Developmental and Injury-induced Changes in DNA Methylation in Regenerative versus Non-regenerative Regions of the Vertebrate Central Nervous System.

Background: Because some of its CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.

Neurophysiological and Behavioral Analysis in .

Because of its resilience to hypoxia and trauma, the frog has long been a favored preparation of neurophysiologists. Its use has led to the discovery of many fundamental properties of neurons and neural circuits. Neurophysiologists were originally attracted to embryos, tadpoles, and frogs because of their ready availability, their external development, and the anatomical accessibility and relatively simple neural circuitry of the visual, locomotory, and vocalization systems.

Comparative gene expression profiling between optic nerve and spinal cord injury in Xenopus laevis reveals a core set of genes inherent in successful regeneration of vertebrate central nervous system axons.

Background: The South African claw-toed frog, Xenopus laevis, is uniquely suited for studying differences between regenerative and non-regenerative responses to CNS injury within the same organism, because some CNS neurons (e.g., retinal ganglion cells after optic nerve crush (ONC)) regenerate axons throughout life, whereas others (e.

Comparisons of SOCS mRNA and protein levels in Xenopus provide insights into optic nerve regenerative success.

In vertebrates from fishes to mammals, optic nerve injury induces increased expression ofSuppressor of Cytokine Signaling 3(SOCS3) mRNA, a modulator of cytokine signaling that is known to inhibit CNS axon regeneration. Unlike amniotes, however, anamniotes successfully regenerate optic axons, despite this increase. To address this seeming paradox, we examined the SOCS3 response to optic nerve injury in the frog,Xenopus laevis, at both the mRNA and protein levels.

Tracing Central Nervous System Axon Regeneration in .

Axonal tracing allows visualizing connectivity between neurons, providing useful information about structure, neuronal location, and function of the nervous system. Identifying regenerating axons and their neuron cell bodies present the particular challenges of labeling the projections of interest while unambiguously demonstrating regrowth of those axons that have been damaged. In the developing brain, an additional labeling challenge arises, as new connections are being made throughout the duration of an experiment.

A novel role for the nuclear localization signal in regulating hnRNP K protein stability in vivo.

hnRNP K is a highly conserved nucleocytoplasmic shuttling protein, which associates with RNAs through synergistic binding via its three KH domains. hnRNP K is required for proper nuclear export and translational control of its mRNA targets, and these processes are controlled by hnRNP K's movement between subcellular compartments. Whereas the nuclear export and localization of hnRNP K that is associated with mRNP complexes has been well studied, the trafficking of hnRNP K that is unbound to mRNA has yet to be elucidated.

Post-transcriptional regulation mediated by specific neurofilament introns in vivo.

Neurons regulate genes post-transcriptionally to coordinate the supply of cytoskeletal proteins, such as the medium neurofilament (NEFM), with demand for structural materials in response to extracellular cues encountered by developing axons. By using a method for evaluating functionality of cis-regulatory gene elements in vivo through plasmid injection into Xenopus embryos, we discovered that splicing of a specific nefm intron was required for robust transgene expression, regardless of promoter or cell type. Transgenes utilizing the nefm 3'-UTR but substituting other nefm introns expressed little or no protein owing to defects in handling of the messenger (m)RNA as opposed to transcription or splicing.

Using Xenopus Embryos to Study Transcriptional and Posttranscriptional Gene Regulatory Mechanisms of Intermediate Filaments.

Intermediate filament genes exhibit highly regulated, tissue-specific patterns of expression during development and in response to injury. Identifying the responsible cis-regulatory gene elements thus holds great promise for revealing insights into fundamental gene regulatory mechanisms controlling tissue differentiation and repair. Because much of this regulation occurs in response to signals from surrounding cells, characterizing them requires a model system in which their activity can be tested within the context of an intact organism conveniently.

Phosphorylation of heterogeneous nuclear ribonucleoprotein K at an extracellular signal-regulated kinase phosphorylation site promotes neurofilament-medium protein expression and axon outgrowth in Xenopus.

Post-transcriptional control of cytoskeletal genes fine-tunes the supply of structural materials to growing axons in response to extracellular cues. In Xenopus, heterogeneous nuclear ribonucleoprotein K (hnRNPK) plays a crucial role in the nuclear export and translation of multiple cytoskeletal-related mRNAs required for axon outgrowth, and as a substrate of multiple kinases, is thus a likely molecular target of cell signaling pathways regulating such outgrowth. To study the role of hnRNPK's phosphorylation by extracellular signal-regulated kinase (ERK) in Xenopus axon outgrowth, we identified the only ERK1 phosphorylation site on Xenopus hnRNPK (S257; homologous with S284 of human hnRNPK) using an in vitro phosphorylation assay and tested its function in vivo by expressing phosphomimetic (S257D) and phosphodeficient (S257A) forms of hnRNPK in Xenopus embryos.

Microtubule-associated protein tau promotes neuronal class II β-tubulin microtubule formation and axon elongation in embryonic Xenopus laevis.

Compared with its roles in neurodegeneration, much less is known about microtubule-associated protein tau's normal functions in vivo, especially during development. The external development and ease of manipulating gene expression of Xenopus laevis embryos make them especially useful for studying gene function during early development. To study tau's functions in axon outgrowth, we characterized the most prominent tau isoforms of Xenopus embryos and manipulated their expression.

A method for using direct injection of plasmid DNA to study cis-regulatory element activity in F0 Xenopus embryos and tadpoles.

The ability to express exogenous reporter genes in intact, externally developing embryos, such as Xenopus, is a powerful tool for characterizing the activity of cis-regulatory gene elements during development. Although methods exist for generating transgenic Xenopus lines, more simplified methods for use with F0 animals would significantly speed the characterization of these elements. We discovered that injecting 2-cell stage embryos with a plasmid bearing a ϕC31 integrase-targeted attB element and two dual β-globin HS4 insulators flanking a reporter transgene in opposite orientations relative to each other yielded persistent expression with sufficiently high penetrance for characterizing the activity of the promoter without having to coinject integrase RNA.

c-Jun N-terminal kinase phosphorylation of heterogeneous nuclear ribonucleoprotein K regulates vertebrate axon outgrowth via a posttranscriptional mechanism.

c-Jun N-terminal kinase (JNK) mediates cell signaling essential for axon outgrowth, but the associated substrates and underlying mechanisms are poorly understood. We identified in Xenopus laevis embryos a novel posttranscriptional mechanism whereby JNK regulates axonogenesis by phosphorylating a specific site on heterogeneous nuclear ribonucleoprotein K (hnRNP K). Both JNK inhibition and hnRNP K knockdown inhibited axon outgrowth and translation of hnRNP K-regulated cytoskeletal RNAs (tau and neurofilament medium), effects that were alleviated by expressing phosphomimetic, but not phosphodeficient, forms of hnRNP K.

Heterogeneous nuclear ribonucleoprotein K, an RNA-binding protein, is required for optic axon regeneration in Xenopus laevis.

Axotomized optic axons of Xenopus laevis, in contrast to those of mammals, retain their ability to regenerate throughout life. To better understand the molecular basis for this successful regeneration, we focused on the role of an RNA-binding protein, heterogeneous nuclear ribonucleoprotein (hnRNP) K, because it is required for axonogenesis during development and because several of its RNA targets are under strong post-transcriptional control during regeneration. At 11 d after optic nerve crush, hnRNP K underwent significant translocation into the nucleus of retinal ganglion cells (RGCs), indicating that the protein became activated during regeneration.

hnRNP K post-transcriptionally co-regulates multiple cytoskeletal genes needed for axonogenesis.

The RNA-binding protein, hnRNP K, is essential for axonogenesis. Suppressing its expression in Xenopus embryos yields terminally specified neurons with severely disorganized microtubules, microfilaments and neurofilaments, raising the hypothesis that hnRNP K post-transcriptionally regulates multiple transcripts of proteins that organize the axonal cytoskeleton. To identify downstream candidates for this regulation, RNAs that co-immunoprecipitated from juvenile brain with hnRNP K were identified on microarrays.

Metamorphosis and the regenerative capacity of spinal cord axons in Xenopus laevis.

Throughout the vertebrate subphylum, the regenerative potential of central nervous system axons is greatest in embryonic stages and declines as development progresses. For example, Xenopus laevis can functionally recover from complete transection of the spinal cord as a tadpole but is unable to do so after metamorphosing into a frog. Neurons of the reticular formation and raphe nucleus are among those that regenerate axons most reliably in tadpole and that lose this ability after metamorphosis.

Post-transcriptional control of neurofilaments: New roles in development, regeneration and neurodegenerative disease.

Neurofilament (NF) protein expression is coupled to axon development and the maintenance of neuronal homeostasis. Here, we present evidence that this tight regulation depends critically on post-transcriptionally regulated changes in NF mRNA transport, translation and stability. Recent studies have shown that post-transcriptional mechanisms modulate increases in NF gene transcription during axon regeneration to yield the final pattern of NF protein expression.

Transcriptional and translational dynamics of light neurofilament subunit RNAs during Xenopus laevis optic nerve regeneration.

Neurofilaments (NFs), which comprise one of three cytoskeletal polymers of vertebrate axons, are heteropolymers of multiple NF subunit proteins. During Xenopus laevis optic nerve regeneration, NF subunit composition undergoes progressive changes that correlate with regenerative success. Understanding the relative contributions of transcriptional and post-transcriptional gene regulatory mechanisms to these changes should therefore provide insights into the control of the axonal growth program.

A crucial role for hnRNP K in axon development in Xenopus laevis.

We report that hnRNP K, an RNA-binding protein implicated in multiple aspects of post-transcriptional gene control, is essential for axon outgrowth in Xenopus. Its intracellular localization was found to be consistent with one of its known roles as an mRNA shuttling protein. In early embryos, it was primarily nuclear, whereas later it occupied both the nucleus and cytoplasm to varying degrees in different neuronal subtypes.

Dynamic regulation of middle neurofilament RNA pools during optic nerve regeneration.

Stereotypical changes in neurofilament subunit expression are highly correlated with the regenerative success of lower vertebrate CNS axons. The phylogenetically conserved binding of ribonucleoproteins to the 3'-untranslated region of the middle neurofilament subunit (NF-M) mRNA suggests that post-transcriptional mechanisms play an important role in the control of NF-M expression. To assess their contribution to the regulated changes in NF-M expression that occur during Xenopus laevis optic axon regeneration, we followed changes in intracellular NF-M RNA pools.

Dynamic endogenous association of neurofilament mRNAs with K-homology domain ribonucleoproteins in developing cerebral cortex.

The low, middle, and high molecular mass neurofilament subunit proteins (NF-L, NF-M, and NF-H) co-polymerize to form neurofilaments (NFs). During development, NF subunit expression is highly regulated, and in neurodegenerative disease, aberrant regulation of this expression can lead to the formation of harmful aggregates. NF expression in both development and disease is under significant post-transcriptional control, but the specific ribonucleoproteins (RNPs) involved are only poorly understood.

Post-transcriptional control of neurofilaments in development and disease.

Tight coordination of the expression of neurofilament subunits is integral to the normal development and function of the nervous system. Imbalances in their expression are increasingly implicated in the induction of neurodegeneration in which formation of neurofilamentous aggregates is central to the pathology. Neurofilament expression can be controlled not only at the transcriptional level but also through post-transcriptional regulation of mRNA localization, stability, and translational efficiency.

A living cell-based biosensor utilizing G-protein coupled receptors: principles and detection methods.

This study explores the feasibility of using a bullfrog fibroblast cell line (FT cells) expressing G protein coupled receptors (GPCRs) as the basis for a living cell-based biosensor. We have fabricated gold microelectrode arrays on a silicon dioxide substrate that supports long term, robust growth of the cells at room temperature and under ambient atmospheric conditions. Activation of an endogenous GPCR to ATP was monitored with an optical method that detects rises in intracellular calcium and with an electrochemical method that monitors the increased secretion of pre-loaded norepinephrine on a MEMS device.

Neurofilament content is correlated with branch length in developing collateral branches of Xenopus spinal cord neurons.

During development, axons form interstitial collateral branches, which are initially dynamic but gradually stabilize as the projection sharpens. The initial outgrowth of collaterals is characterized by transitions in growth dynamics that occur at different lengths. Below 10 microm, collateral branches start out as unstable, thin filopodia.

Regeneration of descending projections in Xenopus laevis tadpole spinal cord demonstrated by retrograde double labeling.

Xenopus laevis tadpoles functionally recover from spinal cord transection. Because this recovery requires the tadpole to metamorphose, it may result from compensatory changes initiated by de novo growth of axons involved in limb dominant locomotion rather than from regeneration of cut axons. To determine whether axonal regrowth contributes to functional recovery, sequential retrograde double labeling with two fluorescent dextran amines was used to identify neurons with regenerated axons.