CRISPR rescues and possible expansion of ICs in Islet>Sp6/7/8.

(A) CryBG>GFP expression in Islet (left) and Foxg (right) CRISPR larvae is rescued by co-electroporation with Islet intron 1 + bpFOG>Flag::Islet-rescue or Foxg>Foxg-rescue constructs, respectively, thanks to silent point mutations disrupting the sgRNA target binding sites. (B) Expression of C11.360>GFP is rescued in Sp6/7/8 CRISPR larvae upon co-electroporation with an Islet intron 1 + bpFOG>Sp6/7/8-rescue construct. (C) Example of expanded IC reporter (C11.360>Unc-76::GFP, green) in larvae (20 hpf/20 °C, ~st. 29) electroporated with Islet intron 1 + -473/-9>Sp6/7/8, as determined by perfect overlap with the Islet intron 1 + -473/-9>H2B::mCherry reporter (pink). See text for more details. See S1 File for exact sequences and detailed electroporation recipes. See S4 Data for the data underlying the graphs and for statistical test details. (TIF)</p

Validation of sgRNAs for CRISPR/Cas9-mediated mutagenesis.

Gene loci diagrams for the 4 transcription factor-encoding genes investigated in this study: Sp6/7/8, Foxg, Islet, and Pou4. Plots underneath each gene show validation by Illumina sequencing (“Next-generation sequencing” or NGS) of amplicons, performed as “Amplicon-EZ” service by Azenta. Mutagenesis efficacies are calculated by this service, and histograms of mapped reads show specificity of indels elicited by each sgRNA. Negative control amplicons are amplified from samples that were electroporated with no sgRNA, U6>Control sgRNA, or sgRNAs targeting unrelated amplicon regions. Note different y axis scales for each plot. Asterisks in Villin exon 5 and Tuba3 amplicon plots indicate naturally occurring indels. Precise calculation of mutagenesis efficacy for Villin.5.105 and Tuba3.3.24 sgRNAs was not given due to these natural indels. (TIF)</p

Genetic perturbations of metamorphosis.

(A) Ciona robusta juvenile undergoing metamorphosis, showing the retracted tail and rotated anterior-posterior body axis (dashed lines). PNs in the former papilla (now substrate attachment stolon, or holdfast) labeled by TGFB>Unc-76::GFP (green). Animal counterstained with DAPI (blue). (B) Scoring of Foxc>H2B::mCherry+ individuals showing tail retraction and/or body rotation at 48 hpf/20 °C in various papilla territory-specific (using Foxc>Cas9) CRISPR-based gene knockouts. Experiments were performed and scored in duplicate and percentages averaged, except for Foxg CRISPR for which a third replicate was performed (see S9 Fig). Number scored individuals in each replicate indicated underneath. “Tailed juveniles” have undergone body rotation but not tail retraction, whereas normally body rotation follows tail retraction. The sgRNA plasmids used for each condition were as follows- Control: U6>Control; Pou4: U6>Pou4.3.21 + U6>Pou4.4.106; Islet: U6>Islet.2; Foxg: U6>Foxg.1.116 + U6>Foxg.5.419; Sp6/7/8: U6>Sp6/7/8.4.29 + U6>Sp6/7/8.8.117. (C) Plot showing lack of any discernable metamorphosis defect after eliminating ACCs using Islet intron 1 + bpFOG>Sp6/7/8 (images not shown). Only Islet intron 1 + bpFOG>H2B::mCherry+ individuals were scored. Experiment was performed and scored in duplicate and averaged (n = 100 each duplicate). ACC specification was scored using the CryBG>Unc-76::GFP reporter. (D) Example of “tailed juveniles” at 47 hpf/20 °C compared to a larva in which no tail retraction or body rotation has occurred, elicited by tissue-specific Foxg CRISPR (Foxc>Cas9 + U6>Foxg.1.116 + U6>Foxg5.419). See S9 Fig for scoring. All error bars denote upper and lower limits. **** p p S4 Data for the data underlying the graphs and for statistical test details. ACC, axial columnar cell; PN, papilla neuron; sgRNA, single-chain guide RNA.</p

Islet is also required for papilla morphogenesis.

(A) Papilla shape is shortened and blunt at the apical end upon tissue-specific CRISPR/Cas9-mediated mutagenesis of Islet. Embryos were electroporated with Islet intron 1 + -473/-9>Unc-76::GFP and Foxc>Cas9. Islet CRISPR was performed using U6>Islet.2 sgRNA plasmid and the negative control used U6>Control. Larvae were imaged at 20 hpf/20 °C (~st. 28). Right: Scoring of percentage of GFP+ larvae classified as having normal “protruding” or blunt papillae, as represented to the left. Experiment was performed and scored in duplicate, using 2 different GFP fusions: Unc-76::GFP and DcxΔC::GFP [88]. Replicate 1: n = 100 for either condition; replicate 2: n = 55 for either condition **** p Islet intron 1 +-473/-9>Unc-76::GFP+) lengths along apical-basal axis in negative control and Islet CRISPR larvae at 18 hpf/20 °C (~st. 28). Both Islet.1 and Islet.2 sgRNAs used in combination. Statistical significance tested by unpaired t test (two-tailed). See S8 Fig for duplicate experiment. (C) In situ mRNA hybridization of Villin, showing expression in Foxg+/Islet+ central papilla cells at 10 hpf/20 °C (st. 21, left) and at larval stage (~st. 27, right). (D) Villin -1978/-1>Unc-76::GFP showing expression in the papilla territory of electroporated larvae (~st. 28), strongest in the central cells. (E) Villin -1978/-1>Unc-76::GFP in st. 28 larvae is down-regulated by tissue-specific CRISPR/Cas9 mutagenesis of Islet (Foxc>Cas9 + U6>Islet.1 + U6>Islet.2, see text for details). (F) Quantification of effect of Islet CRISPR (as in panel E) on Villin -1978/-1>Unc 76::GFP/Foxc>H2B::mCherry mean fluorescence intensity ratios in ROIs defined by the mCherry+ nuclei (see Methods for details). Significance determined by Mann–Whitney test (two-tailed). (G) Villin reporter is up-regulated in st. 28 larvae by overexpressing Islet (Foxc>Islet, see text for details). (H) GFP/mCherry ratio quantification done in identical manner as in F, but comparing Islet overexpression (as in panel G) and control lacZ larvae. (I) Quantification of ACC lengths measured in negative control and papilla-specific Villin CRISPR larvae at 17 hpf/20 °C (~st. 27). Significance tested by unpaired t test (two-tailed). Although no statistically significant difference between control and CRISPR larvae was observed in this replicate, average ACC length was significantly shorter in the CRISPR condition in an additional replicate (S8 Fig). (J) mRNA in situ hybridization for Tuba3, showing enrichment in the central cells of the papillae in st. 27 larvae. (K) Tuba3>Unc-76::GFP reporter plasmid is broadly expressed in the papillae of st. 27 larvae but stronger in central cells. (L) Papilla-specific CRISPR knockout of Tuba3 does not result in decrease of average ACC apical-basal cell length compared to negative control CRISPR using U6>Control sgRNA instead. Significance tested by unpaired t test (two-tailed). ns = not significant. All large bars indicate medians and smaller bars indicate interquartile ranges. See S3 and S4 Data for the data underlying the graphs and for statistical test details. ACC, axial columnar cell; ROI, region of interest; sgRNA, single-chain guide RNA.</p

Effect of various CRISPR knockouts on specification of ACCs, PNs, and OCs.

(A) Scoring of effect of papilla-specific CRISPR knockout of Foxg or Pou4 on specification of ACCs and PNs. Embryos were electroporated with Foxc>H2B::mCherry, Foxc>Cas9, CryBG>Unc-76::GFP (ACC reporter), TGFB>Unc-76::GFP (PN reporter), or L141.36>Unc-76::GFP (OC reporter), and gene-specific sgRNA combinations (see below for specific combinations). All were performed in duplicate and scores averaged, but some replicates and conditions are represented in Figs 3 and 4 also. Total embryos ranged between 76 and 100 per condition per replicate. Specific sgRNAs used: Foxg: U6>Foxg.1.116 + U6>Foxg.5.419; Pou4: U6>Pou4.3.21 + U6>Pou4.4.106; Sp6/7/8: U6>Sp6/7/8.4.29 + U6>Sp6/7/8.8.117; Islet: U6>Islet.2; Control: U6>Control. (B) Foxg, Pou4, and Sp6/7/8 sgRNAs were also tested alone (as opposed to pairs in combination) using reporter assays as in Figs 3 and 4. Those sgRNAs used further are highlighted in blue font. Additional sgRNAs abandoned due to low efficacy indicated in black font. For all plots, only larvae showing Foxc>H2B::mCherry expression in the papillae were scored. See S4 Data for the data underlying the graphs and for statistical test details. (TIF)</p

Finding papilla cell type-specific markers in single-cell RNAseq data.

(A) Cell clusters based from reanalysis and re-clustering of whole-larva single-cell RNA sequencing (scRNAseq) data from Cao and colleagues (see S1 Data). Dashed red box indicated clusters 3 and 33, which appeared to correspond to several papilla cell types. (B) Cells from clusters 3 and 33 from plot A set aside and re-clustered. (C) Differential expression plots showing examples of candidate papilla cell type marker genes mapped onto clusters in B. (D) Fluorescent, whole-mount in situ mRNA hybridization (green) for certain genes plotted in C, labeling different cells in the papillae of Ciona robusta (intestinalis Type A) hatched larvae. (E) Protein domain prediction diagrams for select cell type-specific marker proteins generated by SMART [89]. Unless specifically named, genes are indicated by KyotoHoya (KH) ID numbers (e.g., KH.L96.43). All larvae were fixed at 18 h post-fertilization (hpf), 20 °C, except for C11.360 and C2.1013 (18.5 hpf). Blue counterstain is DAPI. (TIF)</p

Specification of ACCs, ICs, and OCs by a combinatorial logic of Islet and Sp6/7/8.

(A) Overexpression of Sp6/7/8 (using the Islet>Sp6/7/8 plasmid) in all Islet+ papilla cells results in loss of ACCs (assayed by expression of CryBG>Unc-76::GFP, green), but not of ICs (assayed by expression of C11.360>Unc-76::GFP, green). Islet overexpression (with Islet>Flag::Islet-rescue) does not significantly impact the specification of ACCs or ICs. Larvae at 20 hpf/20 °C (~st. 29). (B) Scoring data showing presence or absence of ICs or ACCs in Foxc>H2B::mCherry+ larvae, as represented in panel A. Experiments were performed in duplicate with 99 or 100 larvae in each duplicate. (C) Cell type specification assayed by reporter plasmid expression (green) in larvae subjected to various Islet and/or Sp6/7/8 perturbation conditions (see main text for details). For ICs and ACCs, the “control” condition is negative control CRISPR (U6>Control), while for OCs it is Foxc>lacZ. Overexpression ACC/IC subpanels are at 18.5 hpf/20 °C (~st. 28), all CRISPR and OC panels at 20 hpf/20 °C (~st. 29). (D) Scoring data for most larvae represented in panel C. Foxc>H2B::mCherry+ larvae were scored for cell type-specific GFP reporter expression that was “heterogeneous” (mixed on/off GFP expression, with all “wild type” patterns of expression falling under this category), “predominant” (ectopic/supernumerary GFP+ cells), “sparse” (reduced frequency/intensity of GFP expression), or “absent” (no GFP visible). (E) IC or ACC reporter (C11.360>Unc-76::GFP, CryBG>Unc-76::GFP) expression scored in Foxc>H2B::mCherry+ larvae represented in top 2 panels of right-most column in C. Experiment was performed and scored in duplicate, with number of larvae in each duplicate ranging from 100 to 105. (F) OC-specific reporter (L141.36>Unc-76::GFP) expression scored in Foxc>H2B::mCherry+ larvae represented by the bottom/right-most subpanel in panel B. Scoring strategy same as in Fig 3. Asterisk denotes when a duplicate of the negative control condition was also used for plots in Fig 3, as multiple CRISPR experiments were performed in parallel. All experiments were performed in duplicate, with number of embryos ranging from 76 to 100 per duplicate. Foxc>Cas9 used for all CRISPR/Cas9 experiments. The Islet cis-regulatory sequence used (panels A and B) was always Islet intron 1 + -473/-9. For overexpression conditions, Foxc>lacZ or Islet>LacZ were used to normalize total amount of DNA (see S1 File for detailed electroporation recipes). All error bars indicate upper and lower limits. **** p S4 Data for the data underlying the graphs and statistical test details. ACC, axial columnar cell; IC, inner collocyte; OC, outer collocyte.</p

Summary of statistical tests of proportions (i.e., scoring).

Summary of statistical tests of proportions (i.e., scoring).</p

Differential gene expression table of re-analyzed whole larva single-cell RNAseq data from Cao and colleagues (new clusters 3 and 33 subclustered into 11 subclusters).

Differential gene expression table of re-analyzed whole larva single-cell RNAseq data from Cao and colleagues (new clusters 3 and 33 subclustered into 11 subclusters).</p

Additional marker genes and reporter plasmids expressed in papillae.

(A) TGFB>Unc-76::GFP reporter (green) is not co-expressed in the same cells as the Islet intron 1 + -473/-9>mCherry reporter (pink) at 20.5 hpf (~st. 29). (B) Cross-talk between CryBG>Unc-76::GFP and TGFB>Unc-76::mCherry reporter plasmids at 16 hpf (~st. 26), showing aberrant co-expression in ACCs and/or PNs only when co-electroporated. (C) Mutually exclusive expression of CryBG>lacZ in ACCs (cyan), Gnrh1>Unc-76::GFP in PNs (yellow), and C11.360>Unc-76::mCherry in ICs (magenta), with DAPI counterstained in gray. This larva is the same as in main Fig 2G, with an additional channel and different false coloring. (D) Images from Fig 2 with mCherry and GFP channels displayed separately. (E) C. intestinalis (Type B) larva electroporated with C. robusta C11.360>Unc-76::GFP reporter plasmid, showing specific but weak expression. (F) C. intestinalis (Type B) larva electroporated with C. robusta L96.43>Unc-76::GFP reporter plasmid, also showing weak expression. Papillae in panels E and F outlined by dashed lines. (G) Reporter plasmid containing the first intronic region of Emx drives expression in ICs at 20 hpf (~st. 29), likely corresponding to the “ring” of late Emx expression in Islet+ cells reported in Wagner and colleagues and distinct from earlier Emx expression in the papilla lineage as described in Liu and Satou. (H) C14.116>Unc-76::mCherry reporter expressed in central cells (ACCs+ICs, pink) and basal cells around the 3 papillae at 20.5 hpf (~st. 29). (I) Immunostaining for the Flag epitope tag fused to the Islet-rescue protein used for Islet>Islet experiments in Fig 4. Flag immunostaining in green and Foxc>H2B::mCherry in pink in merged image. Larvae fixed at 19 hpf (~st. 28). DAPI in gray. ACCs, axial columnar cells; PN, papilla neuron. (TIF)</p