Journal: Cell

Article Title: Stepwise emergence of the neuronal gene expression program in early animal evolution

doi: 10.1016/j.cell.2023.08.027

Figure Lengend Snippet: A multi-species placozoan whole-body cell atlas (A) Consensus phylogenetic tree obtained with Bayesian inference under the CAT + GTR + Г4 mixture model on the Metazoa-only 209-markers concatenated aminoacid matrix recoded into 4 categories (SR4). Bayesian posterior probabilities are indicated as supports in key nodes. The cladogram to the right depicts the phylogenetic relationships among placozoans, highlighting the four species here studied. (B) Summary of the statistical support for alternative phylogenetic positions of Placozoa in the different datasets analyzed: (1) only metazoans (63 species) versus metazoans and choanoflagellates as outgroup (81 species); (2) high-information markers (filtered for tree-likeness score with MARE −d 2 parameter) markers filtered for compositional homogeneity (denoted as CH; markers failing the compositional heterogeneity based on simulated alignments using the LG + Γ4 model in p4, at p > 0.01); and (3) original aminoacid multiple sequence alignments versus recoded alignments with three different schemes (SR4, SR6, and Dayhoff6). (C) 2D projection of metacells for each species sampled in this study and pie charts indicating the relative proportion of cells in each broad cell type category, based on a force-directed layout of the metacell co-clustering graph (see ). Right, a broad cell type clustering tree of all four species obtained using the UPGMA average algorithm on Log-Det distance matrices, based on binary ortholog activity in each cell type (fold change ≥ 2). (D) Normalized expression of top variable genes (rows, fold change ≥ 2 with a maximum of 15 genes per metacell) across metacells (columns). Broad cell types are color-coded in the x axis and red squares highlight the peptidergic progenitor metacells. (E) Fluorescent HCR-ISH of Trichoplax sp. H2 specimens showing the expression of an upper epithelia-like marker (calpain-9, top) and the expression of a marker gene for the unknown cell type (β-secretase, bottom). Images correspond to the maximum projection of 183 and 70 optical sections, respectively. The dotted lines indicate the sections used for the extended orthogonal views (45 slices). Arrowheads in the orthogonal views indicate the upper part of the animals. Insets in the bottom image show the detail of cells localized in the rim of the animal. Cells highlighted in the insets were imaged at higher magnification in the portions indicated with a square. Expression of the marker genes is shown to the left of each panel. Scale bars are 50 μm for the general views and 5 μm for the insets. See also Figures S1 , , and .

Article Snippet: We used the metacell normalized gene expression fold change (FC) of each placozoan species to obtain gene modules using the WGCNA algorithm.

Techniques: Sequencing, Activity Assay, Expressing, Marker

Journal: Cell

Article Title: Stepwise emergence of the neuronal gene expression program in early animal evolution

doi: 10.1016/j.cell.2023.08.027

Figure Lengend Snippet: Characterization of intermediate metacells and gene modules, related to Figures 2 and (A) Barplots representing the number of cells classified in each intermediate category (gray) compared with the number of cells doublets in each category (green) that would be expected given the relative frequency of the terminal cell types in each case. We used two-tailed exact binomial tests to determine whether the observed number of intermediate cells significantly differed from the expectation (p values next to each set of bars). (B) Top, barplots representing the number of genes shared in intermediate metacells between the placozoan species where each cell type is found (gray) compared with the number of genes shared by the respective terminal cell types (green). We used one-tailed exact binomial tests to determine whether the number of genes shared across species was higher for terminal than for intermediate cell types (p values shown for each cell type). Notice that in most cases the difference is small and non-significant, indicating that the genes expressed in intermediate cells are conserved across species and not a stochastic sampling of genes expressed in the respective terminal cell types. Bottom, Venn diagrams detailing the number of shared genes across species for lipophil-1/gland intermediate cells (gray) compared with the shared genes by lipophil-1 and gland cell types (green). (C) Intermediate cells exhibit intermediate transcriptional signatures between their terminal cell types. For each pair of cell type in each species, we show the sum of the fraction of UMIs (per 1,000 UMIs) of the top markers (FC ≥ 2). Panels are arranged to indicate the detection of specific intermediate cell types (rows) in each of the species (columns). (D) Flow cytometry scatterplots of Trichoplax sp. H2 cells labeled by HCR-ISH against markers specific for lipophil (fatty acid-binding protein 4, Alexa Fluor-647), gland (chymotrypsin, Alexa Fluor-546) and fiber (angiotensin I-converting enzyme, Alexa Fluor-488) cells. Selected areas in each panel denote the percentage of cells with single or double label, which would correspond to intermediate cells. (E) Heatmaps representing the eigengenes across metacells of gene modules calculated using WGCNA in each placozoan. x axis colors indicate the broad cell type classification of metacells. Module colors (y axis) are arbitrary. (F) Left, gene-gene expression correlation matrix, grouping genes into the same modules as in (E). Right, normalized expression across metacells of genes grouped into modules. Transcription factors are highlighted with a dot to the right of the heatmap. Notice the presence of “lateral” gene modules expressed in individual metacells across cell types. These include, for example, the cell cycle and ciliary apparatus modules. (G) Top 10 gene ontology terms enriched in each multi-species gene module. x axis colors indicate the cell type where each module is most active (manual curation). (H) Fold-change expression of selected genes with immune-related functions across cell types of all four placozoans.

Article Snippet: We used the metacell normalized gene expression fold change (FC) of each placozoan species to obtain gene modules using the WGCNA algorithm.

Techniques: Two Tailed Test, One-tailed Test, Sampling, Flow Cytometry, Labeling, Binding Assay, Gene Expression, Expressing

Journal: Cell

Article Title: Stepwise emergence of the neuronal gene expression program in early animal evolution

doi: 10.1016/j.cell.2023.08.027

Figure Lengend Snippet: Placozoan gene expression programs (A) Multi-species clustering of gene modules across placozoans. Each node represents a gene module (group of genes co-expressed across metacells; see Figures S4 E–S4G), and each node is color-coded according to the species. Edges link modules sharing orthologs across species, and their width reflects the Jaccard index of ortholog overlap between modules (only edges with Jaccard ≥0.125 are shown). We curated 34 multi-species modules, the majority of which are composed of modules from four species (pie plot). Most modules are specific to individual cell types (bar plot), with the exception of cross-cell type modules that include genes related to pan-peptidergic cells, cell cycle (S-phase and G2-phase), meiosis, and the ciliary apparatus. (B) Gene ontology enrichments in selected gene modules (left), and expression of transcription factor (TF) regulators and associated enriched motifs (right). (C) Left, multi-species clustering of non-peptidergic (top) and peptidergic cell types (bottom). The cell type tree has been obtained as in Figure 1 C. Gray boxes list selected TFs specific to various cell type clades. Right, heatmap depicting the fraction of orthologous genes from each gene module expressed across cell types. Modules have been color-coded according to their cell type specificity, with the cross-cell type modules highlighted with asterisks. (D) Number of TFs, GPCRs, and neuropeptides (NPs) expressed (fold change ≥ 2) in each cell type. See also Figures S4 , , and .

Article Snippet: We used the metacell normalized gene expression fold change (FC) of each placozoan species to obtain gene modules using the WGCNA algorithm.

Techniques: Gene Expression, Expressing

Journal: Cell

Article Title: Stepwise emergence of the neuronal gene expression program in early animal evolution

doi: 10.1016/j.cell.2023.08.027

Figure Lengend Snippet: Genetic basis of cell type evolution in Placozoa (A) Aligned genomic region exemplifying different categories of regulatory element (RE) conservation. Each RE is classified according to two criteria: across-species conservation (ancestral/novel) and intra-species sequence dynamics (conserved/neutral/accelerated). (B) Ancestral reconstruction of RE evolution across Placozoa. In extant nodes, REs are classified according to their sequence conservation/acceleration status. (C) Rates of evolution in the transcriptional and regulatory profiles of matched cell types across all four placozoans. For each cell type, we recorded the fraction of specific markers (genes expressed at FC ≥ 1.5) that were gained or lost at least once along the placozoan phylogeny (y axis) and compared them (x axis) with the rate of active RE gain + loss along the same branches (top) or to the fraction of active REs that exhibited signatures of accelerated evolution (x axis, bottom, at phyloP < 0.001) in extant species (bottom). (D) The impact of RE sequence dynamics in gene expression conservation, comparing Trichoplax adhaerens H1 to the other three placozoans. Left, boxplot comparing the expression conservation score of orthologous genes with shared ancestral REs to those of genes with novel REs. Right, boxplot comparing the expression conservation of orthologs with slow-evolving REs to orthologs with one or more accelerated RE. We used one-sided Wilcoxon rank sum tests to test for significant differences in the EC score distributions (p values below each pair of boxplots). (E) Same as (D) but comparing TF-binding motif usage similarity (Spearman correlation of gene-wise maximum motif alignment score). (F) Evolutionary dynamics of various genetic determinants of cell identity at increasing evolutionary distances. The boxplot represents the fraction of shared features for each matched cell type and from the perspective of T. adhaerens H1. Compared features include: conserved genes (genes expressed in a given T. adhaerens cell type with an ortholog in the genome of the other species or reconstructed ancestor), conserved REs (likewise, using sequence conservation of orthologous REs), active genes (genes expressed in a given cell type in both T. adhaerens H1 and the other species), active REs (REs linked to genes expressed in a given cell type in both T. adhaerens H1 and the other species), and used motifs (TF-binding motifs enriched in both T. adhaerens H1 and the other species). The cladogram shows the time-calibrated distances. (G) Distribution of the correlations in gene expression and TF-binding motifs across cell types (both measured as fold-change enrichments) between T. adhaerens H1 and the three other placozoans.

Article Snippet: We used the metacell normalized gene expression fold change (FC) of each placozoan species to obtain gene modules using the WGCNA algorithm.

Techniques: Sequencing, Gene Expression, Expressing, Binding Assay

Journal: Cell

Article Title: Stepwise emergence of the neuronal gene expression program in early animal evolution

doi: 10.1016/j.cell.2023.08.027

Figure Lengend Snippet: Peptidergic cell transcriptional profiles, related to Figures 5 and (A) Expression fold change (FC) of neuropeptide-processing enzymes across species and cell types. Cell types are grouped in four categories, from right to left: peptidergic (light blue), peptidergic progenitors (dark blue), epithelial, and others. Species are indicated with different shapes. (B) Same as (A) for pre-synaptic scaffold genes. (C) Same as (A), for post-synaptic scaffold genes. (D) Identification of H. hongkongensis H13 small peptides. Scatterplot shows the maximum expression of the propeptide gene in any peptidergic cell type (x axis) compared to the abundance of the most common peptide per propeptide as measured by mass spectrometry (y axis). Dot sizes indicate the number of spectra identified for the most common peptide per propeptide. The color code indicates homology of the propeptide and dot border lines indicate the identification of peptide post-translational modifications. (E) Scatterplots showing the two docking scoring metrics (see ) for positive docking peptide receptor pairs shown in Figure 5 E. Barplots on the right show the expression pattern for the corresponding receptor (only for cell types with FC ≥ 1.5). (F) Comparison of global gene expression levels for the three Trichoplax sp. H2 Notch signaling drug treatments (plus DMSO control sample) compared with the reference Trichoplax sp. H2. Scatterplots show the normalized UMI counts per gene in each sample. The Spearman correlation for each comparison is indicated. (G) Expression similarity between placozoan peptidergic progenitor cells (H2 pooled dataset) and cell types assigned to various developmental trajectories in other species (mouse, N. vectensis and H. vulgaris ), measured as weighted Pearson correlation coefficients of cell type-level FC values. Gene markers were selected from ICC-defined ortholog pairs belonging to predicted transcriptional regulator gene families (transcription factors, chromatin regulators, and RNA-binding proteins), and we restricted the analysis to genes with variable expression in both datasets (FC ≥ 1.25 in at least one cell type in both the placozoan reference and the query dataset, totaling 73–207 genes in mouse, 172–367 in N. vectensis , and 318 in H. vulgaris ). For each developmental trajectory (the various neuroectodermal lineages shown in Figure 6 H, plus endoderm, mesoderm, non-neural ectoderm, and endo/mesoderm), we report the the correlation with the most similar cell type in each combination of developmental stage and lineage.

Article Snippet: We used the metacell normalized gene expression fold change (FC) of each placozoan species to obtain gene modules using the WGCNA algorithm.

Techniques: Expressing, Mass Spectrometry, Comparison, Gene Expression, Control, RNA Binding Assay

Journal: Cell

Article Title: Stepwise emergence of the neuronal gene expression program in early animal evolution

doi: 10.1016/j.cell.2023.08.027

Figure Lengend Snippet: Molecular signatures of neurogenesis in placozoans peptidergic cell progenitors (A) 2D projection of metacells of a Trichoplax sp. H2 single-cell pooled transcriptome of individuals grown under four conditions: treatment with the Notch antagonists DAPT (3,453 cells) and LY411575 (4,666 cells), the Notch signaling agonist Yhhu3792 (5,114 cells), and an untreated control (4,765 cells). Metacells have been color-coded by broad cell type based on comparison to the reference Trichoplax sp. H2 dataset ( Figure 1 C). (B) Normalized expression of Delta, Notch, Hes, and Hey in the 2D projection of Trichoplax sp. H2 metacells. (C) Pie plot with cell type proportions among the control cells (top) and fold-change enrichment in cell type fractions for each drug treatment (bottom). p values from a two-sided Fisher’s exact test of cell type counts relative to the control. (D) Differential expression of the Hes, Hey, and Myc TFs in lower epithelial and gland cells (two cell types with broad Notch expression), measured using the difference in UMIs/10 4 between treatment and control. p values indicate significant differential expression based on a two-sided Fisher’s exact test on UMI counts. (E) Expression of selected marker genes related to peptidergic progenitor specification across all four placozoans, including markers used for HCR-ISH experiments. p values from an FDR-adjusted two-sided Fisher’s exact test of UMI counts in a given cell type, relative to the control. (F) Sox TF maximum likelihood phylogenetic analysis supporting the orthology of placozoan Sox1/2/3 and Sox4/11/12. (G) Left, fluorescent HCR-ISH of C. collaboinventa H23 showing the expression of the peptidergic progenitor-specific marker HoiH23_PlH23_008135 (NN peptide, red) in animals with (Gii) and without (Gi) treatment with 10 μM LY411575 for 24 h. Images are maximum projections of 50 (Gi) and 40 (Gii) optical sections. The dotted lines indicate the sections used for the extended orthogonal views (40 slices). Arrowheads in the orthogonal projections indicate the upper part of the animals. Middle, fluorescent HCR-ISH of C. collaboinventa H23 (Giii and Gvi) showing the expression of the peptidergic progenitor-specific markers HoiH23_PlH23_008135 (NN peptide, red) and Klf13 (yellow). Image (Giii) is a maximum projection of 22 optical sections. Images (Giv) to (Gvi) highlight the detail of three individual cells expressing both markers and correspond to the squared sections of image (Giii). Right, fluorescent HCR-ISH of Trichoplax sp. H2 (Gvii and Gviii) showing the expression of the peptidergic progenitor-specific markers Klf13 (red) and Delta receptor (yellow). Image (Gvii) is a maximum projection of 16 optical sections. Inset (Gviii) highlights the detail of a cell expressing both markers. Dotted line depicts the shape of the cell as delineated by the membrane marker (green). Scale bars correspond to 100 μm in i and ii, 10 μm in (Giii) and (Gviii), and 1 μm for (Giv)–(Gvi) and (Gviii). (H) Expression of selected TFs, RNA-binding proteins and chromatin factors specific to placozoan peptidergic progenitors (E) along the neural developmental trajectories described in scRNA-seq experiments in M. musculus (gastrula to pharyngula stage ), N. vectensis (gastrula to adult ), and Hydra vulgaris (regenerating adult ). Genes with expression FC ≥ 1.25 in any cell type of a given developmental trajectory are indicated as colored squares in each (overexpressed genes with FC > 1 and < 1.25 in stages intermediate between two other stages are indicated with a white asterisk). For each developmental trajectory, we also indicate the number of orthologous TFs and RBPs shared with each placozoan species (barplots to the right). See also Figure S7 .

Article Snippet: We used the metacell normalized gene expression fold change (FC) of each placozoan species to obtain gene modules using the WGCNA algorithm.

Techniques: Control, Comparison, Expressing, Quantitative Proteomics, Marker, Membrane, RNA Binding Assay