Basal Vertebrate Research Articles

Sex-dependent pheromonal effects on steroid hormone levels in sea lampreys (Petromyzon marinus)

Sea lampreys (Petromyzon marinus) are basal vertebrates that exhibit reproductive control via a hypothalamic-pituitary-gonadal axis. The function and evolution of the hypothalamic and pituitary peptide hormones are well studied in this species, whereas the functions of classical sex steroid hormones have not been well established due to their low or non-detectable plasma levels. Sea lamprey pheromone 3-keto petromyzonol sulfate (3kPZS) has been shown to increase while 3-keto allocholic acid (3kACA) decreases plasma 15α-hydroxyprogesterone (15αP) levels in prespermiating males (PSM) but not in preovulatory females (POF). However, spermiating male washings that contain both 3kPZS and 3kACA facilitate spawning in both sexes. Therefore, we wondered if the effects of pheromones on POF were elicited by classical steroid hormones such as progesterone, androstenedione, testosterone and estradiol. We hypothesized that waterborne 3kACA and 3kPZS differentially alter steroid hormone levels in prespawning sea lampreys. We determined the sex differences and pheromonal effects on steroid hormone levels in prespawning sea lampreys using sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods. Some PSM became spermiating (SM) at the time of sample collection, and those data were treated as a separate group. We found that males contained more androstenedione and progesterone in the gonad and more estradiol and testosterone in the plasma than POF, whereas POF contained more gonadal testosterone than males (no detectable gonadal testosterone). In POF, 3kPZS decreased gonadal androstenedione but increased gonadal progesterone and plasma estradiol, whereas 3kACA only increased gonadal progesterone levels. Exposure to 3kPZS for 4 h increased plasma 15αP in POF and SM, and gonadal 15αP in POF. Interestingly, 3kACA or 3kPZS depleted gonadal or plasma 15αP in PSM at various time points. On the other hand, both pheromones had no significant effect on androstenedione, progesterone or estradiol levels in males. Plasma testosterone levels did not change after pheromone exposure in both sexes. We conclude that sea lamprey pheromones 3kACA and 3kPZS induced differential steroidal responses in POF, PSM and SM.

Molecular Characterization and Chemotactic Function of CXCL8 in Northeast Chinese Lamprey (Lethenteron morii).

Chemokine-induced chemotaxis of leukocytes is an important part of the innate immunity and has been shown to mediate inflammation in all groups of jawed vertebrates. For jawless vertebrates, hagfish leukocytes are known to show chemotaxis toward mammalian complement anaphylotoxin and Gram-negative bacteria lipopolysaccharide. However, whether chemokines mediate chemotaxis of leukocytes in jawless vertebrates has not been conclusively examined. Here, we show C-X-C motif chemokine ligand 8 (CXCL8, also named interleukin 8) of the Northeast Chinese lamprey (Lethenteron morii) (designated as LmCXCL8) induces chemotaxis in its leukocytes. We identified LmCXCL8 and found it possesses the characteristic N-terminal cysteine residues and GGR (Gly-Gly-Arg) motif. The Lmcxcl8 gene was found to be expressed in all examined tissues, and its expression was inducible in the lamprey challenged by an infectious bacterium, Pseudomonas aeruginosa. A recombinant LmCXCL8 protein elicited concentration-dependent chemotaxis in peripheral blood leukocytes isolated from the Northeast Chinese lamprey. Based on these results, we conclude that LmCXCL8 is a constitutive and inducible acute-phase cytokine that mediates immune defense and trace the chemotactic function of chemokine to basal vertebrates.

11-Deoxycortisol controls hydromineral balance in the most basal osmoregulating vertebrate, sea lamprey (Petromyzon marinus)

It is unknown whether and how osmoregulation is controlled by corticosteroid signaling in the phylogenetically basal vertebrate group Agnatha, including lampreys and hagfishes. It is known that a truncated steroid biosynthetic pathway in lampreys produces two predominant circulating corticosteroids, 11-deoxycortisol (S) and 11-deoxycorticosterone (DOC). Furthermore, lampreys express only a single, ancestral corticosteroid receptor (CR). Whether S and/or DOC interact with the CR to control osmoregulation in lampreys is still unknown. We examined the role of the endogenous corticosteroids in vivo and ex vivo in sea lamprey (Petromyzon marinus) during the critical metamorphic period during which sea lamprey increase osmoregulatory capacity and acquire seawater (SW) tolerance. We demonstrate in vivo that increases in circulating [S] and gill CR abundance are associated with increases in osmoregulatory capacity during metamorphosis. We further show that in vivo and ex vivo treatment with S increases activity and expression of gill active ion transporters and improves SW tolerance, and that only S (and not DOC) has regulatory control over active ion transport in the gills. Lastly, we show that the lamprey CR expresses an ancestral, spironolactone-as-agonist structural motif and that spironolactone treatment in vivo increases osmoregulatory capacity. Together, these results demonstrate that S is an osmoregulatory hormone in lamprey and that receptor-mediated discriminative corticosteroid regulation of hydromineral balance is an evolutionarily basal trait among vertebrates.

The serotonin reuptake blocker citalopram destabilizes fictive locomotor activity in salamander axial circuits through 5-HT1A receptors.

Serotoninergic (5-HT) neurons are powerful modulators of spinal locomotor circuits. Most studies on 5-HT modulation focused on the effect of exogenous 5-HT and these studies provided key information about the cellular mechanisms involved. Less is known about the effects of increased release of endogenous 5-HT with selective serotonin reuptake inhibitors. In mammals, such molecules were shown to destabilize the fictive locomotor output of spinal limb networks through 5-HT1A receptors. However, in tetrapods little is known about the effects of increased 5-HT release on the locomotor output of axial networks, which are coordinated with limb circuits during locomotion from basal vertebrates to mammals. Here, we examined the effect of citalopram on fictive locomotion generated in axial segments of isolated spinal cords in salamanders, a tetrapod where raphe 5-HT reticulospinal neurons and intraspinal 5-HT neurons are present as in other vertebrates. Using electrophysiological recordings of ventral roots, we show that fictive locomotion generated by bath-applied glutamatergic agonists is destabilized by citalopram. Citalopram-induced destabilization was prevented by a 5-HT1A receptor antagonist, whereas a 5-HT1A receptor agonist destabilized fictive locomotion. Using immunofluorescence experiments, we found 5-HT-positive fibers and varicosities in proximity with motoneurons and glutamatergic interneurons that are likely involved in rhythmogenesis. Our results show that increasing 5-HT release has a deleterious effect on axial locomotor activity through 5-HT1A receptors. This is consistent with studies in limb networks of turtle and mouse, suggesting that this part of the complex 5-HT modulation of spinal locomotor circuits is common to limb and axial networks in limbed vertebrates.NEW & NOTEWORTHY Little is known about the modulation exerted by endogenous serotonin on axial locomotor circuits in tetrapods. Using axial ventral root recordings in salamanders, we found that a serotonin reuptake blocker destabilized fictive locomotor activity through 5-HT1A receptors. Our anatomical results suggest that serotonin is released on motoneurons and glutamatergic interneurons possibly involved in rhythmogenesis. Our study suggests that common serotoninergic mechanisms modulate axial motor circuits in amphibians and limb motor circuits in reptiles and mammals.

Morphological and Vascular Study Of The Posterior Lumbar Epidural Fat

Posterior lumbar epidural fat has been described by several authors in various fields: Anesthetic, Radiology and Neurochemistry. In this study, we tried to describe the position of this fat in the lumbar canal (L1-L5), its vascularization and drainage system. 8 Ivorian cadavers are used for the practice of dissection in the laboratory. The anterior abdominal region is chosen for the approach of the lumbar epidural region. The study investigated the situation, morphology and microcirculatory structure of fat at the lumbar level. The fatty clusters were embedded in fine vessels and were better vascularized and drained by veins of large caliber. In the vertebral canal, the fat was struck in the intervertebral foramina and was simply spread on the anterior surface of the ligamentum flavum. The fat as triangular shape with a vertebral base and a foamy summit it wanders in the cerebrospinal fluid. The lumbar epidural fat is better vascularized, and its venous drainage is made by large veins facing the intervertebral foramina embedded in areolar tissue traversed by nerve roots.
 Key Words: vertebral canal, fat, structure, vascularization.

Lessons from the Lamprey on Spinal Cord Regeneration

Unlike mammals, non‐mammalian vertebrates undergo robust, spontaneous regeneration of central nervous system (CNS) structures after injury. Lampreys, which are amongst the most basal vertebrates, provide a unique opportunity to study CNS regeneration at levels ranging from molecules to behavior. After a complete spinal cord transection, lampreys recover nearly normal locomotor behaviors (e.g. swimming, burrowing) over the next 10–12 weeks. This functional recovery is accomplished by robust tissue repair, axon/synapse regeneration, and transcriptional changes in the spinal cord and brain that support CNS repair and growth. Such growth programs are highly conserved across vertebrates, including in mammals. Additionally, lampreys possess a class of large, identified reticulospinal neurons with known regenerative capacities: some are “good regenerators,” while others are “poor regenerators,” allowing us to examine intrinsic factors that govern CNS regeneration on neuron‐by‐neuron basis. This presentation will discuss some of the latest findings in lamprey CNS regeneration, including new molecular strategies with potential for improving CNS regeneration outcomes in other preclinical animal models.Support or Funding InformationNY Spinal Cord Injury Research BoardMarine Biological Laboratory, Woods Hole, MALamprey spinal cord at 11 weeks post‐injury. Many axons (green: neurofilament) are regenerating across the lesion, and the central canal that runs longitudinally down the center of the spinal cord is repaired (blue: DAPI). The original lesion site is in the middle of the image.Figure 1

Divergent genes encoding the putative receptors for growth hormone and prolactin in sea lamprey display distinct patterns of expression

Growth hormone receptor (GHR) and prolactin receptor (PRLR) in jawed vertebrates were thought to arise after the divergence of gnathostomes from a basal vertebrate. In this study we have identified two genes encoding putative GHR and PRLR in sea lamprey (Petromyzon marinus) and Arctic lamprey (Lethenteron camtschaticum), extant members of one of the oldest vertebrate groups, agnathans. Phylogenetic analysis revealed that lamprey GHR and PRLR cluster at the base of gnathostome GHR and PRLR clades, respectively. This indicates that distinct GHR and PRLR arose prior to the emergence of the lamprey branch of agnathans. In the sea lamprey, GHR and PRLR displayed a differential but overlapping pattern of expression; GHR had high expression in liver and heart tissues, whereas PRLR was expressed highly in the brain and moderately in osmoregulatory tissues. Branchial PRLR mRNA levels were significantly elevated by stage 5 of metamorphosis and remained elevated through stage 7, whereas levels of GHR mRNA were only elevated in the final stage (7). Branchial expression of GHR increased following seawater (SW) exposure of juveniles, but expression of PRLR was not significantly altered. The results indicate that GHR and PRLR may both participate in metamorphosis and that GHR may mediate SW acclimation.

Hyperossification in the vertebral column of Devonian placoderm fishes (Arthrodira)

ABSTRACT Skeletal bone enlargement (hyperossification) was thought to only occur in endochondral and dermal bones (e.g., endoskeletons of marine tetrapods and dermal bones of certain jawless vertebrates, placoderms, and teleost fishes). However, in some arthrodiran placoderms (basal jawed vertebrates), i.e., Millerosteus minor, Compagopiscis croucheri, Eastmanosteus calliaspis, several types of bone enlargement also occur within the endoskeleton, affecting the vertebral column. Significantly, placoderm endoskeletal bone is thought to be thin-walled, ossifying only in the fibrous layer surrounding a cartilage precursor (historically called perichondral bone), rather than endochondrally/dermally. Hyperossification differs among these three species, revealing a range of internal and external bone morphologies undescribed for the placoderm endoskeleton. Thus, neural arches of M. minor are swollen in external appearance, in cross-section showing considerable deposition of layered, compact bone. In contrast, the arches of E. calliaspis are unswollen externally, with the neural spine cortex composed of the thin perichondral bone expected for placoderms. Histologically, though, the arch comprises numerous layers of perichondral bone. In C. croucheri, as in E. calliaspis, the spine and arch are unswollen externally, but the perichondral bone is thickened and compact, more similar to the condition in M. minor. Vertebral hyperossification in these taxa differs considerably in the degree and mode of cortical tissue thickening, via addition of tissue to the bone’s external (periosteal) and/or internal (endosteal) surfaces. Hyperossifcation in these arthrodires demonstrates that increases in endoskeletal bone mass are not restricted to crown-group gnathostomes (Chondrichthyes + Actinopterygii), representing a first step in the evolution of this process, involving modifications to cortical bone layers.

Galanin Signaling in the Brain Regulates Color Pattern Formation in Zebrafish.

SummaryColor patterns are prominent features of many animals and are of high evolutionary relevance. In basal vertebrates, color patterns are composed of specialized pigment cells that arrange in multilayered mosaics in the skin. Zebrafish (Danio rerio), the preeminent model system for vertebrate color pattern formation, allows genetic screens as powerful approaches to identify novel functions in a complex biological system. Adult zebrafish display a series of blue and golden horizontal stripes, composed of black melanophores, silvery or blue iridophores, and yellow xanthophores. This stereotyped pattern is generated by self-organization involving direct cell contacts between all three types of pigment cells mediated by integral membrane proteins [1, 2, 3, 4, 5]. Here, we show that neuropeptide signaling impairs the striped pattern in a global manner. Mutations in the genes coding either for galanin receptor 1A (npm/galr1A) or for its ligand galanin (galn) result in fewer stripes, a pale appearance, and the mixing of cell types, thus resembling mutants with thyroid hypertrophy [6]. Zebrafish chimeras obtained by transplantations of npm/galr1A mutant blastula cells indicate that mutant pigment cells of all three types can contribute to a normal striped pattern in the appropriate host. However, loss of galr1A expression in a specific region of the brain is sufficient to cause the mutant phenotype in an otherwise wild-type fish. Increased thyroid hormone levels in mutant fish suggest that galanin signaling through Galr1A in the pituitary is an upstream regulator of the thyroid hormone pathway, which in turn promotes precise interactions of pigment cells during color pattern formation.

Knowing when to stick: touch receptors found in the remora adhesive disc.

Remoras are fishes that piggyback onto larger marine fauna via an adhesive disc to increase locomotor efficiency, likelihood of finding mates and access to prey. Attaching rapidly to a large, fast-moving host is no easy task, and while research to date has focused on how the disc supports adhesion, no attention has been paid to how or if remoras are able to sense attachment. We identified push-rod-like mechanoreceptor complexes embedded in the soft lip of the remora adhesive disc that are known in other organisms to respond to touch and shear forces. This is, to our knowledge, the first time such mechanoreceptor complexes are described in fishes as they were only known previously in monotremes. The presence of push-rod-like mechanoreceptor complexes suggests not only that fishes may be able to sense their environment in ways not heretofore described but that specialized tactile mechanoreceptor complexes may be a more basal vertebrate feature than previously thought.

Osmoregulatory role of the intestine in the sea lamprey (Petromyzon marinus).

Lampreys are the most basal vertebrates with an osmoregulatory strategy. Previous research has established that the salinity tolerance of sea lamprey increases dramatically during metamorphosis, but underlying changes in the gut have not been examined. In the present work, we examined changes in intestinal function during metamorphosis and seawater exposure of sea lamprey (Petromyzon marinus). Fully metamorphosed juvenile sea lamprey had 100% survival after direct exposure to 35 parts per thousand seawater (SW) and only slight elevations in plasma chloride (Cl-) levels. Drinking rates of sea lamprey juveniles in seawater were 26-fold higher than juveniles in freshwater (FW). Na+-K+-ATPase (NKA) activity in the anterior and posterior intestine increased 12- and 3-fold, respectively, during metamorphosis, whereas esophageal NKA activity was lower than in the intestine and did not change with development. Acclimation to SW significantly enhanced NKA activity in the posterior intestine but did not significantly change NKA activity in the anterior intestine, which remained higher than that in the posterior intestine. Intestinal Cl- and water uptake, which were observed in ex vivo preparations of anterior and posterior intestine under both symmetric and asymmetric conditions, were higher in juveniles than in larvae and were similar in magnitude of those of teleost fish. Inhibition of NKA by ouabain in ex vivo preparations inhibited intestinal water absorption by 64%. Our results indicate drinking and intestinal ion and water absorption are important to osmoregulation in SW and that preparatory increases in intestinal NKA activity are important to the development of salinity tolerance that occurs during sea lamprey metamorphosis.

Functional characterization and osmoregulatory role of the Na+-K+-2Cl- cotransporter in the gill of sea lamprey (Petromyzon marinus), a basal vertebrate.

The present study provides molecular and functional characterization of Na+-K+-2Cl- cotransporter (NKCC1/Slc12a2) in the gills of sea lamprey (Petromyzon marinus), the most basal extant vertebrate with an osmoregulatory strategy. We report the full-length peptide sequence for the lamprey Na-K-Cl cotransporter 1 (NKCC1), which we show groups strongly with and occupies a basal position among other vertebrate NKCC1 sequences. In postmetamorphic juvenile lamprey, nkcc1 mRNA was present in many tissues but was fivefold higher in the gill than any other examined tissue, and NKCC1 protein was only detected in the gill. Gill mRNA and protein abundances of NKCC1 and Na+-K+-ATPase (NKA/Atp1a1) were significantly upregulated (20- to 200-fold) during late metamorphosis in fresh water, coinciding with the development of salinity tolerance, and were upregulated an additional twofold after acclimation to seawater (SW). Immunohistochemistry revealed that NKCC1 in the gill is found in filamental ionocytes coexpressing NKA, which develop during metamorphosis in preparation for SW entry. Lamprey treated with bumetanide, a widely used pharmacological inhibitor of NKCC1, exhibited higher plasma Cl- and osmolality as well as reduced muscle water content after 24 h in SW; there were no effects of bumetanide in freshwater-acclimated lamprey. This work provides the first functional characterization of NKCC1 as a mechanism for branchial salt secretion in lampreys, providing evidence that this mode of Cl- secretion has been present among vertebrates for ~550 million years.

Hematopoietic stem cells debut in embryonic lymphomyeloid tissues of elasmobranchs.

The evolutionary initiation of the appearance in lymphomyeloid tissue of the hemopoietic stem cell in the most basal vertebrates model, i.e. the elasmobranch (chondroichthyan) Torpedo marmorata Risso, has been studied. The three consecutive developmental stages of torpedo embryos were obtained by cesarean section from a total of six pregnant torpedoes. Lymphomyeloid tissue was identified in the Leydig organ and epigonal tissue. The sections were treated with monoclonal anti- CD34 and anti-CD38 antibodies to detect hematopoietic stem cells. At stage I (2-cmlong embryos with external gills) and at stage II (3-4 cm-long embryos with a discoidal shape and internal gills), some lymphoid- like cells that do not demonstrate any immunolabeling for these antibodies are present. Neither CD34+ nor CD38+ cells are identifiable in lymphomyeloid tissue of stage I and stage II embryos, while a CD34+CD38- cell was identified in the external yolk sac of stage II embryo. The stage III (10-11-cm-long embryos), the lymphomyeloid tissue contained four cell populations, respectively CD34+CD38-, CD34+CD38+, CD34-CD38+, and CD34- CD38- cells. The spleen and lymphomyeloid tissue are the principal sites for the development of hematopoietic progenitors in embryonic Torpedo marmorata Risso. The results demonstrated that the CD34 expression on hematopoietic progenitor cells and its extraembryonic origin is conserved throughout the vertebrate evolutionary scale.

Nasal Vaccination Drives Modifications of Nasal and Systemic Antibody Repertoires in Rainbow Trout.

Bony fish represent the most basal vertebrate branch with a dedicated mucosal immune system, which comprises immunologically heterogeneous microenvironments armed with innate and adaptive components. In rainbow trout (Oncorhynchus mykiss), a nasopharynx-associated lymphoid tissue (NALT) was recently described as a diffuse network of myeloid and lymphoid cells located in the olfactory organ of fish. Several studies have demonstrated high levels of protection conferred by nasal vaccines against viral and bacterial pathogens; however, the mechanisms underlying the observed protection are not well understood. We applied 5'RACE and a deep sequencing-based approach to investigate the clonal structure of the systemic and mucosal rainbow trout B cell repertoire. The analysis of Ig repertoire in control trout suggests different structures of IgM and IgT spleen and NALT repertoires, with restricted repertoire diversity in NALT. Nasal and injection vaccination with a bacterial vaccine revealed unique dynamics of IgM and IgT repertoires at systemic and mucosal sites and the remarkable ability of nasal vaccines to induce spleen Ig responses. Our findings provide an important immunological basis for the effectiveness of nasal vaccination in fish and other vertebrate animals and will help the design of future nasal vaccination strategies.

Sexual selection and the evolution of sperm morphology in sharks.

Post-copulatory sexual selection, and sperm competition in particular, is a powerful selective force shaping the evolution of sperm morphology. Although mounting evidence suggests that post-copulatory sexual selection influences the evolution of sperm morphology among species, recent evidence also suggests that sperm competition influences variation in sperm morphology at the intraspecific level. However, contradictory empirical results and limited taxonomic scope have led to difficulty in assessing the generality of sperm morphological responses to variation in the strength of sperm competition. Here, we use phylogenetically controlled analyses to explore the effects of sperm competition on sperm morphology and variance in sharks, a basal vertebrate group characterized by wide variation in rates of multiple mating by females, and consequently sperm competition risk. Our analyses reveal that shark species experiencing greater levels of sperm competition produce sperm with longer flagella and that sperm flagellum length is less variable in species under higher sperm competition risk. In contrast, neither the length of the sperm head and midpiece nor variation in sperm head and midpiece length was associated with sperm competition risk. Our findings demonstrate that selection influences both the inter- and intraspecific variation in sperm morphology and suggest that the flagellum is an important target of sexual selection in sharks. These findings provide important insight into patterns of selection on the ejaculate in a basal vertebrate lineage.

Evidence for the Direct Effect of the NPFF Peptide on the Expression of Feeding-Related Factors in Spotted Sea Bass (Lateolabrax maculatus).

Neuropeptide FF (NPFF) is a family member of RF-amide peptides, which are suggested to be involved in the control of vertebrate feeding behavior. However, little is known about the effect of the NPFF peptide on feeding-related processes in basal vertebrates. In this study, four full-length cDNAs, npff, npffr1, npffr2-1, and npffr2-2, were cloned from spotted sea bass and characterized. The conserved NPFF peptide is biologically active because it functionally interacts with different receptors expressed in cultured eukaryotic cells to enhance CRE promoter activity. Tissue distribution analysis showed that the highest npff mRNA expression occurred in the telencephalon, hypothalamus, medulla, gonad and muscle, but the npffrs mRNAs were mainly distributed within the central nervous system (CNS). In situ hybridization (ISH) detected npff-expressing cells in several specific regions ranging across the telencephalon and midbrain to the hypothalamus. Incubation of the spotted sea bass conserved NPFF peptide significantly increased the expression of orexin (orx) and neuropeptide Y (npy) mRNA and decreased the expression of leptin (lep), somatostatin (ss), and cholecystokinin (cck) mRNA in brain cells. Similarly, the conserved NPFF peptide also heightened the expression of gastrin (gas), ghrelin (ghrl), and motilin (mtl) mRNA and significantly reduced the expression of cck mRNA in the intestine and stomach. Taken together, these data suggest that the NPFF peptide may play a stimulating role in regulating feeding-related processes in spotted sea bass.

Dopaminergic modulation of olfactory-evoked motor output in sea lampreys (Petromyzon marinus L.).

Detection of chemical cues is important to guide locomotion in association with feeding and sexual behavior. Two neural pathways responsible for odor‐evoked locomotion have been characterized in the sea lamprey (Petromyzon marinus L.), a basal vertebrate. There is a medial pathway originating in the medial olfactory bulb (OB) and a lateral pathway originating from the rest of the OB. These olfactomotor pathways are present throughout the life cycle of lampreys, but olfactory‐driven behaviors differ according to the developmental stage. Among possible mechanisms, dopaminergic (DA) modulation in the OB might explain the behavioral changes. Here, we examined DA modulation of olfactory transmission in lampreys. Immunofluorescence against DA revealed immunoreactivity in the OB that was denser in the medial part (medOB), where processes were observed close to primary olfactory afferents and projection neurons. Dopaminergic neurons labeled by tracer injections in the medOB were located in the OB, the posterior tuberculum, and the dorsal hypothalamic nucleus, suggesting the presence of both intrinsic and extrinsic DA innervation. Electrical stimulation of the olfactory nerve in an in vitro whole‐brain preparation elicited synaptic responses in reticulospinal cells that were modulated by DA. Local injection of DA agonists in the medOB decreased the reticulospinal cell responses whereas the D2 receptor antagonist raclopride increased the response amplitude. These observations suggest that DA in the medOB could modulate odor‐evoked locomotion. Altogether, these results show the presence of a DA innervation within the medOB that may play a role in modulating olfactory inputs to the motor command system of lampreys.

Genomic sequence analyses of classical and non-classical lamprey progesterone receptor genes and the inference of homologous gene evolution in metazoans

BackgroundNuclear progesterone receptor (nPR) is an evolutionary innovation in vertebrates that mediates genomic responses to progesterone. Vertebrates also respond to progesterone via membrane progesterone receptors (mPRs) or membrane associated progesterone receptors (MAPRs) through rapid nongenomic mechanisms. Lampreys are extant agnathan vertebrates, residing at the evolutionary juncture where vertebrates diverged from invertebrates. A survey of the progesterone receptor (PR) gene sequences in lamprey genomes would inform PR gene evolutionary events during the transition from invertebrates to vertebrates.ResultsIn this study, we annotated sequences of one nPR, four mPR (β, γ, δ and ε) and four MAPR genes from genomes of two lamprey species (Petromyzon marinus and Lethenteron japonicum). To infer the origin and evolutionary history of PR genes, we constructed phylogenetic trees of PR homologous sequences across representative species of metazoans. Phylogenetic analyses revealed that the mPRγ gene first appeared in non-bilaterians, and the mPRβ gene likely arose from a duplication of mPRγ. On the other hand, the mPRγ gene gave rise to the mPRδ and ε genes much later in the vertebrate lineage. In addition, the mPRα gene first appeared in cartilaginous fishes, likely derived from duplication of mPRβ after the agnathan-gnathostome divergence. All known MAPR genes were present in the lamprey genomes. Progesterone receptor membrane component 1 (PGRMC1), neudesin and neuferricin genes probably evolved in parallel in non-bilaterians, whereas two copies of PGRMC genes probably derived from duplication of ancestral PGRMC1 sequence and appeared before the speciation of lampreys.ConclusionsNon-classical mPR and MAPR genes first evolved in non-bilaterians and classical nPR genes evolved later in basal vertebrates. Sequence repertoires for membrane progesterone receptor genes in vertebrates likely originated from an ancestral metazoan sequence and expanded via several duplication events.

Magic roundabout is an endothelial-specific ohnolog of ROBO1 which neo-functionalized to an essential new role in angiogenesis.

BackgroundMagic roundabout (ROBO4) is an unusual endothelial-specific paralog of the family of neuronally-expressed axon guidance receptors called roundabouts. Endothelial cells (ECs), whose uninterrupted sheet delimits the lumen of all vertebrate blood vessels and which are absent from invertebrate species, are a vertebrate-specific evolutionary novelty.ResultsHerein, the evolutionary mechanism of the duplication, retention and divergence of ROBO4 was investigated for the first time. Phylogenetic analyses carried out suggested that ROBO4 is a fast-evolving paralog of ROBO1 formed at the base of vertebrates. The ancestral expression pattern was neuronal. ROBO4 dramatically shifted its expression and became exceptionally specific to ECs. The data-mining of FANTOM5 and ENCODE reveals that ROBO4’s endothelial expression arises from a single transcription start site (TSS), conserved in mouse, controlled by a proximal promoter with a complex architecture suggestive of regulatory neo-functionalization. (An analysis of promoter probabilities suggested the architecture was not due to a chance arrangement of TFBSes). Further evidence for the neo-functionalization of ROBO4 comes from the analysis of its protein interactions, the rates of protein evolution, and of positively selected sites.ConclusionsThe neo-functionalization model explains why ROBO4 protein acquired new context-specific biological functions in the control of angiogenesis. This endothelial-specific roundabout receptor is an illustrative example of the emergence of an essential vertebrate molecular novelty and an endothelial-specific signaling sub-network through 2R-WGD. The emergence of novel cell types, such as ECs, might be a neglected evolutionary force contributing to the high rate of retention of duplicates post-2R-WGD. Crucially, expression neo-functionalization to evolutionarily novel sites of expression conceptually extends the classical model of neo-functionalization.

The Synarcual of the Little Skate, Leucoraja erinacea: Novel Development Among the Vertebrates

Evolutionary variation in anteroposterior patterning of the axial skeleton is a major contributor to the evolution of the vertebrate body plan, with five canonical vertebral types in tetrapods (cervical, thoracic, lumbar, sacral, caudal). However, less is known about the evolutionary origin and variation in vertebral regionalization patterns outside of tetrapods where described vertebral types range from as few as two in some chondrichthyans to eight or more in some teleost fishes. The synarcual is a specialized adaptation of the anterior axial skeleton comprising a putatively fused array of vertebral elements characteristic of jawed vertebrate (gnathostome) clades such as batoid and chimaeroid chondrichthyans where they support enlarged pectoral fins and dorsal fin spines respectively, as well as a fossil group known as the placoderms. Placoderms represent the phylogenetically most basal jawed vertebrates and the presence of a synarcual in these and chondrichthyans may suggest a conserved vertebral type for jawed vertebrates, predating the divergence of stem and crown gnathostomes. Alternatively, synarcuals may have evolved independently in these lineages, exhibiting a remarkable case of morphological convergence. In order to address this question, we investigated the early development of the cervicothoracic synarcual of an emerging model chondrichthyan: the Little skate Leucoraja erinacea. By combining x-ray computed tomography, and classical histology, we show that the skate synarcual is a complex composite element which develops from a hollow, continuous cartilaginous element devoid of any vertebral centra anterior to the pectoral girdle, and fusion/remodeling of initially distinct vertebrae posteriorly. A de novo transcriptome assembly for two developmental stages of the skate synarcual and post-synarcual axial skeletal elements supported this two-phase development, with differences in expression levels of several developmental genes, including Hox family transcription factors, which suggest anterior-posterior regionalization along the vertebral column, potentially linked to the synarcual. In addition, multiple genes related to skeletal remodeling were found to be more highly expressed at stage 33, including genes related to osteoclast activity, normally associated with removal of bone, a tissue absent in chondrichthyans. These genes are potentially related to loss of mineralization as vertebral elements are incorporated into the synarcual.