Roles for ephrins in positionally selective synaptogenesis between motor neurons and muscle fibers.

Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and rostral portions of motor pools innervate rostral fibers within their targets. Here, we implicate A subfamily ephrins in this topographic mapping. First, developing muscles express all five of the ephrin-A genes. Second, rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5. Third, the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles. Fourth, topographic mapping is impaired in muscles of mutant mice lacking ephrin-A2 plus ephrin-A5. Thus, ephrins mediate or modulate positionally selective synapse formation. In addition, the rostrocaudal position of at least one motor pool is altered in ephrin-A5 mutant mice, indicating that ephrins affect nerve-muscle matching by intraspinal as well as intramuscular mechanisms.

Topographic guidance labels in a sensory projection to the forebrain.

Visual connections to the mammalian forebrain are known to be patterned by neural activity, but it remains unknown whether the map topography of such higher sensory projections depends on axon guidance labels. Here, we show complementary expression and binding for the receptor EphA5 in mouse retina and its ligands ephrin-A2 and ephrin-A5 in multiple retinal targets, including the major forebrain target, the dorsal lateral geniculate nucleus (dLGN). These ligands can act in vitro as topographically specific repellents for mammalian retinal axons and are necessary for normal dLGN mapping in vivo. The results suggest a general and economic modular mechanism for brain mapping whereby a projecting field is mapped onto multiple targets by repeated use of the same labels. They also indicate the nature of a coordinate system for the mapping of sensory connections to the forebrain.

Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in multiple aspects of retinocollicular mapping.

Ephrin-A2 and -A5 are thought to be anteroposterior mapping labels for the retinotectal/retinocollicular projection. Here, gene disruptions of both these ephrins are characterized. Focal retinal labeling reveals moderate map abnormalities when either gene is disrupted. Double heterozygotes also have a phenotype, showing an influence of absolute levels. In vitro assays indicate these ephrins are required for repellent activity in the target and also normal responsiveness in the retina. In double homozygotes, anteroposterior order is almost though not completely lost. Temporal or nasal retinal labelings reveal quantitatively similar but opposite shifts, with multiple terminations scattered widely over the target. These results indicate an axon competition mechanism for mapping, with a critical role for ephrins as anteroposterior topographic labels. Dorsoventral topography is also impaired, showing these ephrins are required in mapping both axes.

Enhanced plasticity of retinothalamic projections in an ephrin-A2/A5 double mutant.

Ascending sensory information reaches primary sensory cortical areas via thalamic relay neurons that are organized into modality-specific compartments or nuclei. Although the sensory relay nuclei of the thalamus show consistent modality-specific segregation of afferents, we now show in a wild-type mouse strain that the visual pathway can be surgically "rewired" so as to induce permanent retinal innervation of auditory thalamic cell groups. Applying the same rewiring paradigm to a transgenic mouse lacking the EphA receptor family ligands ephrin-A2 and ephrin-A5 results in more extensive rewiring than in the wild-type strain. We also show for the first time that ephrin-A2 and ephrin-A5 define a distinct border between visual and auditory thalamus. In the absence of this ephrin-A2/A5 border and after rewiring surgery, retinal afferents are better able to invade and innervate the deafferented auditory thalamus. These data suggest that signals that induce retinal axons to innervate the denervated auditory thalamus may compete with barriers, such as the ephrins, that serve to contain them within the normal target. The present findings thus show that the targeting of retinothalamic projections can be surgically manipulated in the mouse and that such plasticity can be controlled by proteins known to regulate topographic mapping.

Altered transcriptional patterns affecting several metabolic pathways in strains of Salmonella typhimurium which overexpress the fructose regulon.

Expression of beta-galactosidase in transcriptional fusions with the pps gene (encoding phosphoenolpyruvate [PEP] synthase), the aceBAK operon (encoding malate synthase, isocitrate lyase, and isocitrate dehydrogenase kinase, respectively), and the phs operon (encoding either thiosulfate reductase or a regulatory protein controlling its expression) was studied in Salmonella typhimurium. beta-Galactosidase synthesis in these strains was repressible either by growth in the presence of glucose or by the presence of a fruR mutation, which resulted in the constitutive expression of the fructose (fru) regulon. Five enzymes of gluconeogenesis (PEP synthase, PEP carboxykinase, isocitrate lyase, malate synthase, and fructose-1,6-diphosphatase) were shown to be repressed either by growth in the presence of glucose or the fruR mutation, while the glycolytic enzymes, enzyme I and enzymes II of the phosphotransferase system as well as phosphofructokinase, were induced either by growth in the presence of glucose or the fruR mutation. Overexpression of the cloned fru regulon genes (not including fruR) resulted in parallel repression of representative gluconeogenic, Krebs cycle, and glyoxylate shunt enzymes. Studies with temperature-sensitive mutants of S. typhimurium which synthesized heat-labile IIIFru proteins provided evidence that this protein plays a role in the regulation of gluconeogenic substrate utilization. Other mutant analyses revealed a complex relationship between fru gene expression and the expression of genes encoding gluconeogenic enzymes. Taken together, the results suggest that a number of genes encoding catabolic, biosynthetic, and amphibolic enzymes in enteric bacteria are transcriptionally regulated by a complex catabolite repression/activation mechanism which may involve enzyme IIIFru of the phosphotransferase system as one component of the regulatory system.

Genetic expression of enzyme I activity of the phosphoenolpyruvate:sugar phosphotransferase system in ptsHI deletion strains of Salmonella typhimurium.

Mutants expressing a novel enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system, termed enzyme I, were isolated from strains of Salmonella typhimurium which were deleted for the HPr and enzyme I structural genes. The mutations lay in a newly defined gene, termed ptsJ, which mapped on the S. typhimurium chromosome between the ptsHI operon and the cysA gene.

Assembly of yeast Sec proteins involved in translocation into the endoplasmic reticulum into a membrane-bound multisubunit complex.

Secretory-protein translocation into the endoplasmic reticulum (ER) is thought to be catalysed by integral membrane proteins. Genetic selections uncovered three Saccharomyces cerevisiae genes (SEC61, SEC62 and SEC63), mutations in which block import of precursor proteins into the ER lumen in vivo and in vitro. The DNA sequences of SEC62 and SEC63 predict multispanning membrane proteins, and biochemical characterization of the SEC62 protein (Sec62) confirms that it is an integral ER membrane protein. Here we show that Sec61, Sec62 and Sec63 are assembled with two additional proteins into a multisubunit membrane-associated complex. These results confirm previous predictions, based upon genetic interactions between the SEC genes, that Sec61, Sec62 and Sec63 act together to facilitate protein translocation into the ER.

Physiological consequences of the complete loss of phosphoryl-transfer proteins HPr and FPr of the phosphoenolpyruvate:sugar phosphotransferase system and analysis of fructose (fru) operon expression in Salmonella typhimurium.

Mutants of Salmonella typhimurium defective in the proteins of the fructose operon [fruB(MH)KA], the fructose repressor (fruR), the energy-coupling enzymes of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) (ptsH and ptsI), and the proteins of cyclic AMP action (cya and crp) were analyzed for their effects on cellular physiological processes and expression of the fructose operon. The fru operon consists of three structural genes: fruB(MH), which encodes the enzyme IIIFru-modulator-FPr tridomain fusion protein of the PTS; fruK, which encodes fructose-1-phosphate kinase; and fruA, which encodes enzyme IIFru of the PTS. Among the mutants analyzed were Tn10 insertion mutants and lacZ transcriptional fusion mutants. It was found that whereas a fruR::Tn10 insertion mutant, several fruB(MH)::Mu dJ and fruK::Mu dJ fusion mutants, and several ptsHI deletion mutants expressed the fru operon and beta-galactosidase at high constitutive levels, ptsH point mutants and fruA::Mu dJ fusion mutants retained inducibility. Inclusion of the wild-type fru operon in trans did not restore fructose-inducible beta-galactosidase expression in the fru::Mu dJ fusion mutants. cya and crp mutants exhibited reduced basal activities of all fru regulon enzymes, but inducibility was not impaired. Surprisingly, fruB::Mu dJ crp or cya double mutants showed over 10-fold inducibility of the depressed beta-galactosidase activity upon addition of fructose, even though this activity in the fruB::Mu dJ fusion mutants that contained the wild-type cya and crp alleles was only slightly inducible. By contrast, beta-galactosidase activity in a fruK::Mu dJ fusion mutant, which was similarly depressed by introduction of a crp or cya mutation, remained constitutive. Other experiments indicated that sugar uptake via the PTS can utilize either FPr-P or HPr-P as the phosphoryl donor, but that FPr is preferred for fructose uptake whereas HPr is preferred for uptake of the other sugars. Double mutants lacking both proteins were negative for the utilization of all sugar substrates of the PTS, were negative for the utilization of several gluconeogenic carbon sources, exhibited greatly reduced adenylate cyclase activity, and were largely nonmotile. These phenotypic properties are more extreme than those observed for tight ptsH and ptsI mutants, including mutants deleted for these genes. A biochemical explanation for this fact is proposed.