Foxn4 acts synergistically with Mash1 to specify subtype identity of V2 interneurons in the spinal cord

Abstract

Neuronal subtype diversification is essential for the establishment of functional neural circuits, and yet the molecular events underlying neuronal diversity remain largely to be defined. During spinal neurogenesis, the p2 progenitor domain, unlike others in the ventral spinal cord, gives rise to two intermingled but molecularly distinct subtypes of interneurons, termed V2a and V2b. We show here that the Foxn4 winged helix͞forkhead transcription factor is coexpressed with the bHLH factor Mash1 in a subset of p2 progenitors. Loss of Foxn4 function eliminates Mash1 expression and V2b neurons and causes a fate-switch to V2a neurons, whereas the absence of Mash1 displays a similar but less severe phenotype. Overexpression of Foxn4 alone in spinal neural progenitors promotes the V2a fate at the expense of the V2b fate, whereas Mash1 suppresses both the V2a and V2b fates. However, coexpression of both Foxn4 and Mash1 promotes the V2b fate while inhibiting the V2a fate, indicating that Foxn4 cooperates with Mash1 to specify the identity of V2b neurons from bipotential p2 progenitors.winged-helix͞forkhead ͉ transcription factor ͉ p2 progenitor ͉ spinal neurogenesis

Lhx3-Chx10 Reticulospinal Neurons in Locomotor Circuits

“…We have demonstrated that Lhx3-Chx10 neurons meet the first five of these criteria, and we thus suggest that they are excellent candidate neurons for mediating locomotor commands. With respect to the first four criteria, we provide data demonstrating that Lhx3/Chx10 neurons are reticulospinal, similar to findings in chick and mouse embryos and larvae (Cepeda-Nieto et al, 2005;Kinkhabwala et al, 2011), and are glutamatergic, key requirements as previously reported in mammals (Douglas et al, 1993;Hägglund et al, 2010;Talpalar et al, 2011), and lower vertebrates (Xenopus tadpole, zebrafish, and lamprey;Brocard and Dubuc, 2003;Li et al, 2004;Kinkhabwala et al, 2011;Kimura et al, 2013). In addition, zebrafish Alx neurons (homologues of Chx10 neurons) were recently identified as hindbrain glutamatergic neurons, some of which are rhythmically active during swimming, project to the spinal cord, and drive spinal locomotor circuits (Kinkhabwala et al, 2011;Kimura et al, 2013).…”

“…In the spinal cord, Lhx3-expressing interneurons (V2 neurons) are known to differentiate into V2a neurons, which are Chx10ϩ, V2b neurons, which express Gata2/3, and a subgroup of V2c neurons, which express Gata2/3 and Sox1 (Karunaratne et al, 2002;Li et al, 2005;Al-Mosawie et al, 2007;Lundfald et al, 2007;Panayi et al, 2010). We therefore asked whether a similar genetic lineage is seen in the brainstem.…”

“…During spinal cord development, cells from the p2 progenitor domain express a combination of transcription factors, including Lhx3 (Zhou et al, 2000;Karunaratne et al, 2002;Li et al, 2005). These Lhx3ϩ V2 cells diversify into several distinct interneuronal populations: Chx10-expressing glutamatergic V2a and Gata2/3-expressing GABA/glycinergic V2b-c interneurons ( , 2008).…”

A Comprehensive Negative Regulatory Program Controlled by Brn3b to Ensure Ganglion Cell Specification from Multipotential Retinal Precursors

“…The preparation of retinal sections, immunofluorescent labeling of retinal sections, and quantification of immunoreactive cells were performed as described previously (Mo et al, 2004;Li et al, 2005). The following primary antibodies were used: anti-tyrosine hydroxylase (TH) (sheep polyclonal; Millipore Bioscience Research Reagents, Temecula, CA); anti-syntaxin (mouse monoclonal; Sigma St. Louis, MO); anti-␤-gal (rabbit polyclonal; Cappel Laboratories, Durham, NC); anti-GABA (rabbit polyclonal; Sigma); anti-GABA transporter 1 (GAT-1) (rabbit polyclonal; Millipore Bioscience Research Reagents); anti-calbindin-D28K (rabbit polyclonal; Swant, Bellizona, Switzerland); anti-Neurod1 (neurogenic differentiation 1) (mouse monoclonal and goat polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA); anti-Lim1/2 (mouse monoclonal; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA); anti-Brn3a (mouse monoclonal; Millipore Bioscience Research Reagents); anti-Brn3b (goat polyclonal; Santa Cruz Biotechnology); anti-Brn3c (rabbit polyclonal) (Xiang et al, 1995); anti-retinoid X receptor ␥ (RXR␥) (rabbit polyclonal; Santa Cruz Biotechnology); anti-Pax6 (paired box gene 6) (rabbit polyclonal; Millipore Bioscience Research Reagents); anti-GFP [rabbit polyclonal (MBL International, Woburn, MA); goat polyclonal (Abcam, Cambridge, MA)]; anti-recoverin (rabbit polyclonal) (Dizhoor et al, 1991); anti-glycine transporter 1 (GLYT1) (goat polyclonal; Millipore Bioscience Research Reagents); anti-Chx10 (ceh-10 homeodomain containing homolog) (sheep polyclonal; Exalpha Biologicals, Maynard, MA); and anti-protein kinase C␣ (PKC␣) (mouse monoclonal; GE Healthcare, Little Chalfont, UK).…”

Generation of V2a Interneurons from Mouse Embryonic Stem Cells

“…Cells in the p2 progenitor domain express Irx3, Lhx3, and Foxn4 [19][20][21][23][24][25] and mature into three distinct interneuron classes, V2a, V2b, and V2c. V2a interneurons are excitatory, glutamatergic, and express Chx10 and Lhx3 [17,18,26], whereas V2b interneurons are inhibitory, GABAergic/glycinergic, and express Gata3 [24,[27][28][29][30][31][32]. Newly identified V2c interneurons arise from a subset of V2b interneurons, and their function in CPG networks is still unknown [33,34].…”