Supplementary Figures

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Logan Shepherd
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1 Supplementary Figures Supplementary Fig. S1: Normal development and organization of the embryonic ventral nerve cord in Platynereis. (A) Life cycle of Platynereis dumerilii. (B-F) Axonal scaffolds and ciliated troches revealed by acetylated-tubulin antibody staining (cyan). Scale bars are 1

2 14, 17, 59 50µm. (B -F ) Schematic drawings of the developing CNS, based on published data and representing the expression of Pdu-soxB (light blue areas), a marker of naive neurectodermal cells; Pdu-elav (orange areas), an early marker of neuron differentiation; and Pdu-synaptotagmin (Pdu-syt; red areas) which is a late marker of neuron differentiation. At 24hpf, the neurectoderm (visualized by Pdu-SoxB expression) is still split into two bilateral domains separated by mesodermal midline cells. Between 24 and 33hpf, neurectodermal cells converge towards the midline resulting in a progressive posterior to anterior fusion (in a zipper-like fashion) of the two halves of the neurectoderm. The fusion is almost complete at 33hpf except in the region of the future mouth and is achieved by 40hpf. At both 24 and 33hpf, the ventral nerve cord (VNC) is composed of only a few primary neurons required for troches motility and swimming. They form two longitudinal connectives that connect the posterior serotoninergic neurons to the anterior prototroche ring nerve and will provide a support to guide the projections of neurons that will differentiate later. Massive neuron differentiation occurs between 40hpf and 72hpf as shown by the expansion of the expression domain of Pdu-elav, in a first time, and of Pdu-syt, in a second time. First commissural projections linking the two longitudinal connectives can be observed from 36-40hpf and their number increases rapidly during subsequent stages. Finally, the embryonic nervous system displays lateral projections that are visible from 48-55hpf and connect the ventral nerve cord to the developing peripheral nervous system. At 72hpf, these lateral projections have increased in number and form the three segmental nerve that innervate the developing parapods. Abbreviations: con = connectives, com = commissures, lat = lateral projections, seg = segmental nerves. 2

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10 Supplementary Fig. S2: Orthology assignment of the newly cloned Platynereis genes. ML trees constructed with PhyML are shown. Statistical supports (alrt) are indicated on the nodes by color circles (color code is indicated in the figure). Nodes without color circles are not statistically supported. When possible, outgroups were used to root the phylogenetic trees; in the other cases, mid-branch rooting was used. Sequences were identified either by their NCBI accession number or the identifier given by the Joint Genome Institute when no accession numbers were available (in the case of Lottia, Capitella and Daphnia). (A) cyclin genes. We included in the analysis cyclin A, B, B3, and E sequences and used cyclin O as outgroup. Single Platynereis genes were found for each class, except for cyclin B where two genes were identified. (B) wntless genes. We found a single Platynereis gene that encodes a protein with the MIG-14_Wnt-bd domain which is specific to Wntless proteins. (C) axin genes. A single gene with high sequence similarity to known axin genes was found in 10

11 Platynereis. (D) flamingo genes. We cloned a single gene in Platynereis with high sequence similarity to Drosophila flamingo/starry night and vertebrates celsr genes. The Platynereis gene encodes a protein with several domains, cadherin repeats, Calcium-binding EGF-like domain, Laminin-type epidermal growth factor-like domain, and Hormone receptor domain, whose presence together is characteristic of Flamingo/Celsr proteins. (E) diego genes. We identified a Platynereis gene with moderate sequence similarity with Drosophila diego and their orthologs in vertebrates, ankyrin 6/diversin. We performed phylogenetic analyses with different samplings of ankyrin proteins, but failed to recover consistent phylogenetic trees (not shown). We however found that the cloned Platynereis gene strongly clusters with Drosophila diego and vertebrate ankyrin 6 genes. For sake of simplicity, we show here a simplified tree only comprising the sequences that cluster with the diego/ankyrin 6 group. Drosophila ankyrin was used as outgroup to root the tree. (F) prickle genes. We found a single Platynereis gene that show high sequence similarity with prickle genes and encodes a protein with PET (Prickle - Espinas-Testin) and Prickle-like LIM domains. Phylogenetic analysis was done using Drosophila paxilin as outgroup. (G) and (H) Rho and Rac genes. These genes belong to a large family that encode proteins characterized by a Rho domain. We identified two genes in Platynereis with high sequence similarities to Rho and Rac genes, respectively. We made a phylogenetic analysis with a large number of Rho and Rac like genes and found that the two Platynereis genes cluster with Drosophila and vertebrate Rac1/2 or Rho1/RhoA genes (not shown). For sake of simplicty, we only show here a subset of the sequences and two separated trees for the two subfamilies. Drosophila Cdc42 and mig-2-like were used as outgroups. (I) rok genes. A single Platynereis gene with high sequence similarity to rok genes and encoding a protein with a Rok-like PKc domain was identified. Phylogenetic analysis was performed with mouse MRCK genes as outgroup. (J) four-jointed genes. We identified two genes that encode proteins with a FAM20_c domain which is 11

12 specific of Four-jointed proteins. We failed to recover any four-jointed genes from non bilaterian species. Phylogenetic analysis showed the existence of two groups of four-jointed genes, one of which containing the Drosophila and vertebrate four-jointed genes as well as genes from most other species including Platynereis. The second group (the genes were named four-jointed-like) only comprises genes from a few species, including Platynereis. (K) and (L) fat and dachsous genes. We found two genes in Platynereis that encode atypical Cadherins and show high sequence similarity with fat and dachsous. 12

13 Supplementary Fig. S3: Expression profile of Pdu-cycB-a in the neurectoderm of Platynereis larvae. Ventral views 24 to 72hpf larvae are shown. (A-E ) WMISH for the PducyclinB-a (cycb-a) gene. At all stages, the expression pattern of Pdu-cycB-a is highly similar to the profile of EdU incorporation showed in Figure 1. Scale bars are 50µm. 13

Supplementary Fig. S4: Cartoons summarizing the architecture of the neurectoderm of 55hpf Platynereis larvae. For the sake of clarity, only one half of the tissue is shown in transverse sections.

14 Supplementary Fig. S4: Cartoons summarizing the architecture of the neurectoderm of 55hpf Platynereis larvae. For the sake of clarity, only one half of the tissue is shown in transverse sections. Apical is up, lateral is on the left, and medial on the right. The dashed line demarcates the CNS from the PNS. At 55hpf the neurectoderm is composed of multiple concentric cell layers organized around the apical ventral midline. Regarding both their mitotic potential and the combination of genes that they express, the succession of these cell layers recapitulates the process of neurogenesis. This architecture suggests that extracellular signals produced by ventral midline cells regulate the balance between proliferation and differentiation of NPCs in a way that is reminiscent to that of the vertebrate neural tube. 14

Supplementary Fig. S5: PNU-74654 treated embryos show defects similar to those observed in embryos treated with endo-iwr1.

15 Supplementary Fig. S5: PNU treated embryos show defects similar to those observed in embryos treated with endo-iwr1. Ventral views of whole embryos and transverse sections through the neurectoderm are shown. See the cartoon in figure 1 for orientations. Embryos were incubated with PNU (5µM) in DMSO (0.05%) or in DMSO only (control group) from 33 to 55hpf. (A-B ) Antibody staining against acetylated tubulin (cyan) showing the axon scaffold of the VNC, coupled with 30min EdU (red) 15

16 incorporation at 55hpf showing the cell proliferation profile and Hoechst nuclear staining (blue). Embryos treated with PNU display an important reduction of the VNC, defects in axon guidance (arrowheads) and an extension of the cell proliferation profile. Abbreviations: con, longitudinal connectives; com, commissural axons; lat, lateral projections. (C) Graph showing the proportions of affected vs unaffected embryos in the control and treated groups (sample sizes are indicated on the graph). The experiment was replicated 3 times. All control embryos and 8% of treated ones are unaffected, 92% of treated embryos are affected. (D-G) WMISH that reveal the expression of several genes involved in the process of neurogenesis. Pdu-neurogenin expression is extended in treated embryos and suggests that the proliferating cells found throughout the whole neurectoderm are NPCs. By contrast, the reduction of Pdu-elav and Pdu-coe expression indicates a decrease in the production of neurons. These results show important similarities with those obtained using endo-iwr1 and therefore reinforce our conclusions about the role of the Wnt/β-catenin pathway in stimulating neuron differentiation. A strong reduction of Pdu-axin expression was also observed, similarly to what was found in endo-iwr1-treated embryos. Gene name abbreviations: Pdu-ngn, neurogenin; Pdu-coe, collier. Scale bars are 50µm for ventral views and 25µm for transverse views. 16

Supplementary Fig. S6: Pharmacological treatments altering Wnt/β-catenin pathway activity do not induce cell death in Platynereis embryos. Ventral views of whole embryos are shown.

17 Supplementary Fig. S6: Pharmacological treatments altering Wnt/β-catenin pathway activity do not induce cell death in Platynereis embryos. Ventral views of whole embryos are shown. See the cartoon in figure 1 for orientations. A TUNEL protocol, revealing the presence of apoptotic cells has been adapted to Platynereis (see methods) and used on embryos treated with endo-iwr1 40µM, PNU µM and azakenpaullone 15µM from 33 to 55hpf, as well as on their respective controls incubated in DMSO. (A) A positive control embryo incubated in DNAseI shows a labelling of all cells following TUNEL assay. (B-D ) The amount of apoptotic cells observed in treated embryos was either equivalent or inferior to that observed in their control counterpart, suggesting that none of the treatments induced cell death in Platynereis embryos. Scale bars are 50µm. 17

Supplementary Fig. S7: Constitutive activation of the Wnt/β-catenin pathway reduces cell proliferation and causes defects in VNC development.

18 Supplementary Fig. S7: Constitutive activation of the Wnt/β-catenin pathway reduces cell proliferation and causes defects in VNC development. Ventral views of whole embryos and transverse sections through the neurectoderm are shown. See the cartoon in figure 1 for orientations. Embryos were incubated with azakenpaullone (15µM) in DMSO (1.5%) or in DMSO only (control group) from 33 to 55hpf. By inhibiting Glycogen-Synthase- Kinase-3 (GSK3), azakenpaullone prevents β-catenin degradation and therefore constitutively activates the Wnt/β-catenin pathway. (A-B ) Antibody staining against acetylated tubulin 18

19 (cyan) showing the axon scaffold of the VNC, coupled with 30min EdU (red) incorporation at 55hpf showing the cell proliferation profile and Hoechst nuclear staining (blue). Embryos treated with Azakenpaullone display an important reduction of the VNC, defects in axon guidance (arrowhead) and an almost complete absence of EdU labeled cells. Abbreviations: con, longitudinal connectives; com, commissural axons; lat, lateral projections. (C) Graph showing the proportions of affected vs unaffected embryos in the control and treated groups (sample sizes are indicated on the graph). The experiment was replicated 3 times. All control embryos are unaffected, and all treated embryos are affected. (D-G) WMISH revealing the expression of several genes involved in neurogenesis. Pdu-neurogenin expression is abolished in the neurectoderm of treated embryos. Together with the strong reduction of the number of EdU incorporating cells, this suggests that the constitutive activation of the Wnt/ -catenin pathway leads to the absence of proliferating NPCs. This effect is opposite to what is observed upon Wnt/β-catenin inhibition using endo-iwr1 or PNU The expression patterns of Pdu-elav and Pdu-coe are reduced, indicating a reduction in the number of neurons. The reduction of the number of both NPCs and neurons is puzzling and could be due to the premature differentiation of the NPCs that would have not stayed in a proliferative state long enough to produce a normal amount of neurons. Alternatively, the constitutive activation of the Wnt/ -catenin pathway may have forced the NPCs to exit the cell cycle while remaining in an undifferentiated state. We observed a very strong increase in the expression of Pdu-axin upon azakenpaullone treatment, reinforcing the hypothesis that the Wnt/β-catenin pathway positively regulates axin expression in Platynereis. Gene name abbreviations: Pdungn, neurogenin; Pdu-coe, collier. Scale bars are 50µm for ventral views and 25µm for transverse views. 19

Figure S1: Phylogenetic analysis of the Platynereis Notch pathway core components, Pdu-

Figure S1: Phylogenetic analysis of the Platynereis Notch pathway core components, Pdu- Electronic supplementary material legends from The Notch pathway in the annelid Platynereis: Insights into chaetogenesis and neurogenesis processes ; Eve Gazave, Quentin I. B. Lemaître and Guillaume Balavoine;