AB10533 Sigma-AldrichAnti-LMX-1 Antibody

Cerebellum development involves the coordinated production of multiple neuronal cell types. The cerebellar primordium contains two germinative zones, the rhombic lip (RL) and the ventricular zone (VZ), which generate different types of glutamatergic and GABAergic neurons, respectively. What regulates the specification and production of glutamatergic and GABAergic neurons as well as the subtypes for each of these two broad classes remains largely unknown. Here we demonstrate with conditional genetic approaches in mice that SMAD4, a major mediator of BMP and TGFβ signaling, is required early in cerebellar development for maintaining the RL and generating subsets of RL-derived glutamatergic neurons, namely neurons of the deep cerebellar nuclei, unipolar brush cells, and the late cohort of granule cell precursors (GCPs). The early cohort of GCPs, despite being deficient for SMAD4, is still generated. In addition, the numbers of GABAergic neurons are reduced in the mutant and the distribution of Purkinje cells becomes abnormal. These studies demonstrate a temporally and spatially restricted requirement for SMAD4 in generating subtypes of cerebellar neurons.

Experiments in mice deficient for Nurr1 or expressing the dominant-negative FGF receptor (FGFR) identified orphan nuclear receptor Nurr1 and FGFR1 as essential factors in development of mesencephalic dopaminergic (mDA) neurons. FGFR1 affects brain cell development by two distinct mechanisms. Activation of cell surface FGFR1 by secreted FGFs stimulates proliferation of neural progenitor cells, whereas direct integrative nuclear FGFR1 signaling (INFS) is associated with an exit from the cell cycle and neuronal differentiation. Both Nurr1 and INFS activate expression of neuronal genes, such as tyrosine hydroxylase (TH), which is the rate-limiting enzyme in dopamine synthesis. Here, we show that nuclear FGFR1 and Nurr1 are expressed in the nuclei of developing TH-positive cells in the embryonic ventral midbrain. Both nuclear receptors were effectively co-immunoprecipitated from the ventral midbrain of FGF-2-deficient embryonic mice, which previously showed an increase of mDA neurons and enhanced nuclear FGFR1 accumulation. Immunoprecipitation and co-localization experiments showed the presence of Nurr1 and FGFR1 in common nuclear protein complexes. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrated the Nurr1-mediated shift of nuclear FGFR1-EGFP mobility toward a transcriptionally active population and that both Nurr1 and FGFR1 bind to a common region in the TH gene promoter. Furthermore, nuclear FGFR1 or its 23-kDa FGF-2 ligand (FGF-2(23)) enhances Nurr1-dependent activation of the TH gene promoter. Transcriptional cooperation of FGFR1 with Nurr1 was confirmed on isolated Nurr1-binding elements. The proposed INFS/Nurr1 nuclear partnership provides a novel mechanism for TH gene regulation in mDA neurons and a potential therapeutic target in neurodevelopmental and neurodegenerative disorders.

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.

The ventral midbrain (vMb) is organized into distinct anatomical domains and contains cohorts of functionally distinct subtypes of midbrain dopamine (mDA) neurons. We tested the hypothesis that genetic history and timing of gene expression within mDA neuron progenitors impart spatial diversity. Using genetic inducible fate mapping to mark the Sonic hedgehog (Shh) and Gli1 lineages at varying embryonic stages, we performed a quantitative and qualitative comparison of the two lineages' contribution to the mDA neuron domains. Dynamic changes in Shh and Gli1 expression in the vMb primordia delineated their spatial contribution to the embryonic day 12.5 vMb: Both lineages first contributed to the medial domain, but subsequently the Gli1 lineage exclusively contributed to the lateral vMb while the Shh lineage expanded more broadly across the vMb. The contribution of both lineages to the differentiated mDA neuron domain was initially biased anteriorly and became more uniform across the anterior/posterior vMb throughout development. Our findings demonstrate that the early Shh and Gli1 lineages specify mDA neurons of the substantia nigra pars compacta while the late Shh and Gli1 lineages maintain their progenitor state longer in the posterior vMb to extend the production of mDA neurons in the ventral tegmental area. Together, our study demonstrates that the timing of gene expression along with the genetic lineage (Shh or Gli1) within the neural progenitors segregate mDA neurons into distinct spatial domains.