Sea lamprey enlightens the origin of the coupling of retinoic acid signaling to vertebrate hindbrain segmentation.

Retinoic acid (RA) is involved in antero-posterior patterning of the chordate body axis and, in jawed vertebrates, has been shown to play a major role at multiple levels of the gene regulatory network (GRN) regulating hindbrain segmentation. Knowing when and how RA became coupled to the core hindbrain GRN is important for understanding how ancient signaling pathways and patterning genes can evolve and generate diversity. Hence, we investigated the link between RA signaling and hindbrain segmentation in the sea lamprey Petromyzon marinus, an important jawless vertebrate model providing clues to decipher ancestral vertebrate features. Combining genomics, gene expression, and functional analyses of major components involved in RA synthesis (Aldh1as) and degradation (Cyp26s), we demonstrate that RA signaling is coupled to hindbrain segmentation in lamprey. Thus, the link between RA signaling and hindbrain segmentation is a pan vertebrate feature of the hindbrain and likely evolved at the base of vertebrates.

Differences in the transcriptome response in the gills of sea lamprey acutely exposed to 3-trifluoromethyl-4-nitrophenol (TFM), niclosamide or a TFM:niclosamide mixture.

Sea lamprey (Petromyzon marinus) control in the Laurentian Great Lakes of North America makes use of two pesticides: 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide, which are often co-applied. Sea lamprey appear to be vulnerable to these agents resulting from a lack of detoxification responses with evidence suggesting that lampricide mixtures produce a synergistic effect. However, there is a lack of information pertaining to the physiological responses of sea lamprey to niclosamide and TFM:niclosamide mixtures. Here, we characterized the transcriptomic responses of the sea lamprey to TFM, niclosamide, and a TFM:niclosamide (1.5 %) mixture in the gill. Along with a control, larval sea lamprey were exposed to each treatment for 6 h, after which gill tissues were extracted for measuring whole-transcriptome responses using RNA sequencing. Differential gene expression patterns were summarized, which included identifying the broad roles of genes and common expression patterns among the treatments. While niclosamide treatment resulted in no differentially expressed genes, TFM- and mixture-treated fish had several differentially expressed genes that were associated with the cell cycle, DNA damage, metabolism, immune function, and detoxification. However, there was no common differential expression among treatments. For the first time, we characterized the transcriptomic response of sea lamprey to niclosamide and a TFM:niclosamide mixture and identified that these agents impact mRNA transcript abundance of genes associated with the cell cycle and cellular death, and immune function, which are likely mediated through mitochondrial dysregulation. These results may help to inform the production of more targeted and effective lampricides in sea lamprey control efforts.

A lamprey neural cell type atlas illuminates the origins of the vertebrate brain.

The vertebrate brain emerged more than ~500 million years ago in common evolutionary ancestors. To systematically trace its cellular and molecular origins, we established a spatially resolved cell type atlas of the entire brain of the sea lamprey-a jawless species whose phylogenetic position affords the reconstruction of ancestral vertebrate traits-based on extensive single-cell RNA-seq and in situ sequencing data. Comparisons of this atlas to neural data from the mouse and other jawed vertebrates unveiled various shared features that enabled the reconstruction of cell types, tissue structures and gene expression programs of the ancestral vertebrate brain. However, our analyses also revealed key tissues and cell types that arose later in evolution. For example, the ancestral brain was probably devoid of cerebellar cell types and oligodendrocytes (myelinating cells); our data suggest that the latter emerged from astrocyte-like evolutionary precursors in the jawed vertebrate lineage. Altogether, our work illuminates the cellular and molecular architecture of the ancestral vertebrate brain and provides a foundation for exploring its diversification during evolution.

An improved germline genome assembly for the sea lamprey Petromyzon marinus illuminates the evolution of germline-specific chromosomes.

Programmed DNA loss is a gene silencing mechanism that is employed by several vertebrate and nonvertebrate lineages, including all living jawless vertebrates and songbirds. Reconstructing the evolution of somatically eliminated (germline-specific) sequences in these species has proven challenging due to a high content of repeats and gene duplications in eliminated sequences and a corresponding lack of highly accurate and contiguous assemblies for these regions. Here, we present an improved assembly of the sea lamprey (Petromyzon marinus) genome that was generated using recently standardized methods that increase the contiguity and accuracy of vertebrate genome assemblies. This assembly resolves highly contiguous, somatically retained chromosomes and at least one germline-specific chromosome, permitting new analyses that reconstruct the timing, mode, and repercussions of recruitment of genes to the germline-specific fraction. These analyses reveal major roles of interchromosomal segmental duplication, intrachromosomal duplication, and positive selection for germline functions in the long-term evolution of germline-specific chromosomes.

N-terminal domain influences steroid activation of the Atlantic sea lamprey corticoid receptor.

Lampreys are jawless fish that evolved about 550 million years ago at the base of the vertebrate line. Modern lampreys contain a corticoid receptor (CR), the common ancestor of the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), which first appear in cartilaginous fish, such as sharks. Until recently, 344 amino acids at the amino terminus of adult lamprey CR were not present in the lamprey CR sequence in GenBank. A search of the recently sequenced lamprey germline genome identified two CR sequences, CR1 and CR2, containing the 344 previously un-identified amino acids. CR1 also contains a novel four amino acid insertion in the DNA-binding domain (DBD). We studied corticosteroid and progesterone activation of CR1 and CR2 and found their strongest response was to 11-deoxycorticosterone and 11-deoxycortisol, the two circulating corticosteroids in lamprey. Based on steroid specificity, both CRs are close to elephant shark MR and distant from elephant shark GR. HEK293 cells that were transfected with full-length CR1 or CR2 and the MMTV promoter have about 3-fold higher steroid-mediated activation compared to HEK293 cells transfected with these CRs and the TAT3 promoter. Deletion of the amino-terminal domain (NTD) of lamprey CR1 and CR2 to form truncated CRs decreased transcriptional activation by about 70% in HEK293 cells that were transfected with MMTV, but increased transcription by about 6-fold in cells transfected with TAT3. This indicated that the promoter has an important effect on NTD regulation of transcriptional activation of the CR by steroids. Our results also indicate that the entire lamprey CR sequence is needed for an accurate determination of steroid-mediated transcription.

Discovery of prolactin-like in lamprey: Role in osmoregulation and new insight into the evolution of the growth hormone/prolactin family.

We used a representative of one of the oldest extant vertebrate lineages (jawless fish or agnathans) to investigate the early evolution and function of the growth hormone (GH)/prolactin (PRL) family. We identified a second member of the GH/PRL family in an agnathan, the sea lamprey (Petromyzon marinus). Structural, phylogenetic, and synteny analyses supported the identification of this hormone as prolactin-like (PRL-L), which has led to added insight into the evolution of the GH/PRL family. At least two ancestral genes were present in early vertebrates, which gave rise to distinct GH and PRL-L genes in lamprey. A series of gene duplications, gene losses, and chromosomal rearrangements account for the diversity of GH/PRL-family members in jawed vertebrates. Lamprey PRL-L is produced in the proximal pars distalis of the pituitary and is preferentially bound by the lamprey PRL receptor, whereas lamprey GH is preferentially bound by the lamprey GH receptor. Pituitary PRL-L messenger RNA (mRNA) levels were low in larvae, then increased significantly in mid-metamorphic transformers (stage 3); thereafter, levels subsided in final-stage transformers and metamorphosed juveniles. The abundance of PRL-L mRNA and immunoreactive protein increased in the pituitary of juveniles under hypoosmotic conditions, and treatment with PRL-L blocked seawater-associated inhibition of freshwater ion transporters. These findings clarify the origin and divergence of GH/PRL family genes in early vertebrates and reveal a function of PRL-L in osmoregulation of sea lamprey, comparable to a role of PRLs that is conserved in jawed vertebrates.

Granulin as an important immune molecule involved in lamprey tissue repair and regeneration by promoting cell proliferation and migration.

Progranulin (PGRN) is an autocrine growth factor that regulates cell proliferation, migration, wound healing, and tissue repair in mammals. Lamprey is the most primitive of the extant vertebrates and is regarded as the survivor of a once flourishing group of paleozoic vertebrates, with a history of more than 500 million years. To date, the evolutionary dynamics and the underlying function of the PGRNs remain largely unclear in lamprey. Here, we screened four genes encoding PGRNs from the genomes of Lethenteron reissneri and Petromyzon marinus, including one long form (named Lr-PGRN-L) and three short forms (named Lr-PGRN-S1, Lr-PGRN-S2, and Lr-PGRN-S3), and performed phylogenetic tree, functional domain, and synteny analyses to identify the evolutionary history of the four Lr-PGRNs. In addition, the expressions of the four Lr-pgrn family genes and the immune response against various pathogenic challenges were also investigated. We found that these genes were widely distributed in various tissues of lamprey and performed a variety of functions. Moreover, our results suggest that Lr-PGRN-S1 induces cell migration and proliferation, and is involved in repair after skin and spinal cord injury under appropriate conditions. Our findings are valuable because they improve the understanding of the evolutionary relationship of vertebrate pgrn genes, as well as providing new insights into the diverse and important roles of Lr-PGRNs.

Lamprey possess both V1R and V2R olfactory receptors, but only V1Rs are expressed in olfactory sensory neurons.

The sense of smell employs some of the largest gene families in the genome to detect and distinguish a multitude of different odors. Within vertebrates, 4 major olfactory receptor families have been described; of which, only 3 (OR, TAAR-like, and V1R) were found already in lamprey, a jawless vertebrate. The forth family (V2R) was believed to have originated later, in jawed vertebrates. Here we have delineated the entire vomeronasal receptor repertoire in 3 lamprey species. We report the presence of 6 v1r and 2 v2r genes in Lethenteron camtschaticum, arctic lamprey, and Lampetra fluviatilis, river lamprey (6 and 1, respectively, in sea lamprey, Petromyzon marinus). Three v1r genes but no v2r genes were found to be expressed in olfactory sensory neurons in the characteristic sparse expression pattern. Our results show the olfactory function of some V1Rs already in lamprey and, unexpectedly, an early origin of the V2R family in the shared ancestor of jawed and jawless vertebrates. However, lamprey v2r genes appear not to have acquired an olfactory function yet, thus dissociating the evolutionary origin of the family from the onset of a function as olfactory receptor.

Pervasive male-biased expression throughout the germline-specific regions of the sea lamprey genome supports key roles in sex differentiation and spermatogenesis.

Sea lamprey undergo programmed genome rearrangement (PGR) in which ∼20% of the genome is jettisoned from somatic cells during embryogenesis. Although the role of PGR in embryonic development has been studied, the role of the germline-specific region (GSR) in gonad development is unknown. We analysed RNA-sequence data from 28 sea lamprey gonads sampled across life-history stages, generated a genome-guided de novo superTranscriptome with annotations, and identified germline-specific genes (GSGs). Overall, we identified 638 GSGs that are enriched for reproductive processes and exhibit 36x greater odds of being expressed in testes than ovaries. Next, while 55% of the GSGs have putative somatic paralogs, the somatic paralogs are not differentially expressed between sexes. Further, putative orthologs of some the male-biased GSGs have known functions in sex determination or differentiation in other vertebrates. We conclude that the GSR of sea lamprey plays an important role in testicular differentiation and potentially sex determination.

A noninvasive eDNA tool for detecting sea lamprey larvae in river sediments: Analytical validation and field testing in a low-abundance ecosystem.

Anthropogenic activities are increasingly threatening aquatic biodiversity, especially anadromous species. Monitoring and conservation measures are thus required to protect, maintain and restore imperilled populations. While many species can be surveyed using traditional capture and visual census techniques, species that use riverine habitats in a less conspicuous manner, such as sea lamprey Petromyzon marinus, can be more challenging to monitor. Sea lamprey larvae (ammocoetes) can spend several years in freshwater burrowed within soft sediments, inhibiting their detection and assessment. Here, we present a qPCR assay based on the detection of environmental DNA (eDNA) to identify the presence of ammocoetes burrowed in the sediment. We present an extensively validated method that ensured both species-specificity of the assay as well as the capacity to detect ammocoetes when abundances are low. Experiments on burrowing activity suggested that most of the DNA released into the sediment occurs during burrowing. Overall, we demonstrate this new molecular-based tool is an efficient and effective complement to traditional monitoring activities targeting larval stages of sea lampreys.

Comparative Analysis of Key Transcription Factors Involved in Development in Sea Lamprey (Petromyzon marinus) through Systematic Mining of Lamprey Gene Expression Dataset.

Transcription factors (TFs), key regulators for gene expression, play varied yet crucial roles throughout cellular polarization, migration, proliferation, differentiation and apoptosis. An important function of TFs is acting in embryogenesis and organogenesis. To identify the candidate TFs in the progression of lamprey early embryogenesis, datasetGSE76037 was downloaded from the Gene Expression Omnibus (GEO) database and an integrated bioinformatic analysis was performed. Our findings revealed a total of 152 TFs in the dataset. The function enrichment analysis showed that these genes were mainly enriched in transcription from RNA polymerase II promoter, cell differentiation, embryonic digestive tract morphogenesis and so on. Hierarchical clustering analysis suggested the expression of TFs was significantly different during early embryogenesis. Moreover, volcano plots and Venn diagrams analysis identified 36 key TFs, which were considered to play an important role during embryogenesis. The weighted correlation network analysis (WGCNA) was constructed and the Pearson correlation coefficient was performed, indicating these TFs might involve in early development of lamprey germline by synergistically regulating each other. The result was confirmed by real-time polymerase chain reaction and Western blotting analysis. In conclusion, differentially expressed genes identified in the present study help us understand the molecular mechanisms underlying the lamprey embryogenesis and provide candidate TFs for further study of vertebrate embryonic development.

Characterization of the variable lymphocyte receptor B genes of a freshwater nonparasitic lamprey species, Lampetra hubbsi.

Agnathans possess a convergent adaptive immune system in comparison to that of jawed vertebrates. In lieu of immunoglobulins, agnathans deploy variable lymphocyte receptors (VLRs), single chain protein effector molecules consisting of leucine rich repeat modules. Foundational work for this discovery utilized the parasitic sea lamprey, Petromyzon marinus. However, for several reasons, it is desirable to employ a local species for further studies of lamprey immunity. A disjunct freshwater species from the Kings River of California, Lampetra hubbsi, was evaluated for this purpose. Validation that its adaptive immune system was analogous to that of P. marinus entailed detailed examination of its immune tissue organization and of its VLRB cDNA transcripts. The VLRB molecules showed high degrees of homology with P. marinus VLRB. Furthermore, hemato-lymphopoietic tissue expression of VLRB protein was confirmed. We conclude that L. hubbsi should be a viable alternative for studying the lamprey adaptive immune system and for generation of monoclonal antibodies.

Tissue and salinity specific Na+/Cl- cotransporter (NCC) orthologues involved in the adaptive osmoregulation of sea lamprey (Petromyzon marinus).

Two orthologues of the gene encoding the Na+-Cl- cotransporter (NCC), termed ncca and nccb, were found in the sea lamprey genome. No gene encoding the Na+-K+-2Cl- cotransporter 2 (nkcc2) was identified. In a phylogenetic comparison among other vertebrate NCC and NKCC sequences, the sea lamprey NCCs occupied basal positions within the NCC clades. In freshwater, ncca mRNA was found only in the gill and nccb only in the intestine, whereas both were found in the kidney. Intestinal nccb mRNA levels increased during late metamorphosis coincident with salinity tolerance. Acclimation to seawater increased nccb mRNA levels in the intestine and kidney. Electrophysiological analysis of intestinal tissue ex vivo showed this tissue was anion absorptive. After seawater acclimation, the proximal intestine became less anion absorptive, whereas the distal intestine remained unchanged. Luminal application of indapamide (an NCC inhibitor) resulted in 73% and 30% inhibition of short-circuit current (Isc) in the proximal and distal intestine, respectively. Luminal application of bumetanide (an NKCC inhibitor) did not affect intestinal Isc. Indapamide also inhibited intestinal water absorption. Our results indicate that NCCb is likely the key ion cotransport protein for ion uptake by the lamprey intestine that facilitates water absorption in seawater. As such, the preparatory increases in intestinal nccb mRNA levels during metamorphosis of sea lamprey are likely critical to development of whole animal salinity tolerance.

Rapid genetic adaptation to recently colonized environments is driven by genes underlying life history traits.

BACKGROUND: Uncovering the mechanisms underlying rapid genetic adaptation can provide insight into adaptive evolution and shed light on conservation, invasive species control, and natural resource management. However, it can be difficult to experimentally explore rapid adaptation due to the challenges associated with propagating and maintaining species in captive environments for long periods of time. By contrast, many introduced species have experienced strong selection when colonizing environments that differ substantially from their native range and thus provide a "natural experiment" for studying rapid genetic adaptation. One such example occurred when sea lamprey (Petromyzon marinus), native to the northern Atlantic, naturally migrated into Lake Champlain and expanded their range into the Great Lakes via man-made shipping canals. RESULTS: Utilizing 368,886 genome-wide single nucleotide polymorphisms (SNPs), we calculated genome-wide levels of genetic diversity (i.e., heterozygosity and π) for sea lamprey collected from native (Connecticut River), native but recently colonized (Lake Champlain), and invasive (Lake Michigan) populations, assessed genetic differentiation between all populations, and identified candidate genes that responded to selection imposed by the novel environments. We observed a 14 and 24% reduction in genetic diversity in Lake Michigan and Lake Champlain populations, respectively, compared to individuals from the Connecticut River, suggesting that sea lamprey populations underwent a genetic bottleneck during colonization. Additionally, we identified 121 and 43 outlier genes in comparisons between Lake Michigan and Connecticut River and between Lake Champlain and Connecticut River, respectively. Six outlier genes that contained synonymous SNPs in their coding regions and two genes that contained nonsynonymous SNPs may underlie the rapid evolution of growth (i.e., GHR), reproduction (i.e., PGR, TTC25, STARD10), and bioenergetics (i.e., OXCT1, PYGL, DIN4, SLC25A15). CONCLUSIONS: By identifying the genomic basis of rapid adaptation to novel environments, we demonstrate that populations of invasive species can be a useful study system for understanding adaptive evolution. Furthermore, the reduction in genome-wide levels of genetic diversity associated with colonization coupled with the identification of outlier genes underlying key life history traits known to have changed in invasive sea lamprey populations (e.g., growth, reproduction) illustrate the utility in applying genomic approaches for the successful management of introduced species.

Lamprey lecticans link new vertebrate genes to the origin and elaboration of vertebrate tissues.

The evolution of vertebrates from an invertebrate chordate ancestor involved the evolution of new organs, tissues, and cell types. It was also marked by the origin and duplication of new gene families. If, and how, these morphological and genetic innovations are related is an unresolved question in vertebrate evolution. Hyaluronan is an extracellular matrix (ECM) polysaccharide important for water homeostasis and tissue structure. Vertebrates possess a novel family of hyaluronan binding proteins called Lecticans, and studies in jawed vertebrates (gnathostomes) have shown they function in many of the cells and tissues that are unique to vertebrates. This raises the possibility that the origin and/or expansion of this gene family helped drive the evolution of these vertebrate novelties. In order to better understand the evolution of the lectican gene family, and its role in the evolution of vertebrate morphological novelties, we investigated the phylogeny, genomic arrangement, and expression patterns of all lecticans in the sea lamprey (Petromyzon marinus), a jawless vertebrate. Though both P. marinus and gnathostomes each have four lecticans, our phylogenetic and syntenic analyses are most consistent with the independent duplication of one of more lecticans in the lamprey lineage. Despite the likely independent expansion of the lamprey and gnathostome lectican families, we find highly conserved expression of lecticans in vertebrate-specific and mesenchyme-derived tissues. We also find that, unlike gnathostomes, lamprey expresses its lectican paralogs in distinct subpopulations of head skeleton precursors, potentially reflecting an ancestral diversity of skeletal tissue types. Together, these observations suggest that the ancestral pre-duplication lectican had a complex expression pattern, functioned to support mesenchymal histology, and likely played a role in the evolution of vertebrate-specific cell and tissue types.

Prediction of ATPase cation transporting 13A2 molecule in Petromyzon marinus and pan-cancer analysis into human tumors from an evolutionary perspective.

The ATPase cation transporting 13A2 protein (ATP13A2), which maintains the homeostasis of mitochondria and lysosomes, plays a significant role in human neurodegenerative diseases and cancer. Through constructing a lamprey proteome database, employing multiple sequence alignment and phylogenetic analysis, 5 ATP13A2 proteins from Petromyzon marinus (Pm-ATP13A2) were identified based on the evolutionary perspective. The motif and domain analysis showed that the ATP13A2 protein was conserved. The multiple phosphorylation sites and transmembrane structures highlighted the characteristics of ATP13A2 as the P-ATPase-V cation transporting protein. Based on the information provided by the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, this study was conducted as a preliminary investigation of the carcinogenic effects of the ATP13A2 gene in a variety of tumors. The ATP13A2 was strongly expressed in most tumors, except in two types of nervous system tumors glioblastoma multiforme (GBM) and brain lower grade glioma (LGG). Moreover, the expression of ATP13A2 was strongly correlated with the prognosis of tumor patients. The high expression of ATP13A2 was obviously related to the poor prognosis of LGG. The poor prognosis of LGG patients may affect the ATP13A2 expression through the immune cells and radiotherapy. Also, cancer-related fibroblast infiltration was observed. All in all, this work offers more insights into the molecular evolution of the ATP13A2 protein and facilitates the understanding of the carcinogenic effects of the ATP13A2 in different tumors. Our discussion also promotes the study into the successful evolution of the vertebrate brain and the mechanism of clinical brain-related diseases.

Paracellular and Transcellular Leukocytes Diapedesis Are Divergent but Interconnected Evolutionary Events.

Infiltration of the endothelial layer of the blood-brain barrier by leukocytes plays a critical role in health and disease. When passing through the endothelial layer during the diapedesis process lymphocytes can either follow a paracellular route or a transcellular one. There is a debate whether these two processes constitute one mechanism, or they form two evolutionary distinct migration pathways. We used artificial intelligence, phylogenetic analysis, HH search, ancestor sequence reconstruction to investigate further this intriguing question. We found that the two systems share several ancient components, such as RhoA protein that plays a critical role in controlling actin movement in both mechanisms. However, some of the key components differ between these two transmigration processes. CAV1 genes emerged during Trichoplax adhaerens, and it was only reported in transcellular process. Paracellular process is dependent on PECAM1. PECAM1 emerged from FASL5 during Zebrafish divergence. Lastly, both systems employ late divergent genes such as ICAM1 and VECAM1. Taken together, our results suggest that these two systems constitute two different mechanical sensing mechanisms of immune cell infiltrations of the brain, yet these two systems are connected. We postulate that the mechanical properties of the cellular polarity is the main driving force determining the migration pathway. Our analysis indicates that both systems coevolved with immune cells, evolving to a higher level of complexity in association with the evolution of the immune system.

Differential expression of somatostatin genes in the central nervous system of the sea lamprey.

The identification of three somatostatin (SST) genes (SSTa, SSTb, and SSTc) in lampreys (Tostivint et al. Gen Comp Endocrinol 237:89-97 https://doi.org/10.1016/j.ygcen.2016.08.006 , 2016) prompted us to study their expression in the brain and spinal cord of the sea lamprey by in situ hybridization. These three genes were only expressed in equivalent neuronal populations in the hypothalamus. In other regions, SST transcripts showed clear differential expression. In the telencephalon, SSTc-positive cells were observed in the medial pallium, ventral part of the lateral pallium, striatum, subhippocampal lobe, and preoptic region. In the diencephalon, SSTa-positive cells were observed in the thalamus and SSTc-positive cells in the prethalamus, posterior tubercle, pretectal area, and nucleus of the medial longitudinal fascicle. In the midbrain, SSTc-positive cells were observed in the torus semicircularis, lateral reticular area, and perioculomotor tegmentum. Different SSTa- and SSTc-positive populations were observed in the isthmus. SSTc neurons were also observed in the rostral octavolateralis area and caudal rhombencephalon. In the spinal cord, SSTa was expressed in cerebrospinal-fluid-contacting (CSF-c) neurons and SSTc in non-CSF-c interneurons. Comparison with previous immunohistochemical studies using anti-SST-14 antibodies strongly suggests that SST-14-like neurons correspond with the SSTa populations. Thus, the SSTc populations were not reported previously in immunohistochemical studies. Cluster-based analyses and alignments of mature peptides suggested that SSTa is an ortholog of SST1 and that SSTb is closely related to SST2 and SST6. These results provide important new insights into the evolution of the somatostatinergic system in vertebrates.

Transcriptomic analysis of gonadal development in parasitic and non-parasitic lampreys (Ichthyomyzon spp.), with a comparison of genomic resources in these non-model species.

Lampreys are jawless fishes that diverged ∼550 million years ago from other vertebrates. Sequencing of the somatic and the germline genomes of the sea lamprey (Petromyzon marinus) in 2013 and 2018, respectively, has helped to improve our understanding of the genes and gene networks that control many aspects of lamprey development. However, little is known about the genetic basis of gonadal differentiation in lampreys, partly due to the prolonged period during which their gonads remain sexually indeterminate. We performed RNA-sequencing on gonadal samples from four chestnut lamprey (Ichthyomyzon castaneus) and six northern brook lamprey (I. fossor) to identify differentially expressed genes (DEG's) and pathways associated with transcriptomic differences in: (1) larvae during early gonadal differentiation versus definitive females (i.e., with oocytes in the slow cytoplasmic growth phase); and (2) females versus definitive males undergoing spermatogonial proliferation. We compared the mapping percentages of these transcriptomes to the two available sea lamprey reference genomes and three annotation files (Ensembl and UCSC for the somatic genome and SIMRbase for the germline genome). We found that mapping the RNA-seq reads to the germline genome gave superior results and, using Trinotate, we provided new putative annotations for 8161 genes in the somatic assembly and 880 genes for the germline assembly. We identified >2000 DEG's between stages and sexes, as well as biological pathways associated with each. Interestingly, some of the upregulated genes (e.g., DEG's associated with spermiation) suggest that changes in gene expression can precede morphological changes by several months. In contrast, only 81 DEG's were evident between the chestnut lamprey (that remains sexually immature during an extended post-metamorphic parasitic feeding phase) and the nonparasitic northern brook lamprey (that undergoes sexual maturation near the end of metamorphosis), but few replicates were available for comparable stages and sexes. This work lays the foundation for identifying and confirming the orthology and the function of genes involved in gonadal development in these and other lamprey species across more developmental stages.

Evolution of the endothelin pathway drove neural crest cell diversification.

Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7-10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR-Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.