Axon Guidance Genes Research Articles

Mesocorticolimbic Connectivity and Volumetric Alterations in DCC Mutation Carriers.

The axon guidance cue receptor DCC (deleted in colorectal cancer) plays a critical role in the organization of mesocorticolimbic pathways in rodents. To investigate whether this occurs in humans, we measured (1) anatomical connectivity between the substantia nigra/ventral tegmental area (SN/VTA) and forebrain targets, (2) striatal and cortical volumes, and (3) putatively associated traits and behaviors. To assess translatability, morphometric data were also collected in Dcc-haploinsufficient mice. The human volunteers were 20 DCC+/- mutation carriers, 16 DCC+/+ relatives, and 20 DCC+/+ unrelated healthy volunteers (UHVs; 28 females). The mice were 11 Dcc+/- and 16 wild-type C57BL/6J animals assessed during adolescence and adulthood. Compared with both control groups, the human DCC+/- carriers exhibited the following: (1) reduced anatomical connectivity from the SN/VTA to the ventral striatum [DCC+/+: p = 0.0005, r(effect size) = 0.60; UHV: p = 0.0029, r = 0.48] and ventral medial prefrontal cortex (DCC+/+: p = 0.0031, r = 0.53; UHV: p = 0.034, r = 0.35); (2) lower novelty-seeking scores (DCC+/+: p = 0.034, d = 0.82; UHV: p = 0.019, d = 0.84); and (3) reduced striatal volume (DCC+/+: p = 0.0009, d = 1.37; UHV: p = 0.0054, d = 0.93). Striatal volumetric reductions were also present in Dcc+/- mice, and these were seen during adolescence (p = 0.0058, d = 1.09) and adulthood (p = 0.003, d = 1.26). Together these findings provide the first evidence in humans that an axon guidance gene is involved in the formation of mesocorticolimbic circuitry and related behavioral traits, providing mechanisms through which DCC mutations might affect susceptibility to diverse neuropsychiatric disorders.SIGNIFICANCE STATEMENT Opportunities to study the effects of axon guidance molecules on human brain development have been rare. Here, the identification of a large four-generational family that carries a mutation to the axon guidance molecule receptor gene, DCC, enabled us to demonstrate effects on mesocorticolimbic anatomical connectivity, striatal volumes, and personality traits. Reductions in striatal volumes were replicated in DCC-haploinsufficient mice. Together, these processes might influence mesocorticolimbic function and susceptibility to diverse neuropsychiatric disorders.

Characterization of a novel OTX2-driven stem cell program in Group 3 and Group 4 medulloblastoma.

Medulloblastoma (MB) is the most common malignant primary pediatric brain cancer. Among the most aggressive subtypes, Group 3 and Group 4 originate from stem/progenitor cells, frequently metastasize, and often display the worst prognosis, yet we know the least about the molecular mechanisms driving their progression. Here, we show that the transcription factor orthodenticle homeobox 2 (OTX2) promotes self‐renewal while inhibiting differentiation in vitro and increases tumor initiation from MB stem/progenitor cells in vivo. To determine how OTX2 contributes to these processes, we employed complementary bioinformatic approaches to characterize the OTX2 regulatory network and identified novel relationships between OTX2 and genes associated with neuronal differentiation and axon guidance signaling in Group 3 and Group 4 MB stem/progenitor cells. In particular, OTX2 levels were negatively correlated with semaphorin (SEMA) signaling, as expression of 9 SEMA pathway genes is upregulated following OTX2 knockdown with some being potential direct OTX2 targets. Importantly, this negative correlation was also observed in patient samples, with lower expression of SEMA4D associated with poor outcome specifically in Group 4 tumors. Functional proof‐of‐principle studies demonstrated that increased levels of select SEMA pathway genes are associated with decreased self‐renewal and growth in vitro and in vivo and that RHO signaling, known to mediate the effects of SEMA genes, is contributing to the OTX2 KD phenotype. Our study provides mechanistic insight into the networks controlled by OTX2 in MB stem/progenitor cells and reveals novel roles for axon guidance genes and their downstream effectors as putative tumor suppressors in MB.

Coordination of olfactory receptor choice with guidance receptor expression and function in olfactory sensory neurons.

Olfactory sensory neurons choose to express a single odorant receptor (OR) from a large gene repertoire and extend axons to reproducible, OR-specific locations within the olfactory bulb. This developmental process produces a topographically organized map of odorant experience in the brain. The axon guidance mechanisms that generate this pattern of connectivity, as well as those that coordinate OR choice and axonal guidance receptor expression, are incompletely understood. We applied the powerful approach of single-cell RNA-seq on newly born olfactory sensory neurons (OSNs) in young zebrafish larvae to address these issues. Expression profiles were generated for 56 individual Olfactory Marker Protein (OMP) positive sensory neurons by single-cell (SC) RNA-seq. We show that just as in mouse OSNs, mature zebrafish OSNs typically express a single predominant OR transcript. Our previous work suggests that OSN targeting is related to the OR clade from which a sensory neuron chooses to express its odorant receptor. We categorized each of the mature cells based on the clade of their predominantly expressed OR. Transcripts expressed at higher levels in each of three clade-related categories were identified using Penalized Linear Discriminant Analysis (PLDA). A genome-wide approach was used to identify membrane-associated proteins that are most likely to have guidance-related activity. We found that OSNs that choose to express an OR from a particular clade also express specific subsets of potential axon guidance genes and transcription factors. We validated our identification of candidate axon guidance genes for one clade of OSNs using bulk RNA-seq from a subset of transgene-labeled neurons that project to a single protoglomerulus. The differential expression patterns of selected candidate guidance genes were confirmed using fluorescent in situ hybridization. Most importantly, we observed axonal mistargeting in knockouts of three candidate axonal guidance genes identified in this analysis: nrp1a, nrp1b, and robo2. In each case, targeting errors were detected in the subset of axons that normally express these transcripts at high levels, and not in the axons that express them at low levels. Our findings demonstrate that specific, functional, axonal guidance related genes are expressed in subsets of OSNs that that can be categorized by their patterns of OR expression.

Highly conserved molecular pathways, including Wnt signaling, promote functional recovery from spinal cord injury in lampreys

In mammals, spinal cord injury (SCI) leads to dramatic losses in neurons and synaptic connections, and consequently function. Unlike mammals, lampreys are vertebrates that undergo spontaneous regeneration and achieve functional recovery after SCI. Therefore our goal was to determine the complete transcriptional responses that occur after SCI in lampreys and to identify deeply conserved pathways that promote regeneration. We performed RNA-Seq on lamprey spinal cord and brain throughout the course of functional recovery. We describe complex transcriptional responses in the injured spinal cord, and somewhat surprisingly, also in the brain. Transcriptional responses to SCI in lampreys included transcription factor networks that promote peripheral nerve regeneration in mammals such as Atf3 and Jun. Furthermore, a number of highly conserved axon guidance, extracellular matrix, and proliferation genes were also differentially expressed after SCI in lampreys. Strikingly, ~3% of differentially expressed transcripts belonged to the Wnt pathways. These included members of the Wnt and Frizzled gene families, and genes involved in downstream signaling. Pharmacological inhibition of Wnt signaling inhibited functional recovery, confirming a critical role for this pathway. These data indicate that molecular signals present in mammals are also involved in regeneration in lampreys, supporting translational relevance of the model.

Correction: The WAGR syndrome gene PRRG4 is a functional homologue of the commissureless axon guidance gene.

[This corrects the article DOI: 10.1371/journal.pgen.1006865.].

The WAGR syndrome gene PRRG4 is a functional homologue of the commissureless axon guidance gene.

WAGR syndrome is characterized by Wilm’s tumor, aniridia, genitourinary abnormalities and intellectual disabilities. WAGR is caused by a chromosomal deletion that includes the PAX6, WT1 and PRRG4 genes. PRRG4 is proposed to contribute to the autistic symptoms of WAGR syndrome, but the molecular function of PRRG4 genes remains unknown. The Drosophila commissureless (comm) gene encodes a short transmembrane protein characterized by PY motifs, features that are shared by the PRRG4 protein. Comm intercepts the Robo axon guidance receptor in the ER/Golgi and targets Robo for degradation, allowing commissural axons to cross the CNS midline. Expression of human Robo1 in the fly CNS increases midline crossing and this was enhanced by co-expression of PRRG4, but not CYYR, Shisa or the yeast Rcr genes. In cell culture experiments, PRRG4 could re-localize hRobo1 from the cell surface, suggesting that PRRG4 is a functional homologue of Comm. Comm is required for axon guidance and synapse formation in the fly, so PRRG4 could contribute to the autistic symptoms of WAGR by disturbing either of these processes in the developing human brain.

PP07. A PML/POLYCOMB/SLIT AXIS REGULATES MIGRATION OF BOTH NORMAL AND NEOPLASTIC CELLS IN THE CNS

INTRODUCTION: The control of SVZ neural progenitor/stem cells (NPCs) migration through the rostral migratory stream (RMS) is essential for normal neurogenesis and serve as the fundamental basis of production/replacement of neurons in the adult brain. Alterations of these processes can lead to neoplastic transformation and glioblastoma multiforme (GBM). The ability of GBM cells to migrate through the brain parenchyma represents a key factor underlying GBM aggressiveness. GBM cells use the same routes used by neuroblasts/immature neurons and neural stem cells upon injury, such as vessels and myelin tracts, suggesting that GBM migration may lay its roots in essential cellular processes controlling migration of immature neurons/neuroblasts and neural stem cells (1). However, our understanding of mechanisms regulating cell migration/invasion during brain tumourigenesis remains limited. METHODS: We combined in vivo and in vitro approaches, including the use of genetically modified mice, primary HGG tissues/NPCs (in collaboration with S Brandner, UCL, C Jones, ICR, S Pollard, Edinburgh, and others). RESULTS: Here we report that the stem cell factor and RAS effector Promyelocytic Leukaemia protein (PML) (2,3) regulates cell migration through the epigenetic control of Slit axon guidance genes in normal and neoplastic cells in the central nervous system. Loss of PML leads to impaired NSC and neuroblast migration and a smaller olfactory bulb in the adult mouse brain. PML controls cell migration via Polycomb Repressive 2-dependent regulation of the Slit axon guidance genes. A similar epigenetically controlled PML/Slit axis is functional also upon RAS-driven neoplastic transformation of NSCs and in primary GBM cells. PML expression inversely correlates with Slit1 expression and patient prognosis in GBM and is enriched in the mesenchymal subtype, which is characterized by activation of the RAS/MAPK pathway. Finally, PML loss impairs tumor growth in an orthotopic animal model. CONCLUSIONS: Taken together, these findings disclose a novel molecular axis at the root of cell migration during normal neurogenesis and brain tumourigenesis. Reference 1. Cuddapah VA et al. Nat Rev Neurosci 15: 455–465. 2. Regad T. et al. Nature neuroscience 12: 132–140. 3. Salomoni P. et al. Cell Res 18: 622–640.

Overexpression of Semaphorin-3E enhances pancreatic cancer cell growth and associates with poor patient survival.

Semaphorin-3E (Sema3E) is a member of an axon guidance gene family, and has recently been reported to contribute to tumor progression and metastasis. However, its role in pancreatic cancer is yet unknown and uncharacterized. In this study, we showed that Sema3E is overexpressed in human pancreatic cancer, and that high Sema3E levels are associated with tumor progression and poor survival. Interestingly, we also observed Sema3E expression in the nucleus, even though Sema3E is reported to be a secreted protein. Overexpression of Sema3E in pancreatic cancer cells promoted cell proliferation and migration in vitro, and increased tumor incidence and growth in vivo. Conversely, knockout of Sema3E suppressed cancer cell proliferation and migration in vitro, and reduced tumor incidence and size in vivo. Moreover, Sema3E induced cell proliferation via acting through the MAPK/ERK pathway. Collectively, these results reveal an undiscovered role of Sema3E in promoting pancreatic cancer pathogenesis, suggesting that Sema3E may be a suitable prognostic marker and therapeutic target for pancreatic cancer.

Dual leucine zipper kinase regulates expression of axon guidance genes in mouse neuronal cells.

BackgroundRecent genetic studies in model organisms, such as Drosophila, C. elegans and mice, have highlighted a critical role for dual leucine zipper kinase (DLK) in neural development and axonal responses to injury. However, exactly how DLK fulfills these functions remains to be determined. Using RNA-seq profiling, we evaluated the global changes in gene expression that are caused by shRNA-mediated knockdown of endogenous DLK in differentiated Neuro-2a neuroblastoma cells.ResultsOur analysis led to the identification of numerous up- and down-regulated genes, among which several were found to be associated with system development and axon guidance according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, respectively. Because of their importance in axonal growth, pruning and regeneration during development and adult life, we then examined by quantitative RT-PCR the mRNA expression levels of the identified axon guidance genes in DLK-depleted cells. Consistent with the RNA-seq data, our results confirmed that loss of DLK altered expression of the genes encoding neuropilin 1 (Nrp1), plexin A4 (Plxna4), Eph receptor A7 (Epha7), Rho family GTPase 1 (Rnd1) and semaphorin 6B (Sema6b). Interestingly, this regulation of Nrp1 and Plxna4 mRNA expression by DLK in Neuro-2a cells was also reflected at the protein level, implicating DLK in the modulation of the function of these axon guidance molecules.ConclusionsCollectively, these results provide the first evidence that axon guidance genes are downstream targets of the DLK signaling pathway, which through their regulation probably modulates neuronal cell development, structure and function.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-016-0068-8) contains supplementary material, which is available to authorized users.

Abstract 694: Axon guidance neuropilin and plexin A2 genes are involved in ERG-associated prostate cancer

Abstract Background: Bone metastases are frequent and severe complications of prostate cancer (PCa), the most common malignancy that affects men in Western countries. Recurrent gene fusions, involving the ERG gene and the androgen-regulated promoter of the TMPRSS2 gene, occur in over 50% of PCa. The abnormal over-expression of the ERG transcription factor disturbs prostate cell transcriptome. We previously identified a series of genes directly and positively regulated by the ERG-fusion in vitro and in vivo. Among them can be found Plexin A2 (PLXNA2), which is a semaphorin receptor family member involved in axon guidance and in many pathophysiological processes, including cancer and bone disorders. We showed that PLXNA2 is involved in TMPRSS2:ERG-mediated enhancements of PC3c cell migration and invasion. These results prompted us to look for genes functionally related to PLXNA2 and involved in ERG-associated PCa. Methods: Using a PCa cell line (PC3c) stably over-expressing the TMPRSS2:ERG fusion, we first used Ingenuity Pathway Analysis® in silico studies to analyze transcriptomic results and identify axon guidance genes associated with ERG-fusion expression in PCa. Their expression was validated in stable clones by RT-PCR and Immunofluorescence. Secondly, using RNAs of a human PCa sample cohort, we performed qRT-PCR to detect and correlate candidate genes to fusion expression. Finally, to validate the relevance of the identified genes, using immunohistochemistry experiments, we studied the expression of these genes in tissue samples, including primary tumors and lymph nodes or bone metastases. Results: Using transcriptomic analyzes and expression validation, we identified neuropilin (NRP1 and NRP2) genes, plexin and semaphorin genes deregulated by the ERG fusion expression in stable clones. In human samples, PLXNA2, NRP1 and NRP2 expression were strongly associated to PCa, and particularly to metastastic PCa. We established a significant correlation between both PLXNA2 and NRP1 gene expression and the presence of TMPRSS2-ERG fusion. At protein level, NRP1 and NRP2 were highly detected in human lymph nodes and bone metastasis samples. Since neuropilin proteins are known to interact with plexin to act as coreceptors for semaphorin ligands, protein colocalization in stable clones suggested a functional association of PLXNA2 and NRPs in tumor cells. Conclusion: Taken together, our results reveal that TMPRSS2-ERG fusion gene expression is able to deregulate axon guidance genes such as NRP1 and PLXNA2 in PCa. Therefore, since the interaction between plexins and their NRP1 and 2 coreceptors often determines the functional specificity of their semaphorin ligand effects, we hypothesize that the ERG fusion expression imbalances the plexin/neuropilin/semaphorin signaling to contribute to PCa progression. These results reinforce the recent interest in targeting neuropilins for cancer therapy. Citation Format: Anthony Turpin, Carine Delliaux, Tian Tian, Nathalie Vanpouille, Anne Flourens, Rachel Deplus, Xavier Leroy, Yvan de Launoit, Martine Duterque-Coquillaud. Axon guidance neuropilin and plexin A2 genes are involved in ERG-associated prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 694.

MicroRNA-338 Attenuates Cortical Neuronal Outgrowth by Modulating the Expression of Axon Guidance Genes.

MicroRNAs (miRs) are small non-coding RNAs that confer robustness to gene networks through post-transcriptional gene regulation. Previously, we identified miR-338 as a modulator of axonal outgrowth in sympathetic neurons. In the current study, we examined the role of miR-338 in the development of cortical neurons and uncovered its downstream mRNA targets. Long-term inhibition of miR-338 during neuronal differentiation resulted in reduced dendritic complexity and altered dendritic spine morphology. Furthermore, monitoring axon outgrowth in cortical cells revealed that miR-338 overexpression decreased, whereas inhibition of miR-338 increased axonal length. To identify gene targets mediating the observed phenotype, we inhibited miR-338 in cortical neurons and performed whole-transcriptome analysis. Pathway analysis revealed that miR-338 modulates a subset of transcripts involved in the axonal guidance machinery by means of direct and indirect gene targeting. Collectively, our results implicate miR-338 as a novel regulator of cortical neuronal maturation by fine-tuning the expression of gene networks governing cortical outgrowth.

Morphogenesis of the C. elegans Intestine Involves Axon Guidance Genes.

Genetic and molecular studies have provided considerable insight into how various tissue progenitors are specified in early embryogenesis, but much less is known about how those progenitors create three-dimensional tissues and organs. The C. elegans intestine provides a simple system for studying how a single progenitor, the E blastomere, builds an epithelial tube of 20 cells. As the E descendants divide, they form a primordium that transitions between different shapes over time. We used cell contours, traced from confocal optical z-stacks, to build a 3D graphic reconstruction of intestine development. The reconstruction revealed several new aspects of morphogenesis that extend and clarify previous observations. The first 8 E descendants form a plane of four right cells and four left cells; the plane arises through oriented cell divisions and VANG-1/Van Gogh-dependent repositioning of any non-planar cells. LIN-12/Notch signaling affects the left cells in the E8 primordium, and initiates later asymmetry in cell packing. The next few stages involve cell repositioning and intercalation events that shuttle cells to their final positions, like shifting blocks in a Rubik’s cube. Repositioning involves breaking and replacing specific adhesive contacts, and some of these events involve EFN-4/Ephrin, MAB-20/semaphorin-2a, and SAX-3/Robo. Once cells in the primordium align along a common axis and in the correct order, cells at the anterior end rotate clockwise around the axis of the intestine. The anterior rotation appears to align segments of the developing lumen into a continuous structure, and requires the secreted ligand UNC-6/netrin, the receptor UNC-40/DCC, and an interacting protein called MADD-2. Previous studies showed that rotation requires a second round of LIN-12/Notch signaling in cells on the right side of the primordium, and we show that MADD-2-GFP appears to be downregulated in those cells.

Targeted and exome sequencing identified somatic mutations in hepatocellular carcinoma.

Genetic analysis has revealed a subset of recurrently mutated genes and aberrant cellular signaling pathways in hepatocellular carcinoma. To investigate genetic alterations and dysregulated pathways in hepatocellular carcinoma, we performed targeted sequencing and exome analysis using next-generation sequencer. We analyzed the somatic mutational profiles of 16 genes in primary hepatocellular carcinoma by targeted ultra-deep sequencing using nine pairs of specimens (tumor and peripheral blood) and whole-exome sequencing using one pair of samples. Overall, somatic mutations with high allele fraction were identified in tumor tissues by targeted deep sequencing. Somatic mutations with high allele fraction were observed in TP53 (3/9; 33%) and CTNNB1 (2/9; 22%) genes in five out of nine (55%) specimens. In vitro analysis showed that CTNNB1 H36P mutant protein identified in tumor samples was resistant to protein degradation and promoted cell proliferation. Exome sequencing identified SLIT3 mutation, implying that dysregulation of axon guidance genes is involved in the development of hepatocellular carcinoma. These results showed that TP53 and WNT/β-catenin signaling pathways were commonly mutated in hepatocellular carcinoma. These results suggest that targeted sequencing and exome sequencing enable the identification of putative oncogenic driver mutations during the development of hepatocarcinoma.

Whole Genome Sequencing of Newly Established Pancreatic Cancer Lines Identifies Novel Somatic Mutation (c.2587G>A) in Axon Guidance Receptor Plexin A1 as Enhancer of Proliferation and Invasion.

The genetic profile of human pancreatic cancers harbors considerable heterogeneity, which suggests a possible explanation for the pronounced inefficacy of single therapies in this disease. This observation has led to a belief that custom therapies based on individual tumor profiles are necessary to more effectively treat pancreatic cancer. It has recently been discovered that axon guidance genes are affected by somatic structural variants in up to 25% of human pancreatic cancers. Thus far, however, some of these mutations have only been correlated to survival probability and no function has been assigned to these observed axon guidance gene mutations in pancreatic cancer. In this study we established three novel pancreatic cancer cell lines and performed whole genome sequencing to discover novel mutations in axon guidance genes that may contribute to the cancer phenotype of these cells. We discovered, among other novel somatic variants in axon guidance pathway genes, a novel mutation in the PLXNA1 receptor (c.2587G>A) in newly established cell line SB.06 that mediates oncogenic cues of increased invasion and proliferation in SB.06 cells and increased invasion in 293T cells upon stimulation with the receptor’s natural ligand semaphorin 3A compared to wild type PLXNA1 cells. Mutant PLXNA1 signaling was associated with increased Rho-GTPase and p42/p44 MAPK signaling activity and cytoskeletal expansion, but not changes in E-cadherin, vimentin, or metalloproteinase 9 expression levels. Pharmacologic inhibition of the Rho-GTPase family member CDC42 selectively abrogated PLXNA1 c.2587G>A-mediated increased invasion. These findings provide in-vitro confirmation that somatic mutations in axon guidance genes can provide oncogenic gain-of-function signals and may contribute to pancreatic cancer progression.

Abstract A48: Overexpression of Semaphorin-3E enhances pancreatic cancer cell proliferation ad is associated with patient poor survival

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death in the US with a 5-year survival rate of less than 6%. There is an urgent need for a better understanding of PDAC in order to find better therapeutic targets. Two recent large-scale genomic analyses of human PDAC have uncovered increased copy numbers of an axon-guidance gene SEMA3E that codes for Semaphorin-3E (Sema3E). Several recent reports have implicated Sema3E in metastasis of breast and colon cancers, particularly in promoting tumor cell migration and invasion, as well as inducing epithelial-to-mesenchymal transition (EMT). Here, we evaluated Sema3E expression levels in human pancreatic cancer tissue samples and correlation with patient survival; we further studied roles of Sema3E in pancreatic cancer cell proliferation. Materials and Methods: To determine expression levels of Sema3E in PDAC, qPCR analysis was performed on resected human PDAC samples and matched tissue controls, and immunohistochemical (IHC) analysis was performed on resected human PDAC tissues and tissues from a mouse model of PDAC. To generate Sema3E-overexpressing stable cells, human PDAC cell lines were transduced with Sema3E-expressing lentivirus or vector control. To generate Sema3E knock-down cell lines, CRISPR/Cas9 system was used to target Sema3E in the genome. MTT assay was performed on Sema3E-overexpressed cells to determine the effects of Sema3E on cell proliferation. Results: Using qPCR, Sema3E was found to be significantly upregulated in 46% of human PDAC samples (n=24), with an average fold-change of 9.54. IHC analyses of human and mouse PDAC revealed higher expression of Sema3E in tumor cells compared to normal cells (n=54). Interestingly, in human PDAC, Sema3E expression was concentrated in the nuclei of tumor cells while it has been reported to be a secreted protein. We found a positive correlation between high N/C ratio of Sema3E expression (i.e. expression in the nucleus relative to the cytoplasm) and poor survival. In addition, we also found overexpression of Sema3E is correlated with patient poor survival by analyzing a TCGA dataset (n=78). Overexpression of Sema3E in human PDAC cell lines enhanced cell proliferation and migration in vitro, while knocking-down Sema3E resulted in reduced cell proliferation. Conclusions: Our findings indicate that Sema3E is overexpressed in PDAC and is correlated with patient poor survival. Sema3E may play a pathogenic role in PDAC progression by enhancing tumor cell proliferation. These findings suggest that Sema3E could be an attractive novel therapeutic target for PDAC. Citation Format: Lin-Kin Yong, Syeling Lai, Zhengdong Liang, Dali Li, Ethan Poteet, William Fisher, Qianxin Mo, Changyi Chen, Qizhi Yao. Overexpression of Semaphorin-3E enhances pancreatic cancer cell proliferation ad is associated with patient poor survival. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A48.

Role of Tet1/3 Genes and Chromatin Remodeling Genes in Cerebellar Circuit Formation

Although mechanisms underlying early steps in cerebellar development are known, evidence is lacking on genetic and epigenetic changes during the establishment of the synaptic circuitry. Using metagene analysis, we report pivotal changes in multiple reactomes of epigenetic pathway genes in cerebellar granule cells (GCs) during circuit formation. During this stage, Tet genes are upregulated and vitamin C activation of Tet enzymes increases the levels of 5-hydroxymethylcytosine (5hmC) at exon start sites of upregulated genes, notably axon guidance genes and ion channel genes. Knockdown of Tet1 and Tet3 by RNAi in exvivo cerebellar slice cultures inhibits dendritic arborization of developing GCs, a critical step in circuit formation. These findings demonstrate a role for Tet genes and chromatin remodeling genes in the formation of cerebellar circuitry.

Abstract 3574: Novel roles of an axon-guidance molecule, semaphorin 3E, in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death in the US with a 5-year survival rate of less than 6%. There is an urgent need for a better understanding of PDAC in order to find better therapeutic targets. Two recent large-scale genomic analyses of human PDAC have uncovered increased copy numbers of an axon-guidance gene SEMA3E that codes for Semaphorin-3E (Sema3E). Several recent reports have implicated Sema3E in metastasis of breast and colon cancers, particularly in promoting tumor cell migration and invasion, as well as inducing epithelial-to-mesenchymal transition (EMT). We hypothesize a possible new role of an axon-guidance gene Sema3E in PDAC progression by promoting cell proliferation, invasion, and metastasis. Materials and Methods: To determine expression levels of Sema3E in human PDAC, qRT-PCR and IHC analysis was performed on resected PDAC samples and matched tissue controls. To determine Sema3E levels in mouse PDAC, IHC analysis was performed on pancreas tissue from a genetically engineered spontaneous PDAC mouse model, the PDX-1-Cre; KrasG12D; P53R172H (KPC) mouse. To generate Sema3E-overexpressing stable cells, human PDAC cell lines Mia PaCa-2 and Panc-48 cells were transduced with Sema3E-expressing lentivirus or vector control. QRT-PCR and western blot analyses were performed to verify the overexpression of Sema3E. Colony formation assay (CFA) and MTT assay were performed on Sema3E-transduced cells to determine the effects of Sema3E on cell survival and proliferation respectively, while a wound healing assay and boyden chamber assay were performed to determine effects of Sema3E on cell migration and invasion respectively. Results: Sema3E was found to be significantly upregulated in 46% of patient PDAC samples (n = 24), with an average fold-change of 9.54 across the samples. IHC analyses of patient and mouse PDAC revealed higher expression of Sema3E in tumor cells compared to normal cells. Interestingly, Sema3E expression was concentrated in the nuclei of tumor cells while it has been reported in current literature to be a secreted protein found in normal tissues. Sema3E-transduced PDAC cell lines Mia PaCa-2-Sema3E and Panc-48-Sema3E had higher Sema3E expression in cell lysates and culture supernatant compared to control cells. Mia PaCa-2-Sema3E had a 60.4% higher rate of colony formation than the control (p = 0.005), while both Mia PaCa-2-Sema3E and Panc-48-Sema3E had higher proliferation rates (58.8%; p<0.0001 and 31.0%; p<0.0001 respectively) compared to controls. Interestingly, both Sema3E-transduced cells did not have a significant difference in migration and invasion compared to controls. Conclusions: Our findings indicate that Sema3E is overexpressed in PDAC and it may play some pathogenic roles in PDAC progression by enhancing tumor cell proliferation. These findings indicate that Sema3E could be an attractive novel therapeutic target in PDAC treatment strategies. Citation Format: Lin-Kin Yong, Dali Li, Ethan Poteet, Zhengdong Liang, William Fisher, Changyi Chen, Qizhi Cathy Yao. Novel roles of an axon-guidance molecule, semaphorin 3E, in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3574. doi:10.1158/1538-7445.AM2015-3574

Binocular vision, the optic chiasm, and their associations with vertebrate motor behavior

Ipsilateral retinal projections (IRP) in the optic chiasm (OC) vary considerably. Most animal groups possess laterally situated eyes and no or few IRP, but, e.g. cats and primates have frontal eyes and high proportions of IRP. The traditional hypothesis that bifocal vision developed to enable predation or to increase perception in restricted light conditions applies mainly to mammals. The eye-forelimb (EF) hypothesis presented here suggests that the reception of visual feedback of limb movements in the limb steering cerebral hemisphere was the fundamental mechanism behind the OC evolution. In other words, that evolutionary change in the OC was necessary to preserve hemispheric autonomy. In the majority of vertebrates, motor processing, tactile, proprioceptive, and visual information involved in steering the hand (limb, paw, fin) is primarily received only in the contralateral hemisphere, while multisensory information from the ipsilateral limb is minimal. Since the involved motor nuclei, somatosensory areas, and vision neurons are situated in same hemisphere, the neuronal pathways involved will be relatively short, optimizing the size of the brain. That would not have been possible without, evolutionary modifications of IRP. Multiple axon-guidance genes, which determine whether axons will cross the midline or not, have shaped the OC anatomy. Evolutionary change in the OC seems to be key to preserving hemispheric autonomy when the body and eye evolve to fit new ecological niches. The EF hypothesis may explain the low proportion of IRP in birds, reptiles, and most fishes; the relatively high proportions of IRP in limbless vertebrates; high proportions of IRP in arboreal, in contrast to ground-dwelling, marsupials; the lack of IRP in dolphins; abundant IRP in primates and most predatory mammals, and why IRP emanate exclusively from the temporal retina. The EF hypothesis seams applicable to vertebrates in general and hence more parsimonious than traditional hypotheses.

Convergent differential regulation of SLIT-ROBO axon guidance genes in the brains of vocal learners.

Only a few distantly related mammals and birds have the trait of complex vocal learning, which is the ability to imitate novel sounds. This ability is critical for speech acquisition and production in humans, and is attributed to specialized forebrain vocal control circuits that have several unique connections relative to adjacent brain circuits. As a result, it has been hypothesized that there could exist convergent changes in genes involved in neural connectivity of vocal learning circuits. In support of this hypothesis, expanding on our related study (Pfenning et al. [2014] Science 346: 1256846), here we show that the forebrain part of this circuit that makes a relatively rare direct connection to brainstem vocal motor neurons in independent lineages of vocal learning birds (songbird, parrot, and hummingbird) has specialized regulation of axon guidance genes from the SLIT–ROBO molecular pathway. The SLIT1 ligand was differentially downregulated in the motor song output nucleus that makes the direct projection, whereas its receptor ROBO1 was developmentally upregulated during critical periods for vocal learning. Vocal nonlearning bird species and male mice, which have much more limited vocal plasticity and associated circuits, did not show comparable specialized regulation of SLIT–ROBO genes in their nonvocal motor cortical regions. These findings are consistent with SLIT and ROBO gene dysfunctions associated with autism, dyslexia, and speech sound language disorders and suggest that convergent evolution of vocal learning was associated with convergent changes in the SLIT–ROBO axon guidance pathway. J. Comp. Neurol. 523:892–906, 2015. © 2014 Wiley Periodicals, Inc.

Molecular Mechanisms Regulating the Defects in Fragile X Syndrome Neurons Derived from Human Pluripotent Stem Cells

SummaryFragile X syndrome (FXS) is caused by the absence of the fragile X mental retardation protein (FMRP). We have previously generated FXS-induced pluripotent stem cells (iPSCs) from patients’ fibroblasts. In this study, we aimed at unraveling the molecular phenotype of the disease. Our data revealed aberrant regulation of neural differentiation and axon guidance genes in FXS-derived neurons, which are regulated by the RE-1 silencing transcription factor (REST). Moreover, we found REST to be elevated in FXS-derived neurons. As FMRP is involved in the microRNA (miRNA) pathway, we employed miRNA-array analyses and uncovered several miRNAs dysregulated in FXS-derived neurons. We found hsa-mir-382 to be downregulated in FXS-derived neurons, and introduction of mimic-mir-382 into these neurons was sufficient to repress REST and upregulate its axon guidance target genes. Our data link FMRP and REST through the miRNA pathway and show a new aspect in the development of FXS.