Retina Restored and Brain Abnormalities Ameliorated by Single-Copy Knock-In of Human NR2E1 in Null Mice

Abstract

gNr2e1 encodes a stem cell fate determinant of the mouse forebrain and retina. Abnormal regulation of this gene results in retinal, brain, and behavioral abnormalities in mice. However, little is known about the functionality of human NR2E1. We investigated this functionality using a novel knock-in humanized-mouse strain carrying a single-copy bacterial artificial chromosome (BAC). We also documented, for the first time, the expression pattern of the human BAC, using an NR2E1-lacZ reporter strain. Unexpectedly, cerebrum and olfactory bulb hypoplasia, hallmarks of the Nr2e1-null phenotype, were not fully corrected in animals harboring one functional copy of human NR2E1. These results correlated with an absence of NR2E1-lacZ reporter expression in the dorsal pallium of embryos and proliferative cells of adult brains. Surprisingly, retinal histology and electroretinograms demonstrated complete correction of the retina-null phenotype. These results correlated with appropriate expression of the NR2E1-lacZ reporter in developing and adult retina. We conclude that the human BAC contained all the elements allowing correction of the mouse-null phenotype in the retina, while missing key regulatory regions important for proper spatiotemporal brain expression. This is the first time a separation of regulatory mechanisms governing NR2E1 has been demonstrated. Furthermore, candidate genomic regions controlling expression in proliferating cells during neurogenesis were identified. During embryogenesis, the proper development of an organism relies on orchestration between proliferation, differentiation, and death of different cell populations. The resulting dynamic balance depends on both cell intrinsic regulators and environmental factors. One such intrinsic regulator is Nr2e1 (also known as Tlx, Tll, and Tailless), which encodes a highly conserved transcription factor known to be a key stem cell fate determinant in both the developing mouse forebrain and retina (18,24,29,39,59). In mouse, abnormal regulation of this gene results in blindness, behavior abnormalities, and brain tumor initiation and progression (27,35,36,41,56,58). In humans, candidate regulatory mutations have been found in patients suffering from microcephaly, bipolar disorder, schizophrenia, and aggression (22, 23). Upregulation of NR2E1 expression was also found in cancer, and a somatic-protein-coding mutation was found in glioblastoma (27,(35)(36)(37)(38)47). These data argue in favor of strengthening our knowledge regarding the regulation and function of human NR2E1 and thereby the potential for this gene to have a role in disease. To date, the main information regarding human NR2E1 expression comes from assembled analyses done on homogenized tissue samples and information from a humanized mouse model (1,18,23). This model demonstrated complete correction of the null-brain phenotype while only ameliorating the eye phenotype. The uncorrected eye phenotype was proposed to be due to gene dosage sensitivity during eye development; a hypothesis that has not bee...

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

“…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, S100b, 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].…”

“…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].…”

“…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].…”

“…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.…”

“…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].…”