ABSTRACT
Mature messenger RNAs (mRNAs) consist of coding sequence (CDS) and 5' and 3' UTRs, typically expected to show similar abundance within a given neuron. Examining mRNA from defined neurons, we unexpectedly show extremely common unbalanced expression of cognate 3' UTR and CDS sequences; many genes show high 3' UTR relative to CDS, others show high CDS to 3' UTR. In situ hybridization (19 of 19 genes) shows a broad range of 3' UTR-to-CDS expression ratios across neurons and tissues. Ratios may be spatially graded or change with developmental age but are consistent across animals. Further, for two genes examined, a 3' UTR-to-CDS ratio above a particular threshold in any given neuron correlated with reduced or undetectable protein expression. Our findings raise questions about the role of isolated 3' UTR sequences in regulation of protein expression and highlight the importance of separately examining 3' UTR and CDS sequences in gene expression analyses.
Subject(s)
3' Untranslated Regions/genetics , Neurons/physiology , Open Reading Frames/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Animals , Base Sequence , Gene Expression Regulation , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , NIH 3T3 CellsABSTRACT
There are two main subgroups of midbrain dopaminergic (DA) neurons: the more medially located ventral tegmental area (VTA) DA neurons, which have axons that innervate the ventral-lateral (VL) striatum, and the more laterally located substantia nigra (SN) DA neurons, which preferentially degenerate in Parkinson's disease (PD) and have axons that project to the dorsal-medial (DM) striatum. DA axonal projections in the striatum are not discretely localized and they arborize widely, however they do not stray from one zone to the other so that VTA axons remain in the VL zone and SN axons in the DM zone. Here we provide evidence that Netrin-1 acts in a novel fashion to topographically pattern midbrain DA axons into these two striatal zones by means of a gradient of Netrin-1 in the striatum and by differential attraction of the axons to Netrin-1. Midbrain DA neurons are attracted to the striatum in culture and this attraction is blocked by an anti-DCC (Netrin receptor) antibody. Mechanistically, outgrowth of both VTA and SN DA axons is stimulated by Netrin-1, but the two populations of DA axons respond optimally to overlapping but distinct concentrations of Netrin-1, with SN axons preferring lower concentrations and VTA axons preferring higher concentrations. In vivo this differential preference is closely mirrored by differences in Netrin-1 expression in their respective striatal target fields. In vivo in mice lacking Netrin-1, DA axons that reach the striatum fail to segregate into two terminal zones and to fully innervate the striatum. Our results reveal novel actions for Netrin-1 and provide evidence for a mechanism through which DA axons can selectively innervate one of two terminal zones in the striatum but have free reign to arborize widely within a terminal zone.
Subject(s)
Axons/physiology , Corpus Striatum/cytology , Dopaminergic Neurons/physiology , Gene Expression Regulation, Developmental/genetics , Nerve Growth Factors/metabolism , Tumor Suppressor Proteins/metabolism , Age Factors , Animals , COS Cells , Chickens , Chlorocebus aethiops , DCC Receptor , Dopamine and cAMP-Regulated Phosphoprotein 32/metabolism , Embryo, Mammalian , In Vitro Techniques , Mice , Mice, Inbred C57BL , Nerve Growth Factors/genetics , Netrin-1 , Organ Culture Techniques , Rats , Rats, Sprague-Dawley , Receptors, Cell Surface/metabolism , Repressor Proteins/metabolism , Tumor Suppressor Proteins/genetics , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Dopaminergic neurons derived from human embryonic stem cells will be useful in future transplantation studies of Parkinson's disease patients. As newly generated neurons must integrate and reconnect with host cells, the ability of hESC-derived neurons to respond to axon guidance cues will be critical. Both Netrin-1 and Slit-2 guide rodent embryonic dopaminergic (DA) neurons in vitro and in vivo, but very little is known about the response of hESC-derived DA neurons to any axonal guidance cues. Here we examined the ability of Netrin-1 and Slit-2 to affect human ESC DA axons in vitro. hESC DA neurons mature over time in culture with the developmental profile of DA neurons in vivo, including expression of the DA neuron markers FoxA2, En-1 and Nurr-1, and receptors for both Netrin and Slit. hESC DA neurons respond to exogenous Netrin-1 and Slit-2, showing an increased responsiveness to Netrin-1 as the neurons mature in culture. These responses were maintained in the presence of pro-inflammatory cytokines that might be encountered in the diseased brain. These studies are the first to evaluate and confirm that suitably matured human ES-derived DA neurons can respond appropriately to axon guidance cues.
