Chromosome 4 from Drosophila melanogaster has several unusual features that distinguish it from the other chromosomes. These include a diffuse appearance in salivary gland polytene chromosomes, an absence of recombination, and the variegated expression of P-element transgenes. As part of a larger project to understand these properties, a physical map of this chromosome is being assembled. The sequence of two cosmids representing approximately 5% of the polytenized region is reported here. Both cosmid clones contain numerous repeated DNA sequences, as identified by cross hybridization with labeled genomic DNA, BLAST searches, and dot matrix analysis. These repeated sequences are positioned between and within the transcribed sequences. The repetitive sequences include three copies of the mobile element Hoppel, one copy of the mobile element HB, and 18 DINE repeats. DINE is a novel, short repeated sequence dispersed throughout both cosmid sequences. One cosmid includes the previously described cubitus interruptus gene and two new genes: one with a predicted amino acid sequence similar to ribosomal protein S3a, which is consistent with the Minute(4)101 locus thought to be in the region, and a second, plexinB, a novel member of the protein family that includes mammalian plexin and met-hepatocyte growth factor receptor. The other cosmid contains only the two short 5'-most exons from the zinc-finger-homolog-2 (zfh-2) gene. This is the first extensive sequence analysis of noncoding DNA from chromosome 4. The distribution of the various repeats suggests its organization is similar to the beta-heterochromatic regions near the base of the major chromosome arms. Such a pattern may account for the diffuse banding of the polytene chromosome 4 and the variegation of many P-element transgenes on the chromosome (Locke, 1999).
Plexins are neuronal receptors for the repulsive axon guidance molecule Semaphorins. Plexin B (PlexB) binds directly to the active, GTP-bound form of the Rac GTPase. A seven amino acid sequence in PlexB is required for RacGTP binding. The interaction of PlexB with RacGTP is necessary for Plexin-mediated axon guidance in vivo. A different region of PlexB binds to RhoA. Dosage-sensitive genetic interactions suggest that PlexB suppresses Rac activity and enhances RhoA activity. Biochemical evidence indicates that PlexB sequesters RacGTP from its downstream effector PAK. These results suggest a model whereby PlexB mediates repulsion by coordinately regulating two small GTPases in opposite directions: PlexB binds to RacGTP and downregulates its output by blocking its access to PAK and, at the same time, binds to and increases the output of RhoA (Hu, 2001).
Plexin B binds to the active form of Rac (RacGTP); the binding maps to a 147 amino acid region, PlexBDelta3 (amino acids 1617 through 1765). To identify the critical binding sequence in PlexBDelta3, small deletions and point mutations were introduced and a seven amino acid sequence NTLAHYG (1722 through 1728) toward the C terminus of PlexBDelta3 has been identified that, when deleted, abolishes Rac binding (PlexBDelta3d7). Deletions in neighboring regions 1743 through 1759 (PlexBDelta3d17) and 1707 through 1714 do not affect Rac binding (Hu, 2001).
The NTLAHYG sequence is highly conserved among Plexin family members. In particular, the tyrosine residue within the sequence is invariable. In human Plexin B1, a putative Cdc42/Rac interactive binding (CRIB)-like motif right after this conserved sequence has been described. Although the CRIB-like motif is not found in Drosophila PlexB, this may reflect a conservation of the binding mechanism at a higher structural level. The Psi blast program predicts two blocks of sequences in the Plexin cytoplasmic domain that share similarity with R-ras family GAP proteins. The sequence needed for RacGTP binding is located between these two GAP-like regions (Hu, 2001).
In the Drosophila genome, there are six Rho family small GTPases: Rac1 (referred to here as Rac), Rac2, Cdc42, RhoA, Mtl, and RhoL. To gain some insight into the specificity of the interaction, the binding of PlexBDelta3 with all six Drosophila Rho-like GTPases was examined. Only Rac and Rac2, which share the highest degree of sequence similarity (93% identity), show strong interactions with the BDelta3 region of PlexB (Hu, 2001).
Several lines of evidence suggest that RhoA is also involved in PlexiB signaling. Clustering of the vertebrate PlexB in Swiss 3T3 cells leads to stress fiber formation, indicative of Rho activation. The response can be blocked by inhibitors of Rho or of its downstream effector Rho kinase. Genetic data also indicate that RhoA mediates part of Plexin B signaling in embryonic axon guidance. It was of interest, then, to enquire whether RhoA may also directly associate with PlexB (Hu, 2001).
PlexBDelta, a larger piece of the PlexB cytoplasmic domain (1617 through 1827) binds to RhoA. In contrast to a preferential binding to GTPgammaS-bound Rac, PlexBDelta binds to the GTPgammaS and GDP-bound forms of RhoA equally well. The binding requires the last 40 amino acids of PlexBDelta. The seven amino acid internal deletion that eliminates PlexBDelta binding to Rac does not affect its binding to RhoA. Thus, two independent regions in PlexB cytoplasmic domain have been defined that are important for PlexB association with Rac and RhoA, respectively. Cdc42, another Rho family GTPase, does not bind to PlexBDelta (Hu, 2001).
Dosage-sensitive genetic interactions suggest Rac antagonizes Plexin B signaling. Since there is no mutant available for PlexB with which to examine genetic interactions, whether the gain of function phenotype of PlexB is sensitive to the level of expression of Rac or RhoA was examined. PlexB is endogenously expressed by CNS neurons. Overexpression of PlexB in all embryonic CNS neurons can be achieved with the UAS-GAL4 binary expression system. Flies containing the UAS-PlexB transgene reporter are crossed to flies carrying a neuron-specific transcriptional control driver, elav-GAL4. With two independent UAS-PlexB transgenic lines, a consistent, GAL4-dependent phenotype was observed in specific motor nerve branches. In particular, a striking defect was observed in the ability of the ISNb (intersegmental nerve b) motor axons to innervate the ventral longitudinal muscles 7, 6, 13, and 12. In wild-type embryos, the ISNb projects into the ventral longitudinal muscles. Particular motor axons innervate specific muscles; for example, the RP3 motor axon innervates muscles 7 and 6, while other ISNb axons innervate muscles 13 and 12. When PlexB is overexpressed in these neurons, two types of phenotypes are observed that are consistent with PlexB being a repulsive guidance receptor for muscle-expressed Semaphorins: (1) the RP3 axon frequently fails to defasciculate from the ISNb motor nerve branch, and as a result muscles 7 and 6 are uninnervated; (2) ISNb axons often fail to reach their distal-most target muscle 12 (scored as 'stall'). The copy number of UAS-PlexB transgene and elav-GAL4 driver was varied to generate embryos with a range of levels of expression of PlexB; 'RP3 missing' and 'stall' phenotypes are dose dependent. This dosage sensitivity suggests that the PlexB gain-of-function phenotype may provide a sensitive background for revealing genetic interactions with genes encoding downstream components involved in Plexin B signaling (Hu, 2001).
Does the binding of PlexB to Rac increase or decrease the output of Rac? It was reasoned that if increasing PlexB expression produces its effect by activating Rac, then genetically limiting Rac gene dose might suppress the PlexB overexpression phenotype. Alternatively, if PlexB signals by turning down Rac activity, then an enhancement of the plexB overexpression phenotypes might result when Rac is reduced. Indeed, the results support the second alternative: PlexB inactivates Rac. Rac protein level was reduced by 50% using a small deficiency line, Df(3L)Ar14-8 (61C04-62A08), in a moderate PlexB overexpression background (one copy transgene, one copy driver). This resulted in a distinct increase in the penetrance of PlexB gain of function phenotypes. A complementary effect results when Rac dosage is increased in the same neurons where PlexB is overexpressed with a UAS-Rac transgene. Under such conditions, a suppression on the PlexB gain of function phenotypes is observed (Hu, 2001).
Consistent with the idea that PlexB signals by downregulating Rac activity, the Plexin gain-of-function stall phenotype is reminiscent of the loss-of-function phenotype of a positive regulator of Rac, the Trio GEF. Trio has been shown to play a role in axon guidance in Drosophila and nematode and has provided additional evidence that, in this capacity, Trio interacts with Rac and regulates PAK activity. Similar to reducing Rac, reducing Trio enhances PlexB gain-of-function phenotype. However, the enhancement caused by reducing Trio is not as great as that caused by reducing Rac. This probably reflects that Trio is not directly coupled to PlexB and is not the only positive regulator (GEF) for Rac in motor axons. Rather, Trio is likely to be one of many positive regulators of Rac in these axons (Hu, 2001).
The role of RhoA in PlexB signaling was examined by reducing RhoA gene dosage with two different RhoA mutant alleles, Rhorev220 and RhoAl(2)k07236. Instead of enhancing the PlexB gain-of-function phenotypes as the Rac deficiency does, partially removing RhoA suppresses the PlexB gain-of-function phenotypes. This result suggests that RhoA acts antagonistically to Rac and, moreover, that RhoA partially mediates Plexin B signaling (Hu, 2001).
To further test the model that PlexB downregulates Rac output, the effect of increasing Plexin was examined in Rac dominant-negative embryos. No mutant for Drosophila Rac has yet been published, but a loss-of-function analysis for Rac, achieved by overexpressing a dominant-negative form of Drac (N17Rac) in neurons, has revealed dramatic defects in motor axon guidance. The same ISNb nerve branch that is affected by PlexB overexpression is also sensitive to overexpression of dominant-negative Rac (N17Rac). The predominant ISNb defect in N17Rac embryos occurs at an earlier target entry point, where the whole ISNb branch normally branches off from ISN nerve. In N17Rac embryos, the ISNb fails to enter the ventral muscles and instead follows the ISN distally toward dorsal muscles (scored as 'bypass'). The difference in the quality of the ISNb phenotype of N17Rac and Plexin B gain of function embryos may likely reflect the fact that Rac is downstream of multiple guidance receptors (Hu, 2001).
The penetrance of the N17Rac bypass phenotype is very sensitive to gene dosage. When the N17Rac transgene is expressed using drivers of different strengths, different frequencies of defects result. This suggests that N17Rac only partially knocks out the wild-type gene function and that expressing N17Rac with a driver of medium strength may provide a sensitized background for testing genes that regulate the remaining Rac activity. It was reasoned that if the Plexin and Rac interaction regulates Rac activity, then it might be possible to alter the penetrance of the N17Rac bypass phenotype by simultaneously increasing PlexB gene dose in the same neurons. Indeed, coexpressing PlexB and RacN17 results in a distinct enhancement of the ISNb bypass phenotypes. N17Rac embryos also show bypass defects in the SNa motor axons that project to lateral muscle targets. This SNa bypass phenotype has never been observed in any other mutant background, and it also turns out to be enhanced by simultaneously overexpressing PlexB in these neurons (Hu, 2001).
In a reciprocal experiment PlexB was reduced in Rac dominant-negative embryos to see if this had an opposite effect. This was done by injecting double-strand RNA of PlexB into N17Rac embryos. N17Rac embryos injected with Plexin B dsRNA show distinct reduction in bypass defects compared with N17Rac embryos injected with buffer. Thus, reducing PlexB and increasing PlexB in Rac dominant embryos produces opposite modulations, consistent with the model that PlexB downregulates Rac activity (Hu, 2001).
To test whether the PlexB gain-of-function and the genetic interactions depend on the direct association between PlexB and Rac, a mutant PlexB transgene, UAS-Plex Bd7, was constructed containing the seven amino acid NTLAHYG deletion in the Rac binding region of an otherwise wild-type PlexB. The same enhancement test on N17Rac embryos was performed with this mutant transgene, and no enhancement was observed. The PlexB gain-of-function phenotypes also seem to be dependent on this Rac binding region. In contrast to wild-type PlexB, when the d7 mutant transgene is overexpressed under the control of the same neuronal GAL4 driver elav, the frequency of ISNb phenotypes is significantly lower. This low-penetrance phenotype is not enhanced by removing one copy of Rac (Hu, 2001).
In vivo expression and targeting of PlexBd7 transgene could not be examined due to the lack of PlexB antibody. Nevertheless, the seven amino acid deletion does not affect protein expression and stability of PlexBDelta3 in in vitro experiments. Three independent lines of PlexBd7 transgenes show consistent behavior when tested for their phenotypes and interactions with Rac, arguing that the negative result is not caused by the insertion site (Hu, 2001).
In light of the genetic interactions between PlexB and Rac, the biochemical nature of this negative regulation was investigated. PlexB was found to compete with the Rac downstream effector PAK (p21-activated kinase) for binding to RacGTP. PAK is a serine/threonine kinase that mediates a major part of Rac signaling output to actin polymerization. Upon binding to RacGTP, PAK undergoes a conformational change that releases an autoinhibition on the kinase domain and becomes active. Since Rac binding is critical for PAK activation and also because PlexB and PAK bind to Rac in the same GTP-dependent manner, it was asked whether PlexB and PAK may bind to the same region of Rac and whether their binding to RacGTP is mutually exclusive (Hu, 2001).
An in vitro pull-down competition assay was used in which in vitro translated L61Rac was incubated with bead-bound GST-PAK1-141 in the presence or absence of soluble PlexB protein fragment: PlexBDelta2(1619-1753). (PlexBDelta2 is a 135 amino acid fragment of PlexB. It binds to RacGTP equally well as PlexBDelta3, but it can be expressed at a higher level.) When Plexin BDelta2 is present in the binding solution, the amount of RacL61 pulled down by PAK1-141 is greatly reduced. The extent of reduction is dependent on the amount of PlexBDelta2 used. At the 48:1 molar ratio of PlexBDelta2 to PAK1-141, the reduction is close to complete. PlexBDelta2d7, a deletion PlexB fragment that is incapable of binding to Rac, does not compete with PAK for the Rac binding. Conversely, the presence of PAK protein fragment PAK78-151 also reduces RacL61 binding to PlexBDelta3. This shows that the binding of the two proteins to RacGTP is indeed mutually exclusive (Hu, 2001).
Does this competition exist in vivo? If it does, then it may be expected that overexpressing PAK together with PlexB in embryos will cancel out PlexB gain-of-function effect. Indeed, overexpressing PAK in a PlexB gain-of-function background suppresses the phenotypes of the latter, demonstrating that PlexB signaling can be antagonized by the Rac effector PAK in vivo (Hu, 2001).
It is concluded that PlexB mediates repulsion in vivo in part by binding to active Rac (RacGTP) and downregulating its effector output and in part by binding to and activating RhoA. Biochemical analysis shows that PlexB binds to RacGTP. A seven amino acid sequence in the cytoplasmic domain of PlexB is required for this binding. Genetic analysis shows that PlexB downregulates the output of RacGTP. Removal of one copy of Rac enhances a PlexB gain-of-function phenotype, while overexpression of PlexB enhances a Rac dominant-negative phenotype in motor axon guidance. Overexpression of a mutant form of PlexB that lacks the seven amino acid sequence required for Rac binding does not generate its own gain-of-function phenotype, and it does not enhance a Rac dominant-negative phenotype. It is also shown that PlexB binds to RhoA through a different region of its cytoplasmic domain. Although the biochemical mechanism is not known, genetic analysis suggests that PlexB increases the output of RhoA (Hu, 2001).
The results presented here allow a confirmation and extension of a current model concerning the role of GTPases in axon guidance. This model suggests that attractive guidance cues locally activate Rac or Cdc42 in the growth cone while repulsive guidance cues locally activate RhoA. It is argued that what is important is the relative balance in the output of Rac versus RhoA. An example is provided in which the PlexiB receptor mediates repulsive axon guidance by downregulating RacGTP output and simultaneously upregulating RhoA output. A coordinate regulation of these two small GTPases may allow the receptor to have a finer control over actin regulatory machinery. Semaphorin signaling can be converted from repulsion to attraction by changes in cGMP level. It would be interesting to test whether and how the cGMP signaling can affect this Rac/Rho balance (Hu, 2001).
Drosophila has two Plexins: A and B. Both Plexin A and B are highly expressed in the central nervous system. The two proteins share high sequence similarity in their cytoplasmic domain, indicating a similar mode of signaling shared by the two. A direct physical association of RacGTP with PlexB but not with PlexA has been demonstrated. However, genetic interactions have been found between Rac and both Plexins. For example, increasing PlexA also enhances the Rac dominant-negative phenotype as does PlexB. In COS cell and DRG neurons, Rac shows coclustering with PlexA upon Sema3A ligand treatment. It is likely that ligand binding to PlexA causes Rac binding (and subsequent inactivation of Rac) just as with PlexB, but it may be that PlexA requires an unknown third protein to help mediate or facilitate this physical interaction. From a genetic perspective, they both appear to function in the same way, mediating repulsion at least in part by inactivating Rac (Hu, 2001).
The transmembrane protein OTK associates with Plexin A and contributes to the Sema 1a/Plexin A signaling pathway. Mammalian Plexin B1 also coimmunoprecipitates with OTK. In the future, it will be interesting to test whether PlexB also interacts with OTK in vivo and to what degree the Rac/Rho GTPases and OTK signaling pathways function together or in parallel downstream of Plexins (Hu, 2001).
The plexin family transmembrane proteins are putative receptors for semaphorins, which are implicated in the morphogenesis of animal embryos, including axonal guidance. Putative null mutants of the C. elegans plexinA gene, plx-1, have been generated and characterized. plx-1 mutants exhibit morphological defects: displacement of ray 1 and discontinuous alae. The epidermal precursors for the affected organs are aberrantly arranged in the mutants, and a plx-1::gfp transgene is expressed in these epidermal precursor cells as they undergo dynamic morphological changes. Suppression of C. elegans transmembrane semaphorins, Ce-Sema-1a and Ce-Sema-1b, by RNA interference causes 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, exhibits phenotypes distinct from those of plx-1 mutants. A heterologous expression system has shown that Ce-Sema-1a, but not Ce-Sema-2a, physically binds to PLX-1. These 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 (Fujii, 2002).
Semaphorins and ephrins are axon guidance cues. In C. elegans, semaphorin-2a/mab-20 and ephrin-4/efn-4/mab-26 also regulate cell sorting to form distinct rays in the male tail. Several erf (enhancer of ray fusion) mutations were identified in a mab-20 enhancer screen. Mutants of plexin-2 (plx-2) and unc-129, which encodes a divergent axon guiding TGF-β, were also found to be erfs. Genetic analyses show that plx-2 and mab-20 function in the same pathway, as expected if PLX-2 is a receptor for MAB-20. Surprisingly, MAB-20 also signals in a parallel pathway that requires efn-4. This signal utilizes a non-plexin receptor. The expression of plx-2, efn-4, and unc-129 in subsets of 3-cell sensory ray clusters likely mediates the ray-specific cell sorting functions of the ubiquitously expressed mab-20. A model is presented for the integrated control of TGF-β, semaphorin, and ephrin signaling in the sorting of cell clusters into distinct rays in the developing male tail (Ikegami, 2004).
The male tail in C. elegans is characterized by nine distinct, linearly arranged sensory rays visible as finger-like protrusions on each side of the animal. Each ray comprises three cells that derive from a common ray precursor cell (Rn cell where n = 1–9). Each Rn cell divides to form Rn.p, a hypodermal cell, and Rn.a. The Rn.a descendants form a pre-ray cluster of three lineally related cells that eventually form two neurons (RnA and RnB) and a structural support cell (Rnst), which encases the sensory endings of these neurons to form an adult ray. Initially, the Rn.a descendants (3-cell cluster) of one ray contact the Rn.a descendants destined to form neighboring rays. They then undergo dynamic shape and position changes that separate each cluster of three lineally related cells from neighboring 3-cell clusters to ultimately form the distinct ray sensillae of the adult male. These changes are orchestrated by active cellular mechanisms involving specific cell-cell interactions within and between the 3-cell clusters destined to form rays. The final positions of the adult rays are determined by the site of attachment of the structural support cell Rnst (one of the Rn.a descendants in each cluster) to the basal surface of the cuticle in L4 male larvae (Ikegami, 2004 and references therein).
In mutants of mab-20, neighboring Rn.a descendants for each ray frequently fail to separate from each other, resulting in fused rays. Mutants of ephrin-4 (efn-4/mab-26) are known to share a similar male tail phenotype with mutants of mab-20. efn-4 encodes an ephrin-related protein that fails to bind to VAB-1, the only known ephrin receptor in C. elegans. As shown by the nonadditive ray fusion defects of mab-20 and efn-4 null mutants, it was proposed that these two genes function in a common pathway to sort the 3-cell clusters destined to form individual rays. The functional relatedness of efn-4 to mab-20 suggests the existence of crosstalk and a possible ancestral link between ephrin and semaphorin signaling mechanisms (Ikegami, 2004 and references therein).
The fact that only mutants of mab-20/sema-2A and efn-4/mab-26 were identified in large-scale screens for male ray fusion defects suggests that the signaling mechanisms that are regulated by MAB-20/Sema-2A and EFN-4/MAB-26 are encoded largely by genes that are either essential for viability or are redundant. Modifier screens have proven to be especially useful for revealing such genes. Therefore, in order to identify novel semaphorin signaling pathways and novel components of known semaphorin signaling pathways, and to better understand how ephrin and semaphorin signaling are integrated, a large-scale screen was undertaken for mutations that enhance a weak allele of mab-20. This screen identified mutants of the C. elegans plexin-2 (plx-2) gene, which encodes a presumed receptor for Sema-2A/MAB-20, plus mutants of at least six erf genes (erf-1 to erf-5 plus plx-2) that enhance both the body morphology and male ray fusion defects of mab-20(bx61ts). unc-129, which encodes a TGF-β that regulates axon guidance in C. elegans, was also found to enhance ray fusion defects of a mab-20 weak allele. Genetic and phenotypic characterization of these mutants has begun to reveal hierarchical pathways of semaphorin and ephrin function involved in several aspects of C. elegans morphogenesis. For example, even though putative null alleles of plx-2 do not cause a male ray fusion phenotype resembling the mab-20 mutants, genetic interactions between plx-2, efn-4, unc-129, and erf mutations have revealed the existence of redundant mechanisms that regulate the signaling of mab-20 and has suggested a role (in the context of a functional network) for efn-4, plx-2, and unc-129 in integrating these pathways. The integrated regulation of semaphorin, ephrin, and TGF-β signaling in the sorting of Rn.a descendants into distinct rays in C. elegans may have implications for regulating differential cell adhesions involved in a variety of cell movements and cell shape changes that occur during animal development (Ikegami, 2004).
Plexins are functional receptors for Semaphorin axon guidance cues. Previous studies have established that some Plexins directly bind RACGTP and RHO. Recent work in C. elegans has shown that semaphorin 1 (smp-1 and smp-2) and plexin 1 (plx-1) are required to prevent anterior displacement of the ray 1 cells in the male tail. plx-1 is shown genetically to be part of the same functional pathway as smp-1 and smp-2 for male ray positioning. RAC GTPase genes mig-2 and ced-10 probably function redundantly, whereas unc-73, which encodes a GEF for both of these GTPases, is required cell autonomously for preventing anterior displacement of ray 1 cells. RNAi analysis indicates that rho-1-encoded RHO GTPase, plus let-502 and K08B12.5-encoded RHO-kinases, are also required to prevent anterior displacement of ray 1 cells, suggesting that different kinds of RHO-family GTPases act similarly in ray 1 positioning. At low doses of wild-type mig-2 and ced-10, the Semaphorin 1 proteins no longer act through PLX-1 to prevent anterior displacements of ray 1, but have the opposite effect, acting through PLX-1 to mediate anterior displacements of ray 1. These results suggest that Plexin 1 senses levels of distinct RHO and RAC GTPases. At normal levels of RHO and RAC, Semaphorin 1 proteins and PLX-1 prevent a forward displacement of ray 1 cells, whereas at low levels of cycling RAC, Semaphorin 1 proteins and PLX-1 actively mediate their anterior displacement. Endogenously and ectopically expressed SMP-1 and SMP-2 suggest that the hook, a major source of Semaphorin 1 proteins in the male tail, normally attracts PLX-1-expressing ray 1 cells (Dalpé, 2004).
Vulva development in C. elegans involves cell fate specification followed by a morphogenesis phase in which homologous mirror image pairs within a linear array of primordial vulva cells form a crescent shape as they move sequentially towards a midline position within the array. The homologous pairs from opposite half vulvae in fixed sequence fuse with one another at their leading tips to form ring-shaped (toroidal) cells stacked in precise alignment one atop the other. The semaphorin 1a SMP-1, and its plexin receptor PLX-1, are required for the movement of homologous pairs of vulva cells towards this midline position. SMP-1 is upregulated on the lumen membrane of each primordial vulva cell as it enters the forming vulva and apparently attracts the next flanking homologous PLX-1-expressing vulva cells towards the lumen surface of the ring. Consequently, a new ring-shaped cell forms immediately ventral to the previously formed ring. This smp-1- and plx-1-dependent process repeats until seven rings are stacked along the dorsoventral axis, creating a common vulva lumen. Ectopic expression of SMP-1 suggests it has an instructive role in vulva cell migration. At least two parallel acting pathways are required for vulva formation: one requires SMP-1, PLX-1 and CED-10; and another requires the MIG-2 Rac GTPase and its putative activator UNC-73 (Dalpe, 2005).
One of the earliest guidance decisions for spinal cord motoneurons occurs when pools of motoneurons orient their growth cones towards a common, segmental exit point. In contrast to later events, remarkably little is known about the molecular mechanisms underlying intraspinal motor axon guidance. In zebrafish sidetracked (set) mutants, motor axons exit from the spinal cord at ectopic positions. By single-cell labeling and time-lapse analysis it was shown that motoneurons with cell bodies adjacent to the segmental exit point properly exit from the spinal cord, whereas those farther away display pathfinding errors. Misguided growth cones either orient away from the endogenous exit point, extend towards the endogenous exit point but bypass it or exit at non-segmental, ectopic locations. Furthermore, sidetracked acts cell autonomously in motoneurons. Positional cloning reveals that sidetracked encodes Plexin A3, a semaphorin guidance receptor for repulsive guidance. Finally, sidetracked (plexin A3) is shown to play an additional role in motor axonal morphogenesis. Together, these data genetically identify the first guidance receptor required for intraspinal migration of pioneering motor axons and implicate the well-described semaphorin/plexin signaling pathway in this poorly understood process. It is proposed that axonal repulsion via Plexin A3 is a major driving force for intraspinal motor growth cone guidance (Palaisa, 2007).
In zebrafish embryos, the axons of the posterior trigeminal (Vp) and facial (VII) motoneurons project stereotypically to a small number of target muscles derived from the first and second branchial arches (BA1, BA2). Use of the Islet1 (Isl1)-GFP transgenic line enabled precise real-time observations of the growth cone behaviour of the Vp and VII motoneurons within BA1 and BA2. Screening for N-ethyl-N-nitrosourea-induced mutants identified seven distinct mutations affecting different steps in the axonal pathfinding of these motoneurons. The class 1 mutations caused severe defasciculation and abnormal pathfinding in both Vp and VII motor axons before they reached their target muscles in BA1. The class 2 mutations caused impaired axonal outgrowth of the Vp motoneurons at the BA1-BA2 boundary. The class 3 mutation caused impaired axonal outgrowth of the Vp motoneurons within the target muscles derived from BA1 and BA2. The class 4 mutation caused retraction of the Vp motor axons in BA1 and abnormal invasion of the VII motor axons in BA1 beyond the BA1-BA2 boundary. Time-lapse observations of the class 1 mutant, vermicelli (vmc), which has a defect in the plexin A3 (plxna3) gene, revealed that Plxna3 acts with its ligand Sema3a1 for fasciculation and correct target selection of the Vp and VII motor axons after separation from the common pathways shared with the sensory axons in BA1 and BA2, and for the proper exit and outgrowth of the axons of the primary motoneurons from the spinal cord (Tanaka, 2007).
Immunohistochemistry, using monoclonal antibodies (named A5 and B2) that specifically recognize cell surface proteins neuropilin and plexin, respectively, has revealed that olfactory axons in Xenopus tadpoles can be classified into several subgroups by virtue of the expression levels of these two cell surface molecules. The vomeronasal axons express plexin but not neuropilin. The plexin-positive and neuropilin-negative vomeronasal axons form a discrete fiber bundle, even after they join with the principal olfactory axons. However, the principal olfactory axons can divided into at least two subclasses; the neuropilin-predominant axons, which express high levels of the neuropilin and low levels of the plexin, and the plexin-predominant axons, which express high levels of the plexin and low levels of the neuropilin. Within the olfactory nerve the pathways for these two principal olfactory axon subclasses are initially intermingled with one another, but gradually segregate throughout their courses from the nose to the cerebrum. Eventually, the neuropilin-predominant and the plexin-predominant principal olfactory axon subclasses project to specified glomeruli in topographically related regions within the main olfactory bulb. Neuroanatomical tracings of the olfactory projection also confirm the gradual segregation of the pathways for the principal olfactory axons. These results allow for the speculation that both the neuropilin and the plexin are involved in axon interactions, and play roles in the organization of the precise patterns of the olfactory pathway and projection (Satoda, 1995).
Plexin is a neuronal cell surface molecule that has been identified in Xenopus. cDNA cloning reveals that plexin has no homology to known neuronal cell surface molecules but possesses, in its extracellular segment, three internal repeats of cysteine clusters that are homologous to the cysteine-rich domain of the c-met proto-oncogene protein product. The exogenous plexin proteins expressed on the surfaces of L cells by cDNA transfection mediate cell adhesion via a homophilic binding mechanism, under the presence of calcium ions. Plexin is expressed in the receptors and neurons of particular sensory systems. These findings indicate that plexin is a novel calcium-dependent cell adhesion molecule and suggest its involvement in specific neuronal cell interaction and/or contact (Ohta, 1995).
By screening E17.5 mouse brain cDNA libraries, two cDNAs encoding new plexin-like proteins were isolated. Sequencing revealed that these two proteins are type 1 membrane proteins that show over 60% identity to mouse plexin 1 at the amino acid level. Moreover, putative extracellular segments of these two proteins have three repeats of a cysteine-rich domain that is a common motif for plexin proteins. Thus, these two proteins have been named mouse plexin 2 and mouse plexin 3. Mouse plexin 3 cDNA clones in which a part of the protein-coding region had been deleted were obtained. Also, Northern blot analysis shows molecular heterogeneity in mouse plexin 2 mRNAs. These findings indicate that in the mouse, plexins comprise a molecular family (the plexin family) (Kameyama, 1996).
The vaccinia virus A39R protein is a member of the semaphorin family. A39R.Fc protein was used to affinity purify an A39R receptor from a human B cell line. Tandem mass spectrometry of receptor peptides yielded partial amino acid sequences that allowed the identification of corresponding cDNA clones. Sequence analysis of this receptor has indicated that it is a novel member of the plexin family and has identified a semaphorin-like domain within this family, thus suggesting an evolutionary relationship between receptor and ligand. A39R up-regulated ICAM-1 on human monocytes, and induced cytokine production from them as well. These data, then, describe a receptor for an immunologically active semaphorin and suggest that it may serve as a prototype for other plexin-semaphorin binding pairs (Comeau, 1998).
In Drosophila, plexin A is a functional receptor for semaphorin-1a. Plexins encode large transmembrane proteins whose cysteine-rich extracellular domains share regions of homology with the scatter factor receptors (encoded by the Met gene family). The extracellular domains of plexins also contain ~500 amino acid semaphorin domains. However, the highly conserved cytoplasmic moieties of plexins (~600 amino acids), have no homology with the Met tyrosine kinase domain nor with any other known protein. Met-like receptors and their ligands, the scatter factors, mediate a complex biological program including dissociation of cell-cell contacts, motility, and invasion. During embryogenesis, scatter factor-1 and Met promote the dissociation of cell layers in the somites and drive the migration of myogenic cells to their appropriate location. Met and scatter factor-1 are also involved in controlling neurite outgrowth and axonal guidance. 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 an unidentified tyrosine kinase activity. Plexins may also act as ligands mediating repulsion in epithelial cells in vitro. It is concluded that plexins are receptors for multiple (and perhaps all) classes of semaphorins, either alone or in combination with neuropilins, and that they trigger a novel signal transduction pathway controlling cell repulsion (Tamagnone, 1999).
Semaphorins and their receptors, plexins, are widely expressed in embryonic and adult tissues. In general, their functions are poorly characterized, but in neurons they provide essential attractive and repulsive cues that are necessary for axon guidance. The Rho family GTPases Rho, Rac, and Cdc42 control signal transduction pathways that link plasma membrane receptors to the actin cytoskeleton and thus regulate many actin-driven processes, including cell migration and axon guidance. Using yeast two-hybrid screening and in vitro interaction assays, it has been shown that Rac in its active, GTP bound state interacts directly with the cytoplasmic domain of mammalian and Drosophila B plexins. Plexin-B1 clustering in fibroblasts does not cause the formation of lamellipodia, which suggests that Rac is not activated. Instead, it results in the assembly of actin:myosin filaments and cell contraction, which indicates Rho activation. Surprisingly, these cytoskeletal changes are both Rac and Rho dependent. Clustering of a mutant plexin, lacking the Rac binding region, induces similar cytoskeletal changes, and this finding indicates that the physical interaction of plexin-B1 with Rac is not required for Rho activation. The findings that plexin-B signaling to the cytoskeleton is both Rac and Rho dependent form a starting point for unraveling the mechanism by which semaphorins and plexins control axon guidance and cell migration (Driessens, 2001).
Many previously identified Rac targets contain a distinctive Rac binding site, the CRIB motif, but sequence analysis does not reveal any obvious CRIB-like sequence in plexin-B1. To identify the region of plexin-B1 that contains the Rac interaction site, a series of truncations were expressed as GST fusion proteins in E. coli. These were used in a dot blot assay. Rac interacts with a region encompassing 180 residues (amino acids 1724-1903) of the receptor (Driessens, 2001).
Plexin-B1 is a member of a large family of transmembrane proteins, and based on sequence alignments, four classes of plexins (A, B, C, and D) have been described. To test whether Rac could interact directly with other members of the family, cDNAs were obtained for human plexin-A2 (kiaa0463), plexin-B2 (kiaa0315), and plexin-D1 (kiaa0620). A region corresponding to amino acids 1724-1903 of plexin-B1 was cloned into the pGEX vector; GST fusion proteins were analyzed in the dot blot assay, but under these conditions only plexin-B1 was found to interact (Driessens, 2001).
In Drosophila, two plexins have been identified: Drosophila plexin-A and Drosophila plexin-B. Recombinant Drosophila plexin-B protein (C-terminal 435 amino acids, similar to plexin-B1 two-hybrid clone) interacts strongly with in vitro translated Drosophila L61Rac1 and weakly with wild-type Drosophila Rac1 in a pull-down experiment. Drosophila plexin-A does not interact with Drosophila Rac1 under the same conditions. A Drosophila plexin-B fragment corresponding to amino acids 1724-1903 of human plexin-B1 interacts similarly with Drosophila Rac1, as does a shorter, 149 amino acid region. Partial binding was observed with a 54 amino acid domain (Driessens, 2001).
Two blocks of sequence similarity, of approximately 320 and 150 amino acids each, have been identified in plexin cytoplasmic domains. These two blocks of sequence similarity are separated by a variable linker. This linker region is most divergent between the plexin subfamilies. The minimal Rac binding region in Drosophila plexin-B consists of the last 149 amino acids of the first conserved block but does not contain the linker region. Alignment of this 149 amino acid region of Drosophila plexin-B with other human plexins reveals a sequence highly conserved among all plexin subfamilies (Driessens, 2001).
A mechanism is proposed for plexin-B signaling to the actin cytoskeleton. In this mechanism, clustering of B plexins induces a Rac-dependent activation of Rho. These results provide a framework for the further exploration of the complex mechanisms by which plexins affect the actin cytoskeleton in different cell types, including neurons (Driessens, 2001).
Classic studies using avian model systems have demonstrated that cardiac neural crest cells are required for proper development of the cardiovascular system. Environmental influences that perturb neural crest development cause congenital heart defects in laboratory animals and in man. However, little progress has been made in determining molecular programs specifically regulating cardiac neural crest migration and function. Only recently have complex transgenic tools become available that confirm the presence of cardiac neural crest cells in the mammalian heart. These studies have relied upon the use of transgenic mouse lines and fate-mapping studies using Cre recombinase and neural crest-specific promoters. In this study, these techniques have been used to demonstrate that PlexinA2 is expressed by migrating and postmigratory cardiac neural crest cells in the mouse. Plexins function as co-receptors for semaphorin signaling molecules and mediate axon pathfinding in the central nervous system. PlexinA2-expressing cardiac neural crest cells are patterned abnormally in several mutant mouse lines with congenital heart disease including those lacking the secreted signaling molecule Semaphorin 3C. These data suggest a parallel between the function of semaphorin signaling in the central nervous system and in the patterning of cardiac neural crest in the periphery (Brown, 2001).
Plexins are receptors implicated in mediating signaling by semaphorins, a family of axonal chemorepellents. The role of specific plexins in mediating semaphorin function in vivo has not, however, yet been examined in vertebrates. Plexin-A3 is the most ubiquitously expressed plexin family member within regions of the developing mammalian nervous system known to contain semaphorin-responsive neurons. Using a chimeric receptor construct, evidence has been provided that plexin-A3 can transduce a repulsive signal in growth cones in vitro. Analysis of plexin-A3 knockout mice shows that plexin-A3 contributes to Sema3F and Sema3A signaling and that plexin-A3 regulates the development of hippocampal axonal projections in vivo (Cheng, 2001).
To test directly for a plexin-A3 signaling function, a gain-of-function approach was taken, asking whether the plexin-A3 cytoplasmic domain could mediate a repulsive response in the context of a chimeric receptor in which the extracellular domain of plexin-A3 is replaced by that of Met, a receptor for hepatocyte growth factor (HGF). This Met-plexin-A3 chimera was then introduced into embryonic Xenopus spinal neurons, which are repelled by Sema3A. Wild-type neurons do not respond to HGF but are attracted to HGF when wild-type Met is introduced in these cells. In contrast, when the chimeric Met-plexin-A3 receptor is introduced into these neurons, HGF elicites a robust repulsive response. This result provides evidence that plexin-A3 can function directly in signaling repulsion (Cheng, 2001).
Tracing studies have shown that developing hippocampal afferent axons invade their appropriate domains and layers in a highly specific fashion. Such stereotyped growth suggests the involvement of short-range cues providing layer-specific targeting information. Studies of reeler mutant mice deficient in reelin implicate a chemorepellent associated with Cajal-Retzius cells that inhibits commissural axon outgrowth into stratum lacunosum moleculare. Moreover, it has been suggested that Sema3F may play an important role in lamina-specific projections of hippocampal afferents. Plexin-A3 mutant commissural axons project to inappropriate laminae within the hippocampus, supporting the idea that the loss of plexin-A3 causes a reduction or loss in response to this Sema3F-based cue. Additionally, the observed switch in laminar termination zones also suggests the unmasking of an attractive cue within SLM. Laminar termination defects were not observed in spinal cord or cerebellum in plexin-A3 mutant mice, suggesting the importance of cues other than Sema3F in directing these terminations (Cheng, 2001).
A new member of the plexin-A subfamily has been identified in mice, plexin-A4: it was expressed in the developing nervous system with a pattern different from that of other members of the plexin-A subfamily (plexin-A1, plexin-A2 and plexin-A3). COS-7 cells coexpressing plexin-A4 with neuropilin-1 were induced by Sema3A, a member of the class 3 semaphorin, to undergo cell contraction. Ectopic expression of plexin-A4 in mitral cells that are originally insensitive to Sema3A results in the collapse of growth cones in the presence of Sema3A. These results suggest that plexin-A4 plays a role in the propagation of Sema3A activities. The strong expression of plexin-A4 in hippocampal neurons suggests the importance of plexin-A4 in hypocampus axon guidance (Suto, 2003).
Semaphorins, originally identified as axon guidance factors in the nervous system, play integral roles in organogenesis. A critical involvement is demonstrated for Sema6D in cardiac morphogenesis. Ectopic expression of Sema6D or RNA interference against Sema6D induces expansion or narrowing of the ventricular chamber, respectively, during chick embryonic development. Sema6D also exerts region-specific activities on cardiac explants, a migration-promoting activity on outgrowing cells from the conotruncal segment, and a migration-inhibitory activity on those from the ventricle. Plexin-A1 mediates these activities as the major Sema6D-binding receptor. Plexin-A1 forms a receptor complex with vascular endothelial growth factor receptor type 2 in the conotruncal segment or with Off-track in the ventricle segment; these complexes are responsible for the effects of Sema6D on the respective regions. Thus, the differential association of Plexin-A1 with additional receptor components entitles Sema6D to exert distinct biological activities at adjacent regions. This is crucial for complex cardiac morphogenesis (Toyofuku, 2004).
Plexin-A1 may form receptor complexes specific for Sema6D containing distinct components to exert opposite effects on different regions of the cardiac tube. The Drosophila Plexin-A receptor associates with OTK, a receptor tyrosine kinase-like transmembrane protein. This complex transduces Sema1a signals. The expression of various receptor kinases and NP1 was examined in outgrowing cells from cardiac explants. Among analyzed molecules, four molecules exhibited unique expression patterns. OTK and Neuropilin-1 (NP1) mRNAs are expressed predominantly in the outgrowing cells from the ventricle. In contrast, VEGFR1 and VEGFR2 mRNAs are strongly expressed in cells of the conotruncal segment. NP1 is known to form the functional Sema3A receptor complex with Plexin-A1. However, in spite of its abundant expression, the interaction of NP1 with Plexin-A1 could not be detected in cells from the ventricle, which is in good agreement with the fact that Sema3A does not show any effect on the ventricle explants. Since Drosophila OTK has been shown to interact with not only invertebrate Plexin-A, but also vertebrate Plexin-A3 and Plexin-B3, the ability of OTK to interact with Plexin-A1 was analyzed. An association between OTK and Plexin-A1 could be demonstrated when these molecules were transfected into HEK293 cells. The functional involvement of vertebrate OTK in Sema6D signaling was examined by knocking down OTK expression by RNAi. Treatment with siRNA specific for chicken OTK (cOTK) significantly reduces the levels of cOTK mRNA without changing any other mRNA tested. RNAi against cOTK blocked Sema6D-induced inhibition of migration of outgrowing cells from ventricle explants, but did not influence the effect of Sema6D on cells from conotruncal segments, indicating that OTK is functionally coupled with Plexin-A1 in the ventricle region. The effect of RNAi against cOTK on cardiac tube formation was also examined. Treatment with siRNA specific for cOTK, which significantly reduces the expression of cOTK mRNA, results in the failure of bending and expansion of the ventricular region. This phenotype is similar to those of chick embryos treated with siRNA specific for cSema6D or cPlexin-A1. These results suggest that the morphological effect of Sema6D on the ventricular region is mediated through the Plexin-A1-OTK receptor complex (Toyofuku, 2004).
Hippocampal mossy fibers project preferentially to the stratum lucidum, the proximal-most lamina of the suprapyramidal region of CA3. The molecular mechanisms that govern this lamina-restricted projection are still unknown. This study examined the projection pattern of mossy fibers in mutant mice for semaphorin receptors plexin-A2 and plexin-A4, and their ligand, the transmembrane semaphorin Sema6A. plexin-A2 deficiency causes a shift of mossy fibers from the suprapyramidal region to the infra- and intrapyramidal regions, while plexin-A4 deficiency induces inappropriate spreading of mossy fibers within CA3. The plexin-A2 loss-of-function phenotype is genetically suppressed by Sema6A loss of function. Based on these results, a model is proposed for the lamina-restricted projection of mossy fibers: the expression of plexin-A4 on mossy fibers prevents them from entering the Sema6A-expressing suprapyramidal region of CA3 and restricts them to the proximal-most part, where Sema6A repulsive activity is attenuated by plexin-A2 (Suto, 2007).
Extending axons in the developing nervous system are guided to their targets through the coordinate actions of attractive and repulsive guidance cues. The semaphorin family of guidance cues comprises several members that can function as diffusible axonal chemorepellents. To begin to elucidate the mechanisms that mediate the repulsive actions of Collapsin-1/Semaphorin III/D (Sema III), a search in embryonic rat sensory neurons (using expression cloning) was carried out for Sema III-binding proteins. Sema III binds with high affinity to the transmembrane protein neuropilin; antibodies to neuropilin block the ability of Sema III to repel sensory axons and to induce the collapse of neuronal growth cones. Both the C domain and the semaphorin domain of Sema II can independently bind neuropilin. Neuropilin is an axonal protein present in the developing Xenopus nervous system. Neuropilin comprises in its extracellular domain two domains with similarity to the C1 and C2 domains of coagulation factors V and VIII; a MAM domain, and two CUB motifs (a CUB domain in the metalloproteinase Tolloid, a relative of BMP-1, is suggested to mediate an interaction with the BMP family member Decapentaplegic). These results provide evidence that neuropilin is a receptor or a component of a receptor complex that mediates the effects of Sema III on these axons (He, 1997).
Semaphorin III (Sema III) is a secreted protein that causes in vitro neuronal growth cone collapse and chemorepulsion of neurites, and in vivo is required for correct sensory afferent innervation and other aspects of development. However, the mechanism of Sema III function remains unknown. Neuropilin, a type I transmembrane protein implicated in aspects of neurodevelopment, is a Sema III receptor. Neuropilin-2, a related neuropilin family member, is described in this study. Both neuropilin and neuropilin-2 are expressed in overlapping, yet distinct, populations of neurons in the rat embryonic nervous system (Kolodkin, 1997).
Neuropilin is a neuronal cell surface protein that has been shown to function as a receptor for a secreted protein, semaphorin III/D, which can induce neuronal growth cone collapse and repulsion of neurites in vitro. Neuropilin is a type I membrane protein that is highly conserved among vertebrates, can mediate cell adhesion by a heterophilic molecular interaction, and can promote neurite outgrowth in vitro. The roles of neuropilin in vivo, however, are unknown. Neuropilin-deficient mutant mice produced by targeted disruption of the neuropilin gene show severe abnormalities in the trajectory of efferent fibers of the PNS. The trajectory of each cranial nerve is severely disorganized in the neuropilin mutant embryos. The ophthalmic nerve is defasciculated, and overshoots far beyond the growing front of the normal nerve. The distal parts of the maxillary and mandibular nerves are also difasciculated in mutants and spread into almost all parts of the maxillae and mandibula, respectively. The distal parts of the facial nerve, glossopharyngeal and vagus nerves also expand beyond their normal extentions. Spinal nerve fibers at the trunk level show abnormal trajectory and projection in Neuropilin mutants, and limb innervation by the fourth to eighth cervical spinal nerves is abnormal. Neuropilin-deprived dorsal root ganglion neurons are protected from growth cone collapse elicited by semaphorin III/D. These results indicate that neuropilin-semaphorin III/D-mediated chemorepulsive signals play a major role in the guidance of PNS efferents (Kitsukawa, 1997).
Neuropilin 1 (NP-1) has been identified as a necessary component of a semaphorin D (SemD) receptor that repulses dorsal root ganglion (DRG) axons during development. SemA and SemE are related to SemD and bind to NP-1, but do not repulse DRG axons. By expressing NP-1 in retinal neurons and NP-2 in DRG neurons, it has been demonstrated that neuropilins are sufficient to determine the functional specificity of semaphorin reponsiveness. SemA and SemE block SemD binding to NP-1 and abolish SemD repulsion in axons expressing NP-1. SemA and SemE seem to have a newly discovered protein antagonist capacity toward NP-1 receptors, whereas they act as agonists at receptors containing NP-2 (Takahashi, 1998).
The collapsin and semaphorin family of extracellular proteins contributes to axonal path finding by repulsing axons and collapsing growth cones. To explore the mechanism of collapsin-1 action, a truncated collapsin-1-alkaline phosphatase fusion protein (CAP-4) was expressed. This protein retains biological activity as a DRG growth cone collapsing agent and saturably binds to DRG neurons with low nanomolar affinity. Specific CAP-4 binding sites are present on DRG neurons, sympathetic neurons, and motoneurons, but not on retinal, cortical, or brainstem neurons. Outside the nervous system, high levels of CAP-4 binding sites are present in the mesenchyme surrounding major blood vessels and developing bone and in lung. These sites provide a substrate for the collapsin-1-dependent patterning of non-neuronal tissues perturbed in sema III (-/-) mice. The staining patterns for mouse semaphorin D/III and chick collapsin-1 fusion proteins are indistinguishable from one another but quite separate from those for semaphorin B and M-semaphorin F fusion proteins. These data imply that there exists a family of high-affinity semaphorin binding sites similar in complexity to the semaphorin ligand family (Takahashi, 1997).
Neuropilin (neuropilin-1) was recently identified as a receptor for Collapsin-1/Semaphorin III/D (Sema III). A related protein has been identified, neuropilin-2, whose mRNA is expressed by developing neurons in a pattern largely, though not completely, nonoverlapping with that of neuropilin-1. Unlike neuropilin-1, which binds with high affinity to the three structurally related semaphorins (Sema III, Sema E, and Sema IV), neuropilin-2 shows high affinity binding only to Sema E and Sema IV, not Sema III. These results identify neuropilins as a family of receptors (or components of receptors) for at least one semaphorin subfamily. They also suggest that the specificity of action of different members of this subfamily may be determined by the complement of neuropilins expressed by responsive cells (Chen, 1997).
Neuropilin-1 and neuropilin-2 show specificity in binding to different class III semaphorins, including Sema III, Sema E, and Sema IV, suggesting that the specificity of action of these semaphorins is dictated by the complement of neuropilins expressed by responsive neurons. In support of this, sympathetic axons have been shown to coexpress neuropilin-1 and -2; their responses to Sema III, Sema E, and Sema IV are affected in predicted ways by antibodies to neuropilin-1, and neuropilin-1 and -2 can form homo- and hetero-oligomers through an interaction involving (at least partly) the neuropilin MAM (meprin, A5, mu) domain. These results support the idea that in sympathetic axons, the Sema III signal is mediated predominantly by neuropilin-1 oligomers; the Sema IV signal by neuropilin-2 oligomers, and the Sema E signal by neuropilin-1 and -2, either as homo- or hetero-oligomers (Chen, 1998).
Collapsin-1, a member of the semaphorin family, activates receptors on specific growth cones, thereby inhibiting their motility. Neuropilin, a previously cloned transmembrane protein, has recently been identified as a candidate receptor for collapsin-1. The cloning of chick collapsin-3 and -5 has been completed, and collapsin-1, -2, -3, and -5 are known to bind to overlapping but distinct axon tracts. In situ, there are inferred to be distinct receptors with different affinities for collapsin-1, -2, -3, and -5. In contrast, these four collapsins all bind recombinant neuropilin with similar affinities. Strong binding to neuropilin is mediated by the carboxy third of the collapsins, while the semaphorin domain confers collapsins' unique binding patterns in situ. It is proposed that neuropilin is a common component of a semaphorin receptor complex, and that additional differentially expressed receptor components interact with the semaphorin domains to confer binding specificity (Feiner, 1997).
To explore a role for chemorepulsive axon guidance mechanisms in the regeneration of primary olfactory axons, the expression of the chemorepellent semaphorin III (sema III), its receptor neuropilin-1, and collapsin response mediator protein-2 (CRMP-2) were examined during regeneration of the olfactory system. In the intact olfactory system, neuropilin-1 and CRMP-2 mRNA expression define a distinct population of olfactory receptor neurons, corresponding to immature (B-50/GAP-43-positive) neurons, and a subset of mature (olfactory marker protein-positive) neurons, located in the lower half of the olfactory epithelium. Sema III mRNA is expressed in pial sheet cells and in second-order olfactory neurons that are the target cells of neuropilin-1-positive primary olfactory axons. These data suggest that in the intact olfactory bulb, sema III creates a molecular barrier, which helps restrict ingrowing olfactory axons to the nerve and glomerular layers of the bulb. Both axotomy of the primary olfactory nerve and bulbectomy induce the formation of new olfactory receptor neurons expressing neuropilin-1 and CRMP-2 mRNA. After axotomy, sema III mRNA is transiently induced in cells at the site of the lesion. These cells align regenerating bundles of olfactory axons. In contrast to the transient appearance of sema III-positive cells at the lesion site after axotomy, sema III-positive cells increase progressively after bulbectomy, apparently preventing regenerating neuropilin-1-positive nerve bundles from growing deeper into the lesion area. The presence of sema III in scar tissue and the concomitant expression of its receptor neuropilin-1 on regenerating olfactory axons suggests that semaphorin-mediated chemorepulsive signal transduction may contribute to the regenerative failure of these axons after bulbectomy (Pasterkamp, 1998).
The semaphorins are the largest family of repulsive axon guidance molecules. Secreted semaphorins bind neuropilin receptors and repel sensory, sympathetic and motor axons. CA1, CA3 and dentate gyrus axons from E15-E17 mouse embryo explants are selectively repelled by entorhinal cortex and neocortex. The secreted semaphorins Sema III and Sema IV and their receptors Neuropilin-1 and -2 are expressed in the hippocampal formation during appropriate stages. Sema III and Sema IV strongly repel CA1, CA3 and dentate gyrus axons; entorhinal axons are only repelled by Sema III. An antibody against Neuropilin-1 blocks the repulsive action of Sema III and the entorhinal cortex, but has no effect on Sema IV-induced repulsion. Thus, chemorepulsion plays a role in axon guidance in the hippocampus; secreted semaphorins are likely to be responsible for this action, and the same axons can be repelled by two distinct semaphorins via two different receptors (Chedotal, 1998).
Somatosensory axon outgrowth is repulsed when soluble semaphorin D (semD) binds to growth cone neuropilin-1 (Npn-1). SemD ligand binding studies of Npn-1 mutants demonstrate that the sema domain binds to the amino-terminal quarter, or complement-binding (CUB) domain, of Npn-1. By herpes simplex virus- (HSV-) mediated expression of Npn-1 mutants in chick retinal ganglion cells, it has been shown that semD-induced growth cone collapse requires two segments of the ectodomain of Npn-1: the CUB domain and the juxtamembrane portion, or MAM (meprin, A5, mu) domain. In contrast, the transmembrane segment and cytoplasmic tail of Npn-1 are not required for biologic activity. These data imply that the CUB and MAM ectodomains of Npn-1 interact with another transmembrane growth cone protein, which in turn transduces a semD signal into axon repulsion (Nakamura, 1998).
Neuropilins bind secreted members of the semaphorin family of proteins. Neuropilin-1 is a receptor for Sema III. Neuropilin-2 is a receptor for the secreted semaphorin Sema IV and acts selectively to mediate repulsive guidance events in discrete populations of neurons. neuropilin-2 and semaIV are expressed in strikingly complementary patterns during neurodevelopment. The extracellular complement-binding (CUB) and coagulation factor domains of neuropilin-2 confer specificity to the Sema IV repulsive response, and these domains of neuropilin-1 are necessary and sufficient for binding of the Sema III semaphorin (sema) domain. The coagulation factor domains alone are necessary and sufficient for binding of the Sema III immunoglobulin- (Ig-) basic domain and the unrelated ligand, vascular endothelial growth factor (VEGF). Lastly, neuropilin-1 can homomultimerize and form heteromultimers with neuropilin-2. These results provide insight into how interactions between neuropilins and secreted semaphorins function to coordinate repulsive axon guidance during neurodevelopment (Giger, 1998).
The olfactory system provides an example of the complex expression pattern of Sema IV and Neuropilin-2. In the developing olfactory system, SemaIV is expressed in regions apical to the VNR neurons of the vomeronasal organ (VNO). Equally strong expression of neuropilin-2 is observed in the V2R neurons of the basal VNO. The V2R neurons initially project basally into the accessory olfactory nerve, away from the region of SemaIV expression in the VNO, commensurate with the idea that Sema IV serves to direct the initial projections of these neurons. The accessory olfactory bulb (OB), unlike the main OB, does not express high levels of SemaIV, and this lack of semaIV expression may serve to help segregate VNO projections to the accessory OB from olfactory epithelium (OE) projections to the main olfactory bulb. Neuropilin-2 is strongly expressed by both VNO neurons and their target region in the accessory OB. It is possible that homophilic Neuropilin-2 interactions may serve to help establish appropriate connectivity between the VNO and the accessory olfactory bulb and may even function to regulate fasciculation of accessory olfactory neurons. SemaIV is expressed in the main OE, with stronger expression observed in the basal OE, and since subsets of main olfactory receptor neurons (ORNs) also express Neuropilin-2, Sema IV may play a role as well in directing the initial outgrowth of ORNs to the olfactory nerve. In addition, SemaIV is expressed in the main OB in the periglomerular, mitral, and tufted cells and could prevent the main ORNs from overshooting their glomerular targets in the main OB (Giger, 1998).
Neuropilin-1 (Npn-1), a receptor for semaphorin III, mediates the guidance of growth cones on extending neurites. The molecular mechanism of Npn-1 signaling remains unclear. A yeast two-hybrid system has been used to isolate a protein that interacts with the cytoplasmic domain of Npn-1. This Npn-1-interacting protein (NIP) contains a central PSD-95/Dlg/ZO-1 (PDZ) domain and a C-terminal acyl carrier protein domain. The physiological interaction of Npn-1 and NIP is supported by co-immunoprecipitation of these two proteins in extracts from a heterologous expression system and from a native tissue. The C-terminal three amino acids of Npn-1 (S-E-A-COOH), which is conserved from Xenopus to human, is responsible for interaction with the PDZ domain-containing C-terminal two-thirds of NIP. NIP as well as Npn-1 are broadly expressed in mice as assayed by Northern and Western analysis. Immunohistochemistry and in situ hybridization experiments reveal that NIP expression overlaps that of Npn-1. NIP has been independently cloned as RGS-GAIP-interacting protein (GIPC): it was identified by virtue of its interaction with the C terminus of RGS-GAIP and has been suggested to participate in clathrin-coated vesicular trafficking. It is suggested that NIP and GIPC may participate in regulation of Npn-1-mediated signaling as a molecular adapter that couples Npn-1 to membrane trafficking machinery in the dynamic axon growth cone (Cai, 1999).
While neuropilin-1 (NP-1) is necessary and sufficient for growth cone binding of Sema3A, NP-1 does not itself transmit a signal to the cytoplasmic domain of the growth cone. The two known semaphorin-binding proteins, plexin 1 (Plex 1) and neuropilin-1, form a stable complex. Plex 1 alone does not bind semaphorin-3A (Sema3A), but the NP-1/Plex 1 complex has a higher affinity for Sema3A than does NP-1 alone. While Sema3A binding to NP-1 does not alter nonneuronal cell morphology, Sema3A interaction with NP-1/Plex 1 complexes induces adherent cells to round up. Expression of a dominant-negative Plex 1 in sensory neurons blocks Sema3A-induced growth cone collapse. Sema3A treatment leads to the redistribution of growth cone NP-1 and plexin into clusters. Thus, physiologic Sema3A receptors consist of NP-1/plexin complexes (Takahashi, 1999).
Several lines of evidence suggest that the signal transducer for the NP-1/Sema3A complex is a plexin: (1) NP-1 and Plex 1 form an immunoprecipitable complex; (2) the complex exhibits an enhanced affinity for Sema3A close to the Sema3A affinity seen in growth cones; (3) the affinity enhancement of the complex requires the signal-transducing MAM domain of NP-1 and is agonist selective; (4) the NP-1/Plex 1 complex is sufficient to mediate morphologic responses to Sema3A in nonneuronal cells; (5) the NP-1 structural requirements for Plex 1-mediated changes in nonneuronal cell morphology are the same as those for growth cone collapse; (6) the cytoplasmic domain of Plex 1 is essential for these Sema3A-induced morphologic changes; (7) plexin and NP-1 cocluster during Sema3A treatment of DRG growth cones, and (8) a dominant-negative form of Plex 1 dramatically reduces Sema3A-induced growth cone collapse. Taken together, the data strongly support the hypothesis that a plexin mediates the actions of Sema3A/NP-1 complexes (Takahashi, 1999).
The idea that the signal transducer for the NP-1/Sema3A complex is a plexin implies a unified scheme for the mechanism of semaphorin action. All semaphorin signals may be mediated via plexins. Soluble class 3 semaphorins utilize NPs as high-affinity binding intermediates in order to access a more general plexin transduction cascade. For class 1 and viral semaphorins, there is convincing evidence that plexins directly bind semaphorin ligands and mediate cellular effects. Other semaphorins may utilize plexin isoforms directly, or additional non-NP accessory components may exist. It is tempting to speculate that class 3 soluble semaphorins require NPs to enhance binding affinities, whereas membrane-associated semaphorins are confined to the plane of the lipid bilayer and require a lower binding affinity to achieve biological specificity, and this lower affinity is provided by plexins directly. This model also implies that plexins are bifunctional: they can stand alone as Sema-1 receptors and also serve as transducing subunits for Sema3A/NP-1 complexes (Takahashi, 1999).
The unified model of semaphorin signaling through plexins presented above emphasizes the paucity of information concerning how the intracellular domain of plexin might transduce a signal. It is noteworthy that the large intracellular domain is highly conserved across the plexin family but does not share strong sequence similarity to another protein, which might suggest an obvious hypothesis for signaling function. There is weak similarity of Plex 1 residues 1667-1825 with a group of R-ras GTPase-activating proteins (GAPs). However, a model for semaphorin signaling based on this similarity is not obvious. It is known that the NPs and some semaphorins self-associate. Therefore, their association with Plex 1 might create higher-order ligand-receptor complexes. Perhaps receptor aggregation activates a signaling function of the plexin intracellular domain. While Sema3A does not regulate the extent of coprecipitation of NP-1 with NP-1, or NP-1 with Plex 1, the immunohistologic data suggest that Sema3A induces higher-order receptor structures that in turn lead to the activation of signaling cascades and morphologic changes. In support of such a model, it has been found that Sema3A-induced NP-1/plexin clusters are associated with Rac1 aggregation, F-actin nucleation, membrane ruffles, and endocytosis (Takahashi, 1999).
Semaphorin 3A (Sema3A) binds to neuropilin-1 (NP1) and activates the transmembrane Plexin to transduce a repulsive axon guidance signal. Sema3 signals are transduced equally effectively by PlexinA1 or PlexinA2, but not by PlexinA3. Deletion analysis of the PlexinA1 ectodomain demonstrates that the sema domain prevents PlexinA1 activation in the basal state. Sema-deleted PlexinA1 is constitutively active, producing cell contraction, growth cone collapse, and inhibition of neurite outgrowth. The sema domain of PlexinA1 physically associates with the remainder of the PlexinA1 ectodomain and can reverse constitutive activation. Both the sema portion and the remainder of the ectodomain of PlexinA1 associate with NP1 in a Sema3A-independent fashion. Plexin A1 is autoinhibited by its sema domain, and Sema3A/NP1 releases this inhibition (Takahashi, 2001).
What is the role of the PlexA1 sema domain in the NP/PlexA1 complex? PlexA1sem associates with both NP1 and the remainder of PlexA1. Because coprecipitation is not modified by the presence of Sema3A, the PlexA1 sema domain must bind to NP1 at a site different from the Sema3A binding site of NP1. Indeed, coexpression of PlexA1sem with NP1 enhances Sema3A avidity for NP1. PlexA1Deltasem also associates with NP1 in a Sema3A-independent fashion and coexpression of PlexA1Deltasem increases Sema3A affinity for NP1. This implies that NP1 interacts with three partners at three distinct sites. PlexA1sem and the remainder of PlexA1 ectodomain cooperate to increase the Sema3A affinity for NP1. In the NP1/PlexA1 complex, PlexA1 sema domain interactions with both NP1 and the remainder of the PlexA1ectodomain may keep PlexA1 silent. Presumably, Sema3A binding to NP1 results in a dramatic conformational change in this complex. The simplest model is that this conformational change physically separates and functionally reduces PlexA1sem effects on the remainder of the PlexA1 ectodomain. In support of such a conformational dissociation of the two PlexA1 domains, excess PlexA1sem can reverse constitutive activation or Sema3A-induced PlexA1 activation. Although the two PlexA1 domains are functionally dissociated by Sema3A binding to NP-1, they remain bound in a NP1-dependent complex of altered conformation. The NP1 antagonists, Sema3C, Sema3B, and Sema3F, are expected to occupy the Sema3A site but not result in this conformational shift and the release of PlexA1sem from the remainder of the PlexA1 ectodomain (Takahashi, 2001).
In the developing nervous system axons navigate with great precision over large distances to reach their target areas. Chemorepulsive signals such as the semaphorins play an essential role in this process. The effects of one of these repulsive cues, semaphorin 3A (Sema3A), are mediated by the membrane protein neuropilin-1 (Npn-1). Neuropilin-1 is essential but not sufficient to form functional Sema3A receptors and indicates that additional components are required to transduce signals from the cell surface to the cytoskeleton. Members of the plexin family interact with the neuropilins and act as co-receptors for Sema3A. Neuropilin/plexin interaction restricts the binding specificity of neuropilin-1 and allows the receptor complex to discriminate between two different semaphorins. Deletion of the highly conserved cytoplasmic domain of Plexin-A1 or -A2 creates a dominant negative Sema3A receptor that renders sensory axons resistant to the repulsive effects of Sema3A when expressed in sensory ganglia. These data suggest that functional semaphorin receptors contain plexins as signal-transducing subunits and neuropilins as ligand-binding subunits (Rohm, 2000).
The class 3 Semaphorins Sema3A and Sema3F are potent axonal repellents that cause repulsion by binding Neuropilin-1 and Neuropilin-2, respectively. Plexins are implicated as signaling coreceptors for the Neuropilins, but the identity of the Plexins that transduce Sema3A and Sema3F responses in vivo is uncertain. This study shows that Plexin-A3 and -A4 are key determinants of these responses, through analysis of a Plexin-A3/Plexin-A4 double mutant mouse. Sensory and sympathetic neurons from the double mutant are insensitive to Sema3A and Sema3F in vitro, and defects in axonal projections in vivo correspond to those seen in Neuropilin-1 and -2 mutants. Interestingly, a differential requirement was found for these two Plexins: signaling via Neuropilin-1 is mediated principally by Plexin-A4, whereas signaling via Neuropilin-2 is mediated principally by Plexin-A3. Thus, Plexin-A3 and -A4 contribute to the specificity of axonal responses to class 3 Semaphorins (Yaron, 2005).
The establishment of functional neural circuits requires the guidance of axons in response to the actions of secreted and cell-surface molecules such as the semaphorins. Semaphorin 3E and its receptor PlexinD1 are expressed in the brain, but their functions are unknown. This study shows that Sema3E/PlexinD1 signaling plays an important role in initial development of descending axon tracts in the forebrain. Early errors in axonal projections are reflected in behavioral deficits in Sema3E null mutant mice. Two distinct signaling mechanisms can be distinguished downstream of Sema3E. On corticofugal and striatonigral neurons expressing PlexinD1 but not Neuropilin-1, Sema3E acts as a repellent. In contrast, on subiculo-mammillary neurons coexpressing PlexinD1 and Neuropilin-1, Sema3E acts as an attractant. The extracellular domain of Neuropilin-1 is sufficient to convert repulsive signaling by PlexinD1 to attraction. These data therefore reveal a 'gating' function of neuropilins in semaphorin-plexin signaling during the assembly of forebrain neuronal circuits (Chauvet, 2007).
Slit is a secreted protein known to repulse the growth cones of commissural neurons. By contrast, Slit also promotes elongation and branching of axons of sensory neurons. The reason why different neurons respond to Slit in different ways is largely unknown. Islet2 is a LIM/homeodomain-type transcription factor that specifically regulates elongation and branching of the peripheral axons of the primary sensory neurons in zebrafish embryos. PlexinA4, a transmembrane protein known to be a co-receptor for class III semaphorins, was shown to act downstream of Islet2 to promote branching of the peripheral axons of the primary sensory neurons. Intriguingly, repression of PlexinA4 function by injection of the antisense morpholino oligonucleotide specific to PlexinA4 or by overexpression of the dominant-negative variant of PlexinA4 counteracts the effects of overexpression of Slit2 to induce branching of the peripheral axons of the primary sensory neurons in zebrafish embryos, suggesting involvement of PlexinA4 in the Slit signaling cascades for promotion of axonal branching of the sensory neurons. Colocalized expression of Robo, a receptor for Slit2, and PlexinA4 is observed not only in the primary sensory neurons of zebrafish embryos but also in the dendrites of the pyramidal neurons of the cortex of the mammals, and may be important for promoting the branching of either axons or dendrites in response to Slit, as opposed to the growth cone collapse (Miyashita, 2004).
The small GTPase Rac has been implicated in growth cone guidance mediated by semaphorins and their receptors. Plexin-B1, a receptor for Semaphorin4D (Sema4D), and p21-activated kinase (PAK) can compete for the interaction with active Rac and plexin-B1 can inhibit Rac-induced PAK activation. Expression of active Rac enhances the ability of plexin-B1 to interact with Sema4D. Active Rac stimulates the localization of plexin-B1 to the cell surface. The enhancement in Sema4D binding depends on the ability of Rac to bind plexin-B1. These observations support a model where signaling between Rac and plexin-B1 is bidirectional; Rac modulates plexin-B1 activity and plexin-B1 modulates Rac function (Vikis, 2002).
Sema4D enhances the interaction between plexin-B1 and active Rac. A model is proposed by which Sema4D binds the plexin-B1 receptor and stimulates the recruitment of Rac-GTP. Sequestration of Rac results in the inactivation of PAK and growth cone collapse/turning. This model conflicts with studies on the role of Rac downstream of the plexin-A1 receptor where dominant negative Rac inhibits collapse in response to Sema3A; this suggests that Rac activation is required for Sema3A-mediated growth cone collapse. Perhaps plexin-A and -B signal via different mechanisms since plexin-A does not interact with active Rac. However, in Drosophila, Rac functions downstream of plexA even though it does not interact with plexA. It is possible that a yet unidentified protein couples plexin-A with Rac (Vikis, 2002).
These results indicate that another consequence of the plexin-B1/Rac interaction is to modulate Sema4D ligand binding. This effectively classifies plexin-B1 as a downstream effector of Rac and is the first example of a small GTPase that directly regulates receptor function. An enhancement in the quantity of receptor at the cell membrane and minor changes in affinity for ligand contribute to this enhancement. Whether this is a result of enhanced recruitment to the cell surface and/or inhibition of receptor endocytosis is presently unclear. RhoA does not interact with plexin-B1 and does not stimulate Sema4D ligand binding, yet it has been reported to be activated by clustering of plexin-B1 receptor. In Drosophila, plexB interacts with Rho and stimulates its activity. It appears that humans and flies use different mechanisms for plexin-B stimulation of Rho activity (Vikis, 2002).
These data also suggest that endogenous Rac-GTP is necessary for the maintenance of plexin-B1 at the cell surface. This is based on the observation that dominant negative Rac (RacN17), which inhibits endogenous Rac activation, effectively inhibits the plexin-B1/Sema4D interaction. Furthermore, the Rac binding defective mutant plexin-B1-GGA is compromised in the interaction with Sema4D. This led the authors to postulate that factors that modulate Rac activation can enhance the sensitivity of the receptor/ligand interaction. It is possible that activation of a Rac-specific GEF and/or inactivation of a GAP may modulate the levels of plexin-B1 at the cell surface and its affinity for ligand. Hence, this suggests that engagement of plexin-B1 by Sema4D may be regulated by intracellular levels of Rac-GTP. What the biological consequence of this is remains unknown, however this may be a mechanism by which Rac activation by one axon guidance cue can modulate the responsiveness of the axon growth cone to another guidance cue, such as Sema4D. It is worth noting that whether this model operates in axon growth cone guidance requires further analysis in neurons. Under physiological conditions the axon growth cone is exposed to multiple guidance cues. Therefore, Rac may act as a mediator for cross-talk between different axon guidance cues. Furthermore, the data suggest that signaling between plexin-B1 and Rac is bidirectional. Ligand-gated plexin-B1 can sequester Rac from activating other downstream effectors whereas active Rac can enhance the activity of plexin-B1 (Vikis, 2002).
Plexins are widely expressed transmembrane proteins that, in the nervous system, mediate repulsive signals of semaphorins. However, the molecular nature of plexin-mediated signal transduction remains poorly understood. Plexin-B family members associate through their C termini with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG (leukemia-associated Rho GEF). The molecular determinants of the interaction between plexin-B1 and PDZ-RhoGEF or LARG were analyzed. PDZ-RhoGEF mutants lacking the RGS domain or the DH/PH domain are still capable of interacting with plexin-B1 as effectively as wild-type, full-length PDZ-RhoGEF. In contrast, the PDZ-RhoGEF mutant lacking the PDZ domain does not interact with plexin-B1, indicating that the PDZ domain of PDZ-RhoGEF is required for its interaction with plexin-B1. Activation of plexin-B1 by semaphorin 4D regulates combined PDZ-RhoGEF and LARG activity leading to RhoA activation. In addition, a dominant-negative form of PDZ-RhoGEF blocks semaphorin 4D-induced growth cone collapse in primary hippocampal neurons. This study indicates that the interaction of mammalian plexin-B family members with the multidomain proteins PDZ-RhoGEF and LARG represents an essential molecular link between plexin-B and localized, Rho-mediated downstream signaling events that underlie various plexin-mediated cellular phenomena including axonal growth cone collapse (Swiercz, 2002).
Plexins represent a novel family of transmembrane receptors that transduce attractive and repulsive signals mediated by the axon-guiding molecules semaphorins. Emerging evidence implicates Rho GTPases in these biological events. However, Plexins lack any known catalytic activity in their conserved cytoplasmic tails, and how they transduce signals from semaphorins to Rho is still unknown. This study shows that Plexin B2 associates directly with two members of a recently identified family of Dbl homology/pleckstrin homology containing guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and Leukemia-associated Rho GEF (LARG). This physical interaction is mediated by their PDZ domains and a PDZ-binding motif found only in Plexins of the B family. In addition, ligand-induced dimerization of Plexin B is sufficient to stimulate endogenous RhoA potently and to induce the reorganization of the cytoskeleton. Moreover, overexpression of the PDZ domain of PDZ-RhoGEF but not its regulator of G protein signaling domain prevents cell rounding and neurite retraction of differentiated PC12 cells induced by activation of endogenous Plexin B1 by semaphorin 4D. The association of Plexins with LARG and PDZ-RhoGEF thus provides a direct molecular mechanism by which semaphorins acting on Plexin B can control Rho, thereby regulating the actin-cytoskeleton during axonal guidance and cell migration (Perrot, 2002).
Semaphorins are axon guidance molecules that signal through the plexin family of receptors. Semaphorins also play a role in other processes such as immune regulation and tumorigenesis. However, the molecular signaling mechanisms downstream of plexin receptors have not been elucidated. Semaphorin 4D is the ligand for the plexin-B1 receptor and stimulation of the plexin-B1 receptor activates the small GTPase RhoA. Using the intracellular domain of plexin-B1 as an affinity ligand, two Rho-specific guanine nucleotide exchange factors, leukemia-associated Rho GEF (LARG; GEF, guanine nucleotide exchange factors) and PSD-95/Dlg/ZO-1 homology (PDZ)-RhoGEF, were isolated from mouse brain as plexin-B1-specific interacting proteins. LARG and PDZ-RhoGEF contain several functional domains, including a PDZ domain. Biochemical characterizations showed that the PDZ domain of LARG is directly involved in the interaction with the carboxy-terminal sequence of plexin-B1. Mutation of either the PDZ domain in LARG or the PDZ binding site in plexin-B1 eliminates the interaction. The interaction between plexin-B1 and LARG is specific for the PDZ domain of LARG and LARG does not interact with plexin-A1. A LARG-interaction defective mutant of the plexin-B1 receptor was created and was unable to stimulate RhoA activation. The data in this report suggest that LARG plays a critical role in plexin-B1 signaling to stimulate Rho activation and cytoskeletal reorganization (Aurandt, 2002).
The identification of new signaling pathways critical for cardiac morphogenesis will contribute to understanding of congenital heart disease (CHD), which remains a leading cause of mortality in newborn children worldwide. Signals mediated by semaphorin ligands and plexin receptors contribute to the intricate patterning of axons in the central nervous system. A related signaling pathway involves secreted class 3 semaphorins, neuropilins, and a plexin receptor, PlexinD1, expressed by endothelial cells. Interruption of this pathway in mice results in CHD and vascular patterning defects. The type of CHD caused by inactivation of PlexinD1 has been previously attributed to abnormalities of neural crest. This study shows that this form of CHD can be caused by cell-autonomous endothelial defects. Thus, molecular programs that mediate axon guidance in the central nervous system also function in endothelial cells to orchestrate critical aspects of cardiac morphogenesis (Gitler, 2004).
The closely related phenotypes involving the heart outflow tract (OT) and aortic arch defects of sema3C, plexinD1, and Npn-1 knockout mice, coupled with biochemical data, strongly suggest that semaphorin signaling mediated by endothelial cells (ECs) expressing PlexinD1 and neuropilins compose a receptor-ligand paracrine signaling pathway that orchestrates septation of the OT and development of aortic arch artery derivatives. Congenital heart defects in humans frequently involve OT and aortic arch defects, and SEMA3C, PLEXIND1, and NPN-1 are candidate genes for congenital heart disease. The ability of neuropilin to bind distinct ligands (VEGF165 and SEMA3 proteins), coupled with elegant genetic studies, has suggested that neuropilin facilitates VEGF signaling in endothelium and semaphorin signaling in non-ECs including cardiac neural crest. Endothelial-specific loss of Npn-1 has been interpreted in terms of loss of VEGF165 signaling in endothelium. However, biochemical and genetic data identify a direct role for neuropilin-mediated semaphorin signaling in endothelium. This result demands reinterpretation of existing data concerning the role of neuropilin, VEGF, and semaphorin signaling in cardiovascular development and led to the proposal of a unifying model. It is suggested that neuropilin, in ECs, functions in both VEGF and semaphorin signaling and that both pathways are required for proper cardiac OT development. Either Npn-1 or Npn-2 is able to cooperate with PlexinD1 to bind Sema3C. This explains why specific inhibition of Npn-1-dependent semaphorin signaling does not result in OT defects unless the redundant Npn-2 is also inactivated, whereas inactivating either the ligand Sema3C or the coreceptor PlexinD1 is sufficient to produce OT defects (Gitler, 2004 and references therein).
Major vessels of the vertebrate circulatory system display evolutionarily conserved and reproducible anatomy, but the cues guiding this stereotypic patterning remain obscure. In the nervous system, axonal pathways are shaped by repulsive cues provided by ligands of the semaphorin family that are sensed by migrating neuronal growth cones through plexin receptors. Proper blood vessel pathfinding requires the endothelial receptor PlexinD1 and semaphorin signals, and mutations have been identified in plexinD1 in the zebrafish vascular patterning mutant out of bounds. These results reveal the fundamental conservation of repulsive patterning mechanisms between axonal migration in the central nervous system and vascular endothelium during angiogenesis (Torres-Vazquez, 2004).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.