Onecut transcription factors are required for the second phase of development of the A13 dopaminergic nucleus in the mouse

Abstract

The A13 dopaminergic nucleus belongs to the incerto-hypothalamic area. It is thought to exert autonomous roles by integrating sensory input to autonomic, neuroendocrine, and motor output. Although its early development has been well characterized, the factors that contribute to later steps of its formation remain unknown. Transcription factors of the Onecut family have been detected in the A13 nucleus, raising the question of possible roles of these factors during A13 development. Using a combination of immunofluorescence analyses on sections and after whole-mount labeling followed by 3D reconstructions, we further characterized the second phase of development of the A13 nucleus in the mouse, described the distribution of the Onecut proteins throughout A13 development, and analyzed the phenotype of this nucleus in single or compound mutant embryos for the Onecut factors. Here we show that A13 development can be divided into two successive phases. First, during radial migration toward the pial surface the A13 cells differentiate into dopaminergic neurons. Second, these cells gather in the vicinity of the third ventricle. Onecut factors are dynamically and differentially expressed in the A13 nucleus during these two phases of development. In Onecut mutant embryos, the A13 neurons differentiate normally but scatter in the diencephalon and fail to properly gather close to the third ventricle. Hence, Onecut factors are markers of the A13 nucleus throughout embryonic development. They are dispensable for the first phase of A13 development but are required for the second phase of development and for maintenance of this nucleus.

The Onecut Transcription Factors Regulate Differentiation and Distribution of Dorsal Interneurons during Spinal Cord Development

“…Primary antibodies against the following proteins were used: Arx (rabbit; 1:1000; kindly provided by J. Chelly), Bhlhb5 (goat; 1:700; Santa Cruz #sc-6045), Brn3 (goat; 1:500; Santa Cruz #sc-6026), Brn3a (guinea pig; 1:2000; kindly provided by J.E. Johnson; or mouse 1:1000; Santa Cruz #sc-8429), Dmrt3 (guinea pig; 1:5000; kindly provided by K. Kullander #170), Foxd3 (guinea pig; 1:5000; or rabbit; 1:2000; kindly provided by T. Müller), Foxp1 (goat; 1:1000; R&D Systems #AF4534), Foxp2 (goat; 1:1000; Abcam #ab1307), β-galactosidase (chicken; 1:2000; Abcam #ab9361), HNF6 (guinea pig; 1:2000; (Espana and Clotman, 2012b); or rabbit; 1:100; Santa Cruz #sc-13050; or sheep; 1:1000 R&D Systems #AF6277), Isl1/2 (goat; 1:3000; Neuromics #GT15051; or mouse; 1:6000; DSHB #39.4D5), Lbx1 (guinea pig; 1:10,000; or rabbit; 1:5000; kindly provided by T. Müller), Lhx1/5 (mouse; 1:1000; DSHB #4F2), Lmx1b (guinea pig; 1:10,000; or rabbit; 1:2000; kindly provided by T. Müller; or armenian hamster; 1:2; kindly provided by Y. Ono), MafB (rabbit; 1:5000; kindly provided by H. Wende), cMaf (rabbit; 1:3000; kindly provided by H. Wende), OC2 (rat; 1:400; (Clotman et al, 2005); or sheep; 1:500; R&D Systems #AF6294), OC3 (guinea pig; 1:6000; (Pierreux et al, 2004)), mOct6/Scip/Pou3f1 (rabbit; 1:50; kindly provided by Dies N. Meijer), Nurr1 (rat; 1:2000; kindly provided by Y. Ono), Olig3 (guinea pig; 1:6000; or rabbit; 1:2000; kindly provided by T. Müller), Otp (rabbit; 1:500; kindly provided by F. Vaccarino), Pax2 (rabbit; 1:1000; Covance PRB-276P), Phox2a (rabbit; 1:500; kindly provided by J.-F. Brunet), Prox1 (rabbit; 1:1000; Covance PRB-238C), Tlx3 (guinea pig; 1:10,000; or rabbit; 1:2000; kindly provided by T. Müller), Wt1 (rabbit; 1:2000; Santa Cruz #sc-192). Secondary antibodies donkey anti-guinea pig/AlexaFluor 488, 594 or 647, anti-mouse/AlexaFluor 488, 594 or 647, anti-rabbit/AlexaFluor 594 or 647, anti-goat/AlexaFluor 488, anti-rat/AlexaFluor 488 or 647, anti-sheep/AlexaFluor 594, anti-armenian hamster/AlexaFluor 594, purchased from ThermoFisher Scientific or Jackson Laboratories were used at dilution 1:2000.…”

“…Onecut factors, namely Hepatocyte Nuclear Factor-6 (HNF-6, or OC-1), OC-2 and OC-3, are transcriptional activators detected in liver, pancreas and CNS during embryonic development (Lemaigre et al, 1996; Landry et al, 1997; Jacquemin et al, 1999, 2003; Vanhorenbeeck et al, 2002). In the CNS, they regulate the production (Espana and Clotman, 2012a), the diversification (Roy et al, 2012; Francius and Clotman, 2014), the distribution (Espana and Clotman, 2012a,b; Audouard et al, 2013) and the maintenance (Espana and Clotman, 2012a,b; Stam et al, 2012) of specific neuronal populations, as well as the formation of neuromuscular junctions (Audouard et al, 2012). In motor neurons, they are required to maintain levels of Isl1 expression necessary to support the diversification of this population into distinct subsets (Roy et al, 2012).…”

Vsx1 Transiently Defines an Early Intermediate V2 Interneuron Precursor Compartment in the Mouse Developing Spinal Cord

“…The following primary antibodies and dilution were used: mouse anti-Ascl1 at 1:200 (BD #556604), guinea-pig anti-Ascl1 at 1:10,000 (Kim et al, 2008), mouse anti-beta III tubulin at 1:5000 (Chemicon #MAB1637), goat anti-BhlhB5 at 1:1000 (Santa Cruz #sc-6045), rabbit or rat anti-BhlhB5 at 1:2000 (Ross et al, 2012), sheep anti-Chx10 (Vsx2) at 1:500 (Exalpha Biologicals #X1179P), goat anti Dll4 at 1:200 (R&D System #AF1389), rabbit or guinea pig anti-Foxd3 at 1:5000 (Müller et al, 2005), rat anti-Gata3 at 1: 20 (Panayi et al, 2010), guinea-pig anti-Insm1 at 1:10,000 (Welcker et al, 2013), mouse anti-Islet 1/2 at 1:6000 (DSHB #39.4D5), mouse anti-Lhx3 at 1:1000 (DSHB #67.4E12), mouse anti-Lhx1/5 at 1:2000 (DSHB # 4F2), rat anti-Nkx6.1 at 1:2 (Ono et al, 2007), guinea pig anti-OC-1 at 1:6000 (Espana and Clotman, 2012), sheep anti-OC-1 at 1:250 (R&D System #AF6277), rat anti-OC-2 at 1:400 (Clotman et al, 2005), mouse anti-p27 kip1 at 1:2000 (BD), mouse anti-Pax6 at 1:1000 (DSHB #PAX6), mouse anti-phospho-Histone H3 at 1:1000 (Abcam ab14955), rabbit or guinea pig anti-Prdm8 at 1:1000 (Ross et al, 2012), goat anti-Prox1 at 1:100 (R&D Systems # AF2727), mouse anti-Shox2 at 1:500 (Abcam #ab55740), goat anti-Sox 1 at 1:500 (Santa Cruz #sc-17318), rabbit anti-Vsx1 at 1:500 (Clark et al, 2008). …”

Onecut transcription factors act upstream of Isl1 to regulate spinal motoneuron diversification

“…These proteins are detected during development in several endodermal derivatives including liver and pancreas, where they redundantly control different aspects of cell fate decision and morphogenetic processes (Jacquemin et al, 2000;Clotman et al, 2002;Jacquemin et al, 2003a;Pierreux et al, 2006;Vanhorenbeeck et al, 2007). They are also present in the embryonic CNS (Landry et al, 1997;Jacquemin et al, 2003b;Vanhorenbeeck et al, 2007), where they regulate the generation, maintenance or the projections of different encephalic structures (Hodge et al, 2007;Chakrabarty et al, 2012;Espana and Clotman, 2012a;Espana and Clotman, 2012b) and of a spinal interneuron population (Stam et al, 2012). In the ventral spinal cord, the three OC genes are differentially and dynamically expressed during the early steps of MN differentiation (Francius and Clotman, 2010), suggesting that OC factors might contribute to spinal MN development.…”