Sox2-Evf2 lncRNA-mediated mechanisms of chromosome topological control in developing forebrain.

The Evf2 long non-coding RNA directs Dlx5/6 ultraconserved enhancer(UCE)-intrachromosomal interactions, regulating genes across a 27 Mb region on chromosome 6 in mouse developing forebrain. Here, we show that Evf2 long-range gene repression occurs through multi-step mechanisms involving the transcription factor Sox2. Evf2 directly interacts with Sox2, antagonizing Sox2 activation of Dlx5/6UCE, and recruits Sox2 to the Dlx5/6eii shadow enhancer and key Dlx5/6UCE interaction sites. Sox2 directly interacts with Dlx1 and Smarca4, as part of the Evf2 ribonucleoprotein complex, forming spherical subnuclear domains (protein pools, PPs). Evf2 targets Sox2 PPs to one long-range repressed target gene (Rbm28), at the expense of another (Akr1b8). Evf2 and Sox2 shift Dlx5/6UCE interactions towards Rbm28, linking Evf2/Sox2 co-regulated topological control and gene repression. We propose a model that distinguishes Evf2 gene repression mechanisms at Rbm28 (Dlx5/6UCE position) and Akr1b8 (limited Sox2 availability). Genome-wide control of RNPs (Sox2, Dlx and Smarca4) shows that co-recruitment influences Sox2 DNA binding. Together, these data suggest that Evf2 organizes a Sox2 PP subnuclear domain and, through Sox2-RNP sequestration and recruitment, regulates chromosome 6 long-range UCE targeting and activity with genome-wide consequences.

The Evf2 Ultraconserved Enhancer lncRNA Functionally and Spatially Organizes Megabase Distant Genes in the Developing Forebrain.

Gene regulation requires selective targeting of DNA regulatory enhancers over megabase distances. Here we show that Evf2, a cloud-forming Dlx5/6 ultraconserved enhancer (UCE) lncRNA, simultaneously localizes to activated (Umad1, 1.6 Mb distant) and repressed (Akr1b8, 27 Mb distant) chr6 target genes, precisely regulating UCE-gene distances and cohesin binding in mouse embryonic forebrain GABAergic interneurons (INs). Transgene expression of Evf2 activates Lsm8 (12 Mb distant) but fails to repress Akr1b8, supporting trans activation and long-range cis repression. Through both short-range (Dlx6 antisense) and long-range (Akr1b8) repression, the Evf2-5'UCE links homeodomain and mevalonate pathway-regulated enhancers to IN diversity. The Evf2-3' end is required for long-range activation but dispensable for RNA cloud localization, functionally dividing the RNA into 3'-activator and 5'UCE repressor and targeting regions. Together, these results support that Evf2 selectively regulates UCE interactions with multi-megabase distant genes through complex effects on chromosome topology, linking lncRNA-dependent topological and transcriptional control with interneuron diversity and seizure susceptibility.

Enhanced susceptibility to stress and seizures in GAD65 deficient mice.

Reduced gamma-aminobutyric acid (GABA) inhibition has been implicated in both anxiety and epilepsy. GAD65-/- (NOD/LtJ) mice have significantly decreased basal GABA levels in the brain and a lowered threshold for seizure generation. One fifth of GAD65 -/- mice experienced stress-induced seizures upon exposure to an open field at 4 weeks of age. In each successive week until 8 weeks of age, the latency to seizures decreased with prior seizure experience. 100% of GAD65-/- mice exhibited stress-induced seizures by the end of 8 weeks. GAD65-/- mice also exhibited marked impairment in open field exploratory behavior and deficits in spatial learning acquisition on a Barnes maze. Anxiety-like behavior in an open field was observed prior to seizure onset and was predictive of subsequent seizures. Immunohistochemical characterization of interneuron subtypes in GAD65-/- mice showed a selective decrease in GABA and neuropeptide Y (NPY) levels and no change in calbindin (CLB) or calretinin (CLR) immunoreactivity in the hippocampus. Stem cells from the medial ganglionic eminence (MGE) were injected into the hippocampal hilus to restore GABAergic interneurons. One week after transplantation, MGE-transplanted mice demonstrated significant seizure resistance compared to sham surgical controls. The percent area of GFP+ MGE graft in the hippocampus correlated significantly with the increase in seizure latency. Our data indicate that impaired GABAergic neurotransmission can cause anxiety-like behavior and stress-induced seizures that can be rescued by MGE stem cell transplantation.

SHH E176/E177-Zn2+ conformation is required for signaling at endogenous sites.

Sonic hedgehog (SHH) is a master developmental regulator. In 1995, the SHH crystal structure predicted that SHH-E176 (human)/E177 (mouse) regulates signaling through a Zn2+-dependent mechanism. While Zn2+ is known to be required for SHH protein stability, a regulatory role for SHH-E176 or Zn2+ has not been described. Here, we show that SHH-E176/177 modulates Zn2+-dependent cross-linking in vitro and is required for endogenous signaling, in vivo. While ectopically expressed SHH-E176A is highly active, mice expressing SHH-E177A at endogenous sites (ShhE177A/-) are morphologically indistinguishable from mice lacking SHH (Shh-/-), with patterning defects in both embryonic spinal cord and forebrain. SHH-E177A distribution along the embryonic spinal cord ventricle is unaltered, suggesting that E177 does not control long-range transport. While SHH-E177A association with cilia basal bodies increases in embryonic ventral spinal cord, diffusely distributed SHH-E177A is not detected. Together, these results reveal a novel role for E177-Zn2+ in regulating SHH signaling that may involve critical, cilia basal-body localized changes in cross-linking and/or conformation.

Evf2 lncRNA/BRG1/DLX1 interactions reveal RNA-dependent inhibition of chromatin remodeling.

Transcription-regulating long non-coding RNAs (lncRNAs) have the potential to control the site-specific expression of thousands of target genes. Previously, we showed that Evf2, the first described ultraconserved lncRNA, increases the association of transcriptional activators (DLX homeodomain proteins) with key DNA enhancers but represses gene expression. In this report, mass spectrometry shows that the Evf2-DLX1 ribonucleoprotein (RNP) contains the SWI/SNF-related chromatin remodelers Brahma-related gene 1 (BRG1, SMARCA4) and Brahma-associated factor (BAF170, SMARCC2) in the developing mouse forebrain. Evf2 RNA colocalizes with BRG1 in nuclear clouds and increases BRG1 association with key DNA regulatory enhancers in the developing forebrain. While BRG1 directly interacts with DLX1 and Evf2 through distinct binding sites, Evf2 directly inhibits BRG1 ATPase and chromatin remodeling activities. In vitro studies show that both RNA-BRG1 binding and RNA inhibition of BRG1 ATPase/remodeling activity are promiscuous, suggesting that context is a crucial factor in RNA-dependent chromatin remodeling inhibition. Together, these experiments support a model in which RNAs convert an active enhancer to a repressed enhancer by directly inhibiting chromatin remodeling activity, and address the apparent paradox of RNA-mediated stabilization of transcriptional activators at enhancers with a repressive outcome. The importance of BRG1/RNA and BRG1/homeodomain interactions in neurodevelopmental disorders is underscored by the finding that mutations in Coffin-Siris syndrome, a human intellectual disability disorder, localize to the BRG1 RNA-binding and DLX1-binding domains.

Evf2 (Dlx6as) lncRNA regulates ultraconserved enhancer methylation and the differential transcriptional control of adjacent genes.

Several lines of evidence suggest that long non-coding RNA (lncRNA)-dependent mechanisms regulate transcription and CpG DNA methylation. Whereas CpG island methylation has been studied in detail, the significance of enhancer DNA methylation and its relationship with lncRNAs is relatively unexplored. Previous experiments proposed that the ultraconserved lncRNA Evf2 represses transcription through Dlx6 antisense (Dlx6as) transcription and methyl-CpG binding protein (MECP2) recruitment to the Dlx5/6 ultraconserved DNA regulatory enhancer (Dlx5/6ei) in embryonic day 13.5 medial ganglionic eminence (E13.5 MGE). Here, genetic epistasis experiments show that MECP2 transcriptional repression of Evf2 and Dlx5, but not Dlx6, occurs through antagonism of DLX1/2 in E13.5 MGE. Analysis of E13.5 MGE from mice lacking Evf2 and of partially rescued Evf2 transgenic mice shows that Evf2 prevents site-specific CpG DNA methylation of Dlx5/6ei in trans, without altering Dlx5/6 expression. Dlx1/2 loss increases CpG DNA methylation, whereas Mecp2 loss does not affect Dlx5/6ei methylation. Based on these studies, we propose a model in which Evf2 inhibits enhancer DNA methylation, effectively modulating competition between the DLX1/2 activator and MECP2 repressor. Evf2 antisense transcription and Evf2-dependent balanced recruitment of activator and repressor proteins enables differential transcriptional control of adjacent genes with shared DNA regulatory elements.

Heat-shock-mediated conditional regulation of hedgehog/gli signaling in zebrafish.

BACKGROUND: Hedgehog (Hh) signaling is required for embryogenesis and continues to play key roles postembryonically in many tissues, influencing growth, stem cell proliferation, and tumorigenesis. Systems for conditional regulation of Hh signaling facilitate the study of these postembryonic Hh functions. RESULTS: We used the hsp70l promoter to generated three heat-shock-inducible transgenic lines that activate Hh signaling and one line that represses Hh signaling. Heat-shock activation of these transgenes appropriately recapitulates early embryonic loss or gain of Hh function phenotypes. Hh signaling remains activated 24 hr after heat shock in the Tg(hsp70l:shha-EGFP) and Tg(hsp70l:dnPKA-BGFP) lines, while a single heat shock of the Tg(hsp70l:gli1-EGFP) or Tg(hsp70l:gli2aDR-EGFP) lines results in a 6- to 12-hr pulse of Hh signal activation or inactivation, respectively. Using both in situ hybridization and quantitative polymerase chain reaction, we show that these lines can be used to manipulate Hh signaling through larval and juvenile stages. A ptch2 promoter element was used to generate new reporter lines that allow clear visualization of Hh responding cells throughout the life cycle, including graded Hh responses in the embryonic central nervous system. CONCLUSIONS: These zebrafish transgenic lines provide important new experimental tools to study the embryonic and postembryonic roles of Hh signaling.

Balanced Shh signaling is required for proper formation and maintenance of dorsal telencephalic midline structures.

BACKGROUND: The rostral telencephalic dorsal midline is an organizing center critical for the formation of the future cortex and hippocampus. While the intersection of WNTs, BMPs, and FGFs establishes boundaries within this critical center, a direct role of Shh signaling in this region remains controversial. In this paper we show that both increased and decreased Shh signaling directly affects boundary formation within the telencephalic dorsal midline. RESULTS: Viral over-expression of Shh in the embryonic telencephalon prevents formation of the cortical hem and choroid plexus, while expanding the roof plate. In a transgenic model where cholesterol-lacking ShhN is expressed from one allele (ShhN/+), genes expressed in all three domains, cortical hem, choroid plexus and roof plate expand. In Gli1/2 -/- mutant brains, where Shh signaling is reduced, the roof plate expands, again at the expense of cortical hem and plexus. Cell autonomous activation of Shh signaling in the dorsal midline through Gdf7-driven activated Smoothened expression results in expansion of the Wnt3a-expressing cortical hem into the plexus domain. In addition, developmental stage determines dorsal midline responsiveness to Shh. CONCLUSIONS: Together, these data demonstrate that balanced Shh signaling is critical for maintaining regional boundaries within the dorsal midline telencephalic organizing center.

Regulatory long non-coding RNAs and neuronal disorders.

Increasing evidence suggests that GABA neuropathies play a major role in a variety of neuronal disorders. In addition, the role of non-coding RNAs in regulating a wide range of cellular processes is an intense area of investigation. This commentary discusses the intersection of these two fields, a corollary to the finding that adult hippocampal GABAergic interneuron development is controlled by an embryonic non-coding RNA during development.

Balanced gene regulation by an embryonic brain ncRNA is critical for adult hippocampal GABA circuitry.

Genomic studies demonstrate that, although the majority of the mammalian genome is transcribed, only about 2% of these transcripts are code for proteins. We investigated how the long, polyadenylated Evf2 noncoding RNA regulates transcription of the homeodomain transcription factors DLX5 and DLX6 in the developing mouse forebrain. We found that, in developing ventral forebrain, Evf2 recruited DLX and MECP2 transcription factors to important DNA regulatory elements in the Dlx5/6 intergenic region and controlled Dlx5, Dlx6 and Gad1 expression through trans and cis-acting mechanisms. Evf2 mouse mutants had reduced numbers of GABAergic interneurons in early postnatal hippocampus and dentate gyrus. Although the numbers of GABAergic interneurons and Gad1 RNA levels returned to normal in Evf2 mutant adult hippocampus, reduced synaptic inhibition occurred. These results suggest that noncoding RNA-dependent balanced gene regulation in embryonic brain is critical for proper formation of GABA-dependent neuronal circuitry in adult brain.

Isolation of rat telencephalic neural explants to assay Shh GABAergic interneuron differentiation-inducing activity.

Multiple assays for Shh activity using cell lines, primary cultures, and explanted tissue have been described. We first described the use of E11.5 rat dorsal telencephalic explants to assay Shh ventralizing and differentiation-inducing activity in Kohtz et al. (1). Using this assay, we subsequently showed that N-lipid modification is critical for Shh activity in the telencephalon (2). In vivo assays for lipid-modified Shh support the results of our E11.5 telencephalic neural explant assay (2). More recently, the method of isolating telencephaic explants was improved by an intraocular grid, increasing both its accuracy and reproducibility (3). Shh induces the expression of the following ventral telencephalic markers: MASH-1, the Dlx's, and Islet 1/2. Therefore, this assay for Shh induction of GABAergic interneurons defines a competent, but naïve region within the E11.5 dorsal telencephalon, allowing the study of GABAergic interneuron induction and differentiation from an unspecified progenitor population.

Patterns of SDF-1alpha and SDF-1gamma mRNAs, migration pathways, and phenotypes of CXCR4-expressing neurons in the developing rat telencephalon.

Cortical GABAergic neurons originate in the ventral telencephalon, invade the cortex via tangential migration, and integrate into the cortical plate by surface-directed and ventricle-directed migration. In mice lacking CXCR4 or SDF-1, GABAergic neurons fail to complete their migration. It is presently unknown which parts of the migration of CXCR4-expressing GABAergic neurons are driven by SDF-1. Here we compared patterns of SDF-1 isoforms and CXCR4 in the developing rat telencephalon. In the ventral telencephalon, radial glia, striatal, and migratory GABAergic neurons expressed CXCR4. Tangentially migrating CXCR4-expressing neurons populated the marginal zone and started to invade the lateral intermediate zone at embryonic day (E)14. Until E17 the spread of CXCR4-expressing neurons in the dorsomedial direction was accompanied by progressive upregulation of SDF-1alpha in the dorsomedial intermediate/subventricular zone. In the meninges, SDF-1alpha and SDF-1gamma were expressed persistently. During invasion of the cortical plate the orientation of CXCR4-immunoreactive neurons changed gradually from tangential (E17/E18) to radial (postnatal day [P] 0), which was paralleled by downregulation of SDF-1alpha in the intermediate/subventricular zone. At E17, CXCR4-immunoreactive cells were colabeled with markers for ventral forebrain-derived neurons (Dlx) but not markers for glutamatergic (Tbr) or subplate (calretinin) neurons. Postnatally, calretinin- and somatostatin-expressing but not parvalbumin-expressing GABAergic neurons or pyramidal cells contained CXCR4. Pyramidal cells and few large blood vessels expressed SDF-1alpha, while microvessels contained SDF-1gamma transcripts. In summary, SDF-1alpha is expressed along cortical but not subcortical migration routes of GABAergic neurons. We propose that regulated expression of SDF-1 in the intermediate/subventricular zone influences lateromedial tangential migration of CXCR4-expressing GABAergic neurons.

The Evf-2 noncoding RNA is transcribed from the Dlx-5/6 ultraconserved region and functions as a Dlx-2 transcriptional coactivator.

The identification of ultraconserved noncoding sequences in vertebrates has been associated with developmental regulators and DNA-binding proteins. One of the first of these was identified in the intergenic region between the Dlx-5 and Dlx-6 genes, members of the Dlx/dll homeodomain-containing protein family. In previous experiments, we showed that Sonic hedgehog treatment of forebrain neural explants results in the activation of Dlx-2 and the novel noncoding RNA (ncRNA), Evf-1. In this report, we show that the Dlx-5/6 ultraconserved region is transcribed to generate an alternatively spliced form of Evf-1, the ncRNA Evf-2. Evf-2 specifically cooperates with Dlx-2 to increase the transcriptional activity of the Dlx-5/6 enhancer in a target and homeodomain-specific manner. A stable complex containing the Evf-2 ncRNA and the Dlx-2 protein forms in vivo, suggesting that the Evf-2 ncRNA activates transcriptional activity by directly influencing Dlx-2 activity. These experiments identify a novel mechanism whereby transcription is controlled by the cooperative actions of an ncRNA and a homeodomain protein. The possibility that a subset of vertebrate ultraconserved regions may function at both the DNA and RNA level to control key developmental regulators may explain why ultraconserved sequences exhibit 90% or more conservation even after 450 million years of vertebrate evolution.

Zebrafish Gli3 functions as both an activator and a repressor in Hedgehog signaling.

Hedgehog (Hh) signaling regulates cell differentiation and patterning in a wide variety of embryonic tissues. In vertebrates, at least three Gli transcription factors (Gli1, Gli2, and Gli3) are involved in Hh signal transduction. Comparative studies have revealed divergent requirements for Gli1 and Gli2 in zebrafish and mouse. Here, we address the question of whether Gli3 function has also diverged in zebrafish and analyze the regulatory interactions between Hh signaling and Gli activity. We find that zebrafish Gli3 has an early function as an activator of Hh target genes that overlaps with Gli1 activator function in the ventral neural tube. In vitro reporter analysis shows that Gli3 cooperates with Gli1 to activate transcription in the presence of high concentrations of Hh. During late somitogenesis stages, Gli3 is required as a repressor of the Hh response. Gli3 shares this repressor activity with Gli2 in the dorsal spinal cord, hindbrain, and midbrain, but not in the forebrain. Consistently, zebrafish Gli3 blocks Gli1-mediated activation of a reporter gene in the absence of Hh in vitro. In the eye, Gli3 is also required for proper ath5 expression and the differentiation of retinal ganglion cells (RGCs). These results reveal a conserved role for Gli3 in vertebrate development and uncover novel regional functions and regulatory interactions among gli genes.

Synergistic and antagonistic roles of the Sonic hedgehog N- and C-terminal lipids.

The Shh protein contains both N-terminal and C-terminal lipids. The functional redundancy of these lipid moieties is presently unclear. Here, we compare the relative roles of the N- and C-terminal lipids in early rat striatal neuronal differentiation, membrane association and multimerization, and ventralizing activity in the zebrafish forebrain. We show that these lipid act synergistically in cell tethering and the formation of a large (L) multimer (669 kDa). However, the C-terminal lipid antagonizes the rat striatal neuronal differentiation-inducing activity of the N-terminal lipid. In addition, multimerization is required but not sufficient for the differentiation-inducing activity. Based on the presence of different N- and C-lipid-containing Shh proteins in the rat embryo, and on their different activities, we propose that both N- and C-terminal lipids are required for the formation of multimers involved in long-range signaling, and that the C-terminal lipid may function in long-range signaling by reducing Shh activity until it reaches its long-range target. Comparative analysis of the ventralizing activities of different N- and C-terminal lipid-containing Shh proteins in the zebrafish forebrain shows that the presence of at least one lipid is required for signaling activity, suggesting that lipid modification of Shh is a conserved requirement for signaling in the forebrain of rodents and zebrafish.

Developmental regulation of EVF-1, a novel non-coding RNA transcribed upstream of the mouse Dlx6 gene.

We previously reported that sonic hedgehog (Shh) induces the differentiation of rat ventral forebrain neurons expressing a novel marker, EVF-1 [Development 125 (1998) 5079]. In this report, we show that EVF-1 is a novel, developmentally regulated, non-coding RNA, with no homology to other known non-coding RNA sequences. Sequence analysis, in vitro translation, and comparison of the rat and mouse EVF-1 sequences suggest that EVF-1 contains no protein coding regions. Chromosomal location indicates that EVF-1 maps adjacent to the Dlx6 gene on mouse chromosome 6. RNA in situ hybridization of the embryonic rat forebrain shows that EVF-1 is expressed by immature neurons in the subventricular zone and its expression decreases during forebrain development. Whole mount in situ hybridization shows that EVF-1 is expressed at high levels in the branchial arches, ventral forebrain, olfactory bulb, and limbs. EVF-1 expression is linked to Shh and the Dlx family of proteins, genes with a demonstrated importance to ventral forebrain and craniofacial development.