Midbrain dopamine neurons (mDA) are important regulators of diverse physiological functions, including movement, attention, and reward behaviors. Accordingly, aberrant function of dopamine neurons underlies a wide spectrum of disorders, such as Parkinson's disease (PD), dystonia, and schizophrenia. The distinct functions of the dopamine system are carried out by neuroanatomically discrete subgroups of dopamine neurons, which differ in gene expression, axonal projections, and susceptibility in PD. The developmental underpinnings of this heterogeneity are undefined. We have recently shown that in the embryonic CNS, mDA originate from the midbrain floor plate, a ventral midline structure that is operationally defined by the expression of the molecule Shh. Here, we develop these findings to reveal that in the embryonic midbrain, the spatiotemporally dynamic Shh domain defines multiple progenitor pools. We deduce 3 distinct progenitor pools, medial, intermediate, and lateral, which contribute to different mDA clusters. The earliest progenitors to express Shh, here referred to as the medial pool, contributes neurons to the rostral linear nucleus and mDA of the ventral tegmental area/interfascicular regions, but remarkably, little to the substantia nigra pars compacta. The intermediate Shh؉ progenitors give rise to neurons of all dopaminergic nuclei, including the SNpc. The last and lateral pool of Shh؉ progenitors generates a cohort that populates the red nucleus, Edinger Westphal nucleus, and supraoculomotor nucleus and cap. Subsequently, these lateral Shh؉ progenitors produce mDA. This refined ontogenetic definition will expand understanding of dopamine neuron biology and selective susceptibility, and will impact stem cell-derived therapies and models for PD.dopamine ͉ Sonic hedgehog ͉ lineage ͉ substantia nigra
The floor plate, an essential ventral midline organizing center that produces the morphogen Shh, has distinct properties along the neuraxis. The neurogenic potential of the floor plate and its underlying mechanisms remain unknown. Using Shh as a driver for lineage analysis, we found that the mouse midbrain, but not the hindbrain, floor plate is neurogenic, giving rise to dopamine (DA) neurons. Distinct spatiotemporal Shh and Wnt expression may distinguish the neurogenetic potential of these structures. We discovered an inhibitory role for Shh: removal of Shh resulted in neurogenesis from the hindbrain midline and, conversely, high doses of Shh inhibited proliferation and DA neuron production in midbrain cultures. We found that Wnt/beta-catenin signaling is necessary and sufficient for antagonizing Shh, DA progenitor marker induction and promotion of dopaminergic neurogenesis. These findings demonstrate how the dynamic interplay of canonical Wnt/beta-catenin signaling and Shh may orchestrate floor plate neurogenesis or a lack thereof.
MicroRNAs, by modulating gene expression, have been implicated as regulators of various cellular and physiological processes, including differentiation, proliferation, and cancer. Here, we study the role of microRNAs in Schwann cell (SC) differentiation by conditional removal of the microRNA processing enzyme Dicer1. We reveal that both male and female mice lacking Dicer1 in SC (Dicer1 conditional knock-outs) display a severe neurological phenotype resembling congenital hypomyelination. Ultrastructural analyses show that many SC lacking Dicer1 are stalled in differentiation at the promyelinating state and fail to myelinate axons. Gene expression analyses reveal a failure to extinguish genes characteristic of the undifferentiated state such as Sox2, Jun, and Ccnd1. Sox2 and Jun are well characterized negative regulators of SC differentiation. Consistent with Sox2/Jun maintenance, Egr2, a master regulator of the myelinating program, is drastically downregulated and likely accounts for the myelination defect. We posit a model wherein microRNAs are critical for downregulation of antecedent programs of gene expression. In SC differentiation, this is particularly relevant in the key developmental transition from a promyelinating to myelinating SC.
MicroRNAs regulate gene expression in diverse physiological scenarios. Their role in the control of morphogen related signaling pathways has been less studied, particularly in the context of embryonic Central Nervous System (CNS) development. Here, we uncover a role for microRNAs in limiting the spatiotemporal range of morphogen expression and function. Wnt1 is a key morphogen in the embryonic midbrain, and directs proliferation, survival, patterning and neurogenesis. We reveal an autoregulatory negative feedback loop between the transcription factor Lmx1b and a newly characterized microRNA, miR135a2, which modulates the extent of Wnt1/Wnt signaling and the size of the dopamine progenitor domain. Conditional gain of function studies reveal that Lmx1b promotes Wnt1/Wnt signaling, and thereby increases midbrain size and dopamine progenitor allocation. Conditional removal of Lmx1b has the opposite effect, in that expansion of the dopamine progenitor domain is severely compromised. Next, we provide evidence that microRNAs are involved in restricting dopamine progenitor allocation. Conditional loss of Dicer1 in embryonic stem cells (ESCs) results in expanded Lmx1a/b+ progenitors. In contrast, forced elevation of miR135a2 during an early window in vivo phenocopies the Lmx1b conditional knockout. When En1::Cre, but not Shh::Cre or Nes::Cre, is used for recombination, the expansion of Lmx1a/b+ progenitors is selectively reduced. Bioinformatics and luciferase assay data suggests that miR135a2 targets Lmx1b and many genes in the Wnt signaling pathway, including Ccnd1, Gsk3b, and Tcf7l2. Consistent with this, we demonstrate that this mutant displays reductions in the size of the Lmx1b/Wnt1 domain and range of canonical Wnt signaling. We posit that microRNA modulation of the Lmx1b/Wnt axis in the early midbrain/isthmus could determine midbrain size and allocation of dopamine progenitors. Since canonical Wnt activity has recently been recognized as a key ingredient for programming ESCs towards a dopaminergic fate in vitro, these studies could impact the rational design of such protocols.
Since their discovery, midbrain dopamine (DA) neurons have been researched extensively, in part because of their diverse functions and involvement in various neuropsychiatric disorders. Over the last few decades, reports have emerged that midbrain DA neurons were not a homogeneous group, but that DA neurons located in distinct anatomical locations within the midbrain had distinctive properties in terms of physiology, function, and vulnerability. Accordingly, several studies focused on identifying heterogeneous gene expression across DA neuron clusters. Here we review the importance of understanding DA neuron heterogeneity at the molecular level, previous studies detailing heterogeneous gene expression in DA neurons, and finally recent work which brings together previous heterogeneous gene expression profiles in a coordinated manner, at single cell resolution.
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