Pbx Regulates Patterning of the Cerebral Cortex in Progenitors and Postmitotic Neurons

SummaryWe demonstrate using conditional mutagenesis that Pbx1, with and without Pbx2+/− sensitization, regulates regional identity and laminar patterning of the developing mouse neocortex in cortical progenitors (Emx1-Cre) and in newly generated neurons (Nex1-Cre). Pbx1/2 mutants have three salient molecular phenotypes of cortical regional and laminar organization: hypoplasia of the frontal cortex, ventral expansion of the dorsomedial cortex, and ventral expansion of Reelin expression in the cortical plate of the frontal cortex, concomitant with an inversion of cortical layering in the rostral cortex. Molecular analyses, including PBX ChIP-seq, provide evidence that PBX promotes frontal cortex identity by repressing genes that promote dorsocaudal fate

Robo1 modulates proliferation and neurogenesis in the developing neocortex.

International audienceThe elaborate cytoarchitecture of the mammalian neocortex requires the timely production of its constituent pyramidal neurons and interneurons and their disposition in appropriate layers. Numerous chemotropic factors present in the forebrain throughout cortical development play important roles in the orchestration of these events. The Roundabout (Robo) family of receptors and their ligands, the Slit proteins, are expressed in the developing forebrain, and are known to play important roles in the generation and migration of cortical interneurons. However, few studies have investigated their function(s) in the development of pyramidal cells. Here, we observed expression of Robo1 and Slit genes (Slit1, Slit2) in cells lining the telencephalic ventricles, and found significant increases in progenitor cells (basal and apical) at embryonic day (E)12.5 and E14.5 in the developing cortex of Robo1(-/-), Slit1(-/-), and Slit1(-/-)/Slit2(-/-), but not in mice lacking the other Robo or Slit genes. Using layer-specific markers, we found that both early- and late-born pyramidal neuron populations were significantly increased in the cortices of Robo1(-/-) mice at the end of corticogenesis (E18.5). The excess number of cortical pyramidal neurons generated prenatally appears to die in early postnatal life. The observed increase in pyramidal neurons was due to prolonged proliferative activity of their progenitors and not due to changes in cell cycle events. This finding, confirmed by in utero electroporation with Robo1 short hairpin RNA (shRNA) or control constructs into progenitors along the ventricular zone as well as in dissociated cortical cell cultures, points to a novel role for Robo1 in regulating the proliferation and generation of pyramidal neurons

Autism risk gene POGZ promotes chromatin accessibility and expression of clustered synaptic genes.

Deleterious genetic variants in POGZ, which encodes the chromatin regulator Pogo Transposable Element with ZNF Domain protein, are strongly associated with autism spectrum disorder (ASD). Although it is a high-confidence ASD risk gene, the neurodevelopmental functions of POGZ remain unclear. Here we reveal the genomic binding of POGZ in the developing forebrain at euchromatic loci and gene regulatory elements (REs). We profile chromatin accessibility and gene expression in Pogz-/- mice and show that POGZ promotes the active chromatin state and transcription of clustered synaptic genes. We further demonstrate that POGZ forms a nuclear complex and co-occupies loci with ADNP, another high-confidence ASD risk gene, and provide evidence that POGZ regulates other neurodevelopmental disorder risk genes as well. Our results reveal a neurodevelopmental function of an ASD risk gene and identify molecular targets that may elucidate its function in ASD

Single cell enhancer activity distinguishes GABAergic and cholinergic lineages in embryonic mouse basal ganglia.

Enhancers integrate transcription factor signaling pathways that drive cell fate specification in the developing brain. We paired enhancer labeling and single-cell RNA-sequencing (scRNA-seq) to delineate and distinguish specification of neuronal lineages in mouse medial, lateral, and caudal ganglionic eminences (MGE, LGE, and CGE) at embryonic day (E)11.5. We show that scRNA-seq clustering using transcription factors improves resolution of regional and developmental populations, and that enhancer activities identify specific and overlapping GE-derived neuronal populations. First, we mapped the activities of seven evolutionarily conserved brain enhancers at single-cell resolution in vivo, finding that the selected enhancers had diverse activities in specific progenitor and neuronal populations across the GEs. We then applied enhancer-based labeling, scRNA-seq, and analysis of in situ hybridization data to distinguish transcriptionally distinct and spatially defined subtypes of MGE-derived GABAergic and cholinergic projection neurons and interneurons. Our results map developmental origins and specification paths underlying neurogenesis in the embryonic basal ganglia and showcase the power of scRNA-seq combined with enhancer-based labeling to resolve the complex paths of neuronal specification underlying mouse brain development

Ultraconserved enhancer function does not require perfect sequence conservation.

Ultraconserved enhancer sequences show perfect conservation between human and rodent genomes, suggesting that their functions are highly sensitive to mutation. However, current models of enhancer function do not sufficiently explain this extreme evolutionary constraint. We subjected 23 ultraconserved enhancers to different levels of mutagenesis, collectively introducing 1,547 mutations, and examined their activities in transgenic mouse reporter assays. Overall, we find that the regulatory properties of ultraconserved enhancers are robust to mutation. Upon mutagenesis, nearly all (19/23, 83%) still functioned as enhancers at one developmental stage, as did most of those tested again later in development (5/9, 56%). Replacement of endogenous enhancers with mutated alleles in mice corroborated results of transgenic assays, including the functional resilience of ultraconserved enhancers to mutation. Our findings show that the currently known activities of ultraconserved enhancers do not necessarily require the perfect conservation observed in evolution and suggest that additional regulatory or other functions contribute to their sequence constraint

Ultraconserved Enhancers Are Required for Normal Development.

Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting

Ultraconserved Enhancers Are Required for Normal Development.

Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting