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COE loss-of-function analysis reveals a genetic program underlying maintenance and regeneration of the nervous system in planarians.

Cowles MW, Omuro KC, Stanley BN, Quintanilla CG, Zayas RM - PLoS Genet. (2014)

Bottom Line: These experiments revealed novel candidate targets of coe in the CNS such as ion channel, neuropeptide, and neurotransmitter genes.Finally, to determine if loss of any of the validated transcripts underscores the coe knockdown phenotype, we knocked down their expression by RNAi and uncovered a set of coe-regulated genes implicated in CNS regeneration and patterning, including orthologs of sodium channel alpha-subunit and pou4.Our study broadens the knowledge of gene expression programs regulated by COE that are required for maintenance of neural subtypes and nervous system architecture in adult animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, San Diego State University, San Diego, California, United States of America.

ABSTRACT
Members of the COE family of transcription factors are required for central nervous system (CNS) development. However, the function of COE in the post-embryonic CNS remains largely unknown. An excellent model for investigating gene function in the adult CNS is the freshwater planarian. This animal is capable of regenerating neurons from an adult pluripotent stem cell population and regaining normal function. We previously showed that planarian coe is expressed in differentiating and mature neurons and that its function is required for proper CNS regeneration. Here, we show that coe is essential to maintain nervous system architecture and patterning in intact (uninjured) planarians. We took advantage of the robust phenotype in intact animals to investigate the genetic programs coe regulates in the CNS. We compared the transcriptional profiles of control and coe RNAi planarians using RNA sequencing and identified approximately 900 differentially expressed genes in coe knockdown animals, including 397 downregulated genes that were enriched for nervous system functional annotations. Next, we validated a subset of the downregulated transcripts by analyzing their expression in coe-deficient planarians and testing if the mRNAs could be detected in coe+ cells. These experiments revealed novel candidate targets of coe in the CNS such as ion channel, neuropeptide, and neurotransmitter genes. Finally, to determine if loss of any of the validated transcripts underscores the coe knockdown phenotype, we knocked down their expression by RNAi and uncovered a set of coe-regulated genes implicated in CNS regeneration and patterning, including orthologs of sodium channel alpha-subunit and pou4. Our study broadens the knowledge of gene expression programs regulated by COE that are required for maintenance of neural subtypes and nervous system architecture in adult animals.

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COE function is required for differentiation and maintenance of diverse neuron types.(A) coe is expressed in lineage-committed neoblasts (smedwi+) and early progeny [24], and diverse neuron types, including cholinergic (ChAT), GABAergic (gad), octopaminergic (tbh), dopaminergic (th), serotonergic (tph), and neuropeptidergic (cpp-1, npl, spp-18, spp-19, spp-2) neurons. Genes in green were identified in [24]. (B) To gain insights into how loss of COE function contributes to defects in nervous system differentiation, we analyzed the function of genes that were downregulated in coe(RNAi) animals. These analyses identified additional genes required for CNS regeneration (gbrb1, npl, scna-2, scna-3, pou4l-1) and patterning (nkx2l). In coe(RNAi) animals, we also detected upregulated genes enriched for GO terms associated with muscle development (Table 1), suggesting that COE may also function to repress the expression of mesoderm-specific genes.
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pgen-1004746-g007: COE function is required for differentiation and maintenance of diverse neuron types.(A) coe is expressed in lineage-committed neoblasts (smedwi+) and early progeny [24], and diverse neuron types, including cholinergic (ChAT), GABAergic (gad), octopaminergic (tbh), dopaminergic (th), serotonergic (tph), and neuropeptidergic (cpp-1, npl, spp-18, spp-19, spp-2) neurons. Genes in green were identified in [24]. (B) To gain insights into how loss of COE function contributes to defects in nervous system differentiation, we analyzed the function of genes that were downregulated in coe(RNAi) animals. These analyses identified additional genes required for CNS regeneration (gbrb1, npl, scna-2, scna-3, pou4l-1) and patterning (nkx2l). In coe(RNAi) animals, we also detected upregulated genes enriched for GO terms associated with muscle development (Table 1), suggesting that COE may also function to repress the expression of mesoderm-specific genes.

Mentions: COE proteins are known to function as terminal selectors of neuronal identity in adult organisms [14], [15], [52], yet the neuronal subtypes and specific genetic programs regulated by COE in the adult CNS are not well understood. In this study, we exploited the high rate of tissue turnover and regenerative capacity of planarians to expand our understanding of how COE may function in the post-embryonic nervous system. We combined RNAi with RNA-seq analysis and identified a set of differentially expressed genes associated with nervous system biological roles. Expression analysis of a subset of these genes revealed novel candidate targets of coe in planarian neurons (Fig. 7A), some of which underscored coe's essential role in maintaining expression of genes vital for neuronal subtype identity and function (such as neurotransmitter receptors, ion channels, and neuropeptide encoding genes) (Fig. 7A–B). Decoding which transcriptional changes are direct or indirect consequences of coe loss in the planarian model will be vital to further elucidate how mutations in COE proteins cause or contribute to disease pathologies in the CNS. The next step will be to find direct COE binding sites genome-wide using in silico and chromatin immunoprecipitation (ChIP) approaches and combining these findings with our differential expression data. In addition, molecular profiling of coe+ cell populations (such as stem cells, postmitotic progeny, and neurons) will be essential to determine how coe function alters in cell type-specific contexts. In conclusion, our study demonstrates the importance of COE family proteins in neuronal turnover and repair of the adult CNS and broadens our understanding of the regulatory programs governed by these factors.


COE loss-of-function analysis reveals a genetic program underlying maintenance and regeneration of the nervous system in planarians.

Cowles MW, Omuro KC, Stanley BN, Quintanilla CG, Zayas RM - PLoS Genet. (2014)

COE function is required for differentiation and maintenance of diverse neuron types.(A) coe is expressed in lineage-committed neoblasts (smedwi+) and early progeny [24], and diverse neuron types, including cholinergic (ChAT), GABAergic (gad), octopaminergic (tbh), dopaminergic (th), serotonergic (tph), and neuropeptidergic (cpp-1, npl, spp-18, spp-19, spp-2) neurons. Genes in green were identified in [24]. (B) To gain insights into how loss of COE function contributes to defects in nervous system differentiation, we analyzed the function of genes that were downregulated in coe(RNAi) animals. These analyses identified additional genes required for CNS regeneration (gbrb1, npl, scna-2, scna-3, pou4l-1) and patterning (nkx2l). In coe(RNAi) animals, we also detected upregulated genes enriched for GO terms associated with muscle development (Table 1), suggesting that COE may also function to repress the expression of mesoderm-specific genes.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214590&req=5

pgen-1004746-g007: COE function is required for differentiation and maintenance of diverse neuron types.(A) coe is expressed in lineage-committed neoblasts (smedwi+) and early progeny [24], and diverse neuron types, including cholinergic (ChAT), GABAergic (gad), octopaminergic (tbh), dopaminergic (th), serotonergic (tph), and neuropeptidergic (cpp-1, npl, spp-18, spp-19, spp-2) neurons. Genes in green were identified in [24]. (B) To gain insights into how loss of COE function contributes to defects in nervous system differentiation, we analyzed the function of genes that were downregulated in coe(RNAi) animals. These analyses identified additional genes required for CNS regeneration (gbrb1, npl, scna-2, scna-3, pou4l-1) and patterning (nkx2l). In coe(RNAi) animals, we also detected upregulated genes enriched for GO terms associated with muscle development (Table 1), suggesting that COE may also function to repress the expression of mesoderm-specific genes.
Mentions: COE proteins are known to function as terminal selectors of neuronal identity in adult organisms [14], [15], [52], yet the neuronal subtypes and specific genetic programs regulated by COE in the adult CNS are not well understood. In this study, we exploited the high rate of tissue turnover and regenerative capacity of planarians to expand our understanding of how COE may function in the post-embryonic nervous system. We combined RNAi with RNA-seq analysis and identified a set of differentially expressed genes associated with nervous system biological roles. Expression analysis of a subset of these genes revealed novel candidate targets of coe in planarian neurons (Fig. 7A), some of which underscored coe's essential role in maintaining expression of genes vital for neuronal subtype identity and function (such as neurotransmitter receptors, ion channels, and neuropeptide encoding genes) (Fig. 7A–B). Decoding which transcriptional changes are direct or indirect consequences of coe loss in the planarian model will be vital to further elucidate how mutations in COE proteins cause or contribute to disease pathologies in the CNS. The next step will be to find direct COE binding sites genome-wide using in silico and chromatin immunoprecipitation (ChIP) approaches and combining these findings with our differential expression data. In addition, molecular profiling of coe+ cell populations (such as stem cells, postmitotic progeny, and neurons) will be essential to determine how coe function alters in cell type-specific contexts. In conclusion, our study demonstrates the importance of COE family proteins in neuronal turnover and repair of the adult CNS and broadens our understanding of the regulatory programs governed by these factors.

Bottom Line: These experiments revealed novel candidate targets of coe in the CNS such as ion channel, neuropeptide, and neurotransmitter genes.Finally, to determine if loss of any of the validated transcripts underscores the coe knockdown phenotype, we knocked down their expression by RNAi and uncovered a set of coe-regulated genes implicated in CNS regeneration and patterning, including orthologs of sodium channel alpha-subunit and pou4.Our study broadens the knowledge of gene expression programs regulated by COE that are required for maintenance of neural subtypes and nervous system architecture in adult animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, San Diego State University, San Diego, California, United States of America.

ABSTRACT
Members of the COE family of transcription factors are required for central nervous system (CNS) development. However, the function of COE in the post-embryonic CNS remains largely unknown. An excellent model for investigating gene function in the adult CNS is the freshwater planarian. This animal is capable of regenerating neurons from an adult pluripotent stem cell population and regaining normal function. We previously showed that planarian coe is expressed in differentiating and mature neurons and that its function is required for proper CNS regeneration. Here, we show that coe is essential to maintain nervous system architecture and patterning in intact (uninjured) planarians. We took advantage of the robust phenotype in intact animals to investigate the genetic programs coe regulates in the CNS. We compared the transcriptional profiles of control and coe RNAi planarians using RNA sequencing and identified approximately 900 differentially expressed genes in coe knockdown animals, including 397 downregulated genes that were enriched for nervous system functional annotations. Next, we validated a subset of the downregulated transcripts by analyzing their expression in coe-deficient planarians and testing if the mRNAs could be detected in coe+ cells. These experiments revealed novel candidate targets of coe in the CNS such as ion channel, neuropeptide, and neurotransmitter genes. Finally, to determine if loss of any of the validated transcripts underscores the coe knockdown phenotype, we knocked down their expression by RNAi and uncovered a set of coe-regulated genes implicated in CNS regeneration and patterning, including orthologs of sodium channel alpha-subunit and pou4. Our study broadens the knowledge of gene expression programs regulated by COE that are required for maintenance of neural subtypes and nervous system architecture in adult animals.

Show MeSH