Miswiring the brain: Δ9‐tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin‐2 degradation pathway

Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly Δ⁹‐tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptor‐driven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB₁ cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC‐sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin‐2, a microtubule‐binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis‐exposed fetuses, defining SCG10 as the first cannabis‐driven molecular effector in the developing cerebrum. CB₁ cannabinoid receptor activation recruits c‐Jun N‐terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring.     

Distinct roles for Robo2 in the regulation of axon and dendrite growth by retinal ganglion cells

Guidance factors act on the tip of a growing axon to direct it to its target. What role these molecules play, however, in the control of the dendrites that extend from that axon’s cell body is poorly understood. Slits, through their Robo receptors, guide many types of axons, including those of retinal ganglion cells (RGCs). Here we assess and contrast the role of Slit/Robo signalling in the growth and guidance of the axon and dendrites extended by RGCs in Xenopus laevis. As Xenopus RGCs extend dendrites, they express robo2 and robo3, while slit1 and slit2 are expressed in RGCs and in the adjacent inner nuclear layer. Interestingly, our functional data with antisense knockdown and dominant negative forms of Robo2 (dnRobo2) and Robo3 (dnRobo3) indicate that Slit/Robo signalling has no role in RGC dendrite guidance, and instead is necessary to stimulate dendrite branching, primarily via Robo2. Our in vitro culture data argue that Slits are the ligands involved. In contrast, both dnRobo2 and dnRobo3 inhibited the extension of axons and caused the misrouting of some axons. Based on these data, we propose that Robo signalling can have distinct functions in the axon and dendrites of the same cell, and that the specific combinations of Robo receptors could underlie these differences. Slit acts via Robo2 in dendrites as a branching/growth factor but not in guidance, while Robo2 and Robo3 function in concert in axons to mediate axonal interactions and respond to Slits as guidance factors. These data underscore the likelihood that a limited number of extrinsic factors regulate the distinct morphologies of axons and dendrites.     

Homoeologous chromosomes of Xenopus laevis are highly conserved after whole-genome duplication

It has been suggested that whole-genome duplication (WGD) occurred twice during the evolutionary process of vertebrates around 450 and 500 million years ago, which contributed to an increase in the genomic and phenotypic complexities of vertebrates. However, little is still known about the evolutionary process of homoeologous chromosomes after WGD because many duplicate genes have been lost. Therefore, Xenopus laevis (2n=36) and Xenopus (Silurana) tropicalis (2n=20) are good animal models for studying the process of genomic and chromosomal reorganization after WGD because X. laevis is an allotetraploid species that resulted from WGD after the interspecific hybridization of diploid species closely related to X. tropicalis. We constructed a comparative cytogenetic map of X. laevis using 60 complimentary DNA clones that covered the entire chromosomal regions of 10 pairs of X. tropicalis chromosomes. We consequently identified all nine homoeologous chromosome groups of X. laevis. Hybridization signals on two pairs of X. laevis homoeologous chromosomes were detected for 50 of 60 (83%) genes, and the genetic linkage is highly conserved between X. tropicalis and X. laevis chromosomes except for one fusion and one inversion and also between X. laevis homoeologous chromosomes except for two inversions. These results indicate that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in this lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD.     

Can the introduction of Xenopus laevis affect native amphibian populations? Reduction of reproductive occurrence in presence of the invasive species

Biological invasions are regarded as a form of global change and potential cause of biodiversity loss. Xenopus laevis is an anuran amphibian native to sub-Saharan Africa with strong invasive capacity, especially in geographic regions with a Mediterranean climate. In spite of the worldwide diffusion of X. laevis, the effective impact on local ecosystems and native amphibian populations is poorly quantified. A large population of X. laevis occurs in Sicily and our main aim of this work was to assess the consequences of introduction of this alien species on local amphibian populations. In this study we compare the occurrence of reproduction of native amphibians in ponds with and without X. laevis, and before and after the alien colonization. The results of our study shows that, when X. laevis establishes a conspicuous population in a pond system, the populations of Discoglossus pictus, Hyla intermedia and Pelophylax synklepton esculentus show clear signs of distress and the occurrence of reproduction of these native amphibians collapses. In contrast, the populations of Bufo bufo do not appear to be affected by the alien species. Since the Sicilian population of X. laevis shows a strong dispersal capacity, proportionate and quick interventions become necessary to bound the detriment to the Sicilian amphibians populations.     

Retinal stem/progenitor cells in the ciliary marginal zone complete retinal regeneration: A study of retinal regeneration in a novel animal model

Our research group has extensively studied retinal regeneration in adult Xenopus laevis. However, X. laevis does not represent a suitable model for multigenerational genetics and genomic approaches. Instead, Xenopus tropicalis is considered as the ideal model for these studies, although little is known about retinal regeneration in X. tropicalis. In the present study, we showed that a complete retina regenerates at approximately 30 days after whole retinal removal. The regenerating retina was derived from the stem/progenitor cells in the ciliary marginal zone (CMZ), indicating a novel mode of vertebrate retinal regeneration, which has not been previously reported. In a previous study, we showed that in X. laevis, retinal regeneration occurs primarily through the transdifferentiation of retinal pigmented epithelial (RPE) cells. RPE cells migrate to the retinal vascular membrane and reform a new epithelium, which then differentiates into the retina. In X. tropicalis, RPE cells also migrated to the vascular membrane, but transdifferentiation was not evident. Using two tissue culture models of RPE tissues, it was shown that in X. laevis RPE culture neuronal differentiation and reconstruction of the retinal three‐dimensional (3‐D) structure were clearly observed, while in X. tropicalis RPE culture neither ßIII tubulin‐positive cells nor 3‐D retinal structure were seen. These results indicate that the two Xenopus species are excellent models to clarify the cellular and molecular mechanisms of retinal regeneration, as these animals have contrasting modes of regeneration; one mode primarily involves RPE cells and the other mode involves stem/progenitor cells in the CMZ. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 739–756, 2014     

TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts

The spindle segregates chromosomes in dividing eukaryotic cells, and its assembly pathway and morphology vary across organisms and cell types. We investigated mechanisms underlying differences between meiotic spindles formed in egg extracts of two frog species. Small Xenopus tropicalis spindles resisted inhibition of two factors essential for assembly of the larger Xenopus laevis spindles: RanGTP, which functions in chromatin-driven spindle assembly, and the kinesin-5 motor Eg5, which drives antiparallel microtubule (MT) sliding. This suggested a role for the MT-associated protein TPX2 (targeting factor for Xenopus kinesin-like protein 2), which is regulated by Ran and binds Eg5. Indeed, TPX2 was threefold more abundant in X. tropicalis extracts, and elevated TPX2 levels in X. laevis extracts reduced spindle length and sensitivity to Ran and Eg5 inhibition. Higher TPX2 levels recruited Eg5 to the poles, where MT density increased. We propose that TPX2 levels modulate spindle architecture through Eg5, partitioning MTs between a tiled, antiparallel array that promotes spindle expansion and a cross-linked, parallel architecture that concentrates MTs at spindle poles.     

Dynamic responses of Xenopus retinal ganglion cell axon growth cones to netrin-1 as they innervate their in vivo target

Netrin-1 influences retinal ganglion cell (RGC) axon pathfinding and also participates in the branching and synaptic differentiation of mature RGC axons at their target. To investigate whether netrin also serves as an early target recognition signal in the brain, we examined the dynamic behavior of Xenopus RGC axons soon after they innervate the optic tectum. Time-lapse confocal microscopy imaging of RGC axons expressing enhanced yellow fluorescent protein demonstrated that netrin-1 is involved in early axon branching, as recombinant netrin-1 halted further advancement of growth cones into the tectum and induced back branching. RGC growth cones exhibited differential responses to netrin-1 that depended on the degree of differentiation of the axon and the developmental stage of the tadpole. Netrin-1 decreased the total number of branches on newly arrived RGC growth cones at the target, but increased the dynamic branching of more mature arbors at the later developmental stage. To further explore the response of axonal growth cones to netrin, Xenopus RGC axons were followed in culture by time-lapse imaging. Exposure to netrin-1 rapidly increased the forward advancement of the axon and decreased the size and expanse of the growth cone, while also inducing back branching. Taken together, the differential in vivo and in vitro responses to netrin-1 suggest that netrin alone is not sufficient to induce the cessation of growth cone advancement in the absence of a target but can independently modulate axon branching. Collectively, our findings reveal a novel role for netrin on RGC axon branch initiation as growth cones innervate their target.     

Unusual evolutionary conservation and further species-specific adaptations of a large family of nonclassical MHC class Ib genes across different degrees of genome ploidy in the amphibian subfamily Xenopodinae

Nonclassical MHC class Ib (class Ib) genes are a family of highly diverse and rapidly evolving genes wherein gene numbers, organization, and expression markedly differ even among closely related species rendering class Ib phylogeny difficult to establish. Whereas among mammals there are few unambiguous class Ib gene orthologs, different amphibian species belonging to the anuran subfamily Xenopodinae exhibit an unusually high degree of conservation among multiple class Ib gene lineages. Comparative genomic analysis of class Ib gene loci of two divergent (~65 million years) Xenopodinae subfamily members Xenopus laevis (allotetraploid) and Xenopus tropicalis (diploid) shows that both species possess a large cluster of class Ib genes denoted as Xenopus/Silurana nonclassical (XNC/SNC). Our study reveals two distinct phylogenetic patterns among these genes: some gene lineages display a high degree of flexibility, as demonstrated by species-specific expansion and contractions, whereas other class Ib gene lineages have been maintained as monogenic subfamilies with very few changes in their nucleotide sequence across divergent species. In this second category, we further investigated the XNC/SNC10 gene lineage that in X. laevis is required for the development of a distinct semi-invariant T cell population. We report compelling evidence of the remarkable high degree of conservation of this gene lineage that is present in all 12 species of the Xenopodinae examined, including species with different degrees of ploidy ranging from 2, 4, 8 to 12 N. This suggests that the critical role of XNC10 during early T cell development is conserved in amphibians.     

Connecting the Retina to the Brain

The visual system is beautifully crafted to transmit information of the external world to visual processing and cognitive centers in the brain. For visual information to be relayed to the brain, a series of axon pathfinding events must take place to ensure that the axons of retinal ganglion cells, the only neuronal cell type in the retina that sends axons out of the retina, find their way out of the eye to connect with targets in the brain. In the past few decades, the power of molecular and genetic tools, including the generation of genetically manipulated mouse lines, have multiplied our knowledge about the molecular mechanisms involved in the sculpting of the visual system. Here, we review major advances in our understanding of the mechanisms controlling the differentiation of RGCs, guidance of their axons from the retina to the primary visual centers, and the refinement processes essential for the establishment of topographic maps and eye-specific axon segregation. Human disorders, such as albinism and achiasmia, that impair RGC axon growth and guidance and, thus, the establishment of a fully functioning visual system will also be discussed.     

miR‐181a/b control the assembly of visual circuitry by regulating retinal axon specification and growth

Connectivity and function of neuronal circuitry require the correct specification and growth of axons and dendrites. Here, we identify the microRNAs miR‐181a and miR‐181b as key regulators of retinal axon specification and growth. Loss of miR‐181a/b in medaka fish (Oryzias latipes) failed to consolidate amacrine cell processes into axons and delayed the growth of retinal ganglion cell (RGC) axons. These alterations were accompanied by defects in visual connectivity and function. We demonstrated that miR‐181a/b exert these actions through negative modulation of MAPK/ERK signaling that in turn leads to RhoA reduction and proper neuritogenesis in both amacrine cells and RGCs via local cytoskeletal rearrangement. Our results identify a new pathway for axon specification and growth unraveling a crucial role of miR‐181a/b in the proper establishment of visual system connectivity and function. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1252–1267, 2015     

The Xenopus FcR family demonstrates continually high diversification of paired receptors in vertebrate evolution

Background  

Recent studies have revealed an unexpected diversity of domain architecture among FcR-like receptors that presumably fulfill regulatory functions in the immune system. Different species of mammals, as well as chicken and catfish have been found to possess strikingly different sets of these receptors. To better understand the evolutionary history of paired receptors, we extended the study of FcR-like genes in amphibian representatives Xenopus tropicalis and Xenopus laevis.     

Matrix metalloproteinases are required for retinal ganglion cell axon guidance at select decision points

Axons receive guidance information from extrinsic cues in their environment in order to reach their targets. In the frog Xenopus laevis, retinal ganglion cell (RGC) axons make three key guidance decisions en route through the brain. First, they cross to the contralateral side of the brain at the optic chiasm. Second, they turn caudally in the mid-diencephalon. Finally, they must recognize the optic tectum as their target. The matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) families are zinc (Zn)-dependent proteolytic enzymes. The latter functions in axon guidance, but a similar role has not yet been identified for the MMP family. Our previous work implicated metalloproteinases in the guidance decisions made by Xenopus RGC axons. To test specifically the importance of MMPs, we used two different in vivo exposed brain preparations in which RGC axons were exposed to an MMP-specific pharmacological inhibitor (SB-3CT), either as they reached the optic chiasm or as they extended through the diencephalon en route to the optic tectum. Interestingly, SB-3CT affected only two of the guidance decisions, with misrouting defects at the optic chiasm and tectum. Only at higher concentrations was RGC axon extension also impaired. These data implicate MMPs in the guidance of vertebrate axons, and suggest that different metalloproteinases function to regulate axon behaviour at distinct choice points: an MMP is important in guidance at the optic chiasm and the target, while either a different MMP or an ADAM is required for axons to make the turn in the mid-diencephalon.     

Transposable elements in fish functional genomics: technical challenges and perspectives

The study of amphibian embryogenesis has provided important insight into the mechanisms of vertebrate development. The frog Xenopus laevis has been an important model of vertebrate cell biology and development for many decades. Genetic studies in this organism are not practical because of the tetraploid nature of the genome and the long generation time of this species. Recently, a closely related frog, namely Xenopus tropicalis, has been proposed as an alternative system; it shares all of the physical characteristics that make X. laevis a useful model but has the advantage of a diploid genome and short generation time. The rapid accumulation of genetic resources for this animal and the success of pilot mutagenesis screens have helped propel this model system forward. Transposable elements will provide invaluable tools for manipulating the frog genome. These integration systems are ideally suited to transgenesis and insertional mutagenesis strategies in the frog. The high fecundity of the frog combined with the ability to remobilize transposon transgenes integrated into frog genome will allow large-scale insertional mutagenesis screens to be performed in laboratories with modest husbandry capacities.

     

Tenascin-R and axon growth-promoting molecules are up-regulated in the regenerating visual pathway of the lizard (Gallotia galloti)

It is currently unclear whether retinal ganglion cell (RGC) axon regeneration depends on down-regulation of axon growth-inhibitory proteins, and to what extent outgrowth-promoting substrates contribute to RGC axon regeneration in reptiles. We performed an immunohistochemical study of the regulation of the axon growth-inhibiting extracellular matrix molecules tenascin-R and chondroitin sulphate proteoglycan (CSPG), the axon outgrowth-promoting extracellular matrix proteins fibronectin and laminin, and the axonal tenascin-R receptor protein F3/contactin during RGC axon regeneration in the lizard, Gallotia galloti. Tenascin-R and CSPG were expressed in an extracellular matrix-, oligodendrocyte/myelin- and neuron-associated pattern and up-regulated in the regenerating optic pathway. The expression pattern of tenascin-R was not indicative of a role in channeling or restriction of re-growing RGC axons. Up-regulation of fibronectin, laminin, and F3/contactin occurred in spatiotemporal patterns corresponding to tenascin-R expression. Moreover, we analyzed the influence of substrates containing tenascin-R, fibronectin, and laminin on outgrowth of regenerating lizard RGC axons. In vitro regeneration of RGC axons was not inhibited by tenascin-R, and further improved on mixed substrates containing tenascin-R together with fibronectin or laminin. These results indicate that RGC axon regeneration in Gallotia galloti does not require down-regulation of tenascin-R or CSPG. Presence of tenascin-R is insufficient to prevent RGC axon growth, and concomitant up-regulation of axon growth-promoting molecules like fibronectin and laminin may override the effects of neurite growth inhibitors on RGC axon regeneration. Up-regulation of contactin in RGCs suggests that tenascin-R may have an instructive function during axon regeneration in the lizard optic pathway.     

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.     

Perturbations of MicroRNA Function in Mouse Dicer Mutants Produce Retinal Defects and Lead to Aberrant Axon Pathfinding at the Optic Chiasm

Background

During development axons encounter a variety of choice points where they have to make appropriate pathfinding decisions. The optic chiasm is a major decision point for retinal ganglion cell (RGC) axons en route to their target in order to ensure the correct wiring of the visual system. MicroRNAs (miRNAs) belong to the class of small non-coding RNA molecules and have been identified as important regulators of a variety of processes during embryonic development. However, their involvement in axon guidance decisions is less clear.

Methodology/Principal Findings

We report here that the early loss of Dicer, an essential protein for the maturation of miRNAs, in all cells of the forming retina and optic chiasm leads to severe phenotypes of RGC axon pathfinding at the midline. Using a conditional deletion approach in mice, we find in homozygous Dicer mutants a marked increase of ipsilateral projections, RGC axons extending outside the optic chiasm, the formation of a secondary optic tract and a substantial number of RGC axons projecting aberrantly into the contralateral eye. In addition, the mutant mice display a microphthalmia phenotype.

Conclusions

Our work demonstrates an important role of Dicer controlling the extension of RGC axons to the brain proper. It indicates that miRNAs are essential regulatory elements for mechanisms that ensure correct axon guidance decisions at the midline and thus have a central function in the establishment of circuitry during the development of the nervous system.