Tcf7l2 transcription factor is required for the maintenance, but not the initial specification, of the neurotransmitter identity in the caudal thalamus.

BACKGROUND: Dysfunction of GABAergic and glutamatergic neurons in the brain, which establish inhibitory and excitatory networks, respectively, may cause diverse neurological disorders. The mechanism underlying the determination of GABAergic vs. glutamatergic neurotransmitter phenotype in the caudal diencephalon remains largely unknown. RESULTS: In this study, we investigated the consequence of Tcf7l2 (transcription factor 7-like 2) ablation on the neurotransmitter identity of GABAergic and glutamatergic neurons in the caudal diencephalon. We identified positive and negative activity in the control of glutamatergic and GABAergic neuronal gene expression by Tcf7l2. Loss of Tcf7l2 did not alter the initial acquisition of the neurotransmitter identity in thalamic neurons. However, glutamatergic thalamic neurons failed to maintain their excitatory neurotransmitter phenotype in the absence of Tcf7l2. Moreover, a subset of Tcf7l2-deficient thalamic neurons underwent a glutamatergic to GABAergic neurotransmitter identity switch. Our data indicate that Tcf7l2 may promote glutamatergic neuronal differentiation and repress GABAergic neurotransmitter identity in the caudal thalamus. CONCLUSIONS: This study provides evidence for a novel and crucial role of Tcf7l2 in the molecular mechanism by which the neurotransmitter identity of glutamatergic thalamic neurons is established. Our findings exemplify a clear case of neurotransmitter identity regulation that is partitioned into initiation and maintenance phases.

Specification of neurotransmitter identity by Tal1 in thalamic nuclei.

BACKGROUND: The neurons contributing to thalamic nuclei are derived from at least two distinct progenitor domains: the caudal (cTH) and rostral (rTH) populations of thalamic progenitors. These neural compartments exhibit unique neurogenic patterns, and the molecular mechanisms underlying the acquisition of neurotransmitter identity remain largely unclear. RESULTS: T-cell acute lymphocytic leukemia protein 1 (Tal1) was expressed in the early postmitotic cells in the rTH domain, and its expression was maintained in mature thalamic neurons in the ventrolateral geniculate nucleus (vLG) and the intergeniculate leaflet (IGL). To investigate a role of Tal1 in thalamic development, we used a newly generated mouse line driving Cre-mediated recombination in the rTH domain. Conditional deletion of Tal1 did not alter regional patterning in the developing diencephalon. However, in the absence of Tal1, rTH-derived thalamic neurons failed to maintain their postmitotic neuronal features, including neurotransmitter profile. Tal1-deficient thalamic neurons lost their GABAergic markers such as Gad1, Npy, and Penk in IGL/vLG. These defects may be associated at least in part with down-regulation of Nkx2.2, which is known as a critical regulator of rTH-derived GABAergic neurons. CONCLUSIONS: Our results demonstrate that Tal1 plays an essential role in regulating neurotransmitter phenotype in the developing thalamic nuclei. Developmental Dynamics 246:749-758, 2017. © 2017 Wiley Periodicals, Inc.

Tcf7l2 plays crucial roles in forebrain development through regulation of thalamic and habenular neuron identity and connectivity.

The thalamus acts as a central integrator for processing and relaying sensory and motor information to and from the cerebral cortex, and the habenula plays pivotal roles in emotive decision making by modulating dopaminergic and serotonergic circuits. These neural compartments are derived from a common developmental progenitor domain, called prosomere 2, in the caudal forebrain. Thalamic and habenular neurons exhibit distinct molecular profile, neurochemical identity, and axonal circuitry. However, the mechanisms of how their progenitors in prosomere 2 give rise to these two populations of neurons and contribute to the forebrain circuitry remains unclear. In this study, we discovered a previously unrecognized role for Tcf7l2, a transcription factor known as the canonical Wnt nuclear effector and diabetes risk-conferring gene, in establishing neuronal identity and circuits of the caudal forebrain. Using genetic and chemical axon tracers, we showed that efferent axons of the thalamus, known as the thalamocortical axons (TCAs), failed to elongate normally and strayed from their normal course to inappropriate locations in the absence of Tcf7l2. Further experiments with thalamic explants revealed that the pathfinding defects of Tcf7l2-deficient TCAs were associated at least in part with downregulation of guidance receptors Robo1 and Robo2 expression. Moreover, the fasciculus retroflexus, the main habenular output tract, was missing in embryos lacking Tcf7l2. These axonal defects may result from dysregulation of Nrp2 guidance receptor. Strikingly, loss of Tcf7l2 caused a post-mitotic identity switch between thalamic and habenular neurons. Despite normal acquisition of progenitor identity in prosomere 2, Tcf7l2-deficient thalamic neurons adopted a molecular profile of a neighboring forebrain derivative, the habenula. Conversely, habenular neurons failed to maintain their normal post-mitotic neuronal identity and acquired a subset of thalamic neuronal features in the absence of Tcf7l2. Our findings suggest a unique role for Tcf7l2 in generating distinct neuronal phenotypes from homogeneous progenitor population, and provide a better understanding of the mechanism underlying neuronal specification, differentiation, and connectivity of the developing caudal forebrain.

Ascl1 and Helt act combinatorially to specify thalamic neuronal identity by repressing Dlxs activation.

The mammalian thalamus is an essential diencephalic derivative that plays unique roles in processing and relaying sensory and motor information to and from the cerebral cortex. The profile of transcription factors and lineage tracing experiments revealed a spatiotemporal relationship between diencephalic progenitor domains and discrete differentiated neurons contributing to thalamic nuclei. However, the precise molecular mechanisms by which heterogeneous thalamic neurons become specified and assemble into distinct thalamic nuclei are still poorly understood. Here, we show that a combinatorial interaction between the bHLH transcription factors Ascl1 and Helt is required for acquiring thalamic progenitor identity. Surprisingly, in the combined absence of Ascl1 and Helt, rostral thalamic progenitors (TH-R) adopt a molecular profile of a more rostral diencephalic derivative, the prethalamus. Furthermore, we show that the prethalamic factors Dlxs upregulated by Ascl1/Helt deficiency play unique roles in regulating thalamic progenitor specification, and that derepression of Dlx2 and Dlx5 suppress generation of TH-R neurons. Taken together, our results suggest a model whereby the combined activity of two distinct bHLH factors plays a key role in the development of discrete classes of thalamic interneurons.

Revisiting de novo drug design: receptor based pharmacophore screening.

De novo drug design methods such as receptor or protein based pharmacophore modeling present a unique opportunity to generate novel ligands by employing the potential binding sites even when no explicit ligand information is known for a particular target. Recent developments in molecular modeling programs have enhanced the ability of early programs such as LUDI or Pocket that not only identify the key interactions or hot spots at the suspected binding site, but also and convert these hot spots into three-dimensional search queries and virtual screening of the property filtered synthetic libraries. Together with molecular docking studies and consensus scoring schemes they would enrich the lead identification processes. In this review, we discuss the ligand and receptor based de novo drug design approaches with selected examples.

Screening and isolation of a natural dopamine D1 receptor antagonist using cell-based assays.

To develop a cell-based assay to screen for human dopamine D(1) receptor agonists or antagonists from medicinal plant extracts, a stable Chinese hamster ovary (CHO) cell line (CHO-D1R) expressing the human dopamine D(1) receptor was established using an expression vector containing a scaffold attachment region (SAR) element. CHO-D1R cells showed specific binding to [(3)H]-SCH23390 with high affinity (K(d)=1.47+/-0.17 nM) and dose-dependent responses for the dopamine-mediated stimulation of cAMP concentrations (EC(50)=20.6+/-1.44 nM). The screening of medicinal plant extracts using cell-based cAMP assays revealed that an extract of Gleditsia sinensis Lam., which is known to be rich in saponin, had strong antagonist activity for the D(1) receptor. From the activity-guided fractionation and chemical structural analysis of the G. sinensis extract, a compound called gleditsioside F was isolated and was identified to have antagonist activity for the D(1) receptor. Gleditsioside F showed very effective D(1) antagonist activity by inhibiting ligand binding to the D(1) receptor as well as by inhibiting dopamine-mediated increases in cAMP concentration.