A novel floor plate boundary defined by adjacentEn1andDbx1microdomains distinguishes midbrain dopamine and hypothalamic neurons

Abstract

The mesodiencephalic floor plate (mdFP) is the source of diverse neuron types. Yet, how this structure is compartmentalized has not been clearly elucidated. Here, we identify a novel boundary subdividing the mdFP into two microdomains, defined by engrailed 1 (En1) and developing brain homeobox 1 (Dbx1). Utilizing simultaneous dual and intersectional fate mapping, we demonstrate that this boundary is precisely formed with minimal overlap between En1 and Dbx1 microdomains, unlike many other boundaries. We show that the En1 microdomain gives rise to dopaminergic (DA) neurons, whereas the Dbx1 microdomain gives rise to subthalamic (STN), premammillary (PM) and posterior hypothalamic (PH) populations. To determine whether En1 is sufficient to induce DA neuron production beyond its normal limit, we generated a mouse strain that expresses En1 in the Dbx1 microdomain. In mutants, we observed ectopic production of DA neurons derived from the Dbx1 microdomain, at the expense of STN and PM populations. Our findings provide new insights into subdivisions in the mdFP, and will impact current strategies for the conversion of stem cells into DA neurons.

Pluripotent Stem Cell Therapies for Parkinson Disease: Present Challenges and Future Opportunities

“…Furthermore, single cell RNA sequencing of Lmx1a+ precursors in the mouse suggests that the midbrain floor-plate markers FOXA2 and LMX1A are not specific to just mDA progenitors, but also mark the more anterior, subthalamic nucleus (STN) precursors which go on to give rise to glutamatergic neurons ( Kee et al, 2017 ). Those results are in agreement with the idea that the midbrain-diencephalic floor-plate region is subdivided into two distinct domains by En1 for posterior, midbrain (mDA) identity, and Dbx1 for STN neuron population ( Nouri and Awatramani, 2017 ). Importantly, lowering CHIR exposure (0.4–0.6 μM) resulted in a bias toward Lmx1a+/Pitx2+ positive cells (STN neuron) while the percentage of STN neurons was greatly decreased at higher concentrations of CHIR (0.8–1 μM) with increased efficiencies of mDA neuron differentiation ( Kee et al, 2017 ).…”

Mapping projections of molecularly defined dopamine neuron subtypes using intersectional genetic approaches

“…The Dat locus has the advantage of being largely restricted to midbrain DA neurons 16 , barring few other neuronal populations (e.g. ventral premmamillary nucleus, PMv 20 ). Together these complementary approaches form a powerful arsenal to target molecularly distinct DA neurons.…”

“…Adults mice (more than 8 weeks of age) were perfused with cold 4% PFA in PBS, the brains were dissected and fixed overnight in the same solution, cryoprotected in 30% sucrose PBS solution, sectioned at 25μm using a freezing microtome, and stored in −20°C in cryoprotectant solution. For immunofluorescence on free-floating sections, we initially rinsed in PBS, blocked in 5% donkey serum, 0.3% Triton X-100 in PBS, and incubated overnight at 4°C with primary antibodies diluted in blocking solution: rabbit anti-ALDH1A1 (Cat# ab23375; Abcam; 1:200) 8 , goat anti-βgal (Cat# 4600–1409; Biogenesis; 1:1500) 20 , rabbit anti-βgal (Cat# 55976; Cappel; 1:1500) 63 , goat anti-FOXA2 (Cat# sc-6554; Santa Cruz; 1:50) 64 , chicken anti-GFP (Cat# ab13970; abcam; 1:1,500) 20 , rabbit anti-NURR1 (Cat# sc-990; Santa Cruz; 1:500) 8 , goat anti-OTX2 (Cat# GT15095; Neuromics; 1:200) 8 , rabbit anti-SOX6 (Cat# ab30455; Abcam; 1:500) 8 , rabbit anti-TH (Cat# P40101; Pel-Freez; 1:500) 20 , sheep anti-TH (Cat# P60101–0; Pel-Freez; 1:500) 8 , and rabbit anti-VIP (Cat# ab43841; Abcam; 1:500) 8 . Sections were rinsed in PBS+0.05% Tween20 solution and incubated with secondary antibodies generated in donkey and conjugated with appropriate fluorophores (Alexa488, Alexa555, Alexa647; Molecular Probes) diluted 1:250 in blocking solution and counterstained with DAPI (Sigma), rinsed in PBS+0.05% Tween20, and finally coverslipped with Gelvatol.…”

Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors

“…The principles discussed here apply to all genetically targeted recombinase systems, including lox variants, Flp-frt, and Drerox systems. For example, the En1-Dre KI mouse line shows variable paternal germline recombination (Nouri and Awatramani, 2017). Moreover, the problem of unwanted recombination may be compounded by intersectional strategies involving multiple recombinases.…”