An Lmx1b-miR135a2 regulatory circuit modulates Wnt1/Wnt signaling and determines the size of the midbrain dopaminergic progenitor pool.

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.

Spatiotemporally separable Shh domains in the midbrain define distinct dopaminergic progenitor pools.

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.

MicroRNA-deficient Schwann cells display congenital hypomyelination.

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.

Wnt antagonism of Shh facilitates midbrain floor plate neurogenesis.

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.

Transcriptional regulation by Pax3 and TGFbeta2 signaling: a potential gene regulatory network in neural crest development.

Pax3 regulates neural crest cell migration and is critical during neural crest development. TGFbs modify neural crest cell migration and differentiation. TGFbeta2 nullizygous embryos (TGFbeta2(-/-)Pax3(+/+)) display open neural tube and bifid spine, whereas in wild type embryos, the neural tube is closed. In previous work, we have demonstrated that Pax3 regulates TGFbeta2 by directly binding to cis-regulatory elements on its promoter. In this study, we found that the TGFbeta2 nullizygous phenotype can be reversed to the wild type phenotype by down-regulating one allele of Pax3, as in TGFbeta2(-/-)Pax3(+/-) embryos obtained through breeding TGFb2(+/-)Pax3(+/-) mice. The data in this paper suggest that Pax3 and TGFbeta2 interact in a coordinated gene regulatory network, linked by common downstream effector genes, to bring about this phenotypic reversal. Downstream effectors may include Hes1, Ngn2 and Sox9, as well as other genes involved in neuronal differentiation.

Regulation of murine TGFbeta2 by Pax3 during early embryonic development.

Previously our laboratory identified TGFbeta2 as a potential downstream target of Pax3 by utilizing microarray analysis and promoter data base mining (Mayanil, C. S. K., George, D., Freilich, L., Miljan, E. J., Mania-Farnell, B. J., McLone, D. G., and Bremer, E. G. (2001) J. Biol. Chem. 276, 49299-49309). Here we report that Pax3 directly regulates TGFbeta2 transcription by binding to cis-regulatory elements within its promoter. Chromatin immunoprecipitation revealed that Pax3 bound to the cis-regulatory elements on the TGFbeta2 promoter (GenBanktrade mark accession number AF118263). Both TGFbeta2 promoter-luciferase activity measurements in transient cotransfection experiments and electromobility shift assays supported the idea that Pax3 regulates TGFbeta2 by directly binding to its cis-regulatory regions. Additionally, by using a combination of co-immunoprecipitation and chromatin immunoprecipitation, we show that the TGFbeta2 cis-regulatory elements between bp 741-940 and bp 1012-1212 bind acetylated Pax3 and are associated with p300/CBP and histone deacetylases. The cis-regulatory elements between bp 741 and 940 in addition to associating with acetylated Pax3 and HDAC1 also associated with SIRT1. Whole mount in situ hybridization and quantitative real time reverse transcription-PCR showed diminished levels of TGFbeta2 transcripts in Pax3(-/-) mouse embryos (whose phenotype is characterized by neural tube defects) as compared with Pax3(+/+) littermates (embryonic day 10.0; 30 somite stage), suggesting that Pax3 regulation of TGFbeta2 may play a pivotal role during early embryonic development.