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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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The expression pattern of nervous system genes downstream of COE is changed or severely reduced following coe RNAi.Control and coe(RNAi) treated animals were processed for in situ hybridization to the genes indicated above each panel (N≥5 animals per treatment). (A–M′) Detection of all genes was reduced following coe knockdown. Numbers in top right corner represent linear fold changes in mRNA expression in coe(RNAi) planarians relative to the controls. Arrows in I′ and J′ point to loss of expression at the midline compared to the controls (I and J). (N) Quantification of spp19+, spp-18+, and npl+ cells (N = 3); the total number of cells counted is indicated within each bar. Error bars in all graphs are s.d. from the mean; *P<0.05, Student's t-test. Anterior is up in A–M′. Scale bar in A = 100 µm.
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pgen-1004746-g004: The expression pattern of nervous system genes downstream of COE is changed or severely reduced following coe RNAi.Control and coe(RNAi) treated animals were processed for in situ hybridization to the genes indicated above each panel (N≥5 animals per treatment). (A–M′) Detection of all genes was reduced following coe knockdown. Numbers in top right corner represent linear fold changes in mRNA expression in coe(RNAi) planarians relative to the controls. Arrows in I′ and J′ point to loss of expression at the midline compared to the controls (I and J). (N) Quantification of spp19+, spp-18+, and npl+ cells (N = 3); the total number of cells counted is indicated within each bar. Error bars in all graphs are s.d. from the mean; *P<0.05, Student's t-test. Anterior is up in A–M′. Scale bar in A = 100 µm.

Mentions: Based on the annotation of differentially expressed genes, we hypothesized that genes predicted to play roles in nervous system functions in the downregulated category likely include direct COE targets. To test our hypothesis and validate genes found in our RNA-seq dataset, we selected 65 genes that were dramatically downregulated, associated with neural functions, or annotated as transcription factor homologs. First, we performed WISH to determine the tissue-specific pattern of expression of all 65 genes (representative examples are shown in Fig. 4). As we expected, the most prominent mRNA expression pattern was in the nervous system (26 of 65 genes; see Table S2), similar to ChAT and cpp-1, which we had previously found to be putative downstream targets of COE [24]. In addition, we observed genes that were expressed broadly in the nervous system (such as neural cell adhesion molecule-2 (ncam-2), vesicle-associated membrane protein like-1 (vamp), gamma-aminobutyric acid receptor subunit beta like-1 (gbrb-1), and voltage-gated sodium channel alpha-1 (scna-1)) or in discrete neuronal subpopulations (such as secreted peptide prohormone-19, -18, -2 (spp-19, -18, -2), neuropeptide like (npl), voltage-gated sodium channel alpha-2 (scna-2), and caveolin-1 (cav-1)) (Fig. 4A–J). Our list also included transcripts that labeled subsets of neurons in the brain (such as netrin-1) (Fig. 4K) [44]. In addition, we found that the transcription factors iroquios-1 (irx-1) and pou class 4 transcription factor 4 like-1 (pou4l-1) were expressed at or near the cephalic ganglia (Fig. 4L–M), and their mRNA was detected in ChAT+ neurons by fluorescent in situ hybridization (FISH) (Fig. S2). Next, we tested the effect of coe RNAi on the expression of 33 genes that could be visualized in discrete cell populations by WISH. Knockdown of coe led to a marked reduction in the expression of 31 genes (Table S2; representative results are shown in Fig. 4A′–H′, K′–M′); for two genes, scna-2 and cav-1, we observed a loss of expression at the midline (Fig. 4I′–J′). Furthermore, we quantified the number of cells labeled by spp-19, spp-18, and npl probes. As expected, we found there was a significant reduction in the number of spp-19+, spp-18+, and npl+ cells following coe RNAi (Fig. 4N).


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)

The expression pattern of nervous system genes downstream of COE is changed or severely reduced following coe RNAi.Control and coe(RNAi) treated animals were processed for in situ hybridization to the genes indicated above each panel (N≥5 animals per treatment). (A–M′) Detection of all genes was reduced following coe knockdown. Numbers in top right corner represent linear fold changes in mRNA expression in coe(RNAi) planarians relative to the controls. Arrows in I′ and J′ point to loss of expression at the midline compared to the controls (I and J). (N) Quantification of spp19+, spp-18+, and npl+ cells (N = 3); the total number of cells counted is indicated within each bar. Error bars in all graphs are s.d. from the mean; *P<0.05, Student's t-test. Anterior is up in A–M′. Scale bar in A = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004746-g004: The expression pattern of nervous system genes downstream of COE is changed or severely reduced following coe RNAi.Control and coe(RNAi) treated animals were processed for in situ hybridization to the genes indicated above each panel (N≥5 animals per treatment). (A–M′) Detection of all genes was reduced following coe knockdown. Numbers in top right corner represent linear fold changes in mRNA expression in coe(RNAi) planarians relative to the controls. Arrows in I′ and J′ point to loss of expression at the midline compared to the controls (I and J). (N) Quantification of spp19+, spp-18+, and npl+ cells (N = 3); the total number of cells counted is indicated within each bar. Error bars in all graphs are s.d. from the mean; *P<0.05, Student's t-test. Anterior is up in A–M′. Scale bar in A = 100 µm.
Mentions: Based on the annotation of differentially expressed genes, we hypothesized that genes predicted to play roles in nervous system functions in the downregulated category likely include direct COE targets. To test our hypothesis and validate genes found in our RNA-seq dataset, we selected 65 genes that were dramatically downregulated, associated with neural functions, or annotated as transcription factor homologs. First, we performed WISH to determine the tissue-specific pattern of expression of all 65 genes (representative examples are shown in Fig. 4). As we expected, the most prominent mRNA expression pattern was in the nervous system (26 of 65 genes; see Table S2), similar to ChAT and cpp-1, which we had previously found to be putative downstream targets of COE [24]. In addition, we observed genes that were expressed broadly in the nervous system (such as neural cell adhesion molecule-2 (ncam-2), vesicle-associated membrane protein like-1 (vamp), gamma-aminobutyric acid receptor subunit beta like-1 (gbrb-1), and voltage-gated sodium channel alpha-1 (scna-1)) or in discrete neuronal subpopulations (such as secreted peptide prohormone-19, -18, -2 (spp-19, -18, -2), neuropeptide like (npl), voltage-gated sodium channel alpha-2 (scna-2), and caveolin-1 (cav-1)) (Fig. 4A–J). Our list also included transcripts that labeled subsets of neurons in the brain (such as netrin-1) (Fig. 4K) [44]. In addition, we found that the transcription factors iroquios-1 (irx-1) and pou class 4 transcription factor 4 like-1 (pou4l-1) were expressed at or near the cephalic ganglia (Fig. 4L–M), and their mRNA was detected in ChAT+ neurons by fluorescent in situ hybridization (FISH) (Fig. S2). Next, we tested the effect of coe RNAi on the expression of 33 genes that could be visualized in discrete cell populations by WISH. Knockdown of coe led to a marked reduction in the expression of 31 genes (Table S2; representative results are shown in Fig. 4A′–H′, K′–M′); for two genes, scna-2 and cav-1, we observed a loss of expression at the midline (Fig. 4I′–J′). Furthermore, we quantified the number of cells labeled by spp-19, spp-18, and npl probes. As expected, we found there was a significant reduction in the number of spp-19+, spp-18+, and npl+ cells following coe RNAi (Fig. 4N).

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