Molecular Organization and Timing of Wnt1 Expression Define Cohorts of Midbrain Dopamine Neuron Progenitors In Vivo

Department of Neuroscience, Brown University, Providence, Rhode Island 02903, USA.
The Journal of Comparative Neurology (Impact Factor: 3.23). 10/2011; 519(15):2978-3000. DOI: 10.1002/cne.22710
Source: PubMed


Midbrain dopamine (MbDA) neurons are functionally heterogeneous and modulate complex functions through precisely organized anatomical groups. MbDA neurons are generated from Wnt1-expressing progenitors located in the ventral mesencephalon (vMes) during embryogenesis. However, it is unclear whether the progenitor pool is partitioned into distinct cohorts based on molecular identity and whether the timing of gene expression uniquely identifies subtypes of MbDA neurons. In this study we show that Wnt1-expressing MbDA progenitors from embryonic day (E)8.5-12.5 have dynamic molecular identities that correlate with specific spatial locations in the vMes. We also tested the hypothesis that the timing of Wnt1 expression in progenitors is related to the distribution of anatomically distinct cohorts of adult MbDA neurons using genetic inducible fate mapping (GIFM). We demonstrate that the Wnt1 lineage contributes to specific cohorts of MbDA neurons during a 7-day epoch and that the contribution to MbDA neurons predominates over other ventral Mb domains. In addition, we show that calbindin-, GIRK2-, and calretinin-expressing MbDA neuron subtypes are derived from Wnt1-expressing progenitors marked over a broad temporal window. Through GIFM and quantitative analysis we demonstrate that the Wnt1 lineage does not undergo progressive lineage restriction, which eliminates a restricted competence model of generating MbDA diversity. Interestingly, we uncover that two significant peaks of Wnt1 lineage contribution to MbDA neurons occur at E9.5 and E11.5. Collectively, our findings delineate the temporal window of MbDA neuron generation and show that lineage and timing predicts the terminal distribution pattern of MbDA neurons.

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    • "It has been shown that when canonical WNT-signaling is stabilized in the FP, SHH expression is abolished and TH+ neurons appear in the hindbrain, suggesting that WNT-signaling may be involved in mdDA neuronal development [38]. As suggested by several studies, the WNT-family member, which could be involved in the early differentiation of mdDA neurons is WNT1 [19], [40], [43], [57]. For instance, in Wnt1 null-mutants, mdDA neurons do not differentiate properly [41] whereas a more caudal expression of WNT1 and stabilized β-catenin in the caudal FP results in an increase in caudal expression of TH, Nurr1, and Pitx3 [38], [41]. "
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    ABSTRACT: Sonic Hedgehog (SHH) and WNT proteins are key regulators in many developmental processes, like embryonic patterning and brain development. In the brain, SHH is expressed in a gradient starting in the floor plate (FP) progressing ventrally in the midbrain, where it is thought to be involved in the development and specification of mesodiencephalic dopaminergic (mdDA) neurons. GLI2A-mediated SHH-signaling induces the expression of Gli1, which is inhibited when cells start expressing SHH themselves. To determine whether mdDA neurons receive GLI2A-mediated SHH-signaling during differentiation, we used a BAC-transgenic mouse model expressing eGFP under the control of the Gli1 promoter. This mouse-model allowed for mapping of GLI2A-mediated SHH-signaling temporal and spatial in the mouse midbrain. Since mdDA neurons are born from E10.5, peaking at E11.0-E12.0, we examined Gli1-eGFP embryos at E11.5, E12.5, and E13.5, indicating whether Gli1 was induced before or during mdDA development and differentiation. Our data indicate that GLI2A-mediated SHH-signaling is not involved in mdDA neuronal differentiation. However, it appears to be involved in the differentiation of neurons which make up a subset of the red nucleus (RN). In order to detect whether mdDA neuronal differentiation may be under the control of canonical WNT-signaling, we used a transgenic mouse-line expressing LacZ under the influence of stable β-catenin. Here, we show that TH+ neurons of the midbrain receive canonical WNT-signaling during differentiation. Therefore, we suggest that early SHH-signaling is indirectly involved in mdDA development through early patterning of the midbrain area, whereas canonical WNT-signaling is directly involved in the differentiation of the mdDA neuronal population.
    Full-text · Article · May 2014 · PLoS ONE
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    • "Genetic fate-mapping experiments have indicated that both SNc and VTA DA neurons are generated by proliferating progenitors in the ventricular zone (VZ) of the mFP, which can be identified by their expression of two key morphogenic factors, Wnt1 (Brown et al., 2011) and another lipid-modified glycoprotein , Sonic hedgehog (Shh) (Joksimovic et al., 2009b; Blaess et al., 2011; Hayes et al., 2011). Interestingly, Wnt1-expressing progenitors labeled from E7.5 to E13.5 contribute to mDA neurons much more than other cell types in the ventral midbrain (VM), with a similar time-line and extent to SNc and VTA mDA subtypes (Brown et al., 2011). Progenitor cells in the VZ of the mFP, including radial glia-like cells (Bonilla et al., 2008), undergo neurogenesis and give rise to postmitotic migratory cells. "
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    ABSTRACT: Wnts are a highly conserved family of lipid-modified glycoproteins that work as morphogens to activate several signaling pathways, leading to remodeling of the cytoskeleton and the regulation of gene transcription. Wnt signaling regulates multiple cellular functions and cell systems, including the development and maintenance of midbrain dopaminergic (mDA) neurons. These neurons are of considerable interest for regenerative medicine because their degeneration results in Parkinson's disease (PD). This review focuses on new advances in understanding key functions of Wnts in mDA neuron development and using novel tools to regulate Wnt signaling in regenerative medicine for PD. Particularly, recent reports indicate that appropriate levels of Wnt signaling are essential to improve the quantity and quality of stem cell- or reprogrammed cell-derived mDA neurons to be used in drug discovery and cell replacement therapy for PD.
    Full-text · Article · Jan 2014 · Journal of Molecular Cell Biology
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    • "Regions 1 (white arrowhead), 2 (red arrowhead), and 3 (blue arrowhead) indicate diencephalon, v. mes flexure, and caudal v. mes, respectively. Note that the Wnt1(GFP) þ/marker þ domains are bilateral stripes (Brown et al., 2011) and one side is shown in these sagittal sections. (C) Mutants had a diminutive v. mes with mosaic clusters of Wnt1(GFP) þ/OTX2 þ mutant progenitors. "
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    ABSTRACT: Midbrain dopamine (MbDA) neurons are partitioned into medial and lateral cohorts that control complex functions. However, the genetic underpinnings of MbDA neuron heterogeneity are unclear. While it is known that Wnt1-expressing progenitors contribute to MbDA neurons, the role of Wnt1 in MbDA neuron development in vivo is unresolved. We show that mice with a spontaneous point mutation in Wnt1 have a unique phenotype characterized by the loss of medial MbDA neurons concomitant with a severe depletion of Wnt1-expressing progenitors and diminished LMX1a-expressing progenitors. Wnt1 mutant embryos also have alterations in a hierarchical gene regulatory loop suggesting multiple gene involvement in the Wnt1 mutant MbDA neuron phenotype. To investigate this possibility, we conditionally deleted Gbx2, Fgf8, and En1/2 after their early role in patterning and asked whether these genetic manipulations phenocopied the depletion of MbDA neurons in Wnt1 mutants. The conditional deletion of Gbx2 did not result in re-positioning or distribution of MbDA neurons. The temporal deletion of Fgf8 did not result in the loss of either LMX1a-expressing progenitors nor the initial population of differentiated MbDA neurons, but did result in a complete loss of MbDA neurons at later stages. The temporal deletion and species specific manipulation of En1/2 demonstrated a continued and species specific role of Engrailed genes in MbDA neuron development. Notably, our conditional deletion experiments revealed phenotypes dissimilar to Wnt1 mutants indicating the unique role of Wnt1 in MbDA neuron development. By placing Wnt1, Fgf8, and En1/2 in the context of their temporal requirement for MbDA neuron development, we further deciphered the developmental program underpinning MbDA neuron progenitors.
    Preview · Article · Oct 2012 · Developmental Biology
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