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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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Related in: MedlinePlus

coe is expressed in the nervous system and a subset of cycling stem cells.(A) In situ hybridization to coe in S. mediterranea (vn, ventral nerve cords; p, pharynx). Dashed boxes show regions imaged in B–C (N≥10). (B–C) Double-fluorescent in situ hybridization to coe and h2b. Arrowheads mark examples of double-labeled cells (N = 14). Anterior is up in all panels. Scale bars, A = 200 µm, B = 100 µm.
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pgen-1004746-g001: coe is expressed in the nervous system and a subset of cycling stem cells.(A) In situ hybridization to coe in S. mediterranea (vn, ventral nerve cords; p, pharynx). Dashed boxes show regions imaged in B–C (N≥10). (B–C) Double-fluorescent in situ hybridization to coe and h2b. Arrowheads mark examples of double-labeled cells (N = 14). Anterior is up in all panels. Scale bars, A = 200 µm, B = 100 µm.

Mentions: Using an optimized whole-mount in situ hybridization protocol (WISH) (see Materials and Methods), we found that coe mRNA was primarily restricted to neurons in S. mediterranea (Fig. 1A). In agreement with our previous findings [24], we also observed coe transcripts in a subset of cycling stem cells (h2b+) (Fig. 1B–C). We previously reported that coe(RNAi) animals regenerate cephalic ganglia that fail to connect at the anterior commissure and have significantly smaller brains with fewer cpp-1+, npp-4+, and npy-2+ neurons when compared to the controls [24]. This defect is not restricted to the anterior portion of the animal. Additional experiments showed coe(RNAi) animals do not properly regenerate their ventral nerve cords (Fig. S1A–B). Moreover, analysis of the brain patterning defect using anti-VC-1, a marker of the photoreceptor neurons and their axons, revealed that the optic chiasm failed to connect at the midline in coe(RNAi) animals (Fig. S1C). These data demonstrate that coe is essential for neuronal regeneration at both anterior and posterior facing wounds and that coe regulates genes required for reestablishing midline patterning following brain amputation.


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 is expressed in the nervous system and a subset of cycling stem cells.(A) In situ hybridization to coe in S. mediterranea (vn, ventral nerve cords; p, pharynx). Dashed boxes show regions imaged in B–C (N≥10). (B–C) Double-fluorescent in situ hybridization to coe and h2b. Arrowheads mark examples of double-labeled cells (N = 14). Anterior is up in all panels. Scale bars, A = 200 µm, B = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004746-g001: coe is expressed in the nervous system and a subset of cycling stem cells.(A) In situ hybridization to coe in S. mediterranea (vn, ventral nerve cords; p, pharynx). Dashed boxes show regions imaged in B–C (N≥10). (B–C) Double-fluorescent in situ hybridization to coe and h2b. Arrowheads mark examples of double-labeled cells (N = 14). Anterior is up in all panels. Scale bars, A = 200 µm, B = 100 µm.
Mentions: Using an optimized whole-mount in situ hybridization protocol (WISH) (see Materials and Methods), we found that coe mRNA was primarily restricted to neurons in S. mediterranea (Fig. 1A). In agreement with our previous findings [24], we also observed coe transcripts in a subset of cycling stem cells (h2b+) (Fig. 1B–C). We previously reported that coe(RNAi) animals regenerate cephalic ganglia that fail to connect at the anterior commissure and have significantly smaller brains with fewer cpp-1+, npp-4+, and npy-2+ neurons when compared to the controls [24]. This defect is not restricted to the anterior portion of the animal. Additional experiments showed coe(RNAi) animals do not properly regenerate their ventral nerve cords (Fig. S1A–B). Moreover, analysis of the brain patterning defect using anti-VC-1, a marker of the photoreceptor neurons and their axons, revealed that the optic chiasm failed to connect at the midline in coe(RNAi) animals (Fig. S1C). These data demonstrate that coe is essential for neuronal regeneration at both anterior and posterior facing wounds and that coe regulates genes required for reestablishing midline patterning following brain amputation.

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
Related in: MedlinePlus