Genetic dissection of plexin signaling in vivo.

Mammalian plexins constitute a family of transmembrane receptors for semaphorins and represent critical regulators of various processes during development of the nervous, cardiovascular, skeletal, and renal system. In vitro studies have shown that plexins exert their effects via an intracellular R-Ras/M-Ras GTPase-activating protein (GAP) domain or by activation of RhoA through interaction with Rho guanine nucleotide exchange factor proteins. However, which of these signaling pathways are relevant for plexin functions in vivo is largely unknown. Using an allelic series of transgenic mice, we show that the GAP domain of plexins constitutes their key signaling module during development. Mice in which endogenous Plexin-B2 or Plexin-D1 is replaced by transgenic versions harboring mutations in the GAP domain recapitulate the phenotypes of the respective null mutants in the developing nervous, vascular, and skeletal system. We further provide genetic evidence that, unexpectedly, the GAP domain-mediated developmental functions of plexins are not brought about via R-Ras and M-Ras inactivation. In contrast to the GAP domain mutants, Plexin-B2 transgenic mice defective in Rho guanine nucleotide exchange factor binding are viable and fertile but exhibit abnormal development of the liver vasculature. Our genetic analyses uncover the in vivo context-dependence and functional specificity of individual plexin-mediated signaling pathways during development.

The role of Abcb5 alleles in susceptibility to haloperidol-induced toxicity in mice and humans.

BACKGROUND: We know very little about the genetic factors affecting susceptibility to drug-induced central nervous system (CNS) toxicities, and this has limited our ability to optimally utilize existing drugs or to develop new drugs for CNS disorders. For example, haloperidol is a potent dopamine antagonist that is used to treat psychotic disorders, but 50% of treated patients develop characteristic extrapyramidal symptoms caused by haloperidol-induced toxicity (HIT), which limits its clinical utility. We do not have any information about the genetic factors affecting this drug-induced toxicity. HIT in humans is directly mirrored in a murine genetic model, where inbred mouse strains are differentially susceptible to HIT. Therefore, we genetically analyzed this murine model and performed a translational human genetic association study. METHODS AND FINDINGS: A whole genome SNP database and computational genetic mapping were used to analyze the murine genetic model of HIT. Guided by the mouse genetic analysis, we demonstrate that genetic variation within an ABC-drug efflux transporter (Abcb5) affected susceptibility to HIT. In situ hybridization results reveal that Abcb5 is expressed in brain capillaries, and by cerebellar Purkinje cells. We also analyzed chromosome substitution strains, imaged haloperidol abundance in brain tissue sections and directly measured haloperidol (and its metabolite) levels in brain, and characterized Abcb5 knockout mice. Our results demonstrate that Abcb5 is part of the blood-brain barrier; it affects susceptibility to HIT by altering the brain concentration of haloperidol. Moreover, a genetic association study in a haloperidol-treated human cohort indicates that human ABCB5 alleles had a time-dependent effect on susceptibility to individual and combined measures of HIT. Abcb5 alleles are pharmacogenetic factors that affect susceptibility to HIT, but it is likely that additional pharmacogenetic susceptibility factors will be discovered. CONCLUSIONS: ABCB5 alleles alter susceptibility to HIT in mouse and humans. This discovery leads to a new model that (at least in part) explains inter-individual differences in susceptibility to a drug-induced CNS toxicity.

The semaphorin 4D-plexin-B signalling complex regulates dendritic and axonal complexity in developing neurons via diverse pathways.

Semaphorins and their receptors, plexins, have emerged as key regulators of various aspects of neuronal development. In contrast to the Plexin-A family, the cellular functions of Plexin-B family proteins in developing neurons are only poorly understood. An activation of Plexin-B1 via its ligand, semaphorin 4D (Sema4D), produces an acute collapse of axonal growth cones in hippocampal and retinal neurons over the early stages of neurite outgrowth. However, the functional role of Sema4D-Plexin-B interactions over subsequent stages of neurite development, differentiation and maturation has not been characterized. Here we addressed this question using morphogenetic assays and time-lapse imaging on developing rat hippocampal neurons as a model system. Interestingly, Sema4D treatment over several hours was observed to promote branching and complexity in hippocampal neurons via the activation of Plexin-B1. The activation of receptor tyrosine kinases and the Rho kinase following Sema4D treatment was found to control dendritic and axonal morphogenesis by differentially regulating branching and extension. Phosphoinositide-3-kinase, but not extracellular signal-regulated kinase 1/2, was observed to be important for the stimulatory effects of Sema4D on dendritic branching. Furthermore, we observed that the mammalian target of rapamycin is activated downstream of Plexin-B1 and contributes to Sema4D-induced effects on dendritic branching. In contrast, glycogen synthase kinase-3 beta, another effector of phosphoinositide-3-kinase signalling, was not involved. Thus, our results show that Sema4D-Plexin-B interactions modulate dendritic and axonal arborizations of developing neurons by co-ordinated and concerted activation of diverse signalling pathways.

Plexin-B2, but not Plexin-B1, critically modulates neuronal migration and patterning of the developing nervous system in vivo.

Semaphorins and their receptors, plexins, have emerged as important cellular cues regulating key developmental processes. B-type plexins directly regulate the actin cytoskeleton in a variety of cell types. Recently, B-type plexins have been shown to be expressed in striking patterns in the nervous system over critical developmental windows. However, in contrast to the well characterized plexin-A family, the functional role of plexin-B proteins in neural development and organogenesis in vertebrates in vivo is not known. Here, we have elucidated the functional contribution of the two neuronally expressed plexin-B proteins, Plexin-B1 or Plexin-B2, toward the development of the peripheral nervous system and the CNS by generating and analyzing constitutive knock-out mice. The development of the nervous system was found to be normal in mice lacking Plexin-B1, whereas mice lacking Plexin-B2 demonstrated defects in closure of the neural tube and a conspicuous disorganization of the embryonic brain. After analyzing mutant mice, which bypassed neural tube defects, we observed a key requirement for Plexin-B2 in proliferation and migration of granule cell precursors in the developing dentate gyrus, olfactory bulb, and cerebellum. Furthermore, we identified semaphorin 4C as a high-affinity ligand for Plexin-B2 in binding and functional assays. Semaphorin 4C stimulated activation of ErbB-2 and RhoA via Plexin-B2 and enhanced proliferation and migration of granule cell precursors. Semaphorin 4C-induced proliferation of ventricular zone neuroblasts was abrogated in mice lacking Plexin-B2. These genetic and functional analyses reveal a key requirement for Plexin-B2, but not Plexin-B1, in patterning of the vertebrate nervous system in vivo.

[Research on genetic variation of heart fatty acid-binding protein gene in ten pig breeds].

The genetic variation of heart fatty acid-binding protein (H-FABP) gene in 561 pigs including Duroc, Landrace, Large Yorkshire, Nanchang white pig, Erhualian, Meishan, Yushan black pig, Leping spotted pig, Jinhua black head-hind pig and Shanggao black head-hind pig were detected by PCR-RFLP with Hinf I, Hae III and Msp I. The results showed as follows: (1) Nanchang white pig presented only HH genotype while other breeds varied at the Hinf I-RFLP site; (2) The exotic breeds including Duroc, Landrace, Large Yorkshire and the native breed Nanchang white pig were proved to be polymorphic while the five Chinese local breeds presented no variation at the Hae III-RFLP site; (3) Among the tested breeds only Duroc presented variation at the Msp I-RFLP site. It is noted that all the Chinese local breeds present as AADD--genotypes.

[Studies of population genetic relationships among 24 Chinese and exotic pig breeds using AFLP analysis].

A total of 12 AFLP primer combinations were used to detect genetic variation of pooled DNA in a sample of 19 Chinese native pig breeds, 1 cultivated pig breed and 4 European and American pig breeds. The genetic similarity coefficient of 24 pig beeds was calculated from AFLP data, UPGMA cluster analysis was also performed. The 12 primer combinations generated more than 1000 bands, of which 208 bands were polymorphic, 17.3 polymorphic markers were detected by one primer combination on the average. Thirteen putative breed specific bands were produced in the pooled DNA of 8 pig breeds. The cultivated pig breed and 4 exotic pig breeds were clustere into one group, while 19 Chinese native pig breeds were gathered into the other group in the UPGMA tree. The result indicated that AFLP analysis had high assay efficiency index (Ai) and provided a valuable tool for assaying genetic diversity and breed characterization in pigs. Chinese native pig breeds and exotic pig breeds show remarkable genetic differentiation, which had farther genetic relationships. Nanchang White pig and Large White pig, Yushan Black pig and Yanshan Black pig had intimate genetic relationships with each other respectively, which were consistent with its breeding history, geographical distribution and RAPD analysis results. In addition, the reasons for cluster results of some pig breeds from AFLP data were not consistent with morphology, geographical distribution and existing classification were discussed.

[Studies of the relationship of melanocortin receptor 1(MC1R) gene with coat color phenotype in pigs].

Although coat color in pigs has no direct relation with economic traits, it affects economic benefit significantly, coat color selection are widely used in pig breeding and production. PCR-Acc II-RFLP, PCR-BspH I-RFLP and PCR-SSCP were used in combination to analyze genotype at MC1R locus among individuals from 16 full-sib pedigrees and 6 Chinese native breeds including Jinhua, Jiaxing Black, Yushan Black, Leping Spotted, Shanggao Spotted and Shengxian Spotted pig. It was found that the Chinese native pig breeds carry a dominant black allele at MC1R at high frequency, this ED1 allele was suggested to be the major allele controlling black coat color in Chinese native pig breed. In addition, the evidence for a new allele was obtained in Shengxian Spotted pigs by PCR-SSCP analysis. It was reconfirmed from the result of pedigree analysis that ED1 was dominant over EP and e, while EP was incompletely dominant over e.

Calcineurin signaling regulates neural induction through antagonizing the BMP pathway.

Development of the nervous system begins with neural induction, which is controlled by complex signaling networks functioning in concert with one another. Fine-tuning of the bone morphogenetic protein (BMP) pathway is essential for neural induction in the developing embryo. However, the molecular mechanisms by which cells integrate the signaling pathways that contribute to neural induction have remained unclear. We find that neural induction is dependent on the Ca(2+)-activated phosphatase calcineurin (CaN). Fibroblast growth factor (FGF)-regulated Ca(2+) entry activates CaN, which directly and specifically dephosphorylates BMP-regulated Smad1/5 proteins. Genetic and biochemical analyses revealed that CaN adjusts the strength and transcriptional output of BMP signaling and that a reduction of CaN activity leads to an increase of Smad1/5-regulated transcription. As a result, FGF-activated CaN signaling opposes BMP signaling during gastrulation, thereby promoting neural induction and the development of anterior structures.

A functional role for semaphorin 4D/plexin B1 interactions in epithelial branching morphogenesis during organogenesis.

Semaphorins and their receptors, plexins, carry out important functions during development and disease. In contrast to the well-characterized plexin A family, however, very little is known about the functional relevance of B-type plexins in organogenesis, particularly outside the nervous system. Here, we demonstrate that plexin B1 and its ligand Sema4d are selectively expressed in epithelial and mesenchymal compartments during key steps in the genesis of some organs. This selective expression suggests a role in epithelial-mesenchymal interactions. Importantly, using the developing metanephros as a model system, we have observed that endogenously expressed and exogenously supplemented Sema4d inhibits branching morphogenesis during early stages of development of the ureteric collecting duct system. Our results further suggest that the RhoA-ROCK pathway, which is activated downstream of plexin B1, mediates these inhibitory morphogenetic effects of Sema4d and suppresses branch-promoting signalling effectors of the plexin B1 signalling complex. Finally, mice that lack plexin B1 show early anomalies in kidney development in vivo. These results identify a novel function for plexin B1 as a negative regulator of branching morphogenesis during kidney development, and suggest that the Sema4d-plexin B1 ligand-receptor pair contributes to epithelial-mesenchymal interactions during organogenesis via modulation of RhoA signalling.