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Segment-specific neuronal subtype specification by the integration of anteroposterior and temporal cues.

ABSTRACT: The generation of distinct neuronal subtypes at different axial levels relies upon both anteroposterior and temporal cues. However, the integration between these cues is poorly understood. In the Drosophila central nervous system, the segmentally repeated neuroblast 5-6 generates a unique group of neurons, the Apterous (Ap) cluster, only in thoracic segments. Recent studies have identified elaborate genetic pathways acting to control the generation of these neurons. These insights, combined with novel markers, provide a unique opportunity for addressing how anteroposterior and temporal cues are integrated to generate segment-specific neuronal subtypes. We find that Pbx/Meis, Hox, and temporal genes act in three different ways. Posteriorly, Pbx/Meis and posterior Hox genes block lineage progression within an early temporal window, by triggering cell cycle exit. Because Ap neurons are generated late in the thoracic 5-6 lineage, this prevents generation of Ap cluster cells in the abdomen. Thoracically, Pbx/Meis and anterior Hox genes integrate with late temporal genes to specify Ap clusters, via activation of a specific feed-forward loop. In brain segments, "Ap cluster cells" are present but lack both proper Hox and temporal coding. Only by simultaneously altering Hox and temporal gene activity in all segments can Ap clusters be generated throughout the neuroaxis. This study provides the first detailed analysis, to our knowledge, of an identified neuroblast lineage along the entire neuroaxis, and confirms the concept that lineal homologs of truncal neuroblasts exist throughout the developing brain. We furthermore provide the first insight into how Hox/Pbx/Meis anteroposterior and temporal cues are integrated within a defined lineage, to specify unique neuronal identities only in thoracic segments. This study reveals a surprisingly restricted, yet multifaceted, function of both anteroposterior and temporal cues with respect to lineage control and cell fate specification.

SUBMITTER: Karlsson D

PROVIDER: S-EPMC2867937 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UNLABELLED:Although comprehensively described during early neuronal development, the role of DNA methylation/demethylation in neuronal lineage and subtype specification is not well understood. By studying two distinct neuronal progenitors as they differentiate to principal neurons in mouse hippocampus and striatum, we uncovered several principles governing neuronal DNA methylation during brain development. (1) The program consists of three stages: an initial genome-wide methylation during progenitor proliferation is followed by loss of methylation during the transition of regional progenitors to "young" hippocampal/striatal neurons, which is then reversed by gain in methylation during maturation to subtype-specific neurons. (2) At the first two stages, gain and loss of methylation are limited to CpGs, whereas during the third maturation stage, methylation also occurs at non-CpG sites in both lineages. (3) Methylation/demethylation, similar to transcription, are initially highly similar in the two lineages, whereas diversification in methylation and transcription during maturation creates subtype-specific methylation differences. (4) Initially, methylation targets all genomic locations, whereas later, during early and late differentiation, the preferred targets are intronic/intergenic sequences with enhancer-like activity. (5) Differentially methylated genes are enriched in sequential neurodevelopmental functions (such as progenitor proliferation, migration, neuritogenesis, and synaptic transmission); upregulated genes represent current and consecutive stage-specific functions, and downregulated genes represent preceding functions that are no longer required. The main conclusion of our work is that the neuronal methylation/demethylation program is predominantly developmental with minimal lineage specificity, except in the final stage of development when neuron subtype-specific differences also emerge. SIGNIFICANCE STATEMENT:Our work is the first to describe a set of relatively simple rules that govern DNA methylation and demethylation in neuronal development in vivo. By dividing neurodevelopment to three major stages and applying rules to each of them, we created a matrix that comprehensively describes DNA methylation/demethylation events in two neuronal lineages, with a total of 10 cell types spanning the entire neurodevelopment. Beyond increasing our understanding of the epigenetic regulation of normal development, our work will be useful in deciphering how environmental perturbations, such as gestational toxins, drugs, stress, infection, and offspring neglect/maltreatment, interfere with the developmental methylation program.

Project description:Cocaine-mediated repression of the histone methyltransferase (HMT) G9a has recently been implicated in transcriptional, morphological, and behavioral responses to chronic cocaine administration. Here, using a ribosomal affinity purification approach, we find that G9a repression by cocaine occurs in both Drd1 (striatonigral)- and Drd2 (striatopallidal)-expressing medium spiny neurons (MSNs). Conditional knockout and overexpression of G9a within these distinct cell types, however, reveals divergent behavioral phenotypes in response to repeated cocaine treatment. Our studies further indicate that such developmental deletion of G9a selectively in Drd2 neurons results in the unsilencing of transcriptional programs normally specific to striatonigral neurons, and the acquisition of Drd1-associated projection and electrophysiological properties. This partial striatopallidal to striatonigral ‘switching’ phenotype in mice indicates a novel role for G9a in contributing to neuronal subtype identity, and suggests a critical function for cell-type specific histone methylation patterns in the regulation of behavioral responses to environmental stimuli. Polyribosome associated mRNAs from 2-5 month old, age and sex matched Drd1-Cre; Drd1-TRAP; G9afl/fl and Drd1-TRAP; G9afl/fl, Drd2-Cre; Drd2-TRAP; G9afl/fl and Drd2-TRAP; G9afl/fl mice (n = 2-4 mice/genotype/drug treatment, 2 hours after the last of eight repeated cocaine injections of 20 mg/kg/day) were obtained as previously described. EGFP labeled ribosomes and associated mRNAs were immunoprecipitated using a mix of two monoclonal anti-GFP antibodies (50 μg of clones #19C8 and #19F7 for each IP, available at Sloan-Kettering Monoclonal Antibody Facility). Purified mRNA was amplified and processed for microarray and qPCR analysis using the Affymetrix two-cycle cDNA Synthesis kit (Affymetrix) as previously described. Affymetrix Mouse Genome 430 2.0 arrays were used in all experiments. Information regarding the array design and features can be found at www.affymetrix.com. Mouse Genome 430 2.0 arrays were scanned using the GeneChip Scanner 3000 (Affymetrix) and globally scaled to 150 using the Affymetrix GeneChip Operating Software (GCOS v1.4).

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Segment-specific neuronal subtype specification by the integration of anteroposterior and temporal cues.

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