of ocular dominance columns in visual cortex is believed to involve the segregation of overlapping geniculocortical axons into eye-specific patches based on patterns of correlated activity. However, we found that total removal of retinal influence early in visual development did not prevent segregation of geniculocortical axons into alternating stripes with periodicity normal for ocular dominance columns. Because the patterning of geniculocortical afferents resists this dramatic change in the level, source and pattern of spontaneous activity, we propose that formation of ocular dominance columns relies on molecular cues present on thalamic axons, cortical cells or both.

Poliomyelitis in intraspinally inoculated poliovirus receptor transgenic mice.

Mice transgenic with the human poliovirus receptor gene develop clinical signs and neuropathology similar to those of human poliomyelitis when neurovirulent polioviruses are inoculated into the central nervous system (CNS). Factors contributing to disease severity and the frequencies of paralysis and mortality include the poliovirus strain, dose, and gender of the mouse inoculated. The more neurovirulent the virus, as defined by monkey challenge results, the higher the rate of paralysis, mortality, and severity of disease. Also, the time to disease onset is shorter for more neurovirulent viruses. Male mice are more susceptible to polioviruses than females. TGM-PRG-3 mice have a 10-fold higher transgene copy number and produce 3-fold more receptor RNA and protein levels in the CNS than TGM-PRG-1 mice. CNS inoculations with type III polioviruses differing in relative neurovirulence show that these mouse lines are similar in disease frequency and severity, demonstrating that differences in receptor gene dosage and concomitant receptor abundance do not affect susceptibility to infection. However, there is a difference in the rate of accumulation of clinical signs. The time to onset of disease is shorter for TGM-PRG-3 than TGM-PRG-1 mice. Thus, receptor dosage affects the rate of appearance of poliomyelitis in these mice.

Foot-and-mouth disease virus 2A protease mediates cleavage in attenuated Sabin 3 poliovirus vectors engineered for delivery of foreign antigens.

Poliovirus vectors are being studied as potential vaccine delivery systems, with foreign genetic sequences incorporated as part of the viral genome. The foreign sequences are expressed as part of the viral polyprotein. Addition of proteolytic cleavage sites at the junction of the foreign polypeptide and the viral proteins results in cleavage during polyprotein processing. The ability of foot-and-mouth disease virus (FMDV) 2A to mediate proteolytic cleavage in the context of poliovirus vectors was studied. The results demonstrate that FMDV 2A is able to generate cleavage of the foreign antigen from the viral polyprotein. A second cleavage event between the FMDV 2A peptide and the foreign protein was also observed.

Factors controlling the dendritic arborization of retinal ganglion cells.

The effects of changing retinal ganglion cell (RGC) density and availability of presynaptic sites on the development of RGC dendritic arbor in the developing chick retina were contrasted. Visual form deprivation was used to induce ocular enlargement and expanded retinal area resulting in a 20-30% decrease in RGC density. In these retinas, RGC dendritic arbors increased in a compensatory manner to maintain the inner nuclear layer to RGC convergence ratio in a way that is consistent with simple stretching; RGC dendritic arbors become larger with increased branch lengths, but without change in the total number of branches. In the second manipulation, partial optic nerve section was used to produce areas of RGC depletion of approximately 60% in the central retina. This reduction in density is comparable to the density of locations in the normal peripheral retina. In RGC depleted retinas, dendritic arbor areas of RGCs in the central retina grow to match the size of normal peripheral arbors. In contrast to the expanded case, two measures of intrinsic arbor structure are changed in RGC-depleted retinas; the branch density of RGC dendrites is greater, and the relative areas of the two arbors of bistratified cells are altered. We discuss the potential roles of retinal growth, local RGC density, and availability of presynaptic terminals in the developmental control of RGC dendritic arbor.

Differential regulation of Ia expression and antigen presentation by listeriolysin-producing versus non-producing strains of Listeria monocytogenes.

Listeria monocytogenes is an intracellular bacterial pathogen. A single gene product, listeriolysin (LLO), is critical for the induction of protective immunity. We now show that listeria that produce functional LLO augment Ia expression by macrophages and are better presented to a Th1, CD4+ anti-listeria T cell line. We used two genetically engineered strains of listeria which differed only in their ability (Ly+) or inability (Ly-) to produce functional LLO. Ia-negative murine macrophages ingested either Ly+ or Ly-, and then were stimulated by interferon-gamma (IFN-gamma). Increasing numbers of live Ly+, but not Ly-, augmented IFN-gamma-induced Ia expression. Ly+ by itself did not induce Ia expression. Heat-killed Ly+ and Ly- did not augment IFN-gamma-induced Ia expression. The abundance of Ia on the macrophage cell surface is one major determinant of antigen presentation to CD4+ T cells. Consistent with their ability to augment la expression, Ly+ were better presented than Ly- to a CD4+, Th1, anti-listeria T cell line. When macrophages and T cells were from different inbred mouse strains, antigen presentation required identity at the class II region of the MHC gene complex. This indicated that antigen presentation occurred via Ia molecules. The increased ability of macrophages to present Ly+ is a product of the macrophage-listeria interaction, not a property of the T cell tine 86. If Ia-negative macrophages ingested Listeria and were then stimulated by IFN-gamma, Ly+ was presented more efficiently than Ly-. On the other hand, if Ia-positive macrophages ingested Listeria, then Ly+ and Ly- were presented equally well to T cells. Altogether our data is consistent with the hypothesis that macrophages interact differently with Ly+, and that this contributes to the ability of only live Ly+ to induce protective immunity.

Strain-specific reverse transcriptase PCR assay: means to distinguish candidate vaccine from wild-type strains of respiratory syncytial virus.

Candidate live-virus vaccines for respiratory syncytial virus are being developed and are beginning to be evaluated in clinical trials. To distinguish candidate vaccine strains from wild-type strains isolated during these trials, we developed PCR assays specific to two sets of candidate vaccine strains. The two sets were a group A strain (3A), its three attenuated, temperature-sensitive variant strains, a group B strain (2B), and its four attenuated, temperature-sensitive variant strains. The PCR assays were evaluated by testing 18 group A wild-type strains, the 3A strains, 9 group B wild-type strains, and the 2B strains. PCR specific to group A wild-type strains amplified only group A wild-type strains, and 3A-specific PCR amplified only 3A strains. PCR specific to group B wild-type strains amplified all group A and group B strains but gave a 688-bp product for group B wild-type strains, a 279-bp product for 2B strains, a 547-bp product for all group A strains, and an additional 688-bp product for some group A strains, including 3A strains. These types of PCR assays can, in conjunction with other methods, be used to efficiently distinguish candidate vaccine strains from other respiratory syncytial virus strains.

Differential regulation of TNF-alpha production by listeriolysin-producing versus nonproducing strains of Listeria monocytogenes.

Only Listeria monocytogenes that produce listeriolysin O (LLO) elicit protective immunity. Given the importance of tumor necrosis factor alpha (TNF-alpha) in anti-Listeria immunity, we have investigated TNF-alpha production by macrophages after they ingested live LLO-producing compared to LLO-non-producing bacteria. We used two genetically engineered strains of Listeria that differed only in their ability (Ly+) or inability (Ly-) to produce LLO. Ly+ and Ly- caused the same kinetics of increased mRNA abundance for TNF-alpha during the first 90 min after phagocytosis. However, only Ly+ caused sustained transcription of TNF-alpha mRNA, and this may account for the increased release of TNF-alpha. The transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) prevented the sustained abundance of cytokine mRNA 20 h after ingestion of Ly+. In addition, nuclear run-on assays indicated sustained transcription of TNF-alpha genes only after ingestion of Ly+. LLO itself was not responsible for the ability of Ly+ to stimulate the sustained transcription of the TNF-alpha genes. Instead, LLO may allow Listeria to survive within macrophages so that other bacterial products cause sustained TNF-alpha gene transcription. Both Ly+ and Ly- produced molecules, isolated by 50% ammonium sulfate, that induced cytokine production. In conclusion, we now report that Ly+ causes sustained transcription of the TNF-alpha gene and production of TNF-alpha by macrophages in vitro. We speculate that the TNF-alpha may activate endothelium and thus allow the recruitment of T cells to sites of infection. This may contribute to the ability of only LLO-producing Listeria to induce protective immunity.

Characterization of recombinant polioviruses expressing regions of rotavirus VP4, hepatitis B surface antigen, and herpes simplex virus type 2 glycoprotein D.

Recombinant polioviruses expressing antigens from rotavirus, herpes simplex virus type 2, and hepatitis B virus were generated. Fusion of the heterologous polypeptides to the amino terminus of the poliovirus polyprotein did not prevent myristylation of VP0, suggesting a novel mechanism of myristylation for these recombinant viruses. The effects of the parental genetic background, different foreign sequences, and different insert sizes on growth characteristics were compared. Both the size and the nature of the heterologous sequence appeared to be factors influencing the growth and stability of recombinant polioviruses. All of the recombinants showed a temperature-sensitive phenotype, regardless of the genetic background (attenuated or wild type) from which they were derived. Preliminary studies with transgenic mice carrying the poliovirus receptor gene are discussed.

Attenuated poliovirus strain as a live vector: expression of regions of rotavirus outer capsid protein VP7 by using recombinant Sabin 3 viruses.

The ability to express heterologous antigens from attenuated poliovirus strains suggests the potential for use as live vectored vaccines. Full- or partial-length sequences of the gene encoding rotavirus major outer capsid protein VP7 were cloned into the open reading frame of a full-length cDNA copy of poliovirus Sabin type 3. They were inserted either at the 5' end or immediately after the capsid protein coding region, at the junction between precursors P1 and P2. A protease cleavage site for 3C protease was introduced 3' to the foreign sequences to enable proteolytic processing of the antigen from the poliovirus polyprotein. Infectious viruses were generated from several of the DNA constructs, and the presence of the foreign gene sequences was confirmed by reverse transcription of the viral RNA and PCR amplification. Viruses with inserts of about 300 bases maintained the foreign sequences during passage in Vero cells. Viruses carrying larger sequences were unstable, and deletions were generated within the foreign sequences. Expression of the VP7 polypeptides was demonstrated by immunoprecipitation with specific antiserum of labeled proteins from cells infected with Sabin 3 recombinant viruses. Comparative studies of RNA synthesis showed similar kinetics for Sabin 3 and the Sabin 3/VP7 recombinants. One-step growth curves showed that production of recombinant viruses was slower than that of Sabin 3 and that the final titers were 1 to 1.5 logs lower. Accumulation of VP7-containing precursors in infected cells suggests that slow cleavage at the engineered 3C protease site may be a limiting step in the growth of these recombinant Sabin polioviruses and may influence the permissible size of foreign sequence to be inserted.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation, characterization and regulation of the SPO7 sporulation gene.

SPO7 is one of several previously identified genes from the yeast Saccharomyces cerevisiae that is required for sporulation but not for vegetative growth. The SPO7 gene has been cloned by functional complementation and physically mapped 15-16 kb to the left of CEN1. Gene-disruption experiments confirmed that the cloned gene was the bona fide SPO7 gene. SPO7 codes for a 0.95-kb transcript that is expressed at approximately the same level in both vegetative and sporulating cells. The gene was sequenced and has the capacity to encode a 259-amino acid protein that does not appear to be related to other known proteins.

Cloning of chromosome I DNA from Saccharomyces cerevisiae: mutational analysis of the FUN2 transcribed region.

To increase the number of mutationally defined genes on chromosome I from Saccharomyces cerevisiae, most of the FUN2 (Function Unknown Now) transcribed region was deleted by gene replacement. Strains containing the deletion were viable, but grew with a 20% longer generation time. The mutation was recessive. Mutant haploids were able to mate, and homozygous mutant diploids were able to sporulate, giving asci containing four viable ascospores. These results indicate FUN2 is dispensable for the life cycle of S. cerevisiae, but required for an optimal growth rate.

Measles virus L protein evidences elements of ancestral RNA polymerase.

We have determined the nucleotide sequence of the measles virus (MV) L gene using a cDNA library encompassing the entire MV genome (J. Crowley et al. (1987) Intervirology, 28, 65-77). The L gene is 6639 nucleotides in length, and contains a single long open reading frame that could code for a protein of 247,611 kDa. Both the L gene and in particular the predicted L protein of MV bear substantial homology to their counterparts in Sendai virus and Newcastle disease virus, suggesting that the multifunctional nature of paramyxovirus L proteins imposes strong evolutionary constraints. The predicted MV L protein also contains distinct elements of a postulated ancestral RNA polymerase.

Sequence variability and function of measles virus 3' and 5' ends and intercistronic regions.

Sequences critical for the activity of the measles virus (MV) RNA polymerase in transcription and replication were analyzed using a MV genomic cDNA library containing overlapping clones encompassing the entire MV genome. Clones corresponding to the 3' and 5' ends of the MV genome were identified and sequenced, and these sequences were confirmed by primer extension experiments. Neither (+) nor (-) strand leader RNAs were detected in MV-infected cell extracts, using high specific activity riboprobes made form these clones. Clones representing each of the MV gene boundaries were also sequenced, and variations including point mutations, insertions, and deletions were noted. Together with the sequence of the MV L gene region, this report completes the sequence determination of the MV genome.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures.

MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16-1 mutation. MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth. As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M1 replication defects of mak16-1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library. The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I. Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade 1, confirming that this clone was derived from the appropriate region of chromosome I. An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis. 185 amino acids of this open reading frame were replaced with a 1.2 kb fragment carrying the S. cerevisiae URA3 gene by a one-step gene disruption. The resulting strains grew at a rate indistinguishable from the wild type at 20 degrees C, 30 degrees C, or 37 degrees C, but could not grow at 8 degrees C. The deleted region is thus essential only at 8 degrees C, and we name this gene LTE1 (low temperature essential).