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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 RNAi strongly inhibits the expression of ChAT in intact planarians.(A–C) coe RNAi-treated animals were processed for fluorescent in situ hybridization (FISH) to ChAT (N = 10 for each treatment), mat (N = 3 control and 4 RNAi planarians), or collagen (N = 7 control and 5 RNAi). White dashed boxes in A denote regions imaged at higher magnification shown in the panels to the right. Black dashed boxes in C denote regions imaged at higher magnification shown in top right insets. (D) RT-qPCR experiments measuring the relative expression of coe, ChAT, mat, or collagen in control(RNAi) or coe(RNAi) planarians following the 6th RNAi treatment. Graph shows the mean ± s.d. expression levels relative to the controls. *P<0.05, Student's t-test.
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pgen-1004746-g002: coe RNAi strongly inhibits the expression of ChAT in intact planarians.(A–C) coe RNAi-treated animals were processed for fluorescent in situ hybridization (FISH) to ChAT (N = 10 for each treatment), mat (N = 3 control and 4 RNAi planarians), or collagen (N = 7 control and 5 RNAi). White dashed boxes in A denote regions imaged at higher magnification shown in the panels to the right. Black dashed boxes in C denote regions imaged at higher magnification shown in top right insets. (D) RT-qPCR experiments measuring the relative expression of coe, ChAT, mat, or collagen in control(RNAi) or coe(RNAi) planarians following the 6th RNAi treatment. Graph shows the mean ± s.d. expression levels relative to the controls. *P<0.05, Student's t-test.

Mentions: In addition, we previously noted that silencing of coe in intact uninjured animals results in a reduction of ChAT+ and pc2+ neurons near the anterior commissure and a loss of cpp-1+ neurons. Following the 6th feeding of coe dsRNA, 100% of the animals exhibited impaired negative phototaxis [24]. To investigate the specificity of the coe knockdown phenotype on the CNS, we examined the effect of coe RNAi on the intestine and muscle as representative endodermal or mesodermal tissues, respectively. We hybridized uninjured control and coe(RNAi) animals with riboprobes specific to ChAT (as a positive control), mat[36], and collagen[37]. As expected, we observed a decrease in ChAT+ neurons in the head [24] and noted a decrease in ChAT expression throughout the animal (Fig. 2A); by contrast, we did not observe a change in the spatial distribution of mat or collagen following coe knockdown (Fig. 2B–C). To quantify the effect of coe RNAi treatments on the expression of ChAT, mat and collagen, we measured relative mRNA levels by reverse transcription quantitative PCR (RT-qPCR). First, we confirmed coe knockdown led to a significant decrease in the relative expression of coe mRNA (down 60%±16% compared to the controls; Fig. 2D). Measurement of ChAT, mat and collagen from coe(RNAi) planarians revealed that ChAT mRNA levels were significantly down (45%±15%) compared to control animals; in contrast to ChAT, the relative mRNA levels of mat or collagen were not affected by coe RNAi treatment (Fig. 2D). Combined with our previous work [24], these results strongly suggest that coe knockdown specifically affects gene transcription in the nervous system and does not cause obvious defects in other tissues such as the intestine or muscle. Furthermore, our results are consistent with reports demonstrating that COE is required to maintain cholinergic and peptidergic neuronal subtype-specific gene expression in Caenorhabditis elegans and Drosophila melanogaster[14], [15].


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 RNAi strongly inhibits the expression of ChAT in intact planarians.(A–C) coe RNAi-treated animals were processed for fluorescent in situ hybridization (FISH) to ChAT (N = 10 for each treatment), mat (N = 3 control and 4 RNAi planarians), or collagen (N = 7 control and 5 RNAi). White dashed boxes in A denote regions imaged at higher magnification shown in the panels to the right. Black dashed boxes in C denote regions imaged at higher magnification shown in top right insets. (D) RT-qPCR experiments measuring the relative expression of coe, ChAT, mat, or collagen in control(RNAi) or coe(RNAi) planarians following the 6th RNAi treatment. Graph shows the mean ± s.d. expression levels relative to the controls. *P<0.05, Student's t-test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214590&req=5

pgen-1004746-g002: coe RNAi strongly inhibits the expression of ChAT in intact planarians.(A–C) coe RNAi-treated animals were processed for fluorescent in situ hybridization (FISH) to ChAT (N = 10 for each treatment), mat (N = 3 control and 4 RNAi planarians), or collagen (N = 7 control and 5 RNAi). White dashed boxes in A denote regions imaged at higher magnification shown in the panels to the right. Black dashed boxes in C denote regions imaged at higher magnification shown in top right insets. (D) RT-qPCR experiments measuring the relative expression of coe, ChAT, mat, or collagen in control(RNAi) or coe(RNAi) planarians following the 6th RNAi treatment. Graph shows the mean ± s.d. expression levels relative to the controls. *P<0.05, Student's t-test.
Mentions: In addition, we previously noted that silencing of coe in intact uninjured animals results in a reduction of ChAT+ and pc2+ neurons near the anterior commissure and a loss of cpp-1+ neurons. Following the 6th feeding of coe dsRNA, 100% of the animals exhibited impaired negative phototaxis [24]. To investigate the specificity of the coe knockdown phenotype on the CNS, we examined the effect of coe RNAi on the intestine and muscle as representative endodermal or mesodermal tissues, respectively. We hybridized uninjured control and coe(RNAi) animals with riboprobes specific to ChAT (as a positive control), mat[36], and collagen[37]. As expected, we observed a decrease in ChAT+ neurons in the head [24] and noted a decrease in ChAT expression throughout the animal (Fig. 2A); by contrast, we did not observe a change in the spatial distribution of mat or collagen following coe knockdown (Fig. 2B–C). To quantify the effect of coe RNAi treatments on the expression of ChAT, mat and collagen, we measured relative mRNA levels by reverse transcription quantitative PCR (RT-qPCR). First, we confirmed coe knockdown led to a significant decrease in the relative expression of coe mRNA (down 60%±16% compared to the controls; Fig. 2D). Measurement of ChAT, mat and collagen from coe(RNAi) planarians revealed that ChAT mRNA levels were significantly down (45%±15%) compared to control animals; in contrast to ChAT, the relative mRNA levels of mat or collagen were not affected by coe RNAi treatment (Fig. 2D). Combined with our previous work [24], these results strongly suggest that coe knockdown specifically affects gene transcription in the nervous system and does not cause obvious defects in other tissues such as the intestine or muscle. Furthermore, our results are consistent with reports demonstrating that COE is required to maintain cholinergic and peptidergic neuronal subtype-specific gene expression in Caenorhabditis elegans and Drosophila melanogaster[14], [15].

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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