Implication of neuropilin 2/semaphorin 3F in retinocollicular map formation

Neural representations of the environment within the brain take the form of topographic maps whose formation relies on graded expression of axon guidance molecules. Retinocollicular map formation, from retinal ganglion cells (RGCs) to the superior colliculus (SC) in the midbrain, is mainly driven by Eph receptors and their ligands ephrins. However, other guidance molecules participate in the formation of this map. Here we demonstrate that the receptor Neuropilin‐2 is expressed in an increasing nasal–temporal gradient in RGCs, whereas one of its ligands, Semaphorin3F, but not other Sema3 molecules, presents a graded low‐rostral to high‐caudal expression in the SC when mapping is underway. Neuropilin‐2 and its coreceptor Plexin A1 are present on RGC growth cones. Collapse assays demonstrate that Semaphorin3F induces significant growth cone collapse of temporal, but not nasal, RGCs expressing high levels of Neuropilin‐2. Our results suggest that Neuropilin‐2/Semaphorin3F are new candidates involved in retinotopy formation within the SC. Developmental Dynamics 237:3394–3403, 2008. © 2008 Wiley‐Liss, Inc.     

Expression of class 3 semaphorins and their receptors in human breast neoplasia

Staton C A, Shaw L A, Valluru M, Hoh L, Koay I, Cross S S, Reed M W & Brown N J
(2011) Histopathology 59 , 274–282 Expression of class 3 semaphorins and their receptors in human breast neoplasia Aims: This study aimed to identify the involvement of class 3 semaphorins (Sema3) and receptors, neuropilins (Np1 and Np2) and plexins (A1–A4) in breast cancer development and angiogenesis. Methods and results: We quantified and correlated Sema3A, Sema3B, Sema3F and their known receptors and coreceptors Plexin‐A1, Plexin‐A3, Np1 and Np2 in sections of normal human breast, benign and pre‐malignant hyperplastic tissue, pre‐invasive and invasive cancer, and compared these findings with our previously published data on vascular endothelial growth factor (VEGF) and microvessel density (MVD) in the same samples. Histological analysis revealed that Sema3B was expressed more strongly and widely than Sema3A and 3F in normal breast tissue and all three semaphorins decreased with the transition from in situ to invasive cancer (P < 0.014). Plexin‐A3 decreased significantly with progression towards invasive cancer (P < 0.045), whereas Plexin‐A1 expression was only significantly reduced once invasion had occurred (P = 0.012). Np1 and Np2 were expressed in both endothelial and epithelial/tumour cells. Np2 expression did not change, but Np1 expression significantly increased in the spectrum from hyperplasia to ductal carcinoma in situ (P < 0.035), but decreased with invasive cancer. Conclusion: These data suggest that a decrease in class 3 semaphorin and their plexin receptors have some relationship with disease progression in ductal breast carcinoma.     

Glial cell adhesive molecule unzipped mediates axon guidance in Drosophila

Axon guidance needs help from the glial cell system during embryogenesis. In the Drosophila embryonic central nervous system (CNS), longitudinal glia (LG) have been implicated in axon guidance but the mechanism remains unclear. We identified the protein encoded by the Drosophila gene unzipped (uzip) as a novel cell adhesion molecule (CAM). Uzip expressed in Drosophila S2 cells triggered cell aggregation through homophilic binding. In the embryonic CNS, Uzip was mainly produced by the LG but was also located at axons, which is consistent with the secretion of Uzip expressed in cultured cells. Although uzip mutants displayed no axonal defect, loss of uzip enhanced the axonal defects in the mutant of N‐cadherin (CadN) and the Wnt gene family member wnt5. Overexpression of uzip could rescue the phenotype in the CadNuzip^D43 mutant. Thus, Uzip is a novel CAM from the LG regulating axon guidance. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

The extracellular matrix proteoglycan perlecan facilitates transmembrane semaphorin-mediated repulsive guidance

The Drosophila transmembrane semaphorin-1a (Sema-1a) is a repulsive guidance cue that uses the Plexin A (PlexA) receptor during neural development. Sema-1a is required in axons to facilitate motor axon defasciculation at guidance choice points. We found that mutations in the trol gene strongly suppress Sema-1a-mediated repulsive axon guidance. trol encodes the phylogenetically conserved secreted heparan sulfate proteoglycan (HSPG) perlecan, a component of the extracellular matrix. Motor axon guidance defects in perlecan mutants resemble those observed in Sema-1a- and PlexA-null mutant embryos, and perlecan mutants genetically interact with PlexA and Sema-1a. Perlecan protein is found in both the CNS and the periphery, with higher expression levels in close proximity to motor axon trajectories and pathway choice points. Restoring perlecan to mutant motor neurons rescues perlecan axon guidance defects. Perlecan augments the reduction in phospho-focal adhesion kinase (phospho-FAK) levels that result from treating insect cells in vitro with Sema-1a, and genetic interactions among integrin, Sema-1a, and FAK in vivo support an antagonistic relationship between Sema-1a and integrin signaling. Therefore, perlecan is required for Sema-1a–PlexA-mediated repulsive guidance, revealing roles for extracellular matrix proteoglycans in modulating transmembrane guidance cue signaling during neural development.     

Neuron‐specific expression of atp6v0c2 in zebrafish CNS

Vacuolar ATPase (V‐ATPase) is a multi‐subunit enzyme that plays an important role in the acidification of a variety of intracellular compartments. ATP6V0C is subunit c of the V₀ domain that forms the proteolipid pore of the enzyme. In the present study, we investigated the neuron‐specific expression of atp6v0c2, a novel isoform of the V‐ATPase c‐subunit, during the development of the zebrafish CNS. Zebrafish atp6v0c2 was isolated from a genome‐wide analysis of the zebrafish mib^ta52b mutant designed to identify genes differentially regulated by Notch signaling. Whole‐mount in situ hybridization revealed that atp6v0c2 is expressed in a subset of CNS neurons beginning several hours after the emergence of post‐mitotic neurons. The ATP6V0C2 protein is co‐localized with the presynaptic vesicle marker, SV2, suggesting that it is involved in neurotransmitter storage and/or secretion in neurons. In addition, the loss‐of‐function experiment suggests that ATP6V0C2 is involved in the control of neuronal excitability. Developmental Dynamics 239:2501–2508, 2010. © 2010 Wiley‐Liss, Inc.     

Expression pattern of Nogo‐A,MAG, and NgR in regenerating urodele spinal cord

Background: The mammalian central nervous system is incapable of substantial axon regeneration after injury partially due to the presence of myelin‐associated inhibitory molecules including Nogo‐A and myelin associated glycoprotein (MAG). In contrast, axolotl salamanders are capable of considerable axon regrowth during spinal cord regeneration. Results: Here, we show that Nogo‐A and MAG, and their receptor, Nogo receptor (NgR), are present in the axolotl genome and are broadly expressed in the central nervous system (CNS) during development, adulthood, and importantly, during regeneration. Furthermore, we show that Nogo‐A and NgR are co‐expressed in Sox2 positive neural progenitor cells. Conclusions: These expression patterns suggest myelin‐associated proteins are permissive for neural development and regeneration in axolotls. Developmental Dynamics 242:847–860, 2013. © 2013 Wiley Periodicals, Inc.     

Expression of strawberry notch family genes during zebrafish embryogenesis

Our previous study suggested a possible role for Sbno1, a mouse homologue of strawberry notch gene during brain development. In this report, we cloned the zebrafish homologues of sbno, and examined their expression pattern during embryogenesis by whole‐mount in situ hybridization. Zebrafish have three sbno genes: one Sbno1 homologue and two Sbno2 homologues, sbno2a and sbno2b. We observed that the expression of sbno1 and sbno2a was initially ubiquitous and gradually became predominant in the central nervous system as development progressed. The expression of sbno2b was observed in non‐neural tissues in contrast to the other two genes. sbno1 and sbno2a exhibited higher expression in distinct regions within the nervous system of pharyngula‐stage embryos, suggesting possible differing roles for sbno1 and sbno2a during later stages of embryogenesis. Together, the observed gene expression patterns suggest an important role of sbno‐family genes during development of the vertebrate central nervous system. Developmental Dynamics 239:1789–1796, 2010. © 2010 Wiley‐Liss, Inc.     

Copy‐number variations (CNVs) of the human sex steroid metabolizing genes UGT2B17 and UGT2B28 and their associations with a UGT2B15 functional polymorphism

UGT2B17 and UGT2B28 are among the most commonly deleted genes in humans and encode members of the uridine diphosphate (UDP)‐glucuronosyltransferase 2B (UGT2B) subfamily. They are involved, along with UGT2B15, in the catabolism of sex‐steroid hormones. Despite the recent biomedical interest in UGT2B17 and UGT2B28 copy‐number variations (CNVs) within human populations, the impact of their gene dosage has been hampered by the lack of precise molecular identification of the common deletion breakpoints within high homology sequence regions on chromosome 4. We have characterized these common deletions and report their coexistence in Caucasians, along with the p.D85Y (rs1902023:G>T) functional polymorphism of UGT2B15. Segmental duplications of 4.9 kb for UGT2B17 and 6.8 kb for UGT2B28 comprise purine‐rich recombination sites located 117 kb and 108 kb apart on both ends of the deletions. CNVs of UGT2B17 and UGT2B28 occur in Caucasians at 27% and 13.5%, respectively. While only 43% have two copies of both genes, 57% harbor at least one deletion. Their co‐occurrence on 5% of chromosomes creates a 225‐kb genomic gap. CNVs of both UGT2B17 and UGT2B28, with the co‐occurrence of UGT2B15:p.D85Y, generate seven distinct haplotypes. Restricting the analyses to CNV of the UGT2B17 gene without evaluating UGT2B28 CNV, along with the genotype of UGT2B15, may over‐ or underestimate the impact of each gene under physiological conditions or disease states. Hum Mutat 30:1–10, 2009. © 2009 Wiley‐Liss, Inc.     

Zebrafish scarb2a insertional mutant reveals a novel function for the Scarb2/Limp2 receptor in notochord development

Background: Scarb2 or Limp2 belong to a subfamily of Scavenger receptors described as lysosomal transmembrane glycosylated receptors, that are mutated in the human syndrome AMRF (action myoclonus‐renal failure). The zebrafish insertional mutant scarb2a^hi1463Tg has notochord defects, the notochord is a defining feature of chordates running along the center of the longitudinal axis and it is essential for forming the spinal column in all vertebrates. Results: There are three paralogous scarb2 genes in zebrafish; scarb2a, scarb2b, and scarb2c. Both Scarb2a and Scarb2b proteins lack the classical di‐leucine motif. We found that scarb2a^hi1463Tg homozygous zebrafish embryos have a null mutation impairing vacuole formation in the notochord and simultaneously disrupting proper formation of the basement membrane resulting in its thickening at the ventral side of the notochord, which may be the cause for the anomalous upward bending observed in the trunk. Through whole‐mount in situ hybridization, we detected scarb2a mRNA expression in the notochord and in the brain early in development. However, it is puzzling that scarb2a notochord mRNA expression is short‐lived in the presumptive notochord and precedes the complete differentiation of the notochord. Conclusions: This work describes a novel function for the Scarb2 receptor as an essential glycoprotein for notochord development. Developmental Dynamics 245:508–519, 2016. © 2015 Wiley Periodicals, Inc.     

Transcriptional regulatory regions of gap43 needed in developing and regenerating retinal ganglion cells

Mammals and fish differ in their ability to express axon growth‐associated genes in response to CNS injury, which contributes to the differences in their ability for CNS regeneration. Previously we demonstrated that for the axon growth‐associated gene, gap43, regions of the rat promoter that are sufficient to promote reporter gene expression in the developing zebrafish nervous system are not sufficient to promote expression in regenerating retinal ganglion cells in zebrafish. Recently, we identified a 3.6‐kb gap43 promoter fragment from the pufferfish, Takifugu rubripes (fugu), that can promote reporter gene expression during both development and regeneration. Using promoter deletion analysis, we have found regions of the 3.6‐kb fugu gap43 promoter that are necessary for expression in regenerating, but not developing, retinal ganglion cells. Within the 3.6‐kb promoter, we have identified elements that are highly conserved among fish, as well as elements conserved among fish, mammals, and birds. Developmental Dynamics 239:482–495, 2010. © 2009 Wiley‐Liss, Inc.     

Expression of the zebrafish CD133/prominin1 genes in cellular proliferation zones in the embryonic central nervous system and sensory organs

The CD133/prominin1 gene encodes a pentamembrane glycoprotein cell surface marker that is expressed in stem cells from neuroepithelial, hematopoietic, and various organ tissues. Here we report the analysis of two zebrafish CD133/prominin1 orthologues, prominin1a and prominin1b. The expression patterns of the zebrafish prominin1a and b genes were analyzed during embryogenesis using whole mount in situ hybridization. prominin1a and b show novel complementary and overlapping patterns of expression in proliferating zones in the developing sensory organs and central nervous system. The expression patterns suggest functional conservation of the zebrafish prominin1 genes. Initial analyses of prominin1a and b in neoplastic tissue show increased expression of both genes in a subpopulation of cells in malignant peripheral nerve sheath tumors in tp53 mutants. Based on these analyses, the zebrafish prominin1 genes will be useful markers for examining proliferating cell populations in adult organs, tissues, and tumors. Developmental Dynamics 239:1849–1857, 2010. © 2010 Wiley‐Liss, Inc.     

Deletional tolerance prevents AQP4‐directed autoimmunity in mice

Neuromyelitis optica (NMO) is an autoimmune disorder of the central nervous system (CNS) mediated by antibodies to the water channel protein AQP4 expressed in astrocytes. The contribution of AQP4‐specific T cells to the class switch recombination of pathogenic AQP4‐specific antibodies and the inflammation of the blood–brain barrier is incompletely understood, as immunogenic naturally processed T‐cell epitopes of AQP4 are unknown. By immunizing Aqp4 ^?/? mice with full‐length murine AQP4 protein followed by recall with overlapping peptides, we here identify AQP4(201‐220) as the major immunogenic IA^b‐restricted epitope of AQP4. We show that WT mice do not harbor AQP4(201–220)‐specific T‐cell clones in their natural repertoire due to deletional tolerance. However, immunization with AQP4(201–220) of Rag1 ^?/? mice reconstituted with the mature T‐cell repertoire of Aqp4 ^?/? mice elicits an encephalomyelitic syndrome. Similarly to the T‐cell repertoire, the B‐cell repertoire of WT mice is “purged” of AQP4‐specific B cells, and robust serum responses to AQP4 are only mounted in Aqp4 ^?/? mice. While AQP4(201–220)‐specific T cells alone induce encephalomyelitis, NMO‐specific lesional patterns in the CNS and the retina only occur in the additional presence of anti‐AQP4 antibodies. Thus, failure of deletional T‐cell and B‐cell tolerance against AQP4 is a prerequisite for clinically manifest NMO.     

The evolving role of semaphorins and plexins in the immune system: Plexin-A1 regulation of dendritic cell function

Semaphorin and plexin proteins were originally identified for their important axon guidance regulation. In recent years, their role within the immune system has been discovered. Semaphorins are considered the ligands to the plexin receptors, and together, they mediate cell–cell communication. Initially, through gene knock-out and knock-down studies, simple ligand–receptor pairings that regulate specific cellular interactions were discovered, such as between T cells and dendritic cells. However, more recent data indicate that individual cell types express multiple semaphorin and plexin proteins, engendering complex interactions involving multiple ligand–receptor pairs and multiple cell types. In this review, we describe contributions from our lab elucidating Plexin-A1 regulation of the immune system and our more recent findings of Semaphorin 6D regulation of T-cell immunity.     

Xenopus sonic hedgehog guides retinal axons along the optic tract

The role of classic morphogens such as Sonic hedgehog (Shh) as axon guidance cues has been reported in a variety of vertebrate organisms (Charron and Tessier‐Lavigne [ 2005 ] Development 132:2251–2262). In this work, we provide the first evidence that Xenopus sonic hedgehog (Xshh) signaling is involved in guiding retinal ganglion cell (RGC) axons along the optic tract. Xshh is expressed in the brain during retinal axon extension, adjacent to these axons in the ventral diencephalon. Retinal axons themselves express Patched 1 and Smoothened co‐receptors during RGC axon growth. Blocking Shh signaling causes abnormal ventral pathfinding, and targeting errors at the optic tectum. Misexpression of exogenous N‐Shh peptide in vivo also causes pathfinding errors. Retinal axons grown in culture respond to N‐Shh in a dose‐dependent manner, either by decreasing extension at lower concentrations, or retracting axons in the presence of higher doses. These data suggest that Shh signaling is required for normal RGC axon pathfinding and tectal targeting in the developing visual system of Xenopus. We propose that Shh serves as a ventral optic tract repellent that helps to define the caudal boundary for retinal axons in the diencephalon, and that this signaling is also required for initial target recognition at the optic tectum. Developmental Dynamics 239:2921–2932, 2010. © 2010 Wiley‐Liss, Inc.     

Ror2 is required for midgut elongation during mouse development

The receptor tyrosine kinase Ror2 acts as a receptor for Wnt5a to mediate noncanonical Wnt signaling, and it plays essential roles in morphogenesis. Ror2^?/? embryos exhibit phenotypes similar to, albeit generally milder than, those of Wnt5a^?/? embryos. During mouse embryogenesis, Ror2 is expressed in various organs and regions, although little is known about its expression pattern and roles in the developing gut, while Wnt5a is expressed in the developing gut, where its absence causes abnormal phenotypes. Here, we demonstrated that Ror2 was strongly and differentially expressed in the rostral and middle midgut endoderm from embryonic day (E) 10.5 through embryonic day (E) 12.5. At E11.5, Ror2^?/? embryos exhibited a shorter middle midgut with a larger diameter and more accumulation of epithelial cells in the middle midgut than control embryos, while the total cell numbers remained unaltered. These findings suggest that Ror2 plays important roles in midgut elongation by means of an epithelial convergent extension mechanism. Developmental Dynamics 239:941–953, 2010. © 2010 Wiley‐Liss, Inc.     

Identification and expression patterns of members of the protease‐activated receptor (par) gene family during zebrafish development

Protease‐activated receptors (PARs) play critical roles in hemostasis in vertebrates including zebrafish. However, the zebrafish gene classification appears to be complex, and the expression patterns of par genes are not established. Based on analyses of genomic organization, phylogenetics, protein primary structure, and protein internalization, we report the identification of four zebrafish PARs: par1, par2a, par2b, and par3. This classification differs from one reported previously. We also show that these genes have distinct spatiotemporal expression profiles in embryos and larvae, with par1, par2a, and par2b expressed maternally and ubiquitously during gastrula stages and their expression patterns refined at later stages, and par3 expressed only in 3‐day‐old larvae. Notably, the expression patterns of zebrafish par1 and par2b resemble those of their mammalian counterparts, suggesting that receptor function is conserved among vertebrates. This conservation is supported by our findings that Par1 and Par2b are internalized following exposure to thrombin and trypsin, respectively. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.