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Background: High nerve density in tumors and metastasis via nerves (perineural invasion—PNI) have been reported extensively in solid tumors throughout the body including pancreatic, head and neck, gastric, prostate, breast, and colorectal cancers. Ablation of tumor nerves results in improved disease outcomes, suggesting that blocking nerve–tumor communication could be a novel treatment strategy. However, the molecular mechanisms underlying this remain poorly understood. Thus, the aim here was to identify molecular pathways underlying nerve–tumor crosstalk and to determine common molecular features between PNI-associated cancers.

Results: Analysis of head and neck (HNSCC), pancreatic, and gastric (STAD) cancer Gene Expression Omnibus datasets was used to identify differentially expressed genes (DEGs). This revealed extracellular matrix components as highly dysregulated. To enrich for pathways associated with PNI, genes previously correlated with PNI in STAD and in 2 HNSCC studies where tumor samples were segregated by PNI status were analyzed. Neurodevelopmental genes were found to be enriched with PNI. In datasets where tumor samples were not segregated by PNI, neurodevelopmental pathways accounted for 12%–16% of the DEGs. Further dysregulation of axon guidance genes was common to all cancers analyzed. By examining paralog genes, a clear pattern emerged where at least one family member from several axon guidance pathways was affected in all cancers examined. Overall 17 different axon guidance gene families were disrupted, including the ephrin–Eph, semaphorin–neuropilin/plexin, and slit–robo pathways. These findings were validated using The Cancer Genome Atlas and cross-referenced to other cancers with a high incidence of PNI including colon, cholangiocarcinoma, prostate, and breast cancers. Survival analysis revealed that the expression levels of neurodevelopmental gene families impacted disease survival.

Conclusion: These data highlight the importance of the tumor as a source of signals for neural tropism and neural plasticity as a common feature of cancer. The analysis supports the hypothesis that dysregulation of neurodevelopmental programs is a common feature associated with PNI. Furthermore, the data suggested that different cancers may have evolved to employ alternative genetic strategies to disrupt the same pathways. Overall, these findings provide potential druggable targets for novel therapies of cancer management and provide multi-cancer molecular biomarkers.

Background: Alternative splicing provides a broad strategy to amplify the genome. Yet how alternative splicing influences neurodevelopment or indeed which variants are translated at developmental choice points remains poorly explored. Here we focused on a gene important for neurodevelopment, the Lim homeodomain transcription factor, Lhx9. Lhx9 has two noncanonical splice variants, Lhx9a and Lhx9b which compared with the canonical variant Lhx9c have a truncated homeodomain and an alternative C-terminal sequence, suggesting that, if translated, these variants could differently impact on cellular function.

Results: We created a unique antibody tool designed to selectively detect noncanonical Lhx9 variants (Lhx9ab) and used this to examine the protein expression dynamics in embryos. Lhx9ab variants were translated and dynamically expressed similarly between mouse and chicken at key developmental choice points in the spinal cord, limbs and urogenital ridge. Within the spinal cord, enrichment of Lhx9c vs Lhx9ab expression was observed during key migration and axonal projection choice points.

Conclusions: These data support the notion that the expression dynamics between canonical and noncanonical Lhx9 variants could play an important role in spinal neuron maturation. More broadly, determining the temporal dynamics of alternative protein variants is a key entry point to understand how splicing influences developmental processes.

Sensory information relayed to the brain is dependent on complex, yet precise spatial organization of neurons. This anatomical complexity is generated during development from a surprisingly small number of neural stem cell domains. This raises the question of how neurons derived from a common precursor domain respond uniquely to their environment to elaborate correct spatial organization and connectivity. We addressed this question by exploiting genetically labeled mouse embryonic dorsal interneuron 1 (dI1) neurons that are derived from a common precursor domain and give rise to spinal projection neurons with distinct organization of cell bodies with axons projecting either commissurally (dI1c) or ipsilaterally (dI1i). In this study, we examined how the guidance receptor, Robo2, which is a canonical Robo receptor, influenced dI1 guidance during embryonic development. Robo2 was enriched in embryonic dI1i neurons, and loss of Robo2 resulted in misguidance of dI1i axons, whereas dI1c axons remained unperturbed within the mantle zone and ventral commissure. Further, Robo2 profoundly influenced dI1 cell body migration, a feature that was partly dependent on Slit2 signaling. These data suggest that dI1 neurons are dependent on Robo2 for their organization. This work integrated with the field support of a model whereby canonical Robo2 vs. non-canonical Robo3 receptor expression facilitates projection neurons derived from a common precursor domain to read out the tissue environment uniquely giving rise to correct anatomical organization.

Understanding the loss of conserved genes is critical for determining how phenotypic diversity is generated. Here we focus on the evolution of DCC, a gene that encodes a highly conserved neural guidance receptor. Disruption of DCC in animal models and humans results in major neurodevelopmental defects including commissural axon defects. Here we examine DCC evolution in birds, which is of particular interest as a major model system in neurodevelopmental research. We found the DCC containing locus was disrupted several times during evolution, resulting in both gene losses and faster evolution rate of salvaged genes. These data suggest that DCC had been lost independently twice during bird evolution, including in chicken and zebra finch, whereas it was preserved in many other closely related bird species, including ducks. Strikingly, we observed that commissural axon trajectory appeared similar regardless of whether DCC could be detected or not. We conclude that the DCC locus is susceptible to genomic instability leading to independent disruptions in different branches of birds and a significant influence on evolution rate. Overall, the phenomenon of loss or molecular evolution of a highly conserved gene without apparent phenotype change is of conceptual importance for understanding molecular evolution of key biological processes.

Unravelling how neurons are guided during vertebrate embryonic development has wide implications for understanding the assembly of the nervous system. During embryogenesis, migration of neuronal cell bodies and axons occurs simultaneously, but to what degree they influence each other's development remains obscure. We show here that within the mouse embryonic spinal cord, commissural axons bisect, delimit or preconfigure ventral interneuron cell body position. Furthermore, genetic disruption of commissural axons results in abnormal ventral interneuron cell body positioning. These data suggest that commissural axonal fascicles instruct cell body position by acting either as border landmarks (axon-restricted migration), which to our knowledge has not been previously addressed, or acting as cellular guides. This study in the developing spinal cord highlights an important function for the interaction of cell bodies and axons, and provides a conceptual proof of principle that is likely to have overarching implications for the development of neuronal architecture.

The establishment of anatomically stereotyped axonal projections is fundamental to neuronal function. While most neurons project their axons within the central nervous system (CNS), only axons of centrally born motoneurons and peripherally born sensory neurons link the CNS and peripheral nervous system (PNS) together by navigating through specialized CNS/PNS transition zones. Such selective restriction is of importance because inappropriate CNS axonal exit could lead to loss of correct connectivity and also to gain of erroneous functions. However, to date, surprisingly little is known about the molecular-genetic mechanisms that regulate how central axons are confined within the CNS during development. Here, we show that netrin 1/Dcc/Unc5 chemotropism contributes to axonal confinement within the CNS. In both Ntn1 and Dcc mutant mouse embryos, some spinal interneuronal axons exit the CNS by traversing the CNS/PNS transition zones normally reserved for motor and sensory axons. We provide evidence that netrin 1 signalling preserves CNS/PNS axonal integrity in three ways: (1) netrin 1/Dcc ventral attraction diverts axons away from potential exit points; (2) a Dcc/Unc5c-dependent netrin 1 chemoinhibitory barrier in the dorsolateral spinal cord prevents interneurons from being close to the dorsal CNS/PNS transition zone; and (3) a netrin 1/Dcc-dependent, Unc5c-independent mechanism that actively prevents exit from the CNS. Together, these findings provide insights into the molecular mechanisms that maintain CNS/PNS integrity and, to the best of our knowledge, present the first evidence that chemotropic signalling regulates interneuronal CNS axonal confinement in vertebrates.

Background: Neuronal circuit assembly comprises a number of developmental processes that ultimately underlie function. Identifying the molecular events that dictate these processes can give key insights into how neuronal circuit formation is coordinated. To begin to identify such molecular mechanisms, we have analysed the expression of a candidate gene of entirely unknown function within the nervous system. Here we reveal the spatial and temporal distribution of Lzts1 in mouse and chick embryonic spinal cord and propose potential biological functions. Results: Lzts1 mRNA is transiently expressed at the border of the ventricular and mantle zones in subsets of sensory and motor spinal neurons. The protein is localized to the cell body, axon, and trailing process of motor, commissural, and dorsal root neurons during development. Conclusions: Taken together, the spatial and temporal distribution of Lzts1 is consistent with a potential function(s) in cell cycle regulation, axon growth or guidance, and/or migration of neurons. Developmental Dynamics 241:984994, 2012. (c) 2012 Wiley Periodicals, Inc.

Despite increasing evidence for transcriptional control of neural connectivity, how transcription factors regulate discrete steps in axon guidance remains obscure. Projection neurons in the dorsal spinal cord relay sensory signals to higher brain centers. Some projection neurons send their axons ipsilaterally, whereas others, commissural neurons, send axons contralaterally. We show that two closely related LIM homeodomain proteins, Lhx2 and Lhx9, are expressed by a set of commissural relay neurons (dI1c neurons) and are required for the dI1c axon projection. Midline crossing by dI1c axons is lost in Lhx2/9 double mutants, a defect that results from loss of expression of Rig-1 from dI1c axons. Lhx2 binds to a conserved motif in the Rig-1 gene, suggesting that Lhx2/9 regulate directly the expression of Rig-1. Our findings reveal a link between the transcriptional programs that define neuronal subtype identity and the expression of receptors that guide distinctive aspects of their trajectory.

Neural induction constitutes the initial step in the generation of the vertebrate nervous system. In attempting to understand the principles that underlie this process, two key issues need to be resolved. When is neural induction initiated, and what is the cellular source and molecular nature of the neural inducing signal(s)? Currently, these aspects of neural induction seem to be very different in amphibian and amniote embryos. Here we highlight the similarities and the differences, and we propose a possible unifying mechanism.