Excessive Wnt/beta-catenin signaling promotes midbrain floor plate neurogenesis, but results in vacillating dopamine progenitors

Abstract

The floor plate (FP), a ventral midline structure of the developing neural tube, has differential neurogenic capabilities along the anterior-posterior axis. The midbrain FP, unlike the hindbrain and spinal cord floor plate, is highly neurogenic and produces midbrain dopaminergic (mDA) neurons. Canonical Wnt/beta–catenin signaling, at least in part, is thought to account for the difference in neurogenic capability. Removal of beta–catenin results in mDA progenitor specification defects as well as a profound reduction of neurogenesis. To examine the effects of excessive Wnt/beta–catenin signaling on mDA specification and neurogenesis, we have analyzed a model wherein beta–catenin is conditionally stabilized in the Shh+ domain. Here, we show that the Foxa2+/Lmx1a+ domain is extended rostrally in mutant embryos, suggesting that canonical Wnt/beta–catenin signaling can drive FP expansion along the rostrocaudal axis. Although excess canonical Wnt/beta–catenin signaling generally promotes neurogenesis at midbrain levels, less tyrosine hydroxylase (Th)+, mDA neurons are generated, particularly impacting the Substantia nigra pars compacta. This is likely because of improper progenitor specification. Excess canonical Wnt/beta–catenin signaling causes downregulation of net Lmx1b, Shh and Foxa2 levels in mDA progenitors. Moreover, these progenitors assume a mixed identity to that of Lmx1a+/Lmx1b+/Nkx6-1+/Neurog1+ progenitors. We also show by lineage tracing analysis that normally, Neurog1+ progenitors predominantly give rise to Pou4f1+ neurons, but not Th+ neurons. Accordingly, in the mutant embryos, Neurog1+ progenitors at the midline generate ectopic Pou4f1+ neurons at the expense of Th+ mDA neurons. Our study suggests that an optimal dose of Wnt/beta–catenin signaling is critical for proper establishment of the mDA progenitor character. Our findings will impact embryonic stem cell protocols that utilize Wnt pathway reagents to derive mDA neuron models and therapeutics for Parkinson’s disease.

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

“…Consistent with the literature and our previous observations Echelard et al, 1993;Nouri et al, 2015;Puelles and Rubenstein, 2015), βgal + /Foxa2 + cells are observed in the midbrain, and well rostral to the ZLI, in the diencephalon (Fig. 1A-B′ At midbrain levels, Shh/Foxa2 derivatives have been previously described, and include DA and RN populations (Blaess et al, 2011;Hayes et al, 2011;Joksimovic et al, 2009).…”

“…For immunolabeling, tissue sections were processed as previously described (Nouri et al, 2015). Antibodies used were guinea-pig anti-Lmx1a (Yong Chao-Ma, Northwestern University, Evanston, IL, USA; 1:20,000), rabbit anti-Lmx1a (Millipore, AB10533; 1:1000), goat anti-Foxa2 (Santa Cruz, SC6554; 1:50), mouse anti-Pou4f1 (Santa Cruz, sc-31984; 1:50), rabbit and sheep anti-Th (Pel-Freeze, P40101-0/P60101-150; 1:500), mouse anti-Th (Sigma, T2928; 1:1000), mouse anti-En1 (DSHB, 4G11; 1:50), chicken anti-GFP (Abcam, ab13970; 1:1500 or 1:20,000), goat antiβgal (Biogenesis, 4600-1409; 1:1500), rabbit anti-βgal (Cappel, 55976; 1:1500), rabbit anti-Nr4a2 (Santa Cruz, SC-991; 1:50), rabbit anti-Pitx2 (Capra Science, PA 1020-100; 1:1000), rabbit anti-Pitx3 (Invitrogen/Zymed, 38-2850; 1:200), rat anti-Slc6a3 (Santa Cruz, sc-32258; 1:50), rabbit anti-Ddc (Abcam, ab3905; 1:500) and rabbit anti-Slc18a2 (EMD Millipore, AB1598P; 1:500).…”

“…Forced ectopic expression of Otx2 in the hindbrain floor plate (FP) results in DA neuron production in the hindbrain (Ono et al, 2007), whereas loss of Otx1/2 results in expanded hindbrain at the expense of midbrain (Omodei et al, 2008;Puelles et al, 2004;Simeone et al, 2002). Along the dorsoventral (D-V) axis, several studies have demonstrated that the Shh + /Foxa2 + domain is subdivided into at least two main domains defined by Lmx1a/b and Nkx6-1/Sim1/ Neurog1 (Andersson et al, 2006;Blaess et al, 2011;Hayes et al, 2011;Joksimovic et al, 2009;Nakatani et al, 2010;Nouri et al, 2015;Prakash et al, 2009). The Lmx1a/b domain is the origin of most DA neurons, whereas the Nkx6-1/Sim1/Neurog1 domain is the source of Pou4f1 + red nucleus (RN) neurons and other smaller populations.…”

“…In the early embryo, differentiating Th + DA neurons are observed in the midbrain with a caudal limit at the MHB. The anterior limit of this Th + DA cohort is approximately near the base of the zona limitans intrathalamica (ZLI), the boundary between presumptive developmental segmental units, prosomeres 2 and 3 (p2 and p3) (Joksimovic et al, 2009;Nouri et al, 2015;Puelles and Rubenstein, 2015). Because these neurons appear to be generated from the midbrain as well as presumptive p1, p2 and p3, some have called these mesodiencephalic DA neurons (Puelles and Rubenstein, 2015;Veenvliet and Smidt, 2014), as opposed to midbrain DA neurons (Arenas et al, 2015;Blaess and Ang, 2015).…”

“…Because these neurons appear to be generated from the midbrain as well as presumptive p1, p2 and p3, some have called these mesodiencephalic DA neurons (Puelles and Rubenstein, 2015;Veenvliet and Smidt, 2014), as opposed to midbrain DA neurons (Arenas et al, 2015;Blaess and Ang, 2015). Interestingly, most of the currently known DA progenitor markers, including Shh, Wnt1, Otx2, Neurog2, Lmx1a/b, Foxa1/2 and Msx1, among others, are expressed rostrally, well beyond the ZLI, into presumptive p3 of the diencephalon Andersson et al, 2006;Ellisor et al, 2012;Lahti et al, 2012;Nouri et al, 2015;Puelles and Rubenstein, 2015). Why DA neurons are not produced from the entire A-P axis of the Shh + /Foxa2 + /Lmx1a + mdFP, despite the presence of key signaling molecules and transcription factors, is a conundrum.…”