Genomic perspectives of transcriptional regulation in forebrain development

Abstract

The forebrain is the seat of higher-order brain functions, and many human neuropsychiatric disorders are due to genetic defects affecting forebrain development, making it imperative to understand the underlying genetic circuitry. Recent progress now makes it possible to begin fully elucidating the genomic regulatory mechanisms that control forebrain gene expression. Herein, we discuss the current knowledge of how transcription factors drive gene expression programs through their interactions with cis-acting genomic elements, such as enhancers; how analyses of chromatin and DNA modifications provide insights into gene expression states; and how these approaches yield insights into the evolution of the human brain.

Figure 1. Types of gene regulatory sequences and methods for their discovery and characterization

A) Schematic view of different chromatin states in genic and intergenic regions, with characteristic classes of epigenomic features and an overview of selected methods for their genome-wide mapping. B) Track-style view of features commonly associated with different types of regulatory sequences. C) Transgenic reporter assays enable the validation and detailed characterization of enhancer activity patterns in vitro and in vivo. D) Massive-parallel reporter assays enable medium- and large-scale function-based enhancer discovery screens.

Figure 2. TFs with known roles in forebrain development

A/B) Models of transcriptional pathways in the developing mouse basal ganglia based on RNA expression analyses in the embryonic brain of loss of function TF mutants. Green arrows: activation; red stop signal: repression. A) Pathways in the caudal, lateral and medial ganglionic eminences (CGE, LGE, MGE) based on data in references in main text B) Pathways in the medial ganglionic eminence (MGE) based on data in references in main text:. C) Expression of TFs in the basal ganglia of Gsx2^−/−, Dlx1^−/−Dlx2^−/−, and Gsx2^−/−Dlx1^−/−Dlx2^−/− mutant mice. Expression changes are reported separately for two different developmental stages (E12.5 and E15.5) in the ventricular zone (VZ), subventricular zone (SVZ) and mantle zone (MZ) of the CGE, LGE, MGE and Septum. Colors indicate the effect of each mutation on TF expression: Black or no square, not analyzed; gray, no obvious expression change in mutant; white, no detectable expression; magenta, severe reduction in expression; orange, moderate/mild reduction in expression; green, ectopic expression; blue, increased expression. In diagonally divided boxes, the top part represents the dorsal region and the bottom the ventral region. Modified from (Wang et al., 2013).

Figure 3. Spatial and temporal specificity of enhancers active in the developing forebrain

A) Subset of forebrain enhancers with a spectrum of subregional specificities at whole-mount resolution. B) Examples of enhancers with restricted pallial activity in the mouse telencephalon at E11.5. C) Multiple enhancers in the larger region surrounding the Arx gene show subregional forebrain activity patterns that recapitulate endogenous Arx mRNA expression in the mouse forebrain. Notably, enhancer activities show partial spatial redundancy. D) Example of an enhancer with activity across multiple developmental stages, labeling cell populations whose location is consistent with migration from the MGE, through the LGE, to the cortex (white arrows). E) Developmental dynamics of enhancer-associated histone mark H3K27ac at candidate forebrain enhancers analyzed by ChIP-seq across seven stages of brain development. Most sites show temporally restricted H3K27ac marks. F,G) Examples of in vivo validated temporally dynamic enhancer activity in the forebrain, as predicted by temporally dynamic H3K27ac signatures. CP, choroid plexus; Cx, cortex; CxP, cortical plate; DP, dorsal pallium; LGE, lateral ganglionic eminence; LP, lateral pallium; MGE, medial ganglionic eminence; MP, medial pallium; MZ, marginal zone; VP, ventral pallium. A-D modified from Visel et al., 2013. E-G modified from Nord et al., 2013.

Figure 4. Enhancers as tools for analyzing forebrain development

A) PAX6 ChIP-seq analysis from E12.5 cortex showing a peak directly over endogenous enhancer 636 (black bar). B) GFP pallial expression driven by enhancer hs636 in E11 cortex and reduced pallial GFP expression in Pax6−/−. C) Schema showing approximate position of GFP expression (red) within flattened view of E11.5 pallial progenitor zones. D) Enhancer hs636 activity in E10.5 telencephalon in stable transgenics (yellow arrowheads: ventrolateral pallial neurons; red arrowheads: ventrolateral pallial progenitors 24127591). E,F) Fate mapping using enhancer hs636. Cre recombination was tamoxifen-induced at E9.5 and brains were analyzed at E17.5 for tdTomato staining (F). Results are summarized in a schematic map showing dtTomato expression within a flattened view of E17.5 pallial subdivisions, color coded according to approximate density of tdTomato+ cells (E). A-F modified from (Pattabiraman et al., 2014). Abbreviations according to region: Ventral Pallium (VPall, allopallium); AO: anterior olfactory nuclei; OB: olfactory bulb; Pir/EPir; piriform and ectopiriform; LERh: lateral entorhinal; MERh: medial entorhinal. Lateral Pallium (LPall, mesopallium): Ins/Cl: insula/claustrum; LO: lateral orbital; PRh: perirhinal; Orb: orbitofrontal. Dorsal Pallium (DPall; neopallium): AU (A); auditory; DPF: dorsal prefrontal; F: frontal; LPF: lateral prefrontal; M: motor; SS: somatosensory; V: visual. Dorsomedial Pallium (DMPall): Cing (C): cingulate gyrus; IL: infralimbic (and PrL: prelimbic); MOrb: medial orbital; RSP: retrosplenial; PoRh: postrhinal. Medial Pallium (MPall): CA1–3: CA fields 1–3; DG: dentate gyrus; fi (F); fimbria; IG: indusium griseum; Sub (S): subiculum; PaS: parasubiculm; PrS: presubiculum; TT: tenia tecta. Dorsal Midline: bac: brachium of the anterior commissure; bcc: brachium of the corpus callosum; bhc: brachium of the hippocampal commissure; ch; choroid plexus; PSe (PS): pallial septum. Pallial Amygdala (Pall Amygd): AA: anterior amygdala; Ahi: amygdalohippocampal area; BM: basomedial; BLA; basolateral; LA: lateral. Subpallium: Acb: accumbens; CGE: caudal ganglionic eminence; Dg: Diagonal area; LGE: lateral ganglionic eminence; MGE: medial ganglionic eminence; Pal: pallidum; SPSe: subpallial septum; St: striatum. Hypothalamus: hp1, 2: hypothalamic prosomere 1 and 2; PHy: peduncular; Thy: hypothalamus. Diencephalon: Hb; habenula; p2, p3: prosomeres 2 and 3; Thy: terminal hypothalamus; PThE: prethalamic eminence; Th: thalamus.

Figure 5. Insights and Challenges in Deciphering the Regulatory Architecture of Forebrain Development

A) Comparative functional genomic studies, such as large-scale studies of gene expression patterns in the developing brain by RNA in situ hybridization, provide insight into general and human-specific aspects of molecular pathways involved in brain development. B) Comparative genomic studies reveal a deeply conserved regulatory framework associated with brain development, but can also identify specific changes in regulatory sequences that underlie structural and functional innovations in the brain observed in vertebrate evolution. Remarkably, regulatory sequences active during early stages of brain development (mid-gestation in mouse) tend to be under higher evolutionary constraint than those active later in development and in the adult brain. C) Genome-wide association, exome sequencing, copy number variation, and whole-genome sequencing studies of patient cohorts are powerful tools for identifying genes and non-coding sequences associated with neurodevelopmental disorders. These studies have revealed a major role for proteins involved in chromatin remodeling, DNA methylation processes, histone modification, and other transcriptional regulatory pathways and processes, with individual genes reported in human genetic studies offered as examples. D) Systems-level analysis integrating expression, genetic, epigenomic and functional data has the potential to elucidate genetic and functional networks required for normal brain development and function, which are thought to be disrupted in neurodevelopmental and neuropsychiatric disorders.