Figure 1.
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.
Figure 2.
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.
Figure 3.
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.
Table 1.
Annotation of genes differentially expressed in coe(RNAi) animals using DAVID software.
Figure 4.
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.
Figure 5.
Identification of genes expressed coe+ neurons.
Fluorescent in situ hybridization to coe and either spp-19, spp18, npl, spp-2, ncam-2, or netrin-1. Percentages indicate the proportion ± s.d. of cells that were also coe+ (N = 110 spp-19+, 319 spp-18+, 173 npl+, 202 spp-2, 236 ncam-2, and 141 netrin-1 cells counted from 2–3 animals per group). Arrowheads mark double-labeled cells. Anterior is up in all panels. Scale bars = 100 µm.
Table 2.
Candidate COE targets genes identified in S. mediterranea.
Figure 6.
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.
Table 3.
Functional analysis of genes downregulated following coe RNAi.
Figure 7.
COE function is required for differentiation and maintenance of diverse neuron types.
(A) coe is expressed in lineage-committed neoblasts (smedwi+) and early progeny [24], and diverse neuron types, including cholinergic (ChAT), GABAergic (gad), octopaminergic (tbh), dopaminergic (th), serotonergic (tph), and neuropeptidergic (cpp-1, npl, spp-18, spp-19, spp-2) neurons. Genes in green were identified in [24]. (B) To gain insights into how loss of COE function contributes to defects in nervous system differentiation, we analyzed the function of genes that were downregulated in coe(RNAi) animals. These analyses identified additional genes required for CNS regeneration (gbrb1, npl, scna-2, scna-3, pou4l-1) and patterning (nkx2l). In coe(RNAi) animals, we also detected upregulated genes enriched for GO terms associated with muscle development (Table 1), suggesting that COE may also function to repress the expression of mesoderm-specific genes.