Gulf pipefish exemplify syngnathid derived traits.

Gulf pipefish have elongate snouts, have lost teeth on their oral and pharyngeal jaws, possess dermal armor, and have brood pouches in males (panel A). Cartilage (alcian) and bone (alizarin) stained clutch siblings of embryos from the two samples are shown in panels B-G. Embryos have cartilaginous craniofacial skeletons (panels B, E; E marks the Mes(ethmoid) cartilage, C indicates the Ceratohyal, H shows the Hyosymplectic cartilage, and M marks the Meckel’s cartilage) with the onset of ossification in the jaw. They have cartilaginous fin radials in the dorsal fin (C and F). The embryos do not have signs of ossification in the trunk where the exoskeleton will form later (panels D,G). Panels B, C, E, and F, scale bar 200 µM; D, G scale bar 100 µM.

Gulf pipefish single cell atlas contains cells from the entire embryo and identifies genetic pathways active in different cell types.

The UMAP plot (panel A) shows all of the cell clusters and their identities reduced to the first two UMAP dimensions. The graph in panel B displays results of the KEGG pathway analysis in cell clusters identified as connective tissue (excluding blood, pigment, digestive, and immune cells). The number of Seurat identified marker genes for each cluster that was a part of each pathway is displayed on the y axis. Bars are colored and labeled by cell cluster.

Weighted Gene Network Analysis (WGCNA) identifies gene modules that define and unite cell clusters.

A) The strength of association between the gene modules and cell clusters is shown in panel A with dendrogram clustering illustrating the distance between modules and cell clusters. Gene modules are represented by rows and cell clusters by columns. The modules and clusters are clustered using the Pearson distance method. The number of genes in each gene module are shown in the right hand bar plots. Cell clusters are colored based on their identity. The asterisks indicate the module-cluster relationships that have a p-value less than .05 from a two-sided permutation test after correction for multiple tests (FDR). The heatmap is colored by t-statistics in a range of -10 to 10, with highly positive values in yellow and highly negative values in black. B) The identified gene modules possess genes from KEGG pathways. The bars are labeled with the gene module and the size of each bar corresponds to the number of genes from the KEGG pathway in the module. Since WGCNA modules do not have p-values, only KEGG pathways with more than two genes included in the gene module are shown on the plot. C) Identified gene modules contain similar KEGG pathways as the cell clusters that correlated with them. These relationships are shown in Upset plots where each row is a cell cluster or gene module, each column represents KEGG pathways shared by the modules and clusters (shared condition is shown filled in black dots connected by lines), the interaction size is the number of pathways in common between the set of modules and clusters, and the set size is the number of pathways that are enriched in each cluster and module. Panel C1 highlights that ’cytokine-cytokine receptor interaction’ and ’ECM receptor interaction’ are present in module 6 as well as 6 and 4 connective cell clusters respectively. Panel C2 shows that ’cell cycle’ and ’senesence’ are present in module 21 as well as clusters 8 and 16, ’Notch signaling’ genes are present in clusters 8 and 25 as well as module 21, and ’DNA replication’ is present in clusters 8, 10, and 16.

Conserved cell types and gene pathways build unique faces of syngnathids.

Three main conserved signaling pathways are enriched in connective cell types, MAPK signaling (including Fgf signaling), TGF-beta signaling (including BMP signaling), and Wnt signaling. Receptors and ligands expression patterns are shown in panel A heatmap from all cell types with cells present in the head. This heatmap features 100 cells downsampled from each cluster and illustrates that many genes from these families are expressed in these cells. Yellow lines indicate high expression of a gene, while hot pink lines indicate no expression. The pathways are boxed in black. Rows representing fgf22, bmp4, and sfrp1a expression are marked with an asterisk and green box for each respective section of signaling (Fgf, BMP, and Wnt). Panels B, C, and D are in situ hybridizations of three marker genes, prdm16, elnb, and tnmd. prdm16 and elnb mark osteochondrogenic mesenchyme and tnmd marks tendons and ligaments. Panels E, F, and G, show expression patterns of three pathway representatives (fgf22, bmp4, and sfrp1a). All three genes are expressed in the face: fgf22 at the tip of the mandible and bmp4 and sfrp1a above the ethmoid and near the ceratohtyal. Staining is circled with dashed lines. The Meckel’s cartilage (M), mes(Ethmoid) cartilage (E), Quadrate (Q), and Ceratohyal (C) are labeled. Panel C is a dorsal view. Panels B, C, E, F, G, H, I are in lateral view. In situs of fgf22 were completed using 10dpf Gulf pipefish. bmp4, sfrp1a, tnmd, elnb, and prdm16 in situs were completed using wild caught bay pipefish at the onset craniofacial elongation. Panels H and I are summary illustrations of our findings, panel H shows where cells from various clusters were present in the developing head and panel I illustrates where bmp4, sfrp1a, and fgf22 were expressed.

Pipefish do not possess identifiable tooth primordium cells, but continue to express tooth development genes in other contexts.

Panel A presents a dotplot of genes classified by the tissue layer in which they are reported to be expressed during tooth development in other vertebrate (Tucker and Sharpe 2004; Gilbert et al. 2019; Kawasaki 2009). The x-axis contains the assayed genes, with asterisks under the genes that were also examined with in situ hybridizations. The y-axis contains all of the cell clusters. The size of the dot is representative of the percentage of cells from the cluster that express the gene. The color of the dots is average expression of the gene in the cluster (darker purples represent higher expression). Panel B includes in situ hybridizations of selected tooth primordia markers (bmp4, pitx2, lhx6a, dlx2a, and dlx3b) and mature tooth marker (scpp1 ). In situs of bmp4, pitx2, lhx6a, dlx2a, and dlx3b were completed with wild caught bay pipefish that had begun craniofacial elongation. scpp1 in situs were completed using 9dpf Gulf pipefish. The scale bars for all images represents 100 µM.

Tooth and bone development genes expressed during exoskeleton development.

We discovered pitx2 (A1 and A2) and dlx3b (B1 and B2) expression during possible emergence of exoskeletal primordium in wild caught bay pipefish. The embryos used for these in situs were the same stage as those from Figure 5, at the beginning of craniofacial elongation. Figures A1 and B1 have 100 µM scale bars, figures A2 and B2 are taken from the same fish and have 50 µM scale bars. We further found scpp1 is exppressed at the mineralization front of the exoskeleton in 12dpf Gulf pipefish (C) and has a 50 µM scale bar. Alizarin and alcian stained pipefish are shown in panels D1 (12dpf), D2 (1dpf), and D3 (adult Gulf pipefish) to illustrate how the exoskeleton forms. Panels D1 and D2 have 100 µM scale bars.

Gene expression signatures suggest embryonic interactions within brood pouch environment.

Epidermal cells (panel A), with pathways that suggest increased endocytosis and metabolism pathways are in bold text. Pathways upregulated in 3dpf zebrafish epidermal cells are in italics. Pipefish epidermal cells also express 15 lectin genes not found in other cell types (panel B). We suggest an epidermal cell model (panel C), in which we predict pipefish have an enrichment of nutrient processing genes and lectins in comparison with zebrafish cells.

Marker genes define cell identity and are a resource for cell cluster exploration.

This dotplot shows the marker genes on the x-axis, and the cell cluster on the y-axis. The size of each dot indicates the percentage of cells that express the gene in each cluster. The darkness of each dot represents the average expression of the gene.

Marker genes examined through in situ hybridization reveal spatial patterning of cell types.

Informative marker gene in situ hybridization results, including slc25a4, scg2a, prdm16, elnb, fndc9, and insm1b. Embryos are staged to highlight staining of each gene. Relavant anatomical features are distinguished with arrows. One day post spawn Gulf pipefish larvae were used for fndc9 in situs, five day post spawn Gulf pipefish larvae were used for insm1b in situs, and wild caught bay pipefish at the onset of craniofacial elongation were used for all other probes.

Marker genes examined through in situ hybridization reveal spatial patterning of cell types.

Informative marker gene in situ hybridization results, including tnmd, myf5, hpbd, ifitm5, plaat4, hyal6, and soat2. Embryos are staged to highlight staining of each gene. Relavant anatomical features are distinguished with arrows. All in situs were completed with wild caught bay pipefish: one day post spawn larvae were used for the hyal6 probe, mid-somitogenesis embryos were used for the hpbd, and embryos at the onset of craniofacial elongation were used for all other probes.

KEGG analysis reveals pathways signficiantly enriched in cell clusters.

KEGG pathways are listed on the x-axis. For each cluster, the number of genes that belong to the specific KEGG pathway is represented by the height of each bar. Cell clusters are labeled by both color and their designated number. For clarity, only cell clusters with more than three annotated genes in each pathway are shown and certain pathways involving infection and metabolism are removed.

Cell clusters drive gene module connectivity.

The heatmap shows the change in gene network connectivity when individual cell clusters are removed. Cell clusters are in the columns and gene modules are in rows. The coloration scale is based on the change in connecitvity when a cell cluster is removed, with yellow indicating a large change in connectivity. The modules and clusters are clustered using the Pearson distance method. Asterisks indicate cluster-module pairs that are significant using a one way permutation test and FDR correction. The size of the gene modules are shown to the right.

Gene module expression comparison with the Pigment Cluster reveals potential elusive cell types within the cluster.

Genes within the network are combined into one feature then plotted on a feature plot. In this case, UMAP dimensions one and two are the x and y-axis accordingly, dots represent each pigment cell, and the color of the dots represents the expression of the module. Cells that express gene modules are either colored red or green (when modules are plotted on separate plots). When plotted on the same plot, cells that express both gene modules are yellow. Panels A through J show each gene module comparison.

Scpp gene cluster analysis identifies most Scpp losses are not unique to syngnathids.

A) denotes the Syngnathoidei lineage which contains syngnathids and blue spotted cornetfish (among other fish) to highlight that this clade contains various tooth alterations. Syngnathids lack teeth completely and cornetfish have small teeth limited to the lower jaw. B) denotes solely the syngnathid clade, which completely lacks teeth. The phylogenetic tree was time calibrated using Hughes et al. 2018 and Stiller et al. 2022.

3dpf Zebrafish do not express lectin genes in epidermal cells.

This dotplot shows the marker genes on the x-axis and y-axis, organized by cell type. The size of the dot represents the percentage of cells that express the gene in each cluster. The darkness of the dot represents the average expression of the genes.