Gbx2 is essential for maintaining thalamic neuron identity and repressing habenular characters in the developing thalamus

“To avoid an arbitrary cut-off in selecting DE genes, we performed Gene Set Analysis (Subramanian et al., 2005) and found that epithalamus-enriched genes compiled from microarray experiments (see details in Methods) were significantly increased in the Gbx2-deficient thalamus (enrichment score=0.67, FDR 0.001).”

“Differentially expressed genes (DE) were identified using the limma package (Ritchie et al., 2015) with a cutoff of adjusted p value (false discovery rate-FDR) equal or less than 0.05 and fold change equal or greater than 1.7 (log2 fold change >0.8).”

“Batch effects were corrected using ComBat (Johnson et al., 2007).”

“Background correction, variance stabilizing transformation and normalization were performed using the background correction, VST and RSN methods implemented in the lumi package (Du et al., 2008).”

“Dysfunction of the habenula has been implicated in psychiatric disorders, such as depression, schizophrenia, and drug-induced psychosis (Hikosaka, 2010).”

“Based on the pattern of gene expression and axonal tracts, the diencephalon is divided into three developmental compartments called prosomeres (Rubenstein et al., 1994; Puelles and Rubenstein, 2003).”

“Gbx2 is one of the earliest genes that are induced as neural cells exit the cell cycle in the thalamus (Bulfone et al., 1993; Nakagawa and O'Leary, 2003; Chen et al., 2009).”

“Based on the pattern of gene expression and axonal tracts, the diencephalon is divided into three developmental compartments called prosomeres (Rubenstein et al., 1994; Puelles and Rubenstein, 2003).”

“[PubMed: 24952961] Visel A, Thaller C, Eichele G. GenePaint.org: an atlas of gene expression patterns in the mouse embryo.”

“The habenula receives input from the forebrain limbic system and the basal ganglia through the stria medullaris, and projects to monoaminergic nuclei in the midbrain and hindbrain via the fasciculus retroflexus, also called the habenulopeduncular tract (Lecourtier and Kelly, 2007; Hikosaka et al., 2008).”

“Gbx2 is essential for repressing Irx genes in postmitotic cells in the thalamus We and others have demonstrated that Gbx2 ablation results in profound defects in the histogenesis of the thalamus and axonal outgrowth and guidance to the neocortex (Miyashita-Lin et al., 1999; Hevner et al., 2002; Chen et al., 2009; Chat…”

“…receives input from the forebrain limbic system and the basal ganglia through the stria medullaris, and projects to monoaminergic nuclei in the midbrain and hindbrain via the fasciculus retroflexus, also called the habenulopeduncular tract (Lecourtier and Kelly, 2007; Hikosaka et al., 2008).”

“Because neurogenesis progresses in a caudal-to-rostral gradient in the developing thalamus (Angevine, 1970; McAllister and Das, 1977), significantly fewer GFP+ cells were detected in the rostral part, compared to the caudal, of the thalamus (Fig.”

“Experiments in C. elegance have identified a class of transcription factors that play an important role in assigning and/or maintaining the identity of postmitotic neurons and are called “terminal selector genes” (Hobert, 2008; Flames and Hobert, 2009).”

“…is traditionally believed that cell fate specification occurs in proliferating progenitors, emerging evidence indicate that additional mechanisms determine and/or consolidate cell identity after neural precursors exit the cell cycle (Belliveau and Cepko, 1999; Seuntjens et al., 2009; Hobert, 2011).”

“Gbx2 deletion results in the loss of thalamic markers and ectopic expression of epithalamus-enriched genes Iroquois transcription factors play important roles in tissue patterning and cell fate specification (Kobayashi et al., 2002; Kiecker and Lumsden, 2004; Robertshaw et al., 2013).”

“Chick electroporation assays were performed as described previously (Olsen et al., 2006).”

“It has been shown that forced expression of Ebf3 inhibits cell proliferation and increase apoptosis or tumor cells in vitro (Garel et al., 1999; Bai et al., 2007).”

“Deletion of Gbx2 severely disrupts histogenesis of the thalamus and abolishes thalamocortical projections (MiyashitaLin et al., 1999; Hevner et al., 2002; Chen et al., 2009; Chatterjee et al., 2012; Li et al., 2012).”

“As described previously (Nakagawa and O'Leary, 2001; Vue et al., 2007), proneural gene Neurog2 (previously named Ngn2) was broadly expressed in the thalamus except for the lateral-most region that is occupied by postmitotic cells at E12.”

“[PubMed: 25605792] Robertshaw E, Matsumoto K, Lumsden A, Kiecker C. Irx3 and Pax6 establish differential competence for Shh-mediated induction of GABAergic and glutamatergic neurons of the thalamus.”

“5 (Vue et al., 2007).”

“, 2011) (Vue et al., 2009), FGF(Kataoka and Shimogori, 2008; Martinez-Ferre and Martinez, 2009), and WNT (Zhou et al.”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript Moreover, Id4, which codes for a basic helix-loop-helix negative regulator of neurogenesis(Yun et al., 2004; Bedford et al., 2005), was specifically expressed in the wildtype but lost in Gbx2-KO thalamus (Fig.”

“Gbx2 is one of the earliest genes that are induced as neural cells exit the cell cycle in the thalamus (Bulfone et al., 1993; Nakagawa and O'Leary, 2003; Chen et al., 2009).”

“Irx genes are important for pattern formation and cell fate decision (Scholpp and Lumsden, 2010; Chatterjee and Li, 2012).”

“Irx1, which encodes a transcription factor of the Iroquois family, was expressed in the dorsal part of the diencephalon (Cohen et al., 2000).”

“Gene sets were downloaded from the monthly updated Baderlab genesets (http://baderlab.org/GeneSets) and an epithalamus-enriched gene set was curated based on published expression data (Quina et al., 2009).”

“…for the profound defects found in the thalamus lacking Gbx2, particularly in the misrouting of thalamic axons to the ventral midbrain and hindbrain (Chatterjee et al., 2012), and in the abnormal contribution of thalamus-derived neurons to the habenula due to the loss of Gbx2 (Chen et al., 2009).”

“Extracellular signals, such as Shh (Kiecker and Lumsden, 2004; Scholpp et al., 2006; Jeong et al., 2011) (Vue et al., 2009), FGF(Kataoka and Shimogori, 2008; Martinez-Ferre and Martinez, 2009), and WNT (Zhou et al., 2004; Bluske et al., 2012), which are produced from localized signaling centers,…”

“In both mice and humans, the six Irx genes are grouped into two genomic clusters, IrxA (Irx1/2/4) and IrxB (Irx3/5/6) (Peters et al., 2000).”

“Transcription factors, such as Pax6, Otx2, Fezf2, and Iroquois proteins, also participate in controlling the cell fate of progenitors in different diencephalic prosomeres (Hirata et al., 2006; Puelles et al., 2006; Scholpp et al., 2007; Robertshaw et al., 2013; Chatterjee et al., 2014).”

“5 in rats (McAllister and Das, 1977).”

“…(DAKO); mouse anti-neurofilaments (DSHB hybridoma bank); rabbit anti-pH3 (Merck Millipore); mouse anti-Pou4f1 (Santa Cruz); goat anti-Ox2 (R&D Systems); rabbit anti-GFP (Invitrogen); mouse anti-BrdU (BD Biosciences); rabbit anti-Dbx1 (Vue et al., 2007); rabbit anti-Islr2 (Mandai et al., 2009).”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript Moreover, Id4, which codes for a basic helix-loop-helix negative regulator of neurogenesis(Yun et al., 2004; Bedford et al., 2005), was specifically expressed in the wildtype but lost in Gbx2-KO thalamus (Fig.”

“, 2009), FGF(Kataoka and Shimogori, 2008; Martinez-Ferre and Martinez, 2009), and WNT (Zhou et al., 2004; Bluske et al., 2012), which are produced from localized signaling centers, act in concert to pattern the diencephalon.”

“Gbx2, which encodes a homeodomain transcription factor, is expressed in thalamic neuronal precursors that have just exited the cell cycle; its expression is maintained in mature neurons of thalamic nuclei in adult mice and monkeys (Jones and Rubenstein, 2004; Chen et al., 2009).”

“In zebrafish embryos, the neurogenetic gradient in the thalamus is controlled by her6, through interacting with neurog1 and coe2 (an Ebf gene) (Scholpp et al., 2009).”

“[PubMed: 24952961] Visel A, Thaller C, Eichele G. GenePaint.org: an atlas of gene expression patterns in the mouse embryo.”

“Prior studies have demonstrated the important roles of Irx1 and Irx3 in development of the diencephalon (Scholpp et al., 2007; Rodriguez-Seguel et al., 2009; Robertshaw et al., 2013).”

“[PubMed: 25605792] Robertshaw E, Matsumoto K, Lumsden A, Kiecker C. Irx3 and Pax6 establish differential competence for Shh-mediated induction of GABAergic and glutamatergic neurons of the thalamus.”

“Prior studies have demonstrated the important roles of Irx1 and Irx3 in development of the diencephalon (Scholpp et al., 2007; Rodriguez-Seguel et al., 2009; Robertshaw et al., 2013).”

“Finally, homeodomain transcription factor Otx2, which is essential for the generation of glutamatergic neurons by repressing GABAergic identity in the thalamus (Puelles et al., 2006), was also detected throughout the ventricular zone of the diencephalon (Fig.”

“…signals, such as Shh (Kiecker and Lumsden, 2004; Scholpp et al., 2006; Jeong et al., 2011) (Vue et al., 2009), FGF(Kataoka and Shimogori, 2008; Martinez-Ferre and Martinez, 2009), and WNT (Zhou et al., 2004; Bluske et al., 2012), which are produced from localized signaling centers, act in…”

“Gbx2 deletion results in the loss of thalamic markers and ectopic expression of epithalamus-enriched genes Iroquois transcription factors play important roles in tissue patterning and cell fate specification (Kobayashi et al., 2002; Kiecker and Lumsden, 2004; Robertshaw et al., 2013).”

“…and Lumsden, 2004; Scholpp et al., 2006; Jeong et al., 2011) (Vue et al., 2009), FGF(Kataoka and Shimogori, 2008; Martinez-Ferre and Martinez, 2009), and WNT (Zhou et al., 2004; Bluske et al., 2012), which are produced from localized signaling centers, act in concert to pattern the diencephalon.”

“In sharp contrast to the neighboring diencephalic structure, the developing thalamus contains a remarkably large number of basal progenitor cells (Smart, 1972; Wang et al., 2011).”

“…for the profound defects found in the thalamus lacking Gbx2, particularly in the misrouting of thalamic axons to the ventral midbrain and hindbrain (Chatterjee et al., 2012), and in the abnormal contribution of thalamus-derived neurons to the habenula due to the loss of Gbx2 (Chen et al., 2009).”

“Significant progress has been made in our understanding of the development of diencephalic prosomeres (Chatterjee and Li, 2012).”

“We have recently reported the characterization of gene expression in the epithalamus, particularly the habenula in mice and zebrafish (Chatterjee et al., 2014).”

“H2afj, which codes for an atypical histone protein (Nishida et al., 2005), is expressed throughout the VZ of the neural tube (Fig.”

“…is traditionally believed that cell fate specification occurs in proliferating progenitors, emerging evidence indicate that additional mechanisms determine and/or consolidate cell identity after neural precursors exit the cell cycle (Belliveau and Cepko, 1999; Seuntjens et al., 2009; Hobert, 2011).”

“We have previously demonstrated that the Gbx2 lineage contributes exclusively to thalamic nuclei that project to the cortex (Chen et al., 2009; Li et al., 2012).”

“This was attributed to the fact that astrocytes and neurons, given their common developmental origin, share expression of region-specific patterning TFs (Herrero-Navarro et al., 2021), such as Gbx2 , a TF expressed by thalamic astrocytes and neurons (Mallika et al., 2015).”

“To directly test this, we examined whether coexpression of a thalamic fate determinant Gbx2 (29), a factor being shared between astrocytes and neurons of the thalamus, could induce an early and fast redirection of neuronal reprogramming of cortical astrocytes toward a thalamic identity (Fig.”

“TCF7L2 motif enrichment in ChIP-seq peak sequences was tested with AME (McLeay and Bailey, 2010) using default options.”

“In all cases GBX2 is essential for axon outgrowth and pathfinding (Miyashita-Lin et al. 1999; Chatterjee et al. 2012) and is required to suppress expression of markers of habenular identity (Chen et al. 2009: Mallika et al. 2015).”

“Conversely, some genes which expression critically depend on Gbx2 are Rora, Id4, Chst1, Glr1, Cd47, Slc18a2 (also known as Vmat2), Gas7, Hs6st2, Epha3, and Prokr2 (Mallika et al., 2015).”

“Genes with roles in nervous system processes hypermethylated above 80% in Creole samples include (Table 2): (i) GBX2, a modulator of thalamus cells development (Mallika et al. 2015); (ii) NKX2-8, a regional homeobox gene with functions in neuronal development (Safra et al.”

“Notably, 13 of them (65%) have been shown to be specific for thalamic neurons according to previous studies (Mallika et al., 2015; Suzuki-Hirano et al., 2011), indicating the accuracy of lineage-specific markers recovered by URD.”

“Similarly, loss of the gastrulation brain homeobox 2 (Gbx2) gene results in the adoption of habenular neuronal fate at the expense of thalamic neuronal identity, apparently by indirectly altering the developmental program of proliferating thalamic progenitors [48].”

“…grouping ( d ) or the clusters achieved using the default parameters of CHOIR ( e ) or Seurat ( f ). g – i , UMAP embeddings for simulated dataset 45, consisting of 20,000 cells and five ground-truth groups of cells colored by the ground-truth groupings ( g ), the clusters achieved using the…”

“Gbx2 regulates the specification, migration, and axon guidance of several neuronal populations in the brain and spinal cord (Chatterjee et al., 2012; Chen et al., 2010; Luu et al., 2011; Mallika et al., 2015).”

“An important regulator for establishing the epithalamic domain within p2 is fibroblast growth factor 8 (Fgf8) (Martinez-Ferre and Martinez, 2009).”

“5 except for one mouse in which very sparse CTand TC axons are detected simultaneously (Mallika et al., 2015).”

“It was previously reported that, in the case of thalamus development in mice, gbx2 functions in a cell non-autonomous manner, supporting the first two possibilities (Mallika et al., 2015).”

“In particular, Otx2 expression in the habenula has been reported (Courtois et al., 2003; Mallika et al., 2015), although little is known about its dynamics during development.”

“For instance, Gbx2 and Lhx2 play a regulatory role by orchestrating the genetic programs required for thalamic neuron identity, differentiation and connectivity 64–66 .”

“5 in a Gbx2-dependent fashion (Mallika et al. 2015).”

“We focused on developing diencephalon, based on a reported regulatory interaction between hs754 and the gene Irx2 [59], which is expressed during diencephalon development [60].”

“In turn, there is a disruption in the histogenesis of the nuclear complexes and a significant loss of thalamocortical projections (Chen et al., 2009; Chatterjee and Li, 2012; Mallika et al., 2015; Nakagawa, 2019).”

“Chatterjee et al. (2015) recently reported that deletion of the homeodomain transcription factor Gbx2 inmouse led to an expanded expression territory of epithalamic markers, Nrp2, Brn3a/Pou4f1, Robo3 and a loss of thalamic neurons.”

“Gastrulation brain homeobox 2 (GBX2), coding for a homeodomain transcription factor, has been demonstrated to possess the transcriptional inhibition function.(10,11) It is expressed in the forebrain during vertebrate brain development, which helps to locate the midbrain– hindbrain boundary that controls the progressio…”

“In the diencephalon, we identified the central and dorsal posterior thalamic nuclei with the expression of the transcription factor gbx2 (Mallika et al., 2015; Wullimann et al., 2024) and the ventromedial thalamus (VM) through high expression of the neuropeptides nts and vipa (Bello et al., 1994;…”

“We focused on developing diencephalon, based on a reported regulatory interaction between hs754 and the gene Irx2 [59], which is expressed during diencephalon development [60].”

“…AC fate and dendrite lamination Previous studies have demonstrated that Gbx2 is an important transcription factor for neural development from neurogenesis to neuronal morphology in the brain and spinal cord (Chatterjee et al., 2012; Chen et al., 2010; Luu et al., 2011; Mallika et al., 2015).”

“Gbx2 , as an essential gene to maintain the thalamic neuron identity 69 , showed high gene activity in the thalamus glutamatergic neurons.”