Class 3 semaphorins and their receptors in physiological and pathological angiogenesis

Class 3 semaphorins (Sema3) are a family of secreted proteins that were originally identified as axon guidance factors mediating their signal transduction by forming complexes with neuropilins and plexins. However, the wide expression pattern of Sema3 suggested additional functions other than those associated with the nervous system, and indeed many studies have now indicated that Sema3 proteins and their receptors play a role in angiogenesis. The present review specifically focuses on recent evidence for this role in both physiological and pathological angiogenesis.     

Discrete roles for secreted and transmembrane semaphorins in neuronal growth cone guidance in vivo

From the initial stages of axon outgrowth to the formation of a functioning synapse, neuronal growth cones continuously integrate and respond to multiple guidance cues. To investigate the role of semaphorins in the establishment of appropriate axon trajectories, we have characterized a novel secreted semaphorin in grasshopper, gSema 2a. Sema 2a is expressed in a gradient in the developing limb bud epithelium during Ti pioneer axon outgrowth. We demonstrate that Sema 2a acts as chemorepulsive guidance molecule critical for axon fasciculation and for determining both the initial direction and subsequent pathfinding events of the Ti axon projection. Interestingly, simultaneous perturbation of both secreted Sema 2a and transmembrane Sema I results in a broader range and increased incidence of abnormal Ti pioneer axon phenotypes, indicating that different semaphorin family members can provide functionally distinct guidance information to the same growth cone in vivo.     

N-cadherin mediates retinal lamination,maintenance of forebrain compartments and patterning of retinal neurites

The complex, yet highly ordered and predictable, structure of the neural retina is one of the most conserved features of the vertebrate central nervous system. In all vertebrate classes, retinal neurons are organized into laminae with each neuronal class adopting specific morphologies and patterns of connectivity. Using genetic analyses in zebrafish, we demonstrate that N-cadherin (Ncad) has several distinct and crucial functions during the establishment of retinal organization. Although the location of cell division is disorganized in embryos with reduced or no Ncad function, different classes of retinal neurons are generated. However, these neurons fail to organize into correct laminae, most probably owing to compromised adhesion between retinal cells. In addition, amacrine cells exhibit exuberant and misdirected outgrowth of neurites that contributes to severe disorganization of the inner plexiform layer. Retinal ganglion cells also exhibit defects in process outgrowth, with axons exhibiting fasciculation defects and adopting incorrect ipsilateral trajectories. At least some of these defects are likely to be due to a failure to maintain compartment boundaries between eye, optic nerve and brain. Although in vitro studies have implicated Fgf receptors in modulating the axon outgrowth promoting properties of Ncad, most aspects of the Ncad mutant phenotype are not phenocopied by treatments that block Fgf receptor function.     

Caenorhabditis elegans PlexinA, PLX-1, interacts with transmembrane semaphorins and regulates epidermal morphogenesis

The plexin family transmembrane proteins are putative receptors for semaphorins, which are implicated in the morphogenesis of animal embryos, including axonal guidance. We have generated and characterized putative null mutants of the C. elegans plexinA gene, plx-1. plx-1 mutants exhibited morphological defects: displacement of ray 1 and discontinuous alae. The epidermal precursors for the affected organs were aberrantly arranged in the mutants, and a plx-1::gfp transgene was expressed in these epidermal precursor cells as they underwent dynamic morphological changes. Suppression of C. elegans transmembrane semaphorins, Ce-Sema-1a and Ce-Sema-1b, by RNA interference caused a displacement of ray 1 similar to that of plx-1 mutants, whereas mutants for the Ce-Sema-2a/mab-20 gene, which encodes a secreted-type semaphorin, exhibited phenotypes distinct from those of plx-1 mutants. A heterologous expression system showed that Ce-Sema-1a, but not Ce-Sema-2a, physically bound to PLX-1. Our results indicate that PLX-1 functions as a receptor for transmembrane-type semaphorins, and, though Ce-Sema-2a and PLX-1 both play roles in the regulation of cellular morphology during epidermal morphogenesis, they function rather independently.     

Mammalian osmolytes and S-nitrosoglutathione promote Delta F508 cystic fibrosis transmembrane conductance regulator (CFTR) protein maturation and function

In cystic fibrosis, the absence of functional CFTR results in thick mucous secretions in the lung and intestines, as well as pancreatic deficiency. Although expressed at high levels in the kidney, mutations in CFTR result in little or no apparent kidney dysfunction. In an effort to understand this phenomenon, we analyzed Delta F508 CFTR maturation and function in kidney cells under conditions that are common to the kidney, namely osmotic stress. Kidney cells were grown in culture and adapted to 250 mM NaCl and 250 mM urea. High performance liquid chromatography analysis of lysates from kidney cells adapted to these conditions identified an increase in the cellular osmolytes glycerophosphorylcholine, myo-inositol, sorbitol, and taurine. In contrast to isoosmotic conditions, hyperosmotic stress led to the proper folding and processing of Delta F508 CFTR. Furthermore, three of the cellular osmolytes, when added individually to cells, proved effective in promoting the proper folding and processing of the Delta F508 CFTR protein in both epithelial and fibroblast cells. Whole-cell patch clamping of osmolyte-treated cells showed that Delta F508 CFTR had trafficked to the plasma membrane and was activated by forskolin. Encouraged by these findings, we looked at other features common to the kidney that may impact Delta F508 maturation and function. Interestingly, a small molecule, S-nitrosoglutathione, which is a substrate for gamma glutamyltranspeptidase, an abundant enzyme in the kidney, likewise promoted Delta F508 CFTR maturation and function. S-Nitrosoglutathione-corrected Delta F508 CFTR exhibited a shorter half-life as compared with wild type CFTR. These results demonstrate the feasibility of a small molecule approach as a therapeutic treatment in promoting Delta F508 CFTR maturation and function and suggest that an additional treatment may be required to stabilize Delta F508 CFTR protein once present at the plasma membrane. Finally, our observations may help to explain why Delta F508 homozygous patients do not present with kidney dysfunction.     

Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates.

In Drosophila, plexin A is a functional receptor for semaphorin-1a. Here we show that the human plexin gene family comprises at least nine members in four subfamilies. Plexin-B1 is a receptor for the transmembrane semaphorin Sema4D (CD100), and plexin-C1 is a receptor for the GPI-anchored semaphorin Sema7A (Sema-K1). Secreted (class 3) semaphorins do not bind directly to plexins, but rather plexins associate with neuropilins, coreceptors for these semaphorins. Plexins are widely expressed: in neurons, the expression of a truncated plexin-A1 protein blocks axon repulsion by Sema3A. The cytoplasmic domain of plexins associates with a tyrosine kinase activity. Plexins may also act as ligands mediating repulsion in epithelial cells in vitro. We conclude that plexins are receptors for multiple (and perhaps all) classes of semaphorins, either alone or in combination with neuropilins, and trigger a novel signal transduction pathway controlling cell repulsion.     

Zebrafish mosaic eyes is a novel FERM protein required for retinal lamination and retinal pigmented epithelial tight junction formation

Polarization is a common feature of many types of cells, and we are beginning to understand how cells become polarized. The role of cell polarity in development and tissue morphogenesis, however, is much less well understood. Our previous analysis of the mosaic eyes (moe) mutations revealed that moe is required for retinal lamination and also suggested that zebrafish moe function is required in the retinal pigmented epithelium (RPE) for the proper localization of adjacent retinal cell divisions at the apical neuroepithelial surface. To understand the function of moe in the RPE, we cloned the moe locus and show that it encodes a novel FERM (for 4.1 protein, ezrin, radixin, moesin) domain-containing protein. Expression of moe in the eye, kidney, and brain reflects phenotypes found in moe(-) mutants, including RPE and retinal lamination defects, edema, and small or absent brain ventricles. We show that moe function is required for tight junction formation in the RPE. We suggest that moe may be a necessary component of the crumbs pathway that regulates apical cell polarity and also may play a role in photoreceptor morphogenesis.     

Reelin regulates cadherin function via Dab1/Rap1 to control neuronal migration and lamination in the neocortex

Neuronal migration is critical for establishing neocortical cell layers and migration defects can cause neurological and psychiatric diseases. Recent studies show that radially migrating neocortical neurons use glia-dependent and glia-independent modes of migration, but the signaling pathways that control different migration modes and the transitions between them are poorly defined. Here, we show that Dab1, an essential component of the reelin pathway, is required in radially migrating neurons for glia-independent somal translocation, but not for glia-guided locomotion. During migration, Dab1 acts in translocating neurons to stabilize their leading processes in a Rap1-dependent manner. Rap1, in turn, controls cadherin function to regulate somal translocation. Furthermore, cell-autonomous neuronal deficits in somal translocation are sufficient to cause severe neocortical lamination defects. Thus, we define the cellular mechanism of reelin function during radial migration, elucidate the molecular pathway downstream of Dab1 during somal translocation, and establish the importance of glia-independent motility in neocortical development.     

Calcium and retinal function

We survey the primary roles of calcium in retinal function, including photoreceptor transduction, transmitter release by different classes of retinal neuron, calcium-mediated regulation of gap-junctional conductance, activation of certain voltage-gated channels for K⁺ and C1⁻, and modulation of postsynaptic potentials in retinal ganglion cells. We discuss three mechanisms for changing [Ca²⁺]_i, which include flux through voltage-gated calcium channels, through ligand-gated channels, and by release from stores. The neuromodulatory pathways affecting each of these routes of entry are considered. The many neuromodulatory mechanisms in which calcium is a player are described and their effects upon retinal function discussed.     

Mammalian social systems: Structure and function

Mammalian societies are complex socio-ecological systems controlled by the interactions of numerous internal constraints and external factors. We present a simple model describing these systems functionally in terms of the adaptive behavioural strategies for resource exploitation, predation avoidance and mating and rearing of young to maturity shown by the individuals that comprise them. The relations between species-specific limitations on the range of potential individual social behaviour and the environmental variables to which the system is responsive are analysed and hypotheses from correlational and analytical field studies examined. We advocate the continued development of sophisticated systems-analytical approaches to societal analysis taking into account the contrasting informational provenance of factors of different types. This is preferred to either an over-emphasis on environmental determination or excessively formalised neo-darwinian modelling based on assumptions from genetics and selection theory alone.     

Mammalian septin function in hemostasis and beyond

Interest in the biology of mammalian septin proteins has undergone a birth in recent years. Originally identified as critical for yeast budding throughout the 1970s, the septin family is now recognized to extend from yeast to humans and is associated with a variety of events ranging from cytokinesis to vesicle trafficking. An emerging theme for septins is their presence at sites where active membrane or cytoplasmic partitioning is occurring. Here, we briefly review the mammalian septin protein family and focus on a prototypic human and mouse septin, termed SEPT5, that is expressed in the brain, heart, and megakaryocytes. Work from neurobiology laboratories has linked SEPT5 to the exocytic complex of neurons, with implications that SEPT5 regulates neurotransmitter release. Striking similarities exist between neurotransmitter release and the platelet-release reaction, which is a critical step in platelet response to vascular injury. Work from our laboratory has characterized the platelet phenotype from mice containing a targeted deletion of SEPT5. Most strikingly, platelets from SEPT5(null) animals aggregate and release granular contents in response to subthreshold levels of agonists. Thus, the characterization of a SEPT5-deficient mouse has linked SEPT5 to the platelet exocytic process and, as such, illustrates it as an important protein for regulating platelet function. Recent data suggest that platelets contain a wide repertoire of different septin proteins and assemble to form macromolecular septin complexes. The mouse platelet provides an experimental framework to define septin function in hemostasis, with implications for neurobiology and beyond.     

The zebrafish Pard3 ortholog is required for separation of the eye fields and retinal lamination

The vertebrate retina develops from a sheet of neuroepithelial cells. Because adherens and tight junctions are critical for epithelial and neuronal differentiation in a variety of eukaryotic systems, we examined the role of Par-3, a PDZ scaffold protein that is critical in cellular membrane junction formation. We cloned the zebrafish Par-3 ortholog (pard3), which encodes two Pard3 proteins (150 and 180 kDa) that differ in their carboxyl-terminus. Immunohistochemistry revealed that Pard3 localized to the apical region of the retinal and brain neuroepithelium, partially overlapping the adherens junction-associated actin bundles. After retinal lamination, the Pard3 protein was restricted to the outer limiting membrane and the outer and inner plexiform layers in the retina. Reducing Pard3 expression with antisense morpholinos caused loss of the retinal pigmented epithelia, disruption of retinal lamination, and cell death in the ventral diencephalon, which resulted in cyclopia. Overexpressing Pard3 by injection of wild-type pard3 mRNA resulted in cyclopia and eyeless embryos. Thus, Pard3 plays a critical role in the origination and separation of zebrafish eye fields and retinal lamination.     

Coupling between transmembrane calcium transport and membrane potential in retinal rod discs

Changes in the transmembrane potential of bovine rod discs were studied by use of the potential-sensitive fluorescence probes diS-C3-(5) and diBA-C4-(5). The disc membrane was shown to be impermeable to potassium ions. Their concentration in the disc is as high as 2.1 +/- 0.3 mM. The permeability of the disc membrane to Ca2+ was shown to be selective. The accumulation and release of Ca2+ were found to be accompanied by the generation of inside positive and inside negative transmembrane potentials, respectively. The uptake of Ca2+ in the discs may operate against the concentration gradient of the ion. The value of the potential developed is directly proportional to the logarithm of free Ca2+ concentration in the medium (delta phi m = 11.2 +/- 1.6 mV at 4.85 microM Ca2+fr). The accumulation of Ca2+ is decreased by sodium ions and totally inhibited by monensin. This indicates that a Na-Ca exchange process participates in Ca2+ uptake of photoreceptor discs.     

Naturally occurring CCR5 extracellular and transmembrane domain variants affect HIV-1 Co-receptor and ligand binding function.

Analysis of CCR5 variants in human immunodeficiency virus, type 1 (HIV-1), high risk cohorts led to the identification of multiple single amino acid substitutions in the amino-terminal third of the HIV-1 co-receptor CCR5 suggesting the possibility of protective and permissive genotypes; unfortunately, the low frequency of these mutations did not led to correlation with function. Therefore, we used analytical methods to assess the functional and structural significance of six of these variant receptors in vitro. These studies showed three categories of effects on CCR5 function. 1) Mutations in the first extracellular domain of CCR5 severely reduce specific ligand binding and chemokine-induced chemotaxis. 2) An extracellular domain variant, A29S, when co-expressed with CD4, supported HIV-1 infection whereas the others do not. 3) The transmembrane region variants of CCR5 support monotropic HIV-1 infection that is blocked by addition of some receptor agonists. Mutations in the first and second transmembrane domains increase RANTES (regulated on activation normal T-cell expressed) binding affinity but did not affect MIP1beta binding affinity presumably based on differences in ligand-receptor interaction sites. Furthermore, the CCR5 transmembrane mutants do not respond to RANTES with the classical bell-shaped chemotactic response curve, suggesting that they are resistant to RANTES-induced desensitization. These data demonstrate that single amino acid changes in the extracellular domains of CCR5 can have profound effects on both HIV-1 co-receptor and specific ligand-induced functions, whereas mutations in the transmembrane domain only affect the response to chemokine ligands.     

Betaretroviral Envelope Subunits Are Noncovalently Associated and Restricted to the Mammalian Class

The structure of the transmembrane subunit (TM) of the retroviral envelope glycoprotein (Env) is highly conserved among most retrovirus genera and includes a pair of cysteines that forms an intramolecular disulfide loop within the ectodomain. Alpha-, gamma-, and deltaretroviruses have a third cysteine, adjacent to the loop, which forms a disulfide bond between TM and the surface subunit (SU) of Env, while lentiviruses, which have noncovalently associated subunits, lack this third cysteine. The Betaretrovirus genus includes Jaagsiekte sheep retrovirus (JSRV) and mouse mammary tumor virus (MMTV), as well as many endogenous retroviruses. Envelope subunit association had not been characterized in the betaretroviruses, but lack of a third cysteine in the TM ectodomain suggested noncovalently associated subunits. We tested the Env proteins of JSRV and MMTV, as well as human endogenous retrovirus K (HERV-K)108—a betaretrovirus-like human endogenous retrovirus—for intersubunit bonding and found that, as in the lentiviruses, the Env subunits lack an intersubunit disulfide bond. Since these results suggest that the number of cysteines in the TM loop region readily distinguishes between covalent and noncovalent structure, we surveyed endogenous retroviral TM sequences in the genomes of vertebrates represented in public databases and found that (i) retroviruses with noncovalently associated subunits have been present during all of anthropoid evolution and (ii) the noncovalent env motif is limited to mammals, while the covalent type is found among five vertebrate classes. We discuss implications of these findings for retroviral evolution, cross-species transmissions, and recombination events involving the env gene.     

Mammalian sex chromosomes: Evolution of organization and function

Comparisons of chromosome size, morphology and gene arrangements between mammals of different species permit us to deduce the genome characteristics of the common ancestor, and to chart the changes that have occurred during the divergence of the two lineages. The more distantly related are the species compared, the more remote the common ancestor whose characteristics can be deduced. This means that, providing there are sufficient similarities to warrant comparison, the more divergent the species compared, the more significant the contribution to our understanding of the organization of an ancestral mammalian genome and the process of mammalian genome evolution. One of the genetic surprises of the last decade was the discovery that, although gross karyotypes of distantly related orders of eutherian mammals (e.g. cat, cow, rabbit, man) have diverged extensively, gene mapping studies reveal the presence of large chromosome segments conserved across at least 60 million years (O'Brien et al. 1988). This finding makes it worthwhile to extend genetic comparisons to the two groups of mammals most distantly related to eutherian mammals--marsupials and monotremes. Here we will review comparisons of the sex chromosomes in these three major groups of extant mammals, and show how they have led us to a new view of the evolution of mammalian sex chromosome organization and function in sex determination and X chromosome inactivation.     

Glutamate metabolic pathways and retinal function

Glutamate is a major neurotransmitter in the CNS but is also a key metabolite intimately coupled to amino acid production/degradation. We consider the effect of inhibition of two key glutamate metabolic enzymes: glutamine synthetase (GS) and aspartate aminotransferase on retinal function assessed using the electroretinogram to consider photoreceptoral (a-wave) and post-receptoral (b-wave) amplitudes. Quantitative immunocytochemistry was used to assess amino acid levels within photoreceptors, ganglion and Müller cells secondary to GS inhibition. Intravitreal injections of methionine sulfoximine reduced GS immunoreactivity in the rat retina. Additionally, glutamate and its precursor aspartate was reduced in photoreceptors and ganglion cells, but elevated in Müller cells. This reduction in neuronal glutamate was consistent with a deficit in neurotransmission (−75% b-wave reduction). Exogenous glutamine supply completely restored the b-wave, whereas other amino acid substrates (lactate, pyruvate, α-ketoglutarate, and succinate) only partially restored the b-wave (16–20%). Inhibition of the aminotranferases using aminooxyacetic acid had no effect on retinal function. However, aminooxyacetic acid application after methionine sulfoximine further reduced the b-wave (from −75% to −92%). The above data suggest that de novo glutamate synthesis involving aspartate aminotransferase can partially sustain neurotransmission when glutamate recycling is impaired. We also show that altered glutamate homeostasis results in a greater change in amino acid distribution in ganglion cells compared with photoreceptors.