DOI: 10.5061/dryad.f7m0cfz60
title: Single cell RNA sequencing provides clues for the developmental genetic basis of Syngnathidae’s evolutionary adaptations

description: Seahorses, pipefishes, and seadragons are fishes from the family
 Syngnathidae that have evolved extraordinary traits including male
 pregnancy, elongated snouts, loss of teeth, and dermal bony armor. The
 developmental genetic and cellular changes that led to the evolution of
 these traits are largely unknown. Recent syngnathid genomes revealed
 suggestive gene content differences and provide the opportunity for
 detailed genetic analyses. We created a single cell RNA sequencing atlas
 of Gulf pipefish embryos to understand the developmental basis of four
 traits: derived head shape, toothlessness, dermal armor, and male
 pregnancy. We completed marker gene analyses, built genetic networks, and
 examined spatial expression of select genes. We identified
 osteochondrogenic mesenchymal cells in the elongating face that express
 regulatory genes bmp4, sfrp1a, and prdm16. We found no evidence for tooth
 primordia cells, and we observed re-deployment of osteoblast genetic
 networks in developing dermal armor. Finally, we found that epidermal
 cells expressed nutrient processing and environmental sensing genes,
 potentially relevant for the brooding environment. The examined pipefish
 evolutionary innovations are composed of recognizable cell types,
 suggesting derived features originate from changes within existing gene
 networks. Future work addressing syngnathid gene networks across multiple
 stages and species is essential for understanding how their novelties
 evolved.
description: The fasta file and annotation file shared here were used in the
 single cell RNA sequencing analysis described in the publication. The
 fasta nuclear genome file was downloaded from NCBI (GCF_024217435.2). The
 mitochondrial genome fasta file was downloaded from NCBI (NC_065499.1).
 The two fasta files were appended together to create the fasta file used
 in the analysis. To lengthen the 3' UTRs of the genome annotations,
 we produced two scISOrSeq libraries (one from a late embryonic stage and
 one from pregnant and non-pregnant brood pouch tissues) that were
 sequenced on seperate lanes on PacBio Sequel II - SMRT cells 8M. Reads
 were processed according to Healey et al. 2022 and aligned with the Gulf
 pipefish genome (GCF_024217435.2). Gene models from the scISOrSeq datasets
 were merged with the Gulf pipefish NCBI models (GCF_024217435.2) using
 TAMA merge. 
description: # Single cell RNA sequencing provides clues for the developmental genetic
 basis of Syngnathidae’s evolutionary adaptations
 [https://doi.org/10.5061/dryad.f7m0cfz60](https://doi.org/10.5061/dryad.f7m0cfz60) ## Description of the data and file structure These files were modified in order to analyze single cell RNA sequencing libraries produced from Syngnathus scovelli embryos  ### Files and variables #### File: GCF\_024217435.1\_RoL\_Ssco\_1.1\_genomic.MT-Lin.fna **Description:** This fasta file contains two fasta files appended together: GCF*024217435.1 (Syngnathus scovelli n*uclear genome) and NC065499.1 (*Syngnathus scovelli* mitochondria genome). #### File: genemodels\_sscncbi\_emb\_pouch\_LINMT.gtf **Description:** The gene models from this file consists of annotations from GCF*024217435.1 (Syngnathus scovelli n*uclear genome annotation), NC065499.1 (*Syngnathus scovelli* mitochondria genome annotation), and annotations derived from scISOrSeq data. These annotations were merged together using TAMA merge. ## Code/software The fasta files were appended together using cat. The merged annotation file was produced following the pipeline from Healey et al. 2022. To analyze the scISOrSeq reads, sequences were processed using scripts from Healey et al. 2022, aligned with minimap v2.9, and gene models were derived with SQANTI3. The annotation files were merged with TAMA merge. ## Access information Other publicly accessible locations of the data: * scRNAseq reads and the processed dataset is available on **[GSE278814](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE278814)** Data was derived from the following sources: * GCA_024217435.2 (*Synganthus scovelli* nuclear genome) * NC_065499.1 (*Syngnathus scovelli* mitochondrial genome)