Differences in the spatiotemporal expression and epistatic gene regulation of the mesodiencephalic dopaminergic precursor markerPITX3during chicken and mouse development

Abstract

Mesodiencephalic dopaminergic (mdDA) neurons are located in the ventral mesencephalon and caudal diencephalon of all tetrapod species studied so far. They are the most prominent DA neuronal population and are implicated in control and modulation of motor, cognitive and rewarding/affective behaviors. Their degeneration or dysfunction is intimately linked to several neurological and neuropsychiatric human diseases. To gain further insights into their generation, we studied spatiotemporal expression patterns and epistatic interactions in chick embryos of selected marker genes and signaling pathways associated with mdDA neuron development in mouse. We detected striking differences in the expression patterns of the chick orthologs of the mouse mdDA marker genes Pitx3 and Aldh1a1, which suggests important differences between the species in the generation/generating of these cells. We also discovered that the sonic hedgehog signaling pathway is both necessary and sufficient for the induction of ectopic PITX3 expression in chick mesencephalon downstream of WNT9A-induced LMX1a transcription. These aspects of early chicken development resemble the ontogeny of zebrafish diencephalic DA neuronal populations, and suggest a divergence between birds and mammals during evolution.

Dose-Dependent and Subset-Specific Regulation of Midbrain Dopaminergic Neuron Differentiation by LEF1-Mediated WNT1/b-Catenin Signaling

“…HEK-293, COS-7 and SH-SY5Y cells (5 × 10 4 cells/well of a 24-well plate) were transfected with 200 ng/well BAT-gal plasmid [Addgene plasmid 20889 (Maretto et al, 2003)] using Lipofectamine 2000 and fixed after 24 h for ICC. Using Lipofectamine 2000 or TurboFect Transfection Reagent (Thermo Fisher Scientific), 5 × 10 4 or 2 × 10 5 HEK-293 cells/well of a 24-well plate were co-transfected with 200 ng/well of each reporter construct [Super8xTOPFlash or Super8xFOPFlash (Lie et al, 2005), or a wild-type or mutagenized 2514 bp mouse Pitx3 promoter fragment (-2425 to +89) in pGL3 basic vector (Peng et al, 2011)], 0.1 ng/well pRL-SV40 (Promega) internal transfection control and pcDNA3.1 (Thermo Fisher Scientific) "empty" vector to adjust for DNA content; alone or together with 200 ng/well or varying amounts of stabilized pcDNA3-S33Y b-catenin [Addgene plasmid 19286, (Kolligs et al, 1999)] with or without 200 ng/well or varying amounts of human LEF1 cDNA (Waterman et al, 1991), and vice versa, 166 ng/well pMES-Lmx1a-IRES-eGFP (Klafke et al, 2016), or 353.8 ng/well LMX1A siRNA (#144, Supplementary Table 1). The human LEF1 cDNA comprises the 399 aa ORF of full-length human LEF1 protein (including the N-terminal b-catenin binding domain) and was confirmed by sequencing (SEQLAB GmbH, Göttingen, Germany).…”

CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids

“…Several, including WNT3A and WNT10B have prominent roles in MBHB patterning in other species ( Lekven et al, 2003 ). WNT9A , whose chick ortholog is expressed in the ventral mesodiencephalic region of the brain and the dorsal midline of the anterior neural tube (fore-, mid-, hindbrain) in mice ( Klafke et al, 2016 ), was also enriched in SIX6-organoids. Conversely, WNT2B , which regulates peripheral retinal growth ( Cho and Cepko, 2006 ), was expressed in SIX6 + /POU4F2- and SIX6 + /POU4F2 + organoids.…”