Tlx1/3 and Ptf1a Control the Expression of Distinct Sets of Transmitter and Peptide Receptor Genes in the Developing Dorsal Spinal Cord

Abstract

Establishing the pattern of expression of transmitters and peptides as well as their receptors in different neuronal types is crucial for understanding the circuitry in various regions of the brain. Previous studies have demonstrated that the transmitter and peptide phenotypes in mouse dorsal spinal cord neurons are determined by the transcription factors Tlx1/3 and Ptf1a. Here we show that these transcription factors also determine the expression of two distinct sets of transmitter and peptide receptor genes in this region. We have screened the expression of 78 receptor genes in the spinal dorsal horn by in situ hybridization. We found that receptor genes Gabra1, Gabra5, Gabrb2, Gria3, Grin3a, Grin3b, Galr1, and Npy1r were preferentially expressed in Tlx3-expressing glutamatergic neurons and their derivatives, and deletion of Tlx1 and Tlx3 resulted in the loss of expression of these receptor genes. Furthermore, we obtained genetic evidence that Tlx3 uses distinct pathways to control the expression of receptor genes. We also found that receptor genes Grm3, Grm4, Grm5, Grik1, Grik2, Grik3, and Sstr2 were mainly expressed in Pax2-expressing GABAergic neurons in the spinal dorsal horn, and their expression in this region was abolished or markedly reduced in Ptf1a and Pax2 deletion mutant mice. Together, our studies indicate that Tlx1/3 and Ptf1a, the key transcription factors for fate determination of glutamatergic and GABAergic neurons in the dorsal spinal cord, are also responsible for controlling the expression of two distinct sets of transmitter and peptide receptor genes.

Control of Axon Guidance and Neurotransmitter Phenotype of dB1 Hindbrain Interneurons by Lim-HD Code

“…For integration of the reporter gene into the chick genome, the PiggyBac (PB) DNA-integration method was applied (Wang et al, 2010), in which a Cre-conditional reporter cassette (mGFP) was cloned between the two PB arms (PB-CAG-LoxP-STOP-LoxP-mGFP-PB) and electroporated along with the Ptf1a enhancer plasmid and the Pbase transposase. For tracing synaptic connections, a reporter plasmid containing SV2-GFP synaptic reporter cassette was cloned between the two PB arms (PB-CAG-LoxP-STOP-LoxP-SV2-GFP-PB) (Kohl et al, 2012;Hadas et al, 2014) and electroporated into the embryonic hindbrain along with the Ptf1a enhancer and the Pbase transposase. To trace synaptic targets in dB1 or dA1 following modification of Lim-HD expression, the Ptf1a enhancer or EdI1 enhancer was cloned upstream to PhiC31 recombinase and electroporated along with a PhiC31-Lhx1/2-conditional Cre cassette (pCAG-AttB-STOP-AttP-Lhx1/2-IRES-Cre), Cre-conditional SV2-GFP cassette, and Pbase transposase.…”

“…To trace synaptic targets in dB1 or dA1 following modification of Lim-HD expression, the Ptf1a enhancer or EdI1 enhancer was cloned upstream to PhiC31 recombinase and electroporated along with a PhiC31-Lhx1/2-conditional Cre cassette (pCAG-AttB-STOP-AttP-Lhx1/2-IRES-Cre), Cre-conditional SV2-GFP cassette, and Pbase transposase. For restricted labeling of ION-derived dB1 interneurons, Pdx1::Cre plasmid (Gu et al, 2002;Song et al, 2010) was electroporated along with the Ptf1a::FLPo recombinase plasmid and a dual-conditional reporter system (PB-CAG-Frt-STOP-Frt-LoxP-StopLoxP-n/cGFP-PB) (Hadas et al, 2014). Schematic description of the plasmids is provided in each figure.…”

“…Individual rhombomeres give rise to distinct neuronal columns, depending on the position of neural progenitors along the dorsoventral (DV) and AP axes (Lumsden and Krumlauf, 1996;Moens and Prince, 2002). In the dorsal hindbrain, interneurons receive, integrate, process, and transmit sensory inputs from the vestibular, auditory, and proprioceptive systems to higher brain regions (Altman and Bayer, 1977;Wang and Zoghbi, 2001;Maklad et al, 2003;Ryugo and Parks, 2003;Oertel and Young, 2004;Guthrie, 2007). The development of hindbrain dorsal interneuron (HDIs) occurs in sync with establishment of peripheral circuitries and is accompanied by axonal projections in specific tracts and somal migration to form distinct brainstem nuclei (Bourrat and Sotelo, 1990;Oertel and Young, 2004;Rubel et al, 2004;Landsberg et al, 2005;Farago et al, 2006;Pasqualetti et al, 2007).…”

“…For instance, the most dorsal group of neurons, termed dA1, expresses the basic helix-loop-helix transcription factor (TF) Atoh1 and the Lim-homeodomain (HD) proteins Lhx2 and Lhx9 (Ben-Arie et al, 1997;Wang et al, 2005), whereas the more ventral dB1 neurons expresses the pancreatic transcription factor 1a (Ptf1a) and Lhx1 and Lhx5 (Glasgow et al, 2005;Hori et al, 2008;Meredith et al, 2009;Storm et al, 2009). Genetic studies revealed the contribution of dB1 inhibitory neurons from rhombomeres 2-6 to the cochlear nuclei (CN), which act together with dA1-excitatory interneurons to transmit auditory information to the inferior colliculi (Farago et al, 2006;Fujiyama et al, 2009).…”

“…Using an advanced enhancer-based conditional expression system in the chick (Kohl et al, 2012(Kohl et al, , 2013Hadas et al, 2014), combined with a Ptf1a enhancer element (Meredith et al, 2009), in this study we targeted dB1 interneurons to perform spatiotemporal tracing of this molecularly defined HDI population. We found dB1 axons to extend in five specific tracts and to form synapses in the Purkinje cerebellar layer, midbrain vestibular, and auditory nuclei and in the medulla, in correspondence with their cell-body migration to form cochlear, vestibular, and ION hindbrain nuclei centers.…”