Basal progenitor cells in the embryonic mouse thalamus - their molecular characterization and the role of neurogenins and Pax6

<p>Abstract</p> <p>Background</p> <p>The size and cell number of each brain region are influenced by the organization and behavior of neural progenitor cells during embryonic development. Recent studies on developing neocortex have revealed the presence of neural progenitor cells that divide away from the ventricular surface and undergo symmetric divisions to generate either two neurons or two progenitor cells. These 'basal' progenitor cells form the subventricular zone and are responsible for generating the majority of neocortical neurons. However, not much has been studied on similar types of progenitor cells in other brain regions.</p> <p>Results</p> <p>We have identified and characterized basal progenitor cells in the embryonic mouse thalamus. The progenitor domain that generates all of the cortex-projecting thalamic nuclei contained a remarkably high proportion of basally dividing cells. Fewer basal progenitor cells were found in other progenitor domains that generate non-cortex projecting nuclei. By using intracellular domain of Notch1 (NICD) as a marker for radial glial cells, we found that basally dividing cells extended outside the lateral limit of radial glial cells, indicating that, similar to the neocortex and ventral telencephalon, the thalamus has a distinct subventricular zone. Neocortical and thalamic basal progenitor cells shared expression of some molecular markers, including <it>Insm1</it>, Neurog1, Neurog2 and NeuroD1. Additionally, basal progenitor cells in each region also expressed exclusive markers, such as Tbr2 in the neocortex and Olig2 and Olig3 in the thalamus. In <it>Neurog1</it>/<it>Neurog2 </it>double mutant mice, the number of basally dividing progenitor cells in the thalamus was significantly reduced, which demonstrates the roles of neurogenins in the generation and/or maintenance of basal progenitor cells. In <it>Pax6 </it>mutant mice, the part of the thalamus that showed reduced Neurog1/2 expression also had reduced basal mitosis.</p> <p>Conclusions</p> <p>Our current study establishes the existence of a unique and significant population of basal progenitor cells in the thalamus and their dependence on neurogenins and Pax6. These progenitor cells may have important roles in enhancing the generation of neurons within the thalamus and may also be critical for generating neuronal diversity in this complex brain region.</p

Neuroepithelial Body Microenvironment Is a Niche for a Distinct Subset of Clara-Like Precursors in the Developing Airways

Clara cells of mammalian airways have multiple functions and are morphologically heterogeneous. Although Notch signaling is essential for the development of these cells, it is unclear how Notch influences Clara cell specification and if diversity is established among Clara cell precursors. Here we identify expression of the secretoglobin Scgb3a2 and Notch activation as early events in a program of secretory cell fate determination in developing murine airways. We show that Scgb3a2 expression in vivo is Notch-dependent at early stages and ectopically induced by constitutive Notch1 activation, and also that in vitro Notch signaling together with the pan-airway transcription factor Ttf1 (Nkx2.1) synergistically regulate secretoglobin gene transcription. Furthermore, we identified a subpopulation of secretory precursors juxtaposed to presumptive neuroepithelial bodies (NEBs), distinguished by their strong Scgb3a2 and uroplakin 3a (Upk3a) signals and reduced Ccsp (Scgb1a1) expression. Genetic ablation of Ascl1 prevented NEB formation and selectively interfered with the formation of this subpopulation of cells. Lineage labeling of Upk3a-expressing cells during development showed that these cells remain largely uncommitted during embryonic development and contribute to Clara and ciliated cells in the adult lung. Together, our findings suggest a role for Notch in the induction of a Clara cell-specific program of gene expression, and reveals that the NEB microenvironment in the developing airways is a niche for a distinct subset of Clara-like precursors.Molecular and Cellular Biolog

Ascl1 (Mash1) Defines Cells with Long-Term Neurogenic Potential in Subgranular and Subventricular Zones in Adult Mouse Brain

Ascl1 (Mash1) is a bHLH transcription factor essential for neural differentiation during embryogenesis but its role in adult neurogenesis is less clear. Here we show that in the adult brain Ascl1 is dynamically expressed during neurogenesis in the dentate gyrus subgranular zone (SGZ) and more rostral subventricular zone (SVZ). Specifically, we find Ascl1 levels low in SGZ Type-1 cells and SVZ B cells but increasing as the cells transition to intermediate progenitor stages. In vivo genetic lineage tracing with a tamoxifen (TAM) inducible Ascl1CreERT2 knock-in mouse strain shows that Ascl1 lineage cells continuously generate new neurons over extended periods of time. There is a regionally-specific difference in neuron generation, with mice given TAM at postnatal day 50 showing new dentate gyrus neurons through 30 days post-TAM, but showing new olfactory bulb neurons even 180 days post-TAM. These results show that Ascl1 is not restricted to transit amplifying populations but is also found in a subset of neural stem cells with long-term neurogenic potential in the adult brain

Ascl1 is present in a subpopulation of Type-1 stem cells and Type-2 progenitors in adult hippocampus.

<p>(A–D) Ascl1 is weakly detected in Nestin::GFP⁺GFAP⁺ Type-1 stem cells (arrowhead) or strongly detected in Nestin::GFP⁺GFAP⁻ Type-2 progenitors (arrow) in SGZ of adult <i>Nestin::GFP</i> mice. (E) Percentage of Ascl1^High or Ascl1^Low cells that express the markers Nestin::GFP and GFAP (Type-1) (dark shaded bars) or just the marker Nestin::GFP (Type-2) (grey shaded bars). 50 Ascl1⁺ cells were counted per mouse, n = 3 <i>Nestin::GFP</i> mice. (F) Ascl1 is in Type-1 and early Type-2 cells based on a current model of adult hippocampal neurogenesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018472#pone.0018472-Kempermann1" target="_blank">[14]</a>. Scale bar  = 20 µm.</p

A subset of Ascl1 lineage cells continue to produce new granule neurons 30 days after initial Ascl1 expression in adult hippocampus.

<p>(A) Targeting strategy for <i>Ascl1^CreERT2/+</i> knock-in mice. (B) Quantification of the percentage of YFP⁺ cells co-labeled with stage-specific markers in hippocampus of adult <i>Ascl1^CreERT2/+;R26R^YFP/YFP</i> mice 7, 30, or 180 days post-TAM. 150–500 YFP⁺ cells per mouse were counted for each marker, n = 2 <i>Ascl1^CreERT2/+;R26R^YFP/YFP</i> mice per time point. (C–F′) 7 days post-TAM YFP⁺ cells co-express GFAP (and have Type-1 morphology), Sox2, or NeuroD1, but not NeuN. (G–J′) 30 days post-TAM YFP⁺ cells overlap with NeuN, but also can co-express GFAP or NeuroD1. (K–N′) 180 days post-TAM a subpopulation of YFP⁺ cells are still Type-1 cells by morphology and express GFAP and Sox2, whereas the majority of YFP⁺ cells express NeuN but not NeuroD1. (O–V) Neurogenesis in the SGZ dramatically decreases between 12 weeks and 34 weeks of age as seen in the decrease in DCX (P,T), NeuroD1 (Q,U) and Ki67 (R,V). Arrowheads indicate the few cells positive for these markers in the 34 week old mice. Notably, Sox2 does not decrease (O,S) so may label quiescent Type-1 like cells. Scale bars  = 50 µm (C,G,K), 10 µm (D–F′, H–J′, I–V).</p

A subset of Ascl1 lineage cells in adult SVZ have long term self renewing properties in the generation of olfactory bulb neurons.

<p>(A–D) Ascl1 is detected in Nestin::GFP⁺GFAP⁺ cells (B cells) in the SVZ (A–B′) and in Nestin::GFP⁺GFAP⁻ C cells in SVZ (A–B′) and RMS (C,C′) in 8 week old <i>Nestin::GFP</i> transgenic mice. (D) Percentage of Ascl1^High or Ascl1^Low cells that express the markers Nestin::GFP and GFAP (dark shaded bars) or just Nestin::GFP (grey shaded bars) in the RMS and the SVZ. 25 Ascl1⁺ cells per mouse were counted in the RMS; 60 Ascl1⁺ cells per mouse in the SVZ, n = 3 <i>Nestin::GFP</i> mice. (E–E′) mRNA in situ with Ascl1 (E) or Cre (E′) probes in the adult SVZ. (F–T′) Immunofluorescence in <i>Ascl1^CreERT2/+</i>;<i>R26R^YFP/YFP</i> mouse brain sections harvested 7, 30, or 180 days post-TAM demonstrates Ascl1 derived cells along the SVZ-RMS-OB pathway (F–N). 7 days post-TAM most YFP⁺ cells were located in the SVZ, or along the RMS (F–H) and express Sox2 (O–O′) or DCX (P–P′). 30 or 180 days post-TAM YFP⁺ cells mature into neurons in the granule cell layer or the periglomerular layer of the OB (I, L, R–R′, and data not shown). However, many YFP⁺ cells remain as Sox2⁺ or DCX⁺ progenitors in the RMS or SVZ (J–K, M–N, Q–Q′, S–T′). Scale bars  = 50 µm (F–N), 10 µm (O–T′).</p

De Novo Mutation in an Enhancer of EBF3 in simplex autism

AbstractPrevious research in autism and other neurodevelopmental disorders (NDDs) has indicated an important contribution of de novo protein-coding variants within specific genes. The role of de novo noncoding variation has been observable as a general increase in genetic burden but has yet to be resolved to individual functional elements. In this study, we assessed whole-genome sequencing data in 2,671 families with autism, with a specific focus on de novo variation in enhancers with previously characterized in vivo activity. We identified three independent de novo mutations limited to individuals with autism in the enhancer hs737. These mutations result in similar phenotypic characteristics, affect enhancer activity in vitro, and preferentially occur in AAT motifs in the enhancer with predicted disruptions of transcription factor binding. We also find that hs737 is enriched for copy number variation in individuals with NDDs, is dosage sensitive in the human population, is brain-specific, and targets the NDD gene EBF3 that is genome-wide significant for protein coding de novo variants, demonstrating the importance of understanding all forms of variation in the genome.One Sentence SummaryWhole-genome sequencing in thousands of families reveals variants relevant to simplex autism in a brain enhancer of the well-established neurodevelopmental disorder gene EBF3

Coding and noncoding variants in EBF3 are involved in HADDS and simplex autism

International audienceBackground: Previous research in autism and other neurodevelopmental disorders (NDDs) has indicated an important contribution of protein-coding (coding) de novo variants (DNVs) within specific genes. The role of de novo noncoding variation has been observable as a general increase in genetic burden but has yet to be resolved to individual functional elements. In this study, we assessed whole-genome sequencing data in 2671 families with autism (discovery cohort of 516 families, replication cohort of 2155 families). We focused on DNVs in enhancers with characterized in vivo activity in the brain and identified an excess of DNVs in an enhancer named hs737. Results; We adapted the fitDNM statistical model to work in noncoding regions and tested enhancers for excess of DNVs in families with autism. We found only one enhancer (hs737) with nominal significance in the discovery (p = 0.0172), replication (p = 2.5 × 10 −3 ), and combined dataset (p = 1.1 × 10 −4 ). Each individual with a DNV in hs737 had shared phenotypes including being male, intact cognitive function, and hypotonia or motor delay. Our in vitro assessment of the DNVs showed they all reduce enhancer activity in a neuronal cell line. By epigenomic analyses, we found that hs737 is brain-specific and targets the transcription factor gene EBF3 in human fetal brain. EBF3 is genome-wide significant for coding DNVs in NDDs (missense p = 8.12 × 10 −35 , loss-of-function p = 2.26 × 10 −13 ) and is widely expressed in the body. Through characterization of promoters bound by EBF3 in neuronal cells, we saw enrichment for binding to NDD genes (p = 7.43 × 10 −6 , OR = 1.87) involved in gene regulation. Individuals with coding DNVs have greater phenotypic severity (hypotonia, ataxia, and delayed development syndrome [HADDS]) in comparison to individuals with noncoding DNVs that have autism and hypotonia. Conclusions: In this study, we identify DNVs in the hs737 enhancer in individuals with autism. Through multiple approaches, we find hs737 targets the gene EBF3 that is genome-wide significant in NDDs. By assessment of noncoding variation and the genes they affect, we are beginning to understand their impact on gene regulatory networks in NDDs

Genomic analyses implicate noncoding de novo variants in congenital heart disease.

A genetic etiology is identified for one-third of patients with congenital heart disease (CHD), with 8% of cases attributable to coding de novo variants (DNVs). To assess the contribution of noncoding DNVs to CHD, we compared genome sequences from 749 CHD probands and their parents with those from 1,611 unaffected trios. Neural network prediction of noncoding DNV transcriptional impact identified a burden of DNVs in individuals with CHD (n = 2,238 DNVs) compared to controls (n = 4,177; P = 8.7 × 10-4). Independent analyses of enhancers showed an excess of DNVs in associated genes (27 genes versus 3.7 expected, P = 1 × 10-5). We observed significant overlap between these transcription-based approaches (odds ratio (OR) = 2.5, 95% confidence interval (CI) 1.1-5.0, P = 5.4 × 10-3). CHD DNVs altered transcription levels in 5 of 31 enhancers assayed. Finally, we observed a DNV burden in RNA-binding-protein regulatory sites (OR = 1.13, 95% CI 1.1-1.2, P = 8.8 × 10-5). Our findings demonstrate an enrichment of potentially disruptive regulatory noncoding DNVs in a fraction of CHD at least as high as that observed for damaging coding DNVs

Single-cell, whole-embryo phenotyping of mammalian developmental disorders.

Mouse models are a critical tool for studying human diseases, particularly developmental disorders1. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse2. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing3 to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic&nbsp;severities, from established multisystem disorders to deletions of individual regulatory regions4,5. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be decomposable through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution