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In JoVE (1)
Other Publications (11)
- Proceedings of the National Academy of Sciences of the United States of America
- Biochemical Pharmacology
- Liver International : Official Journal of the International Association for the Study of the Liver
- Molecular and Cellular Biology
- Molecular and Cellular Neurosciences
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Annual Review of Neuroscience
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Developmental Neurobiology
- Frontiers in Molecular Neuroscience
Articles by Timothy J Petros in JoVE
JoVE General
In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells
1Departments of Pathology and Cell Biology, and Neuroscience, Columbia University College of Physicians and Surgeons, 2Department of Ophthalmology, Columbia University College of Physicians and Surgeons
Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.
Other articles by Timothy J Petros on PubMed
An Endogenous Capsaicin-like Substance with High Potency at Recombinant and Native Vanilloid VR1 Receptors
The vanilloid receptor VR1 is a nonselective cation channel that is most abundant in peripheral sensory fibers but also is found in several brain nuclei. VR1 is gated by protons, heat, and the pungent ingredient of "hot" chili peppers, capsaicin. To date, no endogenous compound with potency at this receptor comparable to that of capsaicin has been identified. Here we examined the hypothesis, based on previous structure-activity relationship studies and the availability of biosynthetic precursors, that N-arachidonoyl-dopamine (NADA) is an endogenous "capsaicin-like" substance in mammalian nervous tissues. We found that NADA occurs in nervous tissues, with the highest concentrations being found in the striatum, hippocampus, and cerebellum and the lowest concentrations in the dorsal root ganglion. We also gained evidence for the existence of two possible routes for NADA biosynthesis and mechanisms for its inactivation in rat brain. NADA activates both human and rat VR1 overexpressed in human embryonic kidney (HEK)293 cells, with potency (EC(50) approximately 50 nM) and efficacy similar to those of capsaicin. Furthermore, NADA potently activates native vanilloid receptors in neurons from rat dorsal root ganglion and hippocampus, thereby inducing the release of substance P and calcitonin gene-related peptide (CGRP) from dorsal spinal cord slices and enhancing hippocampal paired-pulse depression, respectively. Intradermal NADA also induces VR1-mediated thermal hyperalgesia (EC(50) = 1.5 +/- 0.3 microg). Our data demonstrate the existence of a brain substance similar to capsaicin not only with respect to its chemical structure but also to its potency at VR1 receptors.
Regulation of Anandamide Tissue Levels by N-arachidonylglycine
N-arachidonylglycine (NAGly), the carboxylic analog of the endocannabinoid anandamide, occurs in rat and bovine brain as well as in peripheral sites and shows activity against tonic, formalin-induced pain. It was also observed, using cell membrane preparations, that it inhibits the hydrolytic activity of fatty acid amide hydrolase (FAAH) on anandamide (N-arachidonylethanolamide). These data suggested that it may serve as an endogenous regulator of tissue anandamide concentrations. In this report, we show findings derived from mass spectrometric analyses, indicating that blood levels of anandamide in rats given 10 mg/kg p.o. of NAGly were increased significantly by more than 9-fold when compared with vehicle-treated controls. In vitro evidence in RAW 264.7 cells using a deuterium-labeled NAGly demonstrated that it was not a precursor or source of arachidonic acid for the observed 50% rise in anandamide levels, suggesting that the increase was due to some effect other than increased biosynthesis of anandamide. Moreover, the findings presented here suggest that NAGly can serve as a model for the design of agents to provide pharmacological control of tissue anandamide concentrations.
Circulating Endogenous Cannabinoid Anandamide and Portal, Systemic and Renal Hemodynamics in Cirrhosis
Endocannabinoids may participate in the homeostasis of arterial pressure. Recently, anandamide, the most extensively studied endocannabinoid, has been proposed as a key mediator in the peripheral arterial vasodilation of cirrhosis.
Transcription Factor KLF7 is Important for Neuronal Morphogenesis in Selected Regions of the Nervous System
The Krüppel-like transcription factors (KLFs) are important regulators of cell proliferation and differentiation in several different organ systems. The mouse Klf7 gene is strongly active in postmitotic neuroblasts of the developing nervous system, and the corresponding protein stimulates transcription of the cyclin-dependent kinase inhibitor p21waf/cip gene. Here we report that loss of KLF7 activity in mice leads to neonatal lethality and a complex phenotype which is associated with deficits in neurite outgrowth and axonal misprojection at selected anatomical locations of the nervous system. Affected axon pathways include those of the olfactory and visual systems, the cerebral cortex, and the hippocampus. In situ hybridizations and immunoblots correlated loss of KLF7 activity in the olfactory epithelium with significant downregulation of the p21waf/cip and p27kip1 genes. Cotransfection experiments extended the last finding by documenting KLF7's ability to transactivate a reporter gene construct driven by the proximal promoter of p27kip1. Consistent with emerging evidence for a role of Cip/Kip proteins in cytoskeletal dynamics, we also documented p21waf/cip and p27kip1 accumulation in the cytoplasm of differentiating olfactory sensory neurons. KLF7 activity might therefore control neuronal morphogenesis in part by optimizing the levels of molecules that promote axon outgrowth.
Temporal Regulation of EphA4 in Astroglia During Murine Retinal and Optic Nerve Development
Eph receptors and their ephrin ligands play important roles in many aspects of visual system development. In this study, we characterized the spatial and temporal expression pattern of EphA4 in astrocyte precursor cell (APC) and astrocyte populations in the murine retina and optic nerve. EphA4 is expressed by immotile optic disc astrocyte precursor cells (ODAPS), but EphA4 is downregulated as these cells migrate into the retina. Surprisingly, mature astrocytes in the adult retina re-express EphA4. Within the optic nerve, EphA4 is expressed in specialized astrocytes that form a meshwork at the optic nerve head (ONH). Our in vitro and in vivo data indicate that EphA4 is dispensable for retinal ganglion cell (RGC) axon growth and projections through the chiasm. While optic stalk structure, APC proliferation and migration, retinal vascularization, and oligodendrocyte migration appear normal in EphA4 mutants, the expression of EphA4 in APCs and in the astrocyte meshwork at the ONH has implications for optic nerve pathologies.
Zic2 Regulates Retinal Ganglion Cell Axon Avoidance of EphrinB2 Through Inducing Expression of the Guidance Receptor EphB1
The navigation of retinal axons to ipsilateral and contralateral targets in the brain depends on the decision to cross or avoid the midline at the optic chiasm, a critical guidance maneuver that establishes the binocular visual pathway. Previous work has identified a specific guidance receptor, EphB1, that mediates the repulsion of uncrossed axons away from its ligand, ephrinB2, at the optic chiasm midline (Williams et al., 2003), and a transcription factor Zic2, that, like EphB1, is required for formation of the ipsilateral retinal projection (Herrera et al., 2003). Although the reported similarities in localization implicated that Zic2 regulates EphB1 (Herrera et al., 2003; Williams et al., 2003; Pak et al., 2004), whether Zic2 drives expression of EphB1 protein has not been elucidated. Here we show that EphB1 protein is expressed in the growth cones of axons from ventrotemporal (VT) retina that project ipsilaterally and that repulsion by ephrinB2 is determined by the presence of this receptor on growth cones. Moreover, ectopic delivery of Zic2 into explants from non-VT retina induces expression of EphB1 mRNA and protein. The upregulated EphB1 receptor protein is localized to growth cones and is functional, because it is sufficient to change retinal ganglion cell axon behavior from extension onto, to avoidance of, ephrinB2 substrates. Our results demonstrate that Zic2 upregulates EphB1 expression and define a link between a transcription factor and expression of a guidance receptor protein essential for axon guidance at the vertebrate midline.
Retinal Axon Growth at the Optic Chiasm: to Cross or Not to Cross
At the optic chiasm, retinal ganglion cell axons from each eye converge and segregate into crossed and uncrossed projections, a pattern critical for binocular vision. Here, we review recent findings on optic chiasm development, highlighting the specific transcription factors and guidance cues that implement retinal axon divergence into crossed and uncrossed pathways. Although mechanisms underlying the formation of the uncrossed projection have been identified, the means by which retinal axons are guided across the midline are still unclear. In addition to directives provided by transcription factors and receptors in the retina, gene expression in the ventral diencephalon influences chiasm formation. Throughout this review, we compare guidance mechanisms at the optic chiasm with those in other midline models and highlight unanswered questions both for retinal axon growth and axon guidance in general.
Specificity and Sufficiency of EphB1 in Driving the Ipsilateral Retinal Projection
At the optic chiasm, retinal ganglion cell (RGC) axons make the decision to either avoid or traverse the midline, a maneuver that establishes the binocular pathways. In mice, the ipsilateral retinal projection arises from RGCs in the peripheral ventrotemporal (VT) crescent of the retina. These RGCs express the guidance receptor EphB1, which interacts with ephrin-B2 on radial glia cells at the optic chiasm to repulse VT axons away from the midline and into the ipsilateral optic tract. However, because VT RGCs express more than one EphB receptor, the sufficiency and specificity of the EphB1 receptor in directing the ipsilateral projection is unclear. In this study, we use in utero retinal electroporation to demonstrate that ectopic EphB1 expression can redirect RGCs with a normally crossed projection to an ipsilateral trajectory. Moreover, EphB1 is specifically required for rerouting RGC projections ipsilaterally, because introduction of the highly similar EphB2 receptor is much less efficient in redirecting RGC fibers, even when expressed at higher surface levels. Introduction of EphB1-EphB2 chimeric receptors into RGCs reveals that both extracellular and juxtamembrane domains of EphB1 are required to efficiently convert RGC projections ipsilaterally. Together, these data describe for the first time functional differences between two highly similar Eph receptors at a decision point in vivo, with EphB1 displaying unique properties that efficiently drives the uncrossed retinal projection.
Switching Retinogeniculate Axon Laterality Leads to Normal Targeting but Abnormal Eye-specific Segregation That is Activity Dependent
Partial decussation of sensory pathways allows neural inputs from both sides of the body to project to the same target region where these signals will be integrated. Here, to better understand mechanisms of eye-specific targeting, we studied how retinal ganglion cell (RGC) axons terminate in their thalamic target, the dorsal lateral geniculate nucleus (dLGN), when crossing at the optic chiasm midline is altered. In models with gain- and loss-of-function of EphB1, the receptor that directs the ipsilateral projection at the optic chiasm, misrouted RGCs target the appropriate retinotopic zone in the opposite dLGN. However, in EphB1(-/-) mice, the misrouted axons do not intermingle with normally projecting RGC axons and segregate instead into a distinct patch. We also revisited the role of retinal activity on eye-specific targeting by blocking correlated waves of activity with epibatidine into both eyes. We show that, in wild-type mice, retinal waves are necessary during the first postnatal week for both proper distribution and eye-specific segregation of ipsilateral axons in the mature dLGN. Moreover, in EphB1(-/-) mice, refinement of ipsilateral axons is perturbed in control conditions and is further impaired after epibatidine treatment. Finally, retinal waves are required for the formation of the segregated patch of misrouted axons in EphB1(-/-) mice. These findings implicate molecular determinants for targeting of eye-specific zones that are independent of midline guidance cues and that function in concert with correlated retinal activity to sculpt retinogeniculate projections.
Ephrin-B2 Elicits Differential Growth Cone Collapse and Axon Retraction in Retinal Ganglion Cells from Distinct Retinal Regions
The circuit for binocular vision and stereopsis is established at the optic chiasm, where retinal ganglion cell (RGC) axons diverge into the ipsilateral and contralateral optic tracts. In the mouse retina, ventrotemporal (VT) RGCs express the guidance receptor EphB1, which interacts with the repulsive guidance cue ephrin-B2 on radial glia at the optic chiasm to direct VT RGC axons ipsilaterally. RGCs in the ventral retina also express EphB2, which interacts with ephrin-B2, whereas dorsal RGCs express low levels of EphB receptors. To investigate how growth cones of RGCs from different retinal regions respond upon initial contact with ephrin-B2, we utilized time-lapse imaging to characterize the effects of ephrin-B2 on growth cone collapse and axon retraction in real time. We demonstrate that bath application of ephrin-B2 induces rapid and sustained growth cone collapse and axon retraction in VT RGC axons, whereas contralaterally-projecting dorsotemporal RGCs display moderate growth cone collapse and little axon retraction. Dose response curves reveal that contralaterally-projecting ventronasal axons are less sensitive to ephrin-B2 treatment compared to VT axons. Additionally, we uncovered a specific role for Rho kinase signaling in the retraction of VT RGC axons but not in growth cone collapse. The detailed characterization of growth cone behavior in this study comprises an assay for the study of Eph signaling in RGCs, and provides insight into the phenomena of growth cone collapse and axon retraction in general.
Pluripotent Stem Cells for the Study of CNS Development
The mammalian central nervous system is a complex neuronal network consisting of a diverse array of cellular subtypes generated in a precise spatial and temporal pattern throughout development. Achieving a greater understanding of the molecular and genetic mechanisms that direct a relatively uniform population of neuroepithelial progenitors into diverse neuronal subtypes remains a significant challenge. The advent of pluripotent stem cell (PSC) technology allows researchers to generate diverse neural populations in vitro. Although the primary focus of PSC-derived neural cells has been their therapeutic potential, utilizing PSCs to study neurodevelopment is another frequently overlooked and equally important application. In this review, we explore the potential for utilizing PSCs to study neural development. We introduce the types of neurodevelopmental questions that PSCs can help to address, and we discuss the different strategies and technologies that researchers use to generate diverse subtypes of PSC-derived neurons. Additionally, we highlight the derivation of several thoroughly characterized neural subtypes; spinal motoneurons, midbrain dopaminergic neurons and cortical neurons. We hope that this review encourages researchers to develop innovative strategies for using PSCs for the study of mammalian, and specifically human, neurodevelopment.
