MOESM1 of Developmental system drift in motor ganglion patterning between distantly related tunicates

Additional file 1.  Supplemental sequences, information, and figures

Labeling of subsets of neurons in different hsp68-nlacZ lines.

<p>(A) Coronal section of a mouse carrying a single copy of the hsp68-nlacZ transgene that integrated into the coding region of the Npas3 gene (bar = 1 mm). (B) The table shows the genomic insertion site of eight mouse lines carrying a single copy of the hsp68-nlacZ transgene. (C) Sagittal sections of a mouse carrying a single copy of the hsp68-nlacZ transgene that integrated into the coding region of the Phospholipase C-gamma1 gene. Medial to lateral sections of the brain reveal a medio-lateral expression gradient both in the hippocampus and cerebellum (bar = 1mm).</p

Generation of transgenic mice carrying a nlacZ enhancer probe under control of a hsp68 minimal promoter.

<p>Enhancer probes integrate into different sites in the genome. Depending on the site of integration the interaction of the introduced minimal promoter and enhancer elements of the genome results in restricted expression of the transgene.</p

Labeling of subsets of neurons in thy1mp-cre lines.

<p>(A) Sagittal sections of mice carrying a single copy of the thy1mp-cre transgene (lines named FTC.01-FTC.13). A large variety of subsets of neurons were labeled in eight independent mouse lines shown here (bar = 2.5mm). (B) Labeling of olfactory receptor neurons (FTC.07) (bar = 250 um). (C) Labeling of granule cell neurons in the accessory olfactory bulb (FTC.13) (bar = 500um). (D) Sagittal sections of the dorsal hippocampal formation of eight different thy1mp-cre lines reveal recombination restricted to subregions of the hippocampus (CA1, CA3 or dentate gyrus) (bar = 500 um).</p

Novel genetic markers label distinct cell types of the papillae.

(A) GFP reporter plasmid (green) constructed using the cis-regulatory sequences from the KH.L96.43 gene labels basal cells in between and surrounding the protruding papillae labeled by Foxg reporter plasmid (pink). (B) TGFB>GFP reporter (green) labels PNs, the axons of which make contacts with BTN axons labeled by a BTN-specific Islet reporter (pink), at 23.5 h hpf, approximately corresponding to Hotta stage 30. (C) A KH.C4.78 reporter (C4.78>GFP) also labels PNs, which are also labeled by Foxg>H2B::mCherry (mCh) reporter (pink nuclei). (D) Lack of overlap between expression of C4.78>GFP (green) and a papilla-specific Islet reporter plasmid (pink nuclei) showing that PNs do not arise from Islet+ cells. (E, F) Co-electroporation of C11.360>GFP (green) with H2B::mCherry reporter plasmids (pink nuclei) indicates these cells come from Foxg-expressing cells that also express Islet. (G) C11.360>mCherry reporter (pink) labels centrally located ICs adjacent to ACCs labeled by CryBG>LacZ reporter (green). (H, I) L141.36>GFP reporter (green) labels OCs that arise from Foxg+ cells (pink nuclei) but do not express Islet (pink nuclei). (J) ICs and OCs are distinct cells as there is no overlap between C11.360 (green) and L141.36 (pink) reporter plasmid expression. (K) Ciona intestinalis (Type B) larva ICs labeled with a reporter plasmid made from the corresponding cis-regulatory sequence of the C. intestinalis Chr11.1038 gene, orthologous to C. robusta KH.C11.360. (L) C. intestinalis larva OCs labeled by a Chr7.130 reporter, corresponding to the C. robusta ortholog KH.L141.36. (M) Summary of the main marker genes and corresponding reporter plasmids used in this study to label different subsets of papilla progenitors and their derivative cell types. All GFP and mCherry reporters fused to the Unc-76 tag, unless specified (see Methods and supplement for details). Weaker Foxg -2863/-3 promoter used in panel A, all other Foxg reporters used the improved Foxg -2863/+54 sequence instead. All Islet reporters shown correspond to the Islet intron 1 + bpFOG>H2B::mCherry plasmid. White channel shows either DAPI (nuclei) and/or larva outline in brightfield, depending on the panel. All C. robusta raised at 20 °C to 18 hpf (roughly st. 28) except: panel B (23.5 hpf, ~st. 30); panels C–F (17 hpf, ~st. 27); panels H–J (20 hpf, ~st. 29). C. intestinalis raised at 18 °C to 20–22 hpf (Hotta stage 28). ACC, axial columnar cell; BTN, bipolar tail neuron; hpf, hours post-fertilization; IC, inner collocyte; OC, outer collocyte; PN, papilla neuron.</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

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

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

Both types of collocytes contribute to production of adhesive material.

(A) PNA-stained granules (pink) are seen in the hyaline cap and the apical tip of ICs (left panel, white arrow) in a C. intestinalis larva labeled by the C. intestinalis C11.360>Unc-76::GFP reporter (green). PNA-stained granules are also seen in cells not labeled by the IC reporter (right subpanel, hollow arrowhead), suggesting they are localized in a different cell type. Left and right subpanels are from different focal planes of the same papilla. (B) OCs labeled with C. robusta L141.36>Unc-76::GFP (green) in a C. robusta larva, with PNA-stained granules (pink) in both apical and basal positions within the cell (white arrows). DAPI in blue. (C) PNA staining (pink) in C. robusta upon overexpression of Sp6/7/8 alone, showing reduction of IC specification as assayed by C11.360>Unc-76::GFP expression (green). Weak PNA staining and GFP expression are still visible in some papillae (solid arrow), but not others (open arrowhead). (D) PNA staining (pink) and C11.360>Unc-76::GFP expression (green) in C. robusta upon overexpression of both Islet and Sp6/7/8, showing expansion of IC fate in a single large papilla (arrow). PNA staining is similarly expanded over the entire IC cluster, confirming that ICs produce the adhesive glue. Foxc>lacZ expression (β-galactosidase immunostaining) shown in blue in both C and D. (E) Scoring of larvae represented in panels C and D, averaged across duplicates. Weak PNA staining is observed upon partial suppression of IC fate, but strong PNA staining is seen upon expansion of supernumerary ICs, confirming that this cell type is one of the major contributors of PNA-positive adhesive glue. Total larvae (duplicate 1) or β-galactosidase+ larvae (duplicate 2) were scored. **** p S4 Data for sample size, statistical test details, and for the data underlying the graphs. C. intestinalis raised to 20–22 hpf at 18 °C (~st. 28), C. robusta raised to 20 hpf at 20 °C (~st. 29). See Supplemental Movies for full confocal stacks and S6 Fig for single-channel images. IC, inner collocyte; OC, outer collocyte; PNA, peanut agglutinin.</p

Summary diagram.

(A) Updated diagram of the development of the anterior descendants of Row 6 in the neural plate to show the proposed patterns of MAPK and Delta/Notch signaling that set up the 3 Foxg+ clusters and interleaved Foxg-negative neurogenic cells. (B) Diagram proposing the contributions of Foxg+ and Foxg-negative cells to later patterns of transcription factors that specify the different cell types found in each papilla, which is in turn is repeated 3 times, thanks to the process shown in panel A. (C) Papilla development shown as cell lineages, with dashed lines indicating uncertain cell divisions and lineage history. MAPK regulates binary fate choices, promoting Foxg expression in the papillae proper at first but later suppressing it (and Islet). Lastly, Delta-Notch signaling promotes OC fate and limits PN specification through lateral inhibition. Cell type numbers based on [9]. (D) Provisional gene regulatory network diagram of the transcription factors involved in specification and differentiation of the different papilla cell types. Arrowheads indicate activating gene expression or promoting cell fate, while blunt ends indicate repression of gene expression of cell fate. Solid lines indicate regulatory links (direct or indirect) that are supported by the current data and literature. Dashed lines indicate regulatory links that have not been tested, or need to be investigated in more detail. A-P, anterior-posterior; D-V, dorsal-ventral; OC, outer collocyte; OSP, oral siphon primordium; PN, papilla neuron.</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