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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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Identification of genes expressed coe+ neurons.Fluorescent in situ hybridization to coe and either spp-19, spp18, npl, spp-2, ncam-2, or netrin-1. Percentages indicate the proportion ± s.d. of cells that were also coe+ (N = 110 spp-19+, 319 spp-18+, 173 npl+, 202 spp-2, 236 ncam-2, and 141 netrin-1 cells counted from 2–3 animals per group). Arrowheads mark double-labeled cells. Anterior is up in all panels. Scale bars = 100 µm.
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pgen-1004746-g005: Identification of genes expressed coe+ neurons.Fluorescent in situ hybridization to coe and either spp-19, spp18, npl, spp-2, ncam-2, or netrin-1. Percentages indicate the proportion ± s.d. of cells that were also coe+ (N = 110 spp-19+, 319 spp-18+, 173 npl+, 202 spp-2, 236 ncam-2, and 141 netrin-1 cells counted from 2–3 animals per group). Arrowheads mark double-labeled cells. Anterior is up in all panels. Scale bars = 100 µm.

Mentions: As an additional test to validate the in situ hybridization results, we measured the relative expression levels of downregulated genes in control and coe RNAi-treated planarians using RT-qPCR (Fig. S3A). All of the genes we tested showed a decrease in relative expression following coe RNAi (9 of 14 genes were significantly downregulated; P<0.05, Student's t-test). By contrast, when we measured the relative expression of CNS-expressed genes that were not on our list of differentially expressed genes, none were significantly reduced (11 of 11 genes; Fig. S3B–C). Although some of the control genes we selected were reduced near levels comparable to some genes downregulated following coe RNAi (e.g., ncam2, vamp, and gbrb1; Fig. S3A), we noted that isotig13897 and npp-2[30], which are transcripts detected in subsets of neurons or throughout the CNS, respectively, remained unchanged (Fig. S3B–C). It is possible that some changes in gene expression associated with coe RNAi are consequence of a reduction in nervous system tissue. We proceeded to perform double-FISH to coe and validated genes to determine if any were potential genetic targets of COE. Of the 17 genes we were able to reliably detect by FISH (33 genes were tested; see Table S2), 11 were expressed in coe+ cells (representative results are shown in Fig. 5 and Fig. S4), including ChAT and cpp-1[24]. Together, these results identified nine novel candidate targets of COE in the nervous system, including genes important for maintaining neuronal subtype identity such as ion channels, ion channel receptors, and neuropeptide genes (Table 2). In addition, our data suggest that COE is essential to maintain genetic programs in multiple classes of adult neuronal subtypes including excitatory (cholinergic) and inhibitory (GABAergic) neurons.


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)

Identification of genes expressed coe+ neurons.Fluorescent in situ hybridization to coe and either spp-19, spp18, npl, spp-2, ncam-2, or netrin-1. Percentages indicate the proportion ± s.d. of cells that were also coe+ (N = 110 spp-19+, 319 spp-18+, 173 npl+, 202 spp-2, 236 ncam-2, and 141 netrin-1 cells counted from 2–3 animals per group). Arrowheads mark double-labeled cells. Anterior is up in all panels. Scale bars = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004746-g005: Identification of genes expressed coe+ neurons.Fluorescent in situ hybridization to coe and either spp-19, spp18, npl, spp-2, ncam-2, or netrin-1. Percentages indicate the proportion ± s.d. of cells that were also coe+ (N = 110 spp-19+, 319 spp-18+, 173 npl+, 202 spp-2, 236 ncam-2, and 141 netrin-1 cells counted from 2–3 animals per group). Arrowheads mark double-labeled cells. Anterior is up in all panels. Scale bars = 100 µm.
Mentions: As an additional test to validate the in situ hybridization results, we measured the relative expression levels of downregulated genes in control and coe RNAi-treated planarians using RT-qPCR (Fig. S3A). All of the genes we tested showed a decrease in relative expression following coe RNAi (9 of 14 genes were significantly downregulated; P<0.05, Student's t-test). By contrast, when we measured the relative expression of CNS-expressed genes that were not on our list of differentially expressed genes, none were significantly reduced (11 of 11 genes; Fig. S3B–C). Although some of the control genes we selected were reduced near levels comparable to some genes downregulated following coe RNAi (e.g., ncam2, vamp, and gbrb1; Fig. S3A), we noted that isotig13897 and npp-2[30], which are transcripts detected in subsets of neurons or throughout the CNS, respectively, remained unchanged (Fig. S3B–C). It is possible that some changes in gene expression associated with coe RNAi are consequence of a reduction in nervous system tissue. We proceeded to perform double-FISH to coe and validated genes to determine if any were potential genetic targets of COE. Of the 17 genes we were able to reliably detect by FISH (33 genes were tested; see Table S2), 11 were expressed in coe+ cells (representative results are shown in Fig. 5 and Fig. S4), including ChAT and cpp-1[24]. Together, these results identified nine novel candidate targets of COE in the nervous system, including genes important for maintaining neuronal subtype identity such as ion channels, ion channel receptors, and neuropeptide genes (Table 2). In addition, our data suggest that COE is essential to maintain genetic programs in multiple classes of adult neuronal subtypes including excitatory (cholinergic) and inhibitory (GABAergic) neurons.

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