Genetic dissection of midbrain dopamine neuron development in vivo

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.

Dynamic temporal requirement ofWnt1in midbrain dopamine neuron development

“…Genotyping was carried out as previously described . Rosa26 lox-STOP-lox-tdTomato (R26 tdTomato ) mice were used for lineage tracing and Wnt1-Venus mice were used to detect Wnt1 expression (Madisen et al, 2010;Brown et al, 2011;Ellisor et al, 2012). All Cre and reporter lines in this study have been validated and described elsewhere (Kimmel et al, 2000;Li et al, 2002;Zervas et al, 2004;Madisen et al, 2010;Brown et al, 2011;Hagan and Zervas, 2012;Hayes et al, 2011).…”

“…The v.Mes of Wnt1-Venus embryos was microdissected as previously described Ellisor et al, 2012). Briefly, we isolated the v.Mes under a stereofluorescence microscope.…”

“…We investigated Wnt1, which is initially expressed broadly in the mes, but becomes restricted to distinct domains over time (Wilkinson et al, 1987;Echelard et al, 1994;Zervas et al, 2004). Mice with a null allele of Wnt1 or with a spontaneous mutation in Wnt1 do not form the Mb and Cb, demonstrating the importance of WNT1 for these domains (McMahon and Bradley, 1990;McMahon et al, 1992;Thomas et al, 1991;Bally-Cuif et al, 1995;Ellisor et al, 2012). WNT1 functions through β-catenin-mediated WNT signaling and β-catenin regulates cell cycle exit and MbDA neuron development (Tang et al, 2009;Tang et al, 2010).…”

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

“…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.…”

“…Engrailed 1 (En1) is known to be expressed in the midbrain beginning at about embryonic day (E) 8.0 (Chi et al, 2003;Davis and Joyner, 1988) and encompasses the majority of the DA primordium, excluding a small number of Th + neurons at the rostral-most extent (Lahti et al, 2012). Engrailed 1 and 2 (En1/2) are important for DA neuron generation and survival (Alberi et al, 2004;Alvarez-Fischer et al, 2011;Alves dos Santos and Smidt, 2011;Ellisor et al, 2012;Nordström et al, 2015;Rekaik et al, 2015;Simon et al, 2001;Veenvliet et al, 2013). Moreover, engrailed proteins are involved in various boundary formation systems such as the diencephalic-dorsal midbrain boundary (Matsunaga et al, 2000;Scholpp et al, 2003) and a D-V boundary in the limb apical ectodermal ridge (Kimmel et al, 2000).…”