Temporal and spatial transcriptomic dynamics across brain development in Xenopus laevis tadpoles.

Amphibian metamorphosis is a transitional period that involves significant changes in the cell-type populations and biological processes occurring in the brain. Analysis of gene expression dynamics during this process may provide insight into the molecular events underlying these changes. We conducted differential gene expression analyses of the developing Xenopus laevis tadpole brain during this period in two ways: first, over stages of the development in the midbrain and, second, across regions of the brain at a single developmental stage. We found that genes pertaining to positive regulation of neural progenitor cell proliferation as well as known progenitor cell markers were upregulated in the midbrain prior to metamorphic climax; concurrently, expression of cell cycle timing regulators decreased across this period, supporting the notion that cell cycle lengthening contributes to a decrease in proliferation by the end of metamorphosis. We also found that at the start of metamorphosis, neural progenitor populations appeared to be similar across the fore-, mid-, and hindbrain regions. Genes pertaining to negative regulation of differentiation were upregulated in the spinal cord compared to the rest of the brain, however, suggesting that different programs may regulate neurogenesis there. Finally, we found that regulation of biological processes like cell fate commitment and synaptic signaling follow similar trajectories in the brain across early tadpole metamorphosis and mid- to late-embryonic mouse development. By comparing expression across both temporal and spatial conditions, we have been able to illuminate cell-type and biological pathway dynamics in the brain during metamorphosis.

Imaging Mitochondrial Dynamics in the Xenopus Central Nervous System (CNS).

Notable for producing ATP via oxidative phosphorylation, mitochondria also control calcium homeostasis, lipogenesis, the regulation of reactive oxygen species, and apoptosis. Even within relatively simple cells, mitochondria are heterogeneous with regard to their shape, abundance, movement, and subcellular locations. They exist as interconnected, tubular networks and as motile organelles that are transported along the cytoskeleton for distribution throughout cells. These spatial and morphological features reflect variability in the organelle's capacity to synthesize ATP and support cells. Changes to mitochondria are believed to support cell function and fate, and mitochondrial dysfunction underlies disease in the nervous system. Here we describe an in vivo time-lapse imaging approach to monitor and measure the movement and position of the mitochondria in cells of the developing brain in albino Xenopus laevis tadpoles. The unparalleled benefit of using Xenopus for these experiments is that measurements of mitochondrial morphology and distribution in cells can be measured in vivo, where the surrounding neural circuitry and other inputs that influence these critical organelles remain intact. This protocol draws together techniques to label brain cells and capture the morphology of the cells and their mitochondria with 3D time-lapse confocal microscopy. We describe open-source methods to reconstruct cells in order to quantify the features of their mitochondria.

Morpholino Studies in Xenopus Brain Development.

Antisense morpholino oligonucleotides (MOs) have become a valuable method to knockdown protein levels, to block with mRNA splicing and to interfere with miRNA function. MOs are widely used to alter gene expression in development of Xenopus and Zebrafish, where they are typically injected into the fertilized egg or blastomeres. Here we present methods to use electroporation to target delivery of MOs to the central nervous system of Xenopus laevis or Xenopus tropicalis tadpoles. Briefly, MO electroporation is accomplished by injecting MO solution into the brain ventricle and driving the MOs into cells of the brain with current passing between 2 platinum plate electrodes, positioned on either side of the target brain area. The method is relatively straightforward and uses standard equipment found in many neuroscience labs. A major advantage of electroporation is that it allows spatial and temporal control of MO delivery and therefore knockdown. Co-electroporation of MOs with cell type-specific fluorescent protein expression plasmids allows morphological analysis of cellular phenotypes. Furthermore, co-electroporation of MOs with rescuing plasmids allows assessment of specificity of the knockdown and phenotypic outcome. By combining MO-mediated manipulations with sophisticated assays of neuronal function, such as electrophysiological recording, behavioral assays, or in vivo time-lapse imaging of neuronal development, the functions of specific proteins and miRNAs within the developing nervous system can be elucidated. These methods can be adapted to apply antisense morpholinos to study protein and RNA function in a variety of complex tissues.

Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage, despite rapid and sustained mitochondrial DNA depletion, altered energetics and growth.

DNA polymerase gamma (POLG) is essential for replication and repair of mitochondrial DNA (mtDNA). Mutations in POLG cause mtDNA instability and a diverse range of poorly understood human diseases. Here, we created a unique Polg animal model, by modifying polg within the critical and highly conserved polymerase domain in zebrafish. polg(+/-) offspring were indistinguishable from WT siblings in multiple phenotypic and biochemical measures. However, polg(-/-) mutants developed severe mtDNA depletion by one week post-fertilization (wpf), developed slowly and had regenerative defects, yet surprisingly survived up to 4 wpf. An in vivo mtDNA polymerase activity assay utilizing ethidium bromide (EtBr) to deplete mtDNA, showed that polg(+/-) and WT zebrafish fully recover mtDNA content two weeks post-EtBr removal. EtBr further reduced already low levels of mtDNA in polg(-/-) animals, but mtDNA content did not recover following release from EtBr. Despite significantly decreased respiration that corresponded with tissue-specific levels of mtDNA, polg(-/-) animals had WT levels of ATP and no increase in lactate. This zebrafish model of mitochondrial disease now provides unique opportunities for studying mtDNA instability from multiple angles, as polg(-/-) mutants can survive to juvenile stage, rather than lose viability in embryogenesis as seen in Polg mutant mice.

Metalloprotease OMA1 Fine-tunes Mitochondrial Bioenergetic Function and Respiratory Supercomplex Stability.

Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis, and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. One mechanism - intramitochondrial quality control (IMQC) - is represented by conserved proteases distributed across mitochondrial compartments. Many aspects and physiological roles of IMQC components remain unclear. Here, we show that the IMQC protease Oma1 is required for the stability of the respiratory supercomplexes and thus balanced and tunable bioenergetic function. Loss of Oma1 activity leads to a specific destabilization of respiratory supercomplexes and consequently to unbalanced respiration and progressive respiratory decline in yeast. Similarly, experiments in cultured Oma1-deficient mouse embryonic fibroblasts link together impeded supercomplex stability and inability to maintain proper respiration under conditions that require maximal bioenergetic output. Finally, transient knockdown of OMA1 in zebrafish leads to impeded bioenergetics and morphological defects of the heart and eyes. Together, our biochemical and genetic studies in yeast, zebrafish and mammalian cells identify a novel and conserved physiological role for Oma1 protease in fine-tuning of respiratory function. We suggest that this unexpected physiological role is important for cellular bioenergetic plasticity and may contribute to Oma1-associated disease phenotypes in humans.

The cellular and molecular progression of mitochondrial dysfunction induced by 2,4-dinitrophenol in developing zebrafish embryos.

The etiology of mitochondrial disease is poorly understood. Furthermore, treatment options are limited, and diagnostic methods often lack the sensitivity to detect disease in its early stages. Disrupted oxidative phosphorylation (OXPHOS) that inhibits ATP production is a common phenotype of mitochondrial disorders that can be induced in zebrafish by exposure to 2,4-dinitrophenol (DNP), a FDA-banned weight-loss agent and EPA-regulated environmental toxicant, traditionally used in research labs as an uncoupler of OXPHOS. Despite the DNP-induced OXPHOS inhibition we observed using in vivo respirometry, the development of the DNP-treated and control zebrafish were largely similar during the first half of embryogenesis. During this period, DNP-treated embryos induced gene expression of mitochondrial and nuclear genes that stimulated the production of new mitochondria and increased glycolysis to yield normal levels of ATP. DNP-treated embryos were incapable of sustaining this mitochondrial biogenic response past mid-embryogenesis, as shown by significantly lowered ATP production and ATP levels, decreased gene expression, and the onset of developmental defects. Examining neural tissues commonly affected by mitochondrial disease, we found that DNP exposure also inhibited motor neuron axon arbor outgrowth and the proper formation of the retina. We observed and quantified the molecular and physiological progression of mitochondrial dysfunction during development with this new model of OXPHOS dysfunction, which has great potential for use in diagnostics and therapies for mitochondrial disease.

An in vivo screen to identify candidate neurogenic genes in the developing Xenopus visual system.

Neurogenesis in the brain of Xenopus laevis continues throughout larval stages of development. We developed a 2-tier screen to identify candidate genes controlling neurogenesis in Xenopus optic tectum in vivo. First, microarray and NanoString analyses were used to identify candidate genes that were differentially expressed in Sox2-expressing neural progenitor cells or their neuronal progeny. Then an in vivo, time-lapse imaging-based screen was used to test whether morpholinos against 34 candidate genes altered neural progenitor cell proliferation or neuronal differentiation over 3 days in the optic tectum of intact Xenopus tadpoles. We co-electroporated antisense morpholino oligonucleotides against each of the candidate genes with a plasmid that drives GFP expression in Sox2-expressing neural progenitor cells and quantified the effects of morpholinos on neurogenesis. Of the 34 morpholinos tested, 24 altered neural progenitor cell proliferation or neuronal differentiation. The candidates which were tagged as differentially expressed and validated by the in vivo imaging screen include: actn1, arl9, eif3a, elk4, ephb1, fmr1-a, fxr1-1, fbxw7, fgf2, gstp1, hat1, hspa5, lsm6, mecp2, mmp9, and prkaca. Several of these candidates, including fgf2 and elk4, have known or proposed neurogenic functions, thereby validating our strategy to identify candidates. Genes with no previously demonstrated neurogenic functions, gstp1, hspa5 and lsm6, were identified from the morpholino experiments, suggesting that our screen successfully revealed unknown candidates. Genes that are associated with human disease, such as such as mecp2 and fmr1-a, were identified by our screen, providing the groundwork for using Xenopus as an experimental system to probe conserved disease mechanisms. Together the data identify candidate neurogenic regulatory genes and demonstrate that Xenopus is an effective experimental animal to identify and characterize genes that regulate neural progenitor cell proliferation and differentiation in vivo.

Morpholino studies in Xenopus brain development.

Antisense morpholino oligonucleotides (MOs) have become a valuable method to knock down protein levels, to block mRNA splicing, and to interfere with miRNA function. MOs are widely used to alter gene expression during development of Xenopus and zebra fish, where they are typically injected into the fertilized egg or blastomeres. Here, we present methods to use electroporation to target delivery of MOs to the central nervous system of Xenopus laevis or Xenopus tropicalis tadpoles. Briefly, MO electroporation is accomplished by injecting MO solution into the brain ventricle and driving the MOs into cells in the brain with current passing between two platinum plate electrodes, positioned on either side of the target brain area. The method is straightforward and uses standard equipment found in many neuroscience labs. A major advantage of electroporation is that it allows spatial and temporal control of MO delivery and therefore knockdown. Co-electroporation of MOs with cell-type specific fluorescent protein expression plasmids allows morphological analysis of cellular phenotypes. Furthermore, co-electroporation of MOs with rescuing plasmids allows assessment of specificity of the knockdown and phenotypic outcome. By combining MO-mediated manipulations with sophisticated assays of neuronal function, such as electrophysiological recording, behavioral assays, or in vivo time-lapse imaging of neuronal development, the functions of specific proteins and miRNAs within the developing nervous system can be elucidated. These methods can be adapted to apply antisense morpholinos to study protein and RNA function in a variety of complex tissues.

In vivo time-lapse imaging of cell proliferation and differentiation in the optic tectum of Xenopus laevis tadpoles.

We analyzed the function of neural progenitors in the developing central nervous system of Xenopus laevis tadpoles by using in vivo time-lapse confocal microscopy to collect images through the tectum at intervals of 2-24 hours over 3 days. Neural progenitor cells were labeled with fluorescent protein reporters based on expression of endogenous Sox2 transcription factor. With this construct, we identified Sox2-expressing cells as radial glia and as a component of the progenitor pool of cells in the developing tectum that gives rise to neurons and other radial glia. Lineage analysis of individual radial glia and their progeny demonstrated that less than 10% of radial glia undergo symmetric divisions resulting in two radial glia, whereas the majority of radial glia divide asymmetrically to generate neurons and radial glia. Time-lapse imaging revealed the direct differentiation of radial glia into neurons. Although radial glia may guide axons as they navigate to the superficial tectum, we find no evidence that radial glia function as a scaffold for neuronal migration at early stages of tectal development. Over 3 days, the number of labeled cells increased 20%, as the fraction of radial glia dropped and the proportion of neuronal progeny increased to approximately 60% of the labeled cells. Tadpoles provided with short-term visual enhancement generated significantly more neurons, with a corresponding decrease in cell proliferation. Together these results demonstrate that radial glial cells are neural progenitors in the developing optic tectum and reveal that visual experience increases the proportion of neurons generated in an intact animal.

The Relationship between Dendritic Branch Dynamics and CPEB-Labeled RNP Granules Captured in Vivo.

Cytoplasmic Polyadenylation Element Binding protein (CPEB) is an RNA binding protein involved in dendritic delivery of mRNA and activity-dependent, polyadenylation-induced translation of mRNAs in the dendritic arbor. CPEB affects learning and memory and impacts neuronal morphological and synaptic plasticity. In neurons, CPEB is concentrated in ribonucleoprotein (RNP) granules that distribute throughout the dendritic arbor and localize near synapses, suggesting that the trafficking of RNP granules is important for CPEB function. We tagged full-length CPEB and an inactive mutant CPEB with fluorescent proteins, then imaged rapid dendritic branch dynamics and RNP distribution using two-photon time-lapse microscopy of neurons in the optic tectum of living Xenopus laevis tadpoles. Though the inactive CPEB mutant transports mRNA in the dendritic arbor, its expression interferes with CPEB-dependent translation because it is incapable of activity-triggered mRNA polyadenylation. In dendrites, the distributions of the active and inactive CPEB-containing RNP granules do not differ; the RNP granules are dense and their positions do not correlate with sites of rapid dendritic branch dynamics or the eventual fate of the dendritic branches. Because CPEB's sensitivity to activity-dependent signaling does not alter its dendritic distribution, it indicates that active sites in the dendritic arbor are not targeted for RNP granule localization. Nevertheless, inactive CPEB accumulates in granules in terminal dendritic branches, supporting the hypothesis that upon activation CPEB and its mRNA cargo are released from granules and are then available for dendritic translation.

The RNA binding protein CPEB regulates dendrite morphogenesis and neuronal circuit assembly in vivo.

Visual system development requires experience-dependent mechanisms that regulate neuronal structure and function, including dendritic arbor growth, synapse formation, and stabilization. Although RNA binding proteins have been shown to affect some forms of synaptic plasticity in adult animals, their role in the development of neuronal structure and functional circuitry is not clear. Using two-photon time-lapse in vivo imaging and electrophysiology combined with morpholino-mediated knockdown and expression of functional deletion mutants, we demonstrate that the mRNA binding protein, cytoplasmic polyadenylation element binding protein1 (CPEB1), affects experience-dependent neuronal development and circuit formation in the visual system of Xenopus laevis. These data indicate that sensory experience controls circuit development by regulating translational activity of mRNAs.

The control of anterior foregut motility during a larval molt of the moth Manduca sexta involves the modulation of presynaptic activity.

In the moth, Manduca sexta, anterior foregut motility is modulated during the larval-larval molts in order to control the timing of molting fluid (MF) ingestion. MF is the enzymatic mixture that destroys the outer cuticle so that it can be shed at the end of the molt. The onset of the larval-larval molt is characterized by a dramatic decline in the amplitude of the anterior foregut contractions so that MF is not prematurely ingested. As the end of the molt approaches, the robust contractions of the anterior foregut return and the MF is ingested, enabling the larva to free itself from its old cuticle. In the present study we examine possible mechanisms involved in modulating anterior foregut motility during a larval-larval molt. Our results reveal that the release of a blood-borne factor plays a role in the decline in anterior foregut peristaltic activity during the molt. This blood-borne factor reduces the efficacy of the presynaptic endings of the motorneurons, resulting in a reduction in the amplitude of the excitatory junctional potential (EJP) recorded from the anterior foregut musculature. We also present evidence that crustacean cardioactive peptide (CCAP) targets the motorneuron terminals and its actions are sufficient to trigger the dramatic increase in EJP amplitude and anterior foregut contractions. Finally, the surgical ablation of the subesophageal ganglion, which has been previously described to be a source of CCAP neurons and the CCAP projections to the anterior foregut region, blocks both the increase in anterior foregut motility and the ingestion of MF that normally occur at the end of a larval-larval molt.

In vivo single-cell electroporation for transfer of DNA and macromolecules.

Single-cell electroporation allows transfection of plasmid DNA or macromolecules into individual living cells using modified patch electrodes and common electrophysiological equipment. This protocol is optimized for rapid in vivo electroporation of Xenopus laevis tadpole brains with DNA, dextrans, morpholinos and combinations thereof. Experienced users can electroporate roughly 40 tadpoles per hour. The technique can be adapted for use with other charged transfer materials and in other systems and tissues where cells can be targeted with a micropipette. Under visual guidance, an electrode filled with transfer material is placed in a cell body-rich area of the tadpole brain and a train of voltage pulses applied, which electroporates a nearby cell. We show examples of successfully electroporated single cells, instances of common problems and troubleshooting suggestions. Single-cell electroporation is an affordable method to fluorescently label and genetically manipulate individual cells. This powerful technique enables observation of single cells in an otherwise normal environment.

Modulation of foregut synaptic activity controls resorption of molting fluid during larval molts of the moth Manduca sexta.

We examined the role of the foregut in the resorption of molting fluid (MF) from the exuvial space during the last larval-larval molt of the moth Manduca sexta. In intermolt larvae, the activity of the foregut is characterized by robust peristaltic contractions. With the onset of the molt, MF is secreted into the exuvial space where it digests and weakens the old cuticle. The appearance of MF in the exuvial space is accompanied by a dramatic reduction in the amplitude of the foregut contractions. Foregut peristalsis returned about halfway through the molt, followed shortly by the appearance of MF in the gut. These observations suggested that larvae use their foreguts to remove MF from the exuvial space. Animals whose foreguts were surgically inactivated did not resorb their MF and most failed to successfully shed their old cuticles. The reduction in foregut motility at the onset of the molt was correlated with a sharp decline in the amplitude of the excitatory junctional potentials. With the onset of the molt there was also a decline in the number of presynaptic terminals on the foregut that loaded with the activity-dependent dye FM1-43. In the second half of the molt, the appearance of MF in the foregut and the return of foregut motility was correlated with an increase in FM1-43 loading. These data reveal that during a larval-larval molt, vesicle release and/or recycling of the presynaptic endings on the foregut muscles is modulated to assure the proper timing of MF resorption.