The Orphan Nuclear Receptor TLX/NR2E1 in Neural Stem Cells and Diseases

Abstract

The human TLX gene encodes an orphan nuclear receptor predominantly expressed in the central nervous system. Tailess and Tlx, the TLX homologues in Drosophila and mouse, play essential roles in body-pattern formation and neurogenesis during early embryogenesis and perform crucial functions in maintaining stemness and controlling the differentiation of adult neural stem cells in the central nervous system, especially the visual system. Multiple target genes and signaling pathways are regulated by TLX and its homologues in specific tissues during various developmental stages. This review aims to summarize previous studies including many recent updates from different aspects concerning TLX and its homologues in Drosophila and mouse.

Introduction

Nuclear receptors (NRs) are a group of transcriptional regulators with broad functions in physiological and developmental processes, including metabolism, xenobiotic defense, and reproduction [1]. So far, 18, 49, and 48 NRs in six subfamilies have been identified in Drosophila melanogaster, mice, and humans, respectively [2–4]. TLX, also known as NR2E1 (nuclear receptor subfamily 2, group E, member 1), encodes an orphan NR with no identified ligand. TLX is the human homologue of the Drosophila tailless (tll) gene and is predominantly expressed in the developing brain and retina [5–7]. Human TLX and its homologues in Drosophila, zebrafish, and mouse have conserved functions in brain and retinal development [8–10]. TLX as well as its homologues in other species predominantly acts as a transcriptional repressor, although a function in transcriptional activation has also been suggested in some studies [11, 12].

The functional equivalence between human TLX and mouse Tlx has been supported by the finding that the expression of human TLX reverses the brain and eye defects and aggressive behavior of mice with a deletion of Tlx [13, 14]. Given the validated function of TLX in neural stem cells (NSCs) and brain development, it has been suggested that defects in TLX are associated with several human mental illnesses such as schizophrenia and bipolar disorders [15]. TLX has also been associated with NSC expansion and brain tumorigenesis [16].

Protein Structure of TLX

The human TLX gene was first cloned in a 24-kb region on the chromosomal band 6q21 [6]. This gene includes 9 exons and encodes a protein of 385 amino-acids. Like its homologues in Drosophila, zebrafish, and mouse, the human TLX protein contains a highly-conserved N-terminal DNA-binding domain (DBD), a hinge domain, and a moderately-conserved C-terminal ligand-binding domain (LBD) based on sequence homology analysis [5, 7, 17]. The TLX DBD contains two C4 zinc-finger motifs and includes a P Box, a D Box, and a T/A box involved in DNA binding and dimerization (Fig. 1) [11]. It is notable that TLX contains a ligand-dependent C-terminal activation function 2 (AF-2) domain but not a ligand-independent N-terminal activation function 1 (AF-1) domain, which is found in many NR family members [18]. In order to explore the ligand-binding potential of TLX, Benod et al. recently developed three homology models for the TLX LBD in a transcriptionally-active conformation using a chimera LBD template based on the crystal structures of two human NRs, COUP-TFII (COUP transcription factor II) and RXRα (retinoid X receptor α) [18–20]. This study confirmed that the TLX LBD structure resembles other NR LBD conformations as a canonical helical sandwich, and also revealed notable differences, in that the TLX LBD lacks the first helices α1 and α2 and contains a unique 5-amino-acid insertion between helices α8 and α9. In contrast to the previous description of the full-length TLX-forming monomer [21], Benod et al. suggested that the recombinant TLX LBD can form a homodimer in solution [18]. More interestingly, three compounds were shown to be able to bind the recombinant TLX LBD, indicating that TLX functions can be manipulated using selected compounds [18]. Most recently, Xu et al. have reported the crystal structure of the corepressor assembly in TLX. They found that TLX adopts an auto-repressed conformation in which its helix H12 occupies the coactivator-binding groove, and that, unexpectedly, H12 in this auto-repressed conformation forms a novel binding pocket with residues from helix H3 that accommodates a short helix formed by the conserved ALXXLXXY motif of the Atro box [22].

Fig. 1.

Schematic diagram of basic TLX structure. DBD DNA-binding domain, LBD ligand-binding domain, AF activation function.

TLX Expression, Its Targets, and Related Signal Pathways

The expression patterns of TLX and its homologues in other species have been extensively investigated. The Drosophila tll mutant was identified during large-scale screening for genes associated with pattern-formation in the early embryo [23]. Tll plays important roles in establishing the segmentation pattern during early embryogenesis. It is expressed at both ends of early embryos and functions to suppress the expression of other genes such as Krüpple and Knirps in both the anterior and posterior caps of early embryos; this is indispensable for the appropriate specification of terminal cells [9, 24, 25]. Most of the posterior structures and some portions of the anterior structures including the brain are missing in tll mutant embryos [9, 26–28]. Embryonic cells expressing tll in the head region develop an optic lobe instead of a Bolwig’s organ because tll antagonizes EGFR signaling, which is required for cells to develop into Bolwig’s organ [29]. Tll is also expressed in undifferentiated proliferating neural precursor cells at the third instar larval stage, the last larval stage before eclosion into adult flies, indicating that tll may have an essential function in neuronal stem cells [11]. Consistent with the findings in Drosophila, mouse Tlx is expressed in the developing central nervous system (CNS) from embryonic day 8 (E8) until E13.5 and in retinal progenitor cells (RPCs) from E11.5 until E17.5 during retinal development [5, 30–32]. At the embryonic stage, Tlx expression presents a graded pattern in the telencephalon along the dorsal–ventral axis with a high level in the dorsal-lateral region and a low level in the ventral-medial region [33]. Tlx is also expressed in postnatal NSCs and may activate neurogenesis in the subventricular zone of the lateral ventricle based on the findings that Tlx mutant brains have severely reduced hippocampal dentate gyri and greatly expanded lateral ventricles in adult mice [34–36]. Tlx expression is also detectable in the ventricular zone enriched with NSCs at the postnatal stage [30, 37, 38]. In adults, TLX is detectable in the subependymal zone of the CNS [38]. Tlx overexpression promotes NSC expansion and results in the development of gliomas in mouse [16]. Interestingly, that study also found that TLX expression is elevated in human glioblastomas. These studies suggest that the fruit fly and mouse homologues of human TLX are predominantly expressed in NSCs or neuronal precursor cells and are important for the proliferation and self-renewal of NSCs. Human TLX expression is detectable in multiple organs and tissues including the adrenal gland, brain, cerebellum, testis, placenta, and bone marrow [6, 15, 39]. According to the RNA-seq data from the Illumina Human Body Map 2.0 project (http://www.ensembl.org/info/genome/genebuild/rnaseq_annotation.html), TLX expression in brain is significantly higher than in other organs and tissues, indicating its evolutionarily conserved function in the CNS.

Studies of Drosophila embryogenesis have suggested that Tll acts as a transcriptional repressor to directly suppress the expression of target genes such as knirps, ems, and otd, which are required for the formation of segmented regions in early embryonic development [28, 40, 41]. Prospero, which encodes a homeodomain transcription factor associated with the differentiation of ganglion mother cells after asymmetric stem cell division of neuroblasts in the central brain [42, 43], is the only target gene that has been found to be directly regulated by Tll in post-embryonic stages [44]. In the mouse, multiple genes involved in the development of the CNS and visual system have been identified as Tlx targets, such as Gsh2, Pax2, Pten, p21, p57, S100β, Aqp4, Plce1, Wnt7a, microRNA-9, Bmp4, and GFAP [21, 32–37, 45–50] (Table 1). Interestingly, Tlx may act as a transcriptional activator for the expression of sirt1, Wnt7a, Mash1, and Oct-3/4 based on in vitro luciferase assays and chromatin immunoprecipitation assays [16, 21, 51–53]. A consensus Nr2e1 binding sequence within the promoter region of its target gene has been proposed to contain AAGTCA, which has been identified in the promoters of Wnt7a, GFAP, S100β, Oct3/4, p21, Pax2, Plce1, and Pten [21, 32, 34, 45, 46, 53, 54]. Given the functional equivalence and the highly-conserved DBDs between human TLX and mouse Tlx [13, 14], TLX may have the capacity to bind a similar consensus promoter sequence and suppress or activate the expression of multiple human counterparts of the identified mouse Tlx targets. In addition, a recent study using human neuroblastoma cell lines showed that human TLX, through binding the von Hippel-Lindau protein and stabilizing hydroxylated hypoxia-inducible factor, may bind the VEGF (vascular endothelial factor) promoter and induce VEGF expression to promote angiogenesis under hypoxic conditions [55]. A recent study [55] also reported that TLX binds the promoters of MMP-2 (matrix metalloproteinase-2) and Oct-4 and activates the expression of these genes in neuroblastoma cells [56]. This study further identified two TLX-binding sites with an AAGTCA sequence 1.2-kb upstream of the MMP-2 transcription start site. Apparently, TLX has either a positive or a negative effect on target gene expression in different types of cells and tissues.

Table 1.

Candidate target genes of Tlx

Target genes	Effect of Tlx	Conserved	DNA binding assay			Reference
		AAGTCA motif	Luciferase	EMSA	ChIP
Pten	Repress	+	+	+	+	Sun et al. 2007 [46]
P21	Repress	+	+	+	+	Sun et al. 2010 [54]
Pax2	Repress	+	+	+	+	Yu et al. 2000 [32]
BMP4	Repress			+	+	Qin et al. 2014 [48]
miR-9/miR-137	Repress	+				Zhao et al. 2009 [47]
Gfap	Repress			+		Shi et al. 2004 [34]
Oct3/4 (Pou5f1)	Promote	+	+	+	+	Chavali et al. 2011 [53]
MMP-2	Promote	+	+			Chavali et al. 2014 [56]
SIRT1	Promote	+				Iwahar 2009 [52]
AscL1 (Mash1)	Promote		+			Elmi et al. 2010 [51]
Wnt7a	Promote	+	+			Qu et al. 2010 [21]

The identification of target genes regulated by TLX homologues in fruit fly, mouse, and other species indicates that TLX has important functions in various cellular and developmental processes. Recent studies by Qin et al. showed that mouse Tlx controls the timing of postnatal astrogenesis by regulating the BMP-SMAD rather than the JAK-STAT signaling pathway [48]. The expression of BMP signaling genes such as Bmp4, Hes1, and Id3 and astrocyte marker genes such as GFAP and S100β is up-regulated in the postnatal brain of the Tlx-deleted mouse [48]. Tlx has been reported to interact with BMP7 and/or Sonic Hedgehog (SHH) and relieve the repression of Pax2 expression in mouse retinal astrocytes [50]. Mouse Tlx also regulates the Wnt-Frizzled signaling pathway by directly activating Wnt7a expression [21]. Niu et al. showed that mouse Tlx is involved in regulating DNA replication and cell-cycle signaling in postnatal NSCs [49]. They found that Tlx directly suppresses the expression of p21, a critical cell-cycle gene, via a p53-dependent mechanism [49]. This is consistent with the previous finding of a Tlx-binding site in the p21 promoter region [46]. In contrast to the antagonizing effect of tll on EGFR signaling in Drosophila embryonic optic lobe development, overexpression of mouse Tlx promotes EGFR signaling in cells within the subventricular zone of the adult brain [16]. Tlx may also modulate the mitogen-activated protein kinase pathways to regulate RPC differentiation [45]. In addition, mammalian target of rapamycin signaling and transforming growth factor-β signaling are significantly perturbed in the retina of the Tlx-null mouse [45].

These distinct findings from different types of cells/tissues at different developmental stages suggest the temporal and spatial specificity of the Tlx regulation of gene expression and signaling pathways. Such temporal and spatial specificity probably depends on other interactive proteins that function together with Tlx (or TLX in humans) to form a transcriptional complex in specific cells at certain developmental stages. Atrophin1, BCL11A, LSD1 (a histone demethylase), histone deacetylase 3 (HDAC3), and HDAC5 have been reported to interact with Tlx to regulate the expression of target genes [45, 54, 57, 58]. The interactions among TLX, LSD1, and Atrophin1 have been validated in human Y79 retinoblastoma cells [57]. In that study, TLX, LSD1, CoREST, and HDAC1/2 were suggested to form a transrepression complex at the Pten promoter to suppress Pten expression in retinal cells. Pax6 also genetically interacts with Tlx to regulate the dorsal–ventral patterning of the mouse telencephalon [33].

Roles of TLX Homologues in the Regulation of NSCs and Brain Development

Evidence from the TLX expression profile, TLX target genes, and signaling pathways involving TLX clearly demonstrated that TLX plays important roles both in embryonic neurogenesis and in maintaining the stemness of adult NSCs. As noted above, Drosophila tll is required for both anterior and posterior body pattern formation during early embryogenesis and is important for maintaining the proliferating and undifferentiated state of neuroblasts, Drosophila NSCs, in the late larval stage [9, 12, 25, 28]. Appropriate tll expression is crucial for the suppression of prospero expression and maintenance of the proliferation capacity in neuroblasts. In contrast, in the ganglion mother cells derived from neuroblasts through asymmetric division, a lack of tll expression and elevated prospero expression promote differentiation [42, 43]. It has been reported that ectopic expression of tll in ganglion mother cells inhibits apoptosis, promotes cell-cycle progression, and results in tumor formation in the Drosophila brain [44].

As noted above, mouse Tlx is expressed mainly in the CNS and NSC niche during all developmental stages. Tlx functions to maintain the self-renewal capacity of NSCs in mouse brain from the embryonic to the adult stage [11, 33, 49, 59]. Studies of Tlx-knockout mice have shown that Tlx is required for neurogenesis in the superficial cortical layers and for the normal development of zinc-containing cortical circuits [60, 61]. Tlx is also important for normal development of the cerebral hemispheres [62]. Roy et al. found that Tlx regulates the timing of neurogenesis in the cortex [38]. The temporal and regional requirements for Tlx expression in neuronal progenitor cells have been reported. In Tlx-deficient mice, CNS structures that are generated at a later developmental stage seem to be affected more severely than structures formed during early- or mid-stage neurogenesis. This timing effect may be related to the consequence of a shortened cell cycle due to loss of Tlx. Li et al. also found elevated p21 expression, decreased cyclin D1 expression, prolonged cell cycles, and increased cell-cycle exit in Tlx-null embryonic brains, showing that Tlx functions to regulate the cell cycle of NSCs in the developing mouse brain [37].

In addition to its functions in NSCs in embryonic brains, Tlx also maintains adult NSCs in an undifferentiated, proliferative state [34]. In that study, Tlx-null cells isolated from mutant adult mouse brains presented proliferation defects that were rescued by reintroducing Tlx expression. The essential role of mouse Tlx in activating neurogenesis from adult NSCs has been validated by several studies [35, 49]. Zhang et al. further showed that Tlx is indispensable for stem-cell proliferation in the adult mouse brain and for certain cognitive functions such as spatial learning [36]. A later study using cultured adult rat hippocampus-derived progenitors showed that Tlx not only inhibits glial differentiation but also promotes neuronal lineage commitment by activating expression of the pro-neural gene Mash1 in these adult progenitor cells [51]. Recently, Tlx has been shown to restrain senescence in NSCs and cancer cells [63, 64]. As discussed above, Tlx may maintain the stemness of mouse adult NSCs by activating autonomous Wnt/beta-catenin signaling [21].

Evidence supporting the function of human TLX in brain development is limited. However, the rescue effect of human TLX on brain abnormalities and fierce behavior in Tlx-null mice suggests that human TLX has functions similar to those of mouse Tlx in normal brain development [13, 14].

Roles of TLX Homologues in Regulation of Retinal Progenitor Cell Differentiation and Visual System Development

Another essential function of TLX homologues is regulating RPC differentiation during visual system development. Drosophila tll drives embryonic cells into the fate of developing into optic lobes but not Bolwig’s organ via antagonizing EGFR signaling [29]. Mouse Tlx is involved in eye development from early RPC differentiation to later astrocyte maturation [31]. It controls the generation of proper numbers of RPCs by activating cyclin-D1 and p27 during early retinal layer formation [31]. Further, Tlx expression has been found in Müller glial cells and mature astrocytes of the inner nuclear layer after birth and temporarily in immature astrocytes before migration from the optic nerve to the inner nuclear layer [31]. Consistently, reduced retinal size and cell number, optic nerve degeneration, and visual impairment have been reported in Tlx-deficient mice [32, 45, 65]. It has been suggested that Tlx regulates retinogenesis either through direct suppression of GFAP expression or by modulating upstream BMP signaling [34, 48].

Again, human TLX may serve a function similar to that of mouse Tlx in eye development based on rescue experiments in which retinal defects in Tlx-deficient mice are corrected after introduction of the human LTX gene [14].

TLX and Human Diseases

Given the wide range of mutant phenotypes such as brain abnormalities, visual impairments, violent behavior, and learning disabilities, it is reasonable to suspect that human TLX mutations may be associated with some pathophysiological conditions. Kumar et al. conducted a series of studies to investigate whether human TLX mutations are associated with abnormal brain-behavior conditions such as microcephaly, bipolar disorder, schizophrenia, and aggression [15, 66]. These studies showed that TLX mutations are associated with bipolar disorder [15], and four candidate mutations in the regulatory region of TLX have been associated with microcephaly [66].

Tlx has been reported to interact with Pax6 during mouse brain development [33]. Although human PAX6 mutations are known to cause several disorders including aniridia, a recent study did not find TLX mutations in patients with this condition [67].

There is still a major lack of evidence establishing an association between loss-of-function mutations of TLX and human disorders. On the contrary, elevated expression of TLX has been reported in various types of human glioma [68–73] and glioma cell lines [74], and TLX overexpression seems to be associated with a poor prognosis for patients with glioma [74]. A recent study further reported that TLX promotes the progression of neuroblastoma and is correlated with poor survival [56]. These studies suggested that uncontrolled TLX expression contributes to tumorigenesis. On the other hand, TLX could be a therapeutic target for cancer treatment.

Summary and Future Prospects

TLX and its homologues in other species have been increasingly investigated during the past 15 years. The recent discovery of the involvement of TLX in NSC self-renewal elucidated its functions at the molecular and genetic levels. It is notable that TLX and its homologues, as a key component of a transcription complex, regulate various target genes and modulate multiple signaling pathways in specific tissues from embryonic stages through to adulthood. TLX may have distinct regulatory effects on target genes or signaling pathways depending on the specific internal molecular environment and external stimuli. Thus, the molecular mechanisms of TLX function identified in vitro should be cautiously examined in vivo with the consideration of unique temporal and spatial conditions. It is encouraging that the functional conservation between mouse Tlx and human TLX has been validated by the finding that the ectopic expression of the genomic region of human TLX can correct brain, retinal, and behavioral abnormalities in Tlx-null mice [13, 14]. However, recent initial screening for TLX mutations in several human disorders resulted in only limited findings. Nevertheless, we should remain optimistic regarding the discovery of pathological TLX mutations in the near future with increasing applications of whole-exon and whole-genome sequencing in identifying etiologic mutations in patients with genetic disorders. On the other hand, future studies will focus on how TLX or its regulators such as stem cell-specific transcriptional factors contribute to brain tumorigenesis and provide novel insights into the histogenesis and molecular pathogenesis of primary brain tumors. Moreover, further resolving the structure of TLX protein may help better understand its functional mechanisms and develop potential drugs to modulate them.