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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 maintenance of nervous system architecture in uninjured planarians.(A) Head or tail images from an animal stained with anti-CRMP-2 and processed for FISH to ChAT. CRMP-2 is expressed in axon projections (white arrows) and neuronal cell bodies (yellow arrows; N = 7). (B) Higher magnification image of region denoted by white box in D shows CRMP-2 is detected in ChAT+ cell bodies (arrowhead). Nuclei were stained with DAPI (blue). (C–D) Uninjured control and coe(RNAi) planarians labeled with anti-CRMP-2 and anti-β-TUBULIN or processed for in situ hybridization to cintillo. White and yellow arrows point to axon projections and cell bodies, respectively. N = 8 animals for each treatment; 412 and 290 cintillo+ cells were counted from control and coe(RNAi) animals, respectively. The number in the top right corner indicates the mean ± s.d. of cintillo+ cells; *P<0.05, Student's t-test. Anterior is up in all panels. Scale bars, A = 200 µm, D = 100 µm, E = 50 µm, and G = 200 µm.
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pgen-1004746-g003: COE function is required for maintenance of nervous system architecture in uninjured planarians.(A) Head or tail images from an animal stained with anti-CRMP-2 and processed for FISH to ChAT. CRMP-2 is expressed in axon projections (white arrows) and neuronal cell bodies (yellow arrows; N = 7). (B) Higher magnification image of region denoted by white box in D shows CRMP-2 is detected in ChAT+ cell bodies (arrowhead). Nuclei were stained with DAPI (blue). (C–D) Uninjured control and coe(RNAi) planarians labeled with anti-CRMP-2 and anti-β-TUBULIN or processed for in situ hybridization to cintillo. White and yellow arrows point to axon projections and cell bodies, respectively. N = 8 animals for each treatment; 412 and 290 cintillo+ cells were counted from control and coe(RNAi) animals, respectively. The number in the top right corner indicates the mean ± s.d. of cintillo+ cells; *P<0.05, Student's t-test. Anterior is up in all panels. Scale bars, A = 200 µm, D = 100 µm, E = 50 µm, and G = 200 µm.

Mentions: To investigate if the inhibition of coe perturbs nervous system architecture downstream of gene expression changes, we labeled neuronal cell bodies and their projections using anti-CRMP-2, which labels a subset of neuronal cell bodies and their axon projections, and anti-β-tubulin to visualize nerve projections (Fig. 3A–C). In coe(RNAi) animals, we observed a striking decrease in axon projections labeled by anti-CRMP-2 and anti-β-tubulin compared to the controls; however, expression of CRMP-2 was retained in the cell bodies (Fig. 3C). In addition, when we labeled sensory neurons using cintillo[38], coe(RNAi) animals exhibited significantly fewer cintillo+ cells (Fig. 3D). Our results strongly suggest that nervous system architecture is severely reduced or lost in the absence of coe. These structural defects likely underlie the behavioral abnormalities observed in coe-deficient planarians.


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 maintenance of nervous system architecture in uninjured planarians.(A) Head or tail images from an animal stained with anti-CRMP-2 and processed for FISH to ChAT. CRMP-2 is expressed in axon projections (white arrows) and neuronal cell bodies (yellow arrows; N = 7). (B) Higher magnification image of region denoted by white box in D shows CRMP-2 is detected in ChAT+ cell bodies (arrowhead). Nuclei were stained with DAPI (blue). (C–D) Uninjured control and coe(RNAi) planarians labeled with anti-CRMP-2 and anti-β-TUBULIN or processed for in situ hybridization to cintillo. White and yellow arrows point to axon projections and cell bodies, respectively. N = 8 animals for each treatment; 412 and 290 cintillo+ cells were counted from control and coe(RNAi) animals, respectively. The number in the top right corner indicates the mean ± s.d. of cintillo+ cells; *P<0.05, Student's t-test. Anterior is up in all panels. Scale bars, A = 200 µm, D = 100 µm, E = 50 µm, and G = 200 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004746-g003: COE function is required for maintenance of nervous system architecture in uninjured planarians.(A) Head or tail images from an animal stained with anti-CRMP-2 and processed for FISH to ChAT. CRMP-2 is expressed in axon projections (white arrows) and neuronal cell bodies (yellow arrows; N = 7). (B) Higher magnification image of region denoted by white box in D shows CRMP-2 is detected in ChAT+ cell bodies (arrowhead). Nuclei were stained with DAPI (blue). (C–D) Uninjured control and coe(RNAi) planarians labeled with anti-CRMP-2 and anti-β-TUBULIN or processed for in situ hybridization to cintillo. White and yellow arrows point to axon projections and cell bodies, respectively. N = 8 animals for each treatment; 412 and 290 cintillo+ cells were counted from control and coe(RNAi) animals, respectively. The number in the top right corner indicates the mean ± s.d. of cintillo+ cells; *P<0.05, Student's t-test. Anterior is up in all panels. Scale bars, A = 200 µm, D = 100 µm, E = 50 µm, and G = 200 µm.
Mentions: To investigate if the inhibition of coe perturbs nervous system architecture downstream of gene expression changes, we labeled neuronal cell bodies and their projections using anti-CRMP-2, which labels a subset of neuronal cell bodies and their axon projections, and anti-β-tubulin to visualize nerve projections (Fig. 3A–C). In coe(RNAi) animals, we observed a striking decrease in axon projections labeled by anti-CRMP-2 and anti-β-tubulin compared to the controls; however, expression of CRMP-2 was retained in the cell bodies (Fig. 3C). In addition, when we labeled sensory neurons using cintillo[38], coe(RNAi) animals exhibited significantly fewer cintillo+ cells (Fig. 3D). Our results strongly suggest that nervous system architecture is severely reduced or lost in the absence of coe. These structural defects likely underlie the behavioral abnormalities observed in coe-deficient planarians.

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