Limits...
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.

Show MeSH

Related in: MedlinePlus

CNS regeneration defects following knockdown of COE-regulated genes.(A–D) Animals were fed control, scna-2, nkx2l and pou4l-1 bacterially-expressed dsRNA (indicated to the left of each panel), amputated pre-pharyngeally and allowed to regenerate. Ten-day regenerates were imaged live (A–D), killed and immunostained with anti-SYNAPSIN or processed for fluorescent in situ hybridization to ChAT or npl (N≥4). (E–F) Brain size estimated by measuring head area stained by anti-SYNAPSIN or in situ hybridization to ChAT and normalized by the length of animal for control, scna-2, nkx2l, and pou4l-1 RNAi planarians. (G) Quantification of npl+ cells normalized by brain size measured from ChAT stain in F (N≥4 animals in each group); the total number of npl+ 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–D. Scale bars = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214590&req=5

pgen-1004746-g006: CNS regeneration defects following knockdown of COE-regulated genes.(A–D) Animals were fed control, scna-2, nkx2l and pou4l-1 bacterially-expressed dsRNA (indicated to the left of each panel), amputated pre-pharyngeally and allowed to regenerate. Ten-day regenerates were imaged live (A–D), killed and immunostained with anti-SYNAPSIN or processed for fluorescent in situ hybridization to ChAT or npl (N≥4). (E–F) Brain size estimated by measuring head area stained by anti-SYNAPSIN or in situ hybridization to ChAT and normalized by the length of animal for control, scna-2, nkx2l, and pou4l-1 RNAi planarians. (G) Quantification of npl+ cells normalized by brain size measured from ChAT stain in F (N≥4 animals in each group); the total number of npl+ 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–D. Scale bars = 100 µm.

Mentions: Our RNA-seq dataset revealed that coe is essential to maintain the expression of hundreds of genes in the adult animal. This change in the neuronal gene expression landscape led to abnormal CNS structure and behavior. To identify genes downstream of coe that contribute to CNS differentiation, we took advantage of the experimental ease in examination of gene function in planarian regeneration and analyzed the role of 11 downregulated genes that were expressed in neurons or predicted to encode transcription factors (Table 3). Following RNAi, animals were amputated pre- and post-pharyngeally and allowed to regenerate for 10 days. We found that 6 out of 11 genes resulted in defective brain regeneration (see Table 3); scna-2, pou4l-1, and nkx2l caused the strongest phenotypes. Compared to the controls, scna-2(RNAi) animals had less eye pigmentation or developed a single eyespot; nkx2l(RNAi) animals exhibited photoreceptor defects; and pou4l-1(RNAi) animals had less photoreceptor pigment (Fig. 6A–D). To examine CNS architecture, we stained scna-2, nkx2l, and pou4l-1 RNAi treated planarians with anti-SYNAPSIN and the coe-regulated genes ChAT and npl. Although subtle, all three showed abnormalities in brain morphology (Fig. 6A–D). However, when we measured the area of the brain stained by anti-SYNAPSIN, only scna-2 and pou4l-1 RNAi animals had a significant reduction in neuropil density (Fig. 6E). Consistent with this observation, the ChAT+ brain areas were smaller in scna-2(RNAi) and pou4l-1(RNAi) animals (Fig. 6F) but not in nkx2l(RNAi) animals. The smaller brain phenotype was accompanied by fewer npl+ neurons in scna-2(RNAi) animals; however, despite their smaller brains, pou4l-1(RNAi) animals regenerated significantly more npl+ cells than controls (Fig. 6G). These findings demonstrate that scna-2 is required for CNS regeneration and highlight the importance of ion channels in neurogenesis regulation during CNS development, maintenance, and repair [45]–[47]. Interestingly, these data suggest that pou4l-1 plays a role in the specification of certain neuronal lineages. It is possible that in the absence of pou4l-1, planarians regenerate the incorrect proportion of neuronal subtypes and have disorganized brains, but this possibility will require further analysis with additional neuronal subtype-specific markers. By contrast, our results suggest nkx2l is not required for CNS regeneration per se. Following coe RNAi, nkx2l expression was reduced by in situ hybridization and RT-qPCR (Table S2 and Fig. S3A), but nkx2l, which is primarily expressed in stem cells and in progeny [48], was not detected in the nervous system (Fig. S5A). We hypothesize nkx2l functions in early regeneration to establish patterning, which is consistent with the observation that nkx2l(RNAi) planarians fail to regenerate properly patterned head (Fig. 6C) and tail tissues (Fig. S5B).


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)

CNS regeneration defects following knockdown of COE-regulated genes.(A–D) Animals were fed control, scna-2, nkx2l and pou4l-1 bacterially-expressed dsRNA (indicated to the left of each panel), amputated pre-pharyngeally and allowed to regenerate. Ten-day regenerates were imaged live (A–D), killed and immunostained with anti-SYNAPSIN or processed for fluorescent in situ hybridization to ChAT or npl (N≥4). (E–F) Brain size estimated by measuring head area stained by anti-SYNAPSIN or in situ hybridization to ChAT and normalized by the length of animal for control, scna-2, nkx2l, and pou4l-1 RNAi planarians. (G) Quantification of npl+ cells normalized by brain size measured from ChAT stain in F (N≥4 animals in each group); the total number of npl+ 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–D. Scale bars = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004746-g006: CNS regeneration defects following knockdown of COE-regulated genes.(A–D) Animals were fed control, scna-2, nkx2l and pou4l-1 bacterially-expressed dsRNA (indicated to the left of each panel), amputated pre-pharyngeally and allowed to regenerate. Ten-day regenerates were imaged live (A–D), killed and immunostained with anti-SYNAPSIN or processed for fluorescent in situ hybridization to ChAT or npl (N≥4). (E–F) Brain size estimated by measuring head area stained by anti-SYNAPSIN or in situ hybridization to ChAT and normalized by the length of animal for control, scna-2, nkx2l, and pou4l-1 RNAi planarians. (G) Quantification of npl+ cells normalized by brain size measured from ChAT stain in F (N≥4 animals in each group); the total number of npl+ 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–D. Scale bars = 100 µm.
Mentions: Our RNA-seq dataset revealed that coe is essential to maintain the expression of hundreds of genes in the adult animal. This change in the neuronal gene expression landscape led to abnormal CNS structure and behavior. To identify genes downstream of coe that contribute to CNS differentiation, we took advantage of the experimental ease in examination of gene function in planarian regeneration and analyzed the role of 11 downregulated genes that were expressed in neurons or predicted to encode transcription factors (Table 3). Following RNAi, animals were amputated pre- and post-pharyngeally and allowed to regenerate for 10 days. We found that 6 out of 11 genes resulted in defective brain regeneration (see Table 3); scna-2, pou4l-1, and nkx2l caused the strongest phenotypes. Compared to the controls, scna-2(RNAi) animals had less eye pigmentation or developed a single eyespot; nkx2l(RNAi) animals exhibited photoreceptor defects; and pou4l-1(RNAi) animals had less photoreceptor pigment (Fig. 6A–D). To examine CNS architecture, we stained scna-2, nkx2l, and pou4l-1 RNAi treated planarians with anti-SYNAPSIN and the coe-regulated genes ChAT and npl. Although subtle, all three showed abnormalities in brain morphology (Fig. 6A–D). However, when we measured the area of the brain stained by anti-SYNAPSIN, only scna-2 and pou4l-1 RNAi animals had a significant reduction in neuropil density (Fig. 6E). Consistent with this observation, the ChAT+ brain areas were smaller in scna-2(RNAi) and pou4l-1(RNAi) animals (Fig. 6F) but not in nkx2l(RNAi) animals. The smaller brain phenotype was accompanied by fewer npl+ neurons in scna-2(RNAi) animals; however, despite their smaller brains, pou4l-1(RNAi) animals regenerated significantly more npl+ cells than controls (Fig. 6G). These findings demonstrate that scna-2 is required for CNS regeneration and highlight the importance of ion channels in neurogenesis regulation during CNS development, maintenance, and repair [45]–[47]. Interestingly, these data suggest that pou4l-1 plays a role in the specification of certain neuronal lineages. It is possible that in the absence of pou4l-1, planarians regenerate the incorrect proportion of neuronal subtypes and have disorganized brains, but this possibility will require further analysis with additional neuronal subtype-specific markers. By contrast, our results suggest nkx2l is not required for CNS regeneration per se. Following coe RNAi, nkx2l expression was reduced by in situ hybridization and RT-qPCR (Table S2 and Fig. S3A), but nkx2l, which is primarily expressed in stem cells and in progeny [48], was not detected in the nervous system (Fig. S5A). We hypothesize nkx2l functions in early regeneration to establish patterning, which is consistent with the observation that nkx2l(RNAi) planarians fail to regenerate properly patterned head (Fig. 6C) and tail tissues (Fig. S5B).

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