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.

Functional re-organization of the gills of metamorphosing sea lamprey (Petromyzon marinus): preparation for a blood diet and the freshwater to seawater transition.

Sea lamprey (Petromyzon marinus) begin life as filter-feeding larvae (ammocoetes) before undergoing a complex metamorphosis into parasitic juveniles, which migrate to the sea where they feed on the blood of large-bodied fishes. The greater protein intake during this phase results in marked increases in the production of nitrogenous wastes (N-waste), which are excreted primarily via the gills. However, it is unknown how gill structure and function change during metamorphosis and how it is related to modes of ammonia excretion, nor do we have a good understanding of how the sea lamprey's transition from fresh water (FW) to sea water (SW) affects patterns and mechanisms of N-waste excretion in relation to ionoregulation. Using immunohistochemistry, we related changes in the gill structure of larval, metamorphosing, and juvenile sea lampreys to their patterns of ammonia excretion (Jamm) and urea excretion (Jurea) in FW, and following FW to artificial seawater (ASW) transfer. Rates of Jamm and Jurea were low in larval sea lamprey and increased in feeding juvenile, parasitic sea lamprey. In freshwater-dwelling ammocoetes, immunohistochemical analysis revealed that Rhesus glycoprotein C-like protein (Rhcg-like) was diffusely distributed on the lamellar epithelium, but following metamorphosis, Rhcg-like protein was restricted to SW mitochondrion-rich cells (MRCs; ionocytes) between the gill lamellae. Notably, these interlamellar Rhcg-like proteins co-localized with Na+/K+-ATPase (NKA), which increased in expression and activity by almost tenfold during metamorphosis. The distribution of V-type H+-ATPase (V-ATPase) on the lamellae decreased following metamorphosis, indicating it may have a more important role in acid-base regulation and Na+ uptake in FW, compared to SW. We conclude that the re-organization of the sea lamprey gill during metamorphosis not only plays a critical role in allowing them to cope with greater salinity following the FW-SW transition, but that it simultaneously reflects fundamental changes in methods used to excrete ammonia.

Control of invasive sea lampreys using the piscicides TFM and niclosamide: Toxicology, successes & future prospects.

The invasion of the Laurentian Great Lakes of North America by sea lampreys (Petromyzon marinus) in the early 20th century contributed to the depletion of commercial, recreational and culturally important fish populations, devastating the economies of communities that relied on the fishery. Sea lamprey populations were subsequently controlled using an aggressive integrated pest-management program which employed barriers and traps to prevent sea lamprey from migrating to their spawning grounds and the use of the piscicides (lampricides) 3-trifluoromethyl-4-nitrophenol (TFM) and niclosamide to eliminate larval sea lampreys from their nursery streams. Although sea lampreys have not been eradicated from the Great Lakes, populations have been suppressed to less than 10% of their peak numbers in the mid-1900s. The ongoing use of lampricides provides the foundation for sea lamprey control in the Great Lakes, one of the most successful invasive species control programs in the world. Yet, significant gaps remain in our understanding of how lampricides are taken-up and handled by sea lampreys, how lampricides exert their toxic effects, and how they adversely affect non-target invertebrate and vertebrates species. In this review we examine what has been learned about the uptake, handling and elimination, and the mode of TFM and niclosamide toxicity in lampreys and in non-target animals, particularly in the last 10 years. It is now clear that the mode of TFM toxicity is the same in non-target fishes and lampreys, in which TFM interferes with oxidative phosphorylation by the mitochondria leading to decreased ATP production. Vulnerability to TFM is related to abiotic factors such as water pH and alkalinity, which we propose changes the relative amounts of the bioavailable un-ionized form of TFM in the gill microenvironment. Niclosamide, which is also a molluscicide used to control snails in areas prone to schistosomiasis infections of humans, also likely works by uncoupling oxidative phosphorylation, but less is known about other aspects of its toxicology. The effects of TFM include reductions in energy stores, particularly glycogen and high energy phosphagens. However, non-target fishes readily recover from sub-lethal TFM exposure as demonstrated by the rapid restoration of energy stores and clearance of TFM. Although both TFM and niclosamide are non-persistent in the environment and critical for sea lamprey control, increasing public and institutional concerns about pesticides in the environment makes it imperative to explore other means of sea lamprey control. Accordingly, we also address possible "next-generation" strategies of sea lamprey control including genetic tools such as RNA interference and CRISPR-Cas9 to impair critical physiological processes (e.g. reproduction, digestion, metamorphosis) in lamprey, and the use of green chemistry to develop more environmentally benign chemical methods of sea lamprey control.

Diel activity of newly metamorphosed juvenile sea lamprey (Petromyzon marinus).

Timing of activity, especially for juvenile anadromous fishes undertaking long migrations can be critical for survival. River-resident larval sea lamprey metamorphose into juveniles and migrate from their larval stream habitats in fall through spring, but diel timing of this migratory behavior is not well understood. Diel activity was determined for newly metamorphosed sea lamprey using day/night net sampling and passive integrated transponder (PIT) telemetry in two natural streams and PIT telemetry in an artificial stream. Downstream migration was primarily nocturnal in all studies. All but one of 372 sea lamprey were captured during night sampling in the day/night net collections and all detections (N = 56) for the in-stream PIT telemetry occurred within a few hours after sunset. Most (81% of 48) tagged lamprey moved downstream during the first night following release and moved at speeds consistent with observed water velocities. During long-term observation of behavior in the artificial stream most sea lamprey movement occurred during the night with limited occurrence of movement during daylight hours. Understanding seasonal and diel timing of downstream migration behavior may allow more effective management of sea lamprey for both conservation and control.

Muscle fatty acid profiles of sea lamprey (Petromyzon marinus L.) indicate the use of fast metabolized energy during ontogenesis.

A comprehensive characterization of muscle's FA composition of sea lamprey ammocoetes and adults was performed to test the hypothesis that larvae, and early spawning migrants have a similar FA profile prior to metamorphosis and to spawning migration. Subsequently, the role played by FA signature in these two highly demanding stages of life cycle was inferred. The results confirm that muscle represents an important fat reservoir, and the FA trophic markers revealed the importance of bacteria as sources of iso and anteiso FA and the strong trophic representation of benthic phytoplankton (diatoms) to larvae muscle FA profile. In early spawning migrants, the significance of marine food web to FA muscle profile is highlighted by the presence of FA signatures characteristics of herbivorous calanoid copepods. Although both life cycle phases studied do not share the same muscle FA signature, there is a part of the profile that is common, which is characterized by FA used in ß-oxidation, such as C18:1ω9 but also by medium chain FA and PUFA which points that PUFA are spared as fuel to ß-oxidation process and probably used to the development of tissues membranes (ammocoetes) and gonadal development and eicosanoid production among others (early spawning migrants). Further studies on FA profile are necessary to elucidate the FA role either during different life stages (ontogeny) or in the distinct habitats frequented (freshwater versus marine) by this diadromous species.

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Influence of body size, metabolic rate and life history stage on the uptake and excretion of the lampricide 3-trifluoromethyl-4-nitrophenol (TFM) by invasive sea lampreys (Petromyzon marinus).

Invasive sea lamprey (Petromyzon marinus) are controlled in the Great Lakes using the lampricide 3-trifluoromethyl-4-nitrophenol (TFM), which is applied to streams infested with larval lamprey. However, lamprey that survive treatments (residuals) remain a challenge because they may subsequently undergo metamorphosis into parasitic juvenile animals that migrate downstream to the Great Lakes, where they feed on important sport and commercial fishes. The goal of this study was to determine if body size and life stage could potentially influence sea lamprey tolerance to TFM by influencing patterns of TFM uptake and elimination. Because mass specific rates of oxygen consumption (M˙O2) are lower in larger compared to smaller lamprey, we predicted that TFM uptake would be negatively correlated to body size, suggesting that large larvae would be more tolerant to TFM exposure. Accordingly, TFM uptake and M˙O2 were measured in larvae ranging in size from 0.2-4.2g using radio-labelled TFM (14C-TFM) and static respirometry. Both were inversely proportional to wet mass (M), and could be described usingthe allometric power relationship: Y=aMb, in which M˙O2=1.86M0.53 and TFM Uptake=7.24M0.34. We also predicted that body size would extend to rates of TFM elimination, which was measured following the administration of 14C-TFM (via intraperitoneal injection). However, there were no differences in the half-lives of elimination of TFM (T 1/2-TFM). There were also no differences in M˙O2 or TFM uptake amongst size-matched larval, metamorphosing (stages 6-7), or post-metamorphic (juvenile) sea lamprey. However, the T1/2-TFM was significantly lower in larval than post-metamorphic lamprey (juvenile), indicating the larval lamprey cleared TFM more efficiently than juvenile lamprey. We conclude that larger larval sea lamprey are more likely to survive TFM treatments suggesting that body size might be an important variable to consider when treating streams with TFM to control these invasive species.

Indirect photodegradation of the lampricides TFM and niclosamide.

3-Trifluromethyl-4-nitrophenol (TFM) and 2',5-dichloro-4'-nitrosalicylanilide (niclosamide) are lampricides used in tributaries of the Great Lakes to kill the invasive parasitic sea lamprey (Petromyzon marinus). Although the lampricides have been applied since the late 1950s, their photochemical behavior in natural environments is still not well understood. This study examines the indirect photodegradation of these two compounds and the resulting yields of organic and inorganic photoproducts in water samples collected from five tributaries of Lake Michigan. The tributaries were selected to span the length of Lake Michigan and its natural carbonate geologic gradient. In the presence of dissolved organic matter (DOM), the niclosamide photodegradation rate triples, while the rate of TFM photodegradation is unchanged. Additionally, the yield of lampricide organic products is influenced by DOM because many of the organic photoproducts themselves are prone to DOM-mediated indirect photodegradation. The indirect photodegradation of niclosamide is primarily mediated by reaction with singlet oxygen, which accounts for more than 50% of the increased photodegradation rate. Additionally, hydroxyl radicals and carbonate radicals (CO3-˙) influence niclosamide indirect photolysis, and their contribution is dependent on the specific river water chemistry. For example, CO3-˙ contribution to niclosamide photodegradation, while small, is greater in southern tributaries where there is higher carbonate alkalinity.

A field analysis of lampricide photodegradation in Great Lakes tributaries.

The lampricides 3-trifluoromethyl-4-nitrophenol (TFM) and 2',5-dichloro-4'-nitrosalicylanilide (niclosamide) are added to Great Lakes tributaries to target the sea lamprey, an invasive parasitic fish. This study examines the photochemical behavior of the lampricides in Carpenter Creek, Sullivan Creek, and the Manistique River. The observed loss of TFM in Carpenter and Sullivan Creeks (i.e., 34 and 19%) was similar to the loss of bromide in parallel time of passage studies (i.e., 30 and 29%), demonstrating that TFM photodegradation was minimal in both tributaries during the lampricide application. Furthermore, the absence of inorganic and organic photoproducts in the Manistique River demonstrates that TFM and niclosamide photodegradation was minimal in this large tributary, despite its long residence time (i.e., 3.3 days). Kinetic modeling was used to identify environmental variables primarily responsible for the limited photodegradation of TFM in the field compared to estimates from laboratory data. This analysis demonstrates that the lack of TFM photodegradation was attributable to the short residence times in Carpenter and Sullivan Creeks, while depth, time of year, time of day, and cloud cover influenced photochemical fate in the Manistique River. The modeling approach was extended to assess how many of the 140 United States tributaries treated with lampricides in 2015 and 2016 were amenable to TFM photolysis. While >50% removal of TFM due to photolysis could occur in 13 long and shallow tributaries, in most systems lampricides will reach the Great Lakes untransformed.

Morphological and functional aspects of the epidermis of the sea lamprey Petromyzon marinus throughout development.

The development of the epidermis of sea lamprey Petromyzon marinus along the whole life cycle was studied using conventional staining techniques and lectin histochemistry. The epidermis undergoes variations in morphology and thickness throughout development. The simple cuboidal epithelium found in the epidermis of prolarvae becomes stratified cubic in the adult by increasing the number of cell layers. The cuticle thickness undergoes a steady increase during the larval period. There are changes in the glycoconjugate composition of the three main cell types of the P. marinus epidermis, mucous, granular and skein cells, which are more pronounced after metamorphosis. The Alcian blue-periodic acid Schiff (AB-PAS) histochemical method shows the presence of both acidic and neutral glycoconjugates in the mucous cells, indicating their secretory function. Moreover, lectin analysis reveals a mucous secretion containing glycoconjugates such as sulphated glycosaminoglycans (N-acetylglucosamine and N-acetylgalactosamine) and N-glycoproteins rich in mannose. Although granular cells are AB-PAS negative, they exhibit a similar glycoconjugate composition to the mucous cells. Moreover, granular cells show sialic acid positivity in larvae but this monosaccharide residue is not detected after metamorphosis. The skein cells, a unique cell of lampreys, are negative to AB-PAS staining but they mostly contain l-fucose and sialic acid residues, which also disappear after metamorphosis. The function of the granular and skein cells is still unknown but the role of their glycoconjugate composition is discussed. In addition, a different cellular origin is suggested for these two types of cells.

DNA methylation in a sea lamprey vasotocin receptor gene promoter correlates with tissue- and life-stage-specific mRNA expression.

The jawless vertebrate sea lamprey (Petromyzon marinus) genome has a different structure from both invertebrates and jawed vertebrates featuring high guanine-cytosine (GC) content. This raises the question of whether DNA methylation of cytosine-guanine (CpG) dinucleotides could function to regulate lamprey gene transcription. We previously characterized a lamprey arginine vasotocin (AVT) receptor gene (Pm807) possessing characteristics of both arginine vasopressin (AVP) V1A and oxytocin (OXT) receptor genes of jawed vertebrates. Lamprey Pm807 mRNA is highly expressed in adult heart and larval liver but not expressed in adult liver. Using high-resolution melt (HRM) PCR on bisulfite-converted DNA, we pinpointed a region with tissue-specific differences in DNA melt characteristics, indicating differences in methylation level. Sequencing revealed a pattern of methylation at specific CpGs at consistently higher levels in adult heart and larval liver than adult liver. These CpGs are associated with putative transcription factor binding sequences organized similarly to functional OXTR promoters in mammals, suggesting functional similarity in lamprey gene transcription regulation.

Evolution of the vertebrate claudin gene family: insights from a basal vertebrate, the sea lamprey.

Claudins are major constituents of tight junctions, contributing both to their intercellular sealing and selective permeability properties. While claudins and claudin-like molecules are present in some invertebrates, the association of claudins with tight junctions has been conclusively documented only in vertebrates. Here we report the sequencing, phylogenetic analysis and comprehensive spatiotemporal expression analysis of the entire claudin gene family in the basal extant vertebrate, the sea lamprey. Our results demonstrate that clear orthologues to about half of all mammalian claudins are present in the lamprey, suggesting that at least one round of whole genome duplication contributed to the diversification of this gene family. Expression analysis revealed that claudins are expressed in discrete and specific domains, many of which represent vertebrate-specific innovations, such as in cranial ectodermal placodes and the neural crest; whereas others represent structures characteristic of chordates, e.g. pronephros, notochord, somites, endostyle and pharyngeal arches. By comparing the embryonic expression of claudins in the lamprey to that of other vertebrates, we found that ancestral expression patterns were often preserved in higher vertebrates. Morpholino mediated loss of Cldn3b demonstrated a functional role for this protein in placode and pharyngeal arch morphogenesis. Taken together, our data provide novel insights into the origins and evolution of the claudin gene family and the significance of claudin proteins in the evolution of vertebrates.

Convergent evolution of hemoglobin switching in jawed and jawless vertebrates.

BACKGROUND: During development, humans and other jawed vertebrates (Gnathostomata) express distinct hemoglobin genes, resulting in different hemoglobin tetramers. Embryonic and fetal hemoglobin have higher oxygen affinities than the adult hemoglobin, sustaining the oxygen demand of the developing organism. Little is known about the expression of hemoglobins during development of jawless vertebrates (Agnatha). RESULTS: We identified three hemoglobin switches in the life cycle of the sea lamprey. Three hemoglobin genes are specifically expressed in the embryo, four genes in the filter feeding larva (ammocoete), and nine genes correspond to the adult hemoglobin chains. During the development from the parasitic to the reproductive adult, the composition of hemoglobin changes again, with a massive increase of chain aHb1. A single hemoglobin chain is expressed constitutively in all stages. We further showed the differential expression of other globin genes: Myoglobin 1 is most highly expressed in the reproductive adult, myoglobin 2 expression peaks in the larva. Globin X1 is restricted to the embryo; globin X2 was only found in the reproductive adult. Cytoglobin is expressed at low levels throughout the life cycle. CONCLUSION: Because the hemoglobins of jawed and jawless vertebrates evolved independently from a common globin ancestor, hemoglobin switching must also have evolved convergently in these taxa. Notably, the ontogeny of sea lamprey hemoglobins essentially recapitulates their phylogeny, with the embryonic hemoglobins emerging first, followed by the evolution of larval and adult hemoglobins.

Exposure to the lampricide 3-trifluoromethyl-4-nitrophenol results in increased expression of carbohydrate transporters in Saccharomyces cerevisiae.

The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) is used to control sea lamprey (Petromyzon marinus) populations in freshwater lakes. Although TFM can have sublethal and lethal effects, little is known about gene expression changes with TFM exposure. Microarray analysis was used to determine differential gene expression over 4 h of exposure in Saccharomyces cerevisiae. Among the most significantly up-regulated genes were regulators of carbohydrate transport, including HXT1, HXT3, HXT4, IMA5, MIG2, and YKR075C. Environ Toxicol Chem 2016;35:1727-1732. © 2015 SETAC.

Neuropeptide Y family receptors Y1 and Y2 from sea lamprey, Petromyzon marinus.

The vertebrate gene family for neuropeptide Y (NPY) receptors expanded by duplication of the chromosome carrying the ancestral Y1-Y2-Y5 gene triplet. After loss of some duplicates, the ancestral jawed vertebrate had seven receptor subtypes forming the Y1 (including Y1, Y4, Y6, Y8), Y2 (including Y2, Y7) and Y5 (only Y5) subfamilies. Lampreys are considered to have experienced the same chromosome duplications as gnathostomes and should also be expected to have multiple receptor genes. However, previously only a Y4-like and a Y5 receptor have been cloned and characterized. Here we report the cloning and characterization of two additional receptors from the sea lamprey Petromyzon marinus. Sequence phylogeny alone could not with certainty assign their identity, but based on synteny comparisons of P. marinus and the Arctic lamprey, Lethenteron camtschaticum, with jawed vertebrates, the two receptors most likely are Y1 and Y2. Both receptors were expressed in human HEK293 cells and inositol phosphate assays were performed to determine the response to the three native lamprey peptides NPY, PYY and PMY. The three peptides have similar potencies in the nanomolar range for Y1. No obvious response to the three peptides was detected for Y2. Synteny analysis supports identification of the previously cloned receptor as Y4. No additional NPY receptor genes could be identified in the presently available lamprey genome assemblies. Thus, four NPY-family receptors have been identified in lampreys, orthologs of the same subtypes as in humans (Y1, Y2, Y4 and Y5), whereas many other vertebrate lineages have retained additional ancestral subtypes.

Does DNA methylation regulate metamorphosis? The case of the sea lamprey (Petromyzon marinus) as an example.

Lampreys represent one of the most ancient vertebrate lineages enclosing a special interest for genetic and epigenetic studies. The sea lamprey (Petromyzon marinus) is an anadromous species that experiences metamorphosis all the way up to the adult stage. Although representing a gradual process, metamorphosis in this species involves dramatic conversions with regard to physiological together with structural body changes preparing individuals for a marine and parasitic life; in consequence, multiple gene expression modifications are expected. The implications of thyroid hormones and HOX gene expression changes have previously been reported in this species and also in other vertebrate species. Nonetheless, information lacks on how these genes are regulated in lampreys. We here report about the existence of methylation pattern differences between the adult and the larvae sea lamprey life cycle stages making use of the Methylation-Sensitive Amplified Polymorphism (MSAP) technique. Differentially methylated fragment sequencing allowed to establish homologous identities with HOX genes involved in morphogenesis, along with genes related to the water balance and to the osmotic homoeostasis, all associated to a marine environment adaptation. These results provide evidences revealing that DNA methylation plays a role in the epigenetic regulation of the P. marinus post-natal development representing a starting point for future studies. To the best of our knowledge, this is the first study which detects DNA methylation changes associated with metamorphosis in lampreys.

Iso-petromyroxols: novel dihydroxylated tetrahydrofuran enantiomers from sea lamprey (Petromyzon marinus).

An enantiomeric pair of new fatty acid-derived hydroxylated tetrahydrofurans, here named iso-petromyroxols, were isolated from sea lamprey larvae-conditioned water. The relative configuration of iso-petromyroxol was elucidated with 1D and 2D NMR spectroscopic analyses. The ratio of enantiomers (er) in the natural sample was measured by chiral-HPLC-MS/MS to be ca. 3:1 of (-)- to (+)-antipodes.

Evidence that progestins play an important role in spermiation and pheromone production in male sea lamprey (Petromyzon marinus).

Progestins (progestogens, C21 steroids) have been shown to regulate key physiological activities for reproduction in both sexes in all classes of vertebrates except for Agnathans. Progesterone (P) and 15α-hydroxyprogesterone (15α-P) have been detected in sea lamprey (Petromyzon marinus) plasma, but the expression patterns and functions of putative progestin receptor genes have not yet been investigated. The first objective of this study was to determine the differences in mRNA expression levels of nuclear progestin receptor (nPR) and the membrane receptor adaptor protein 'progesterone receptor membrane component 1' (pgrmc1) in putative target tissues in males at different life stages, with and without lamprey GnRH-I and -III treatment. The second objective was to demonstrate the function of progestins by implanting prespermiating males (PSM) with time-release pellets of P and measuring the latency to the onset of spermiation and plasma concentrations of sex pheromones and steroids. The third objective was to measure the binding affinity of P in the nuclear and membrane fractions of the target tissues. Expression levels of nPR and pgrmc1 differed between life stages and tissues, and in some cases were differentially responsive to lamprey GnRH-I and -III. Increases in nPR and pgrmc1 gene expressions were correlated to the late stages of sexual maturation in males. The highest expression levels of these genes were found in the liver and gill of spermiating males. These organs are, respectively, the site of production and release of the sex pheromone 3 keto-petromyzonol sulfate (3kPZS). The hypothesis that pheromone production may be under hormonal control was tested in vivo by implanting PSM with time-release pellets of P. Concentrations of 3kPZS in plasma after 1week were 50-fold higher than in controls or in males that had been implanted with androstenedione, supporting the hypothesis that P is responsible for regulating the production of the sex pheromone. P treatment also accelerated the onset of spermiation. Saturation and Scatchard analyses of the target tissues showed that both nuclear and membrane fractions bound P with high affinity and low capacity (KD 0.53pmol/g testis and 0.22 pmol/g testis, and Bmax 1.8 and 5.7 nM, respectively), similar to the characteristics of nPR and mPR in other fish. The fact that a high proportion of P was also converted in vivo to 15α-P means that it is not yet possible to determine which of these two steroids is the natural ligand in the sea lamprey.

A new clarification method to visualize biliary degeneration during liver metamorphosis in Sea Lamprey (Petromyzon marinus).

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called "CLARITY" was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

Glutamatergic neuronal populations in the brainstem of the sea lamprey, Petromyzon marinus: an in situ hybridization and immunocytochemical study.

Glutamate is the major excitatory neurotransmitter in vertebrates, and glutamatergic cells probably represent a majority of neurons in the brain. Physiological studies have demonstrated a wide presence of excitatory (glutamatergic) neurons in lampreys. The present in situ hybridization study with probes for the lamprey vesicular glutamate transporter (VGLUT) provides an anatomical basis for the general distribution and precise localization of glutamatergic neurons in the sea lamprey brainstem. Most glutamatergic neurons were found within the periventricular gray layer throughout the brainstem, with the following regions being of particular interest: the optic tectum, torus semicircularis, isthmus, dorsal and medial nuclei of the octavolateral area, dorsal column nucleus, solitary tract nucleus, motoneurons, and reticular formation. The reticular population revealed a high degree of cellular heterogeneity including small, medium-sized, large, and giant glutamatergic neurons. We also combined glutamate immunohistochemistry with neuronal tract-tracing methods or γ-aminobutyric acid (GABA) immunohistochemistry to better characterize the glutamatergic populations. Injection of Neurobiotin into the spinal cord revealed that retrogradely labeled small and medium-sized cells of some reticulospinal-projecting groups were often glutamate-immunoreactive, mostly in the hindbrain. In contrast, the large and giant glutamatergic reticulospinal perikarya mostly lacked glutamate immunoreactivity. These results indicate that glutamate immunoreactivity did not reveal the entire set of glutamatergic populations. Some spinal-projecting octaval populations lacked both VGLUT and glutamate. As regards GABA and glutamate, their distribution was largely complementary, but colocalization of glutamate and GABA was observed in some small neurons, suggesting that glutamate immunohistochemistry might also detect non-glutamatergic cells or neurons that co-release both GABA and glutamate.