Non-ammocoete larvae of Palaeozoic stem lampreys.

Ammocoetes-the filter-feeding larvae of modern lampreys-have long influenced hypotheses of vertebrate ancestry1-7. The life history of modern lampreys, which develop from a superficially amphioxus-like ammocoete to a specialized predatory adult, appears to recapitulate widely accepted scenarios of vertebrate origin. However, no direct evidence has validated the evolutionary antiquity of ammocoetes, and their status as models of primitive vertebrate anatomy is uncertain. Here we report larval and juvenile forms of four stem lampreys from the Palaeozoic era (Hardistiella, Mayomyzon, Pipiscius, and Priscomyzon), including a hatchling-to-adult growth series of the genus Priscomyzon from Late Devonian Gondwana. Larvae of all four genera lack the defining traits of ammocoetes. They instead display features that are otherwise unique to adult modern lampreys, including prominent eyes, a cusped feeding apparatus, and posteriorly united branchial baskets. Notably, phylogenetic analyses find that these non-ammocoete larvae occur in at least three independent lineages of stem lamprey. This distribution strongly implies that ammocoetes are specializations of modern-lamprey life history rather than relics of vertebrate ancestry. These phylogenetic insights also suggest that the last common ancestor of hagfishes and lampreys was a macrophagous predator that did not have a filter-feeding larval phase. Thus, the armoured 'ostracoderms' that populate the cyclostome and gnathostome stems might serve as better proxies than living cyclostomes for the last common ancestor of all living vertebrates.

Evidence from cyclostomes for complex regionalization of the ancestral vertebrate brain.

The vertebrate brain is highly complex, but its evolutionary origin remains elusive. Because of the absence of certain developmental domains generally marked by the expression of regulatory genes, the embryonic brain of the lamprey, a jawless vertebrate, had been regarded as representing a less complex, ancestral state of the vertebrate brain. Specifically, the absence of a Hedgehog- and Nkx2.1-positive domain in the lamprey subpallium was thought to be similar to mouse mutants in which the suppression of Nkx2-1 leads to a loss of the medial ganglionic eminence. Here we show that the brain of the inshore hagfish (Eptatretus burgeri), another cyclostome group, develops domains equivalent to the medial ganglionic eminence and rhombic lip, resembling the gnathostome brain. Moreover, further investigation of lamprey larvae revealed that these domains are also present, ruling out the possibility of convergent evolution between hagfish and gnathostomes. Thus, brain regionalization as seen in crown gnathostomes is not an evolutionary innovation of this group, but dates back to the latest vertebrate ancestor before the divergence of cyclostomes and gnathostomes more than 500 million years ago.

Immunohistochemical Characterization of the Development of Long Photoreceptor Cells in the Lamprey Retina.

The adult lamprey retina has two types of photoreceptor cells, short and long photoreceptor cells, which are equivalent to rods and cones of other vertebrates. In contrast, the retina of lamprey larvae only contains a single type of photoreceptor cell, which appears to correspond to the short photoreceptor cell. However, the developmental pattern of the long photoreceptor cell is unknown. Previously, we reported that antibodies against rhodopsin and iodopsin (the chicken red cone opsin) could discriminate between the outer segments of short and long photoreceptor cells, respectively, in the retina of adult Japanese river lamprey (Lethenteron camtschaticum). Here, we immunohistochemically investigate the appearance of long photoreceptor cells in the larval and adult retinas of the Far Eastern brook lamprey (Lethenteron reissneri), which is a close relative of the Japanese river lamprey, by using anti-iodopsin antibody. We found that iodopsin immunoreactivity was localized not only in the adult retina but also in the larval retina. Moreover, we examined the immunohistochemical localization of signal transduction molecules, such as transducin and arrestin, in the iodopsin-immunoreactive cells of the larval retina. The iodopsin-immunoreactive cells also contained both transducin and arrestin, suggesting that long photoreceptor cells are already functional in the larval stage before the acquisition of visual function. Our results suggest that the iodopsin-immunoreactive cells may be related to not only cone vision in the adult but also photoreception in the larval lamprey.

Novel developmental bases for the evolution of hypobranchial muscles in vertebrates.

BACKGROUND: Vertebrates are characterized by possession of hypobranchial muscles (HBMs). Cyclostomes, or modern jawless vertebrates, possess a rudimentary and superficial HBM lateral to the pharynx, whereas the HBM in jawed vertebrates is internalized and anteroposteriorly specified. Precursor cells of the HBM, marked by expression of Lbx1, originate from somites and undergo extensive migration before becoming innervated by the hypoglossal nerve. How the complex form of HBM arose in evolution is relevant to the establishment of the vertebrate body plan, but despite having long been assumed to be similar to that of limb muscles, modification of developmental mechanisms of HBM remains enigmatic. RESULTS: Here we characterize the expression of Lbx genes in lamprey and hagfish (cyclostomes) and catshark (gnathostome; jawed vertebrates). We show that the expression patterns of the single cyclostome Lbx homologue, Lbx-A, do not resemble the somitic expression of mammalian Lbx1. Disruption of Lbx-A revealed that LjLbx-A is required for the formation of both HBM and body wall muscles, likely due to the insufficient extension of precursor cells rather than to hindered muscle differentiation. Both homologues of Lbx in the catshark were expressed in the somitic muscle primordia, unlike in amniotes. During catshark embryogenesis, Lbx2 is expressed in the caudal HBM as well as in the abdominal rectus muscle, similar to lamprey Lbx-A, whereas Lbx1 marks the rostral HBM and pectoral fin muscle. CONCLUSIONS: We conclude that the vertebrate HBM primarily emerged as a specialized somatic muscle to cover the pharynx, and the anterior internalized HBM of the gnathostomes is likely a novelty added rostral to the cyclostome-like HBM, for which duplication and functionalization of Lbx genes would have been a prerequisite.

Neural crest and placode roles in formation and patterning of cranial sensory ganglia in lamprey.

Vertebrates possess paired cranial sensory ganglia derived from two embryonic cell populations, neural crest and placodes. Cranial sensory ganglia arose prior to the divergence of jawed and jawless vertebrates, but the developmental mechanisms that facilitated their evolution are unknown. Using gene expression and cell lineage tracing experiments in embryos of the sea lamprey, Petromyzon marinus, we find that in the cranial ganglia we targeted, development consists of placode-derived neuron clusters in the core of ganglia, with neural crest cells mostly surrounding these neuronal clusters. To dissect functional roles of neural crest and placode cell associations in these developing cranial ganglia, we used CRISPR/Cas9 gene editing experiments to target genes critical for the development of each population. Genetic ablation of SoxE2 and FoxD-A in neural crest cells resulted in differentiated cranial sensory neurons with abnormal morphologies, whereas deletion of DlxB in cranial placodes resulted in near-total loss of cranial sensory neurons. Taken together, our cell-lineage, gene expression, and gene editing results suggest that cranial neural crest cells may not be required for cranial ganglia specification but are essential for shaping the morphology of these sensory structures. We propose that the association of neural crest and placodes in the head of early vertebrates was a key step in the organization of neurons and glia into paired sensory ganglia.

Lamprey VLRB participates in pathogen detection, VLRB/L-BLNK/L-NF-κB (B-like cells) signal transduction, and development.

In jawed vertebrates, B cell receptors (BCR) are primary pathogen detectors that activate downstream signaling pathways to express adaptive immune effectors. In jawless vertebrates, the variable lymphocyte receptors (VLR) B positive lymphocytes can express and secrete specific VLRB molecules in an analogous manner to that of immunoglobulins by B cells in jawed vertebrates. Our study is the first to demonstrate the possibility of incubation of fertilized eggs and artificial breeding of Lampetra morii larvae throughout their life cycle under laboratory condition. We also found that VLRB, lamprey B-cell linker (L-BLNK), and lamprey nuclear factor-kappa B (L-NF-κB) play key roles in early larval development. Aeromonas hydrophila was found to be a lethal pathogen of L. morii larvae causing rapid infection at a concentration of 107 cfu/mL qRT-PCR results revealed that gene expression levels of VLRB, L-BLNK, and L-NF-κB were up-regulated significantly. Ten-day infection trials showed that VLRB, L-BLNK, and L-NF-κB are crucial for lamprey immune response. Furthermore, the expression levels of L-BLNK and L-NF-κB were down-regulated drastically both at mRNA and protein levels after bacterial infection than in the naive group of VLRB morphants. A similar expression pattern of VLRB and L-BLNK was found in L-NF-κB morphants post bacterial infection. The results were strikingly different in the other two morphants. The VLRB and L-NF-κB expression levels were found to be down-regulated at mRNA and protein levels by less than 30% and 45%, respectively, in L-BLNK morphants compared to those in the naive group. These results indicate that L-BLNK and L-NF-κB might participate in VLRB-mediated immune response. Additionally, in VLRB morphants, the mRNA expression levels of some genes, especially the ones expressed in VLRB+ lymphocytes but not in VLRA+ lymphocytes, were found to be affected. Therefore, these findings of B-like lymphocytes in lamprey offer key evidence with regard to the evolution of adaptive immunity.

Predicting larval metamorphosis of Pacific lamprey Entosphenus tridentatus through measurements of length and mass.

The utility of length and mass measurements to predict the larval metamorphosis of Pacific lamprey Entosphenus tridentatus was evaluated. During 2004-2008, larval E. tridentatus were collected from Cedar Creek (Washington, USA) in either the spring or autumn, measured for total length and total mass, reared in captivity and monitored for metamorphosis. The minimum total length, total mass and condition factor of larvae that were observed to go through metamorphosis were 102 mm, 2.0 g and 1.52, respectively. Logistic models indicated that total length and condition factor in both spring and autumn were the most significant variables for predicting metamorphosis of Pacific lamprey during the subsequent summer. Mass in the autumn also appeared important to predict whether metamorphosis occurred in the subsequent summer. Collectively, all models using specific minimums of total length, total mass or condition factor of larvae as criteria for them to metamorphose were sometimes (5 of 14 cases) able to predict the percentage of larvae that would metamorphose but rarely (1 of 12 cases) able to predict which individual larvae would metamorphose. Similar to other anadromous species of lampreys, the size and condition of larval E. tridentatus have utility for predicting metamorphic fate.

Thrust generation during steady swimming and acceleration from rest in anguilliform swimmers.

Escape swimming is a crucial behavior by which undulatory swimmers evade potential threats. The hydrodynamics of escape swimming have not been well studied, particularly for anguilliform swimmers, such as the sea lamprey Petromyzon marinus For this study, we compared the kinematics and hydrodynamics of larval sea lampreys with those of lampreys accelerating from rest during escape swimming. We used experimentally derived velocity fields to calculate pressure fields and distributions of thrust and drag along the body. Lampreys initiated acceleration from rest with the formation of a high-amplitude body bend at approximately one-quarter body length posterior to the head. This deep body bend produced two high-pressure regions from which the majority of thrust for acceleration was derived. In contrast, steady swimming was characterized by shallower body bends and negative-pressure-derived thrust, which was strongest near the tail. The distinct mechanisms used for steady swimming and acceleration from rest may reflect the differing demands of the two behaviors. High-pressure-based mechanisms, such as the one used for acceleration from rest, could also be important for low-speed maneuvering during which drag-based turning mechanisms are less effective. The design of swimming robots may benefit from the incorporation of such insights from unsteady swimming.

Regulatory evolution of Tbx5 and the origin of paired appendages.

The diversification of paired appendages has been a major factor in the evolutionary radiation of vertebrates. Despite its importance, an understanding of the origin of paired appendages has remained elusive. To address this problem, we focused on T-box transcription factor 5 (Tbx5), a gene indispensable for pectoral appendage initiation and development. Comparison of gene expression in jawless and jawed vertebrates reveals that the Tbx5 expression in jawed vertebrates is derived in having an expression domain that extends caudal to the heart and gills. Chromatin profiling, phylogenetic footprinting, and functional assays enabled the identification of a Tbx5 fin enhancer associated with this apomorphic pattern of expression. Comparative functional analysis of reporter constructs reveals that this enhancer activity is evolutionarily conserved among jawed vertebrates and is able to rescue the finless phenotype of tbx5a mutant zebrafish. Taking paleontological evidence of early vertebrates into account, our results suggest that the gain of apomorphic patterns of Tbx5 expression and regulation likely contributed to the morphological transition from a finless to finned condition at the base of the vertebrate lineage.

Cloning and characterization of a second lamprey pituitary glycoprotein hormone, thyrostimulin (GpA2/GpB5).

A novel heterodimeric glycoprotein hormone (GpH) comprised of alpha (GpA2) and beta (GpB5) subunits was discovered in 2002 and called thyrostimulin for its ability to activate the TSH receptor in mammals, but its central function in vertebrates has not been firmly established. We report here the cloning and expression of lamprey (l)GpB5, and its ability to heterodimerize with lGpA2 to form a functional l-thyrostimulin. The full-length cDNA of lGpB5 encodes 174 amino acids with ten conserved cysteine residues and one glycosylation site that is conserved with other vertebrate GpB5 sequences. Phylogenetic and synteny analyses support that lGpB5 belongs to the vertebrate GpB5 clade. Heterodimerization of lGpB5 and lGpA2 was shown by nickel pull-down of histidine-tagged recombinant subunits. RNA transcripts of lGpB5 were detected in the pituitary of lampreys during both parasitic and adult life stages. Intraperitoneal injection with lGnRH-III (100 µg/kg) increased pituitary lGpA2, lGpB5, and lGpHß mRNA expression in sexually mature, adult female lampreys. A recombinant l-thyrostimulin produced by expression of a fusion gene in Pichia pastoris activated lamprey GpH receptors I and II as measured by cAMP enzymeimmunoassay. In contrast to jawed vertebrates that have pituitary LH, FSH, and TSH, our data support that lampreys only have two functional pituitary GpHs, lGpH and l-thyrostimulin, which consist of lGpA2 and unique beta subunits. It is hypothesized that lGpH and l-thyrostimulin differentially regulate reproductive and thyroid activities in some unknown way(s) in lampreys.

Genetic variation and population structure among larval Lethenteron spp. within the Yukon River drainage, Alaska.

The absence of information on genetic variation and population structure of lampreys Lethenteron spp. in the eastern part of their distribution limits our understanding of the migration ecology and spatial population genetic structure of the species. We examined genetic variation within and among three aggregations of Lethenteron spp. larvae in the Yukon River drainage, Alaska, using microsatellite genotypes. A total of 120 larval lampreys were genotyped at eight microsatellite loci. Global FST was 0.053 (95% CI 0.021-0.086), while pairwise FST values ranged from 0.048-0.057. Model-based Bayesian clustering analyses with sample locality priors (LOCPRIOR) identified three distinct, but admixed, genetic clusters that corresponded with the three aggregations. Estimates of contemporary gene flow indicate substantial reciprocal migration among sites consistent with no or low-fidelity natal homing. These results are largely in agreement with previous reports of historic and contemporary gene flow among Lethenteron spp. in other parts of their geographic distribution.

The structure, splicing, synteny and expression of lamprey COE genes and the evolution of the COE gene family in chordates.

COE genes encode transcription factors that have been found in all metazoans examined to date. They possess a distinctive domain structure that includes a DNA-binding domain (DBD), an IPT/TIG domain and a helix-loop-helix (HLH) domain. An intriguing feature of the COE HLH domain is that in jawed vertebrates it is composed of three helices, compared to two in invertebrates. We report the isolation and expression of two COE genes from the brook lamprey Lampetra planeri and compare these to COE genes from the lampreys Lethenteron japonicum and Petromyzon marinus. Molecular phylogenetic analyses do not resolve the relationship of lamprey COE genes to jawed vertebrate paralogues, though synteny mapping shows that they all derive from duplication of a common ancestral genomic region. All lamprey genes encode conserved DBD, IPT/TIG and HLH domains; however, the HLH domain of lamprey COE-A genes encodes only two helices while COE-B encodes three helices. We also identified COE-B splice variants encoding either two or three helices in the HLH domain, along with other COE-A and COE-B splice variants affecting the DBD and C-terminal transactivation regions. In situ hybridisation revealed expression in the lamprey nervous system including the brain, spinal cord and cranial sensory ganglia. We also detected expression of both genes in mesenchyme in the pharyngeal arches and underlying the notochord. This allows us to establish the primitive vertebrate expression pattern for COE genes and compare this to that of invertebrate chordates and other animals to develop a model for COE gene evolution in chordates.

Discovery of fossil lamprey larva from the Lower Cretaceous reveals its three-phased life cycle.

Lampreys are one of the two surviving jawless vertebrate groups and one of a few vertebrate groups with the best exemplified metamorphosis during their life cycle, which consists of a long-lasting larval stage, a peculiar metamorphosis, and a relatively short adulthood with a markedly different anatomy. Although the fossil records have revealed that many general features of extant lamprey adults were already formed by the Late Devonian (ca. 360 Ma), little is known about the life cycle of the fossil lampreys because of the lack of fossilized lamprey larvae or transformers. Here we report the first to our knowledge discovery of exceptionally preserved premetamorphic and metamorphosing larvae of the fossil lamprey Mesomyzon mengae from the Lower Cretaceous of Inner Mongolia, China. These fossil ammocoetes look surprisingly modern in having an eel-like body with tiny eyes, oral hood and lower lip, anteriorly positioned branchial region, and a continuous dorsal skin fin fold and in sharing a similar feeding habit, as judged from the detritus left in the gut. In contrast, the larger metamorphosing individuals have slightly enlarged eyes relative to large otic capsules, thickened oral hood or pointed snout, and discernable radials but still anteriorly extended branchial area and lack a suctorial oral disk, which characterize the early stages of the metamorphosis of extant lampreys. Our discovery not only documents the larval conditions of fossil lampreys but also indicates the three-phased life cycle in lampreys emerged essentially in their present mode no later than the Early Cretaceous.

Using stable isotopes and C:N ratios to examine the life-history strategies and nutritional sources of larval lampreys.

Natural abundance stable-isotope analysis (δ(13)C and δ(15)N) and C:N ratios were used to study the ammocoete phase of two common non-parasitic lamprey species (least brook lamprey Lampetra aepyptera and American brook lamprey Lethenteron appendix) in two tributaries of the Ohio River (U.S.A.). The C:N ratios suggest that each species employs different lipid accumulation strategies to support its metamorphosis and recruitment into an adult animal. Ammocoete δ(13)C values generally increased with increasing C:N values. In contrast to δ(13)C, ammocoete δ(15)N values were weakly related to the total length (LT) in L. aepyptera, but positively correlated to both LT and C:N ratios in L. appendix. In L. appendix, C:N also correlated positively with LT, and presumably age. A Bayesian mixing model using δ(13)C and δ(15)N was used to estimate nutritional subsidies of different potential food resources to ammocoetes at each site. The models suggested that although nutritional subsidies to ammocoetes varied as a function of site, ammocoetes were generally reliant on large contributions (42-62% at three sites) from aquatic plants. Contributions from aquatic sediment organic matter were also important at all sites (32-63%) for ammocoetes, with terrestrially derived plant materials contributing smaller amounts (4-33%). These findings provide important insights into the feeding ecology and nutrition of two species of lampreys. They also suggest that similar and other quantitative approaches are required to (1) fully understand how the observed stable-isotopes ratios are established in ammocoetes and (2) better assess ammocoete nutritional subsidies in different natal streams.

Evolutionary crossroads in developmental biology: cyclostomes (lamprey and hagfish).

Lampreys and hagfish, which together are known as the cyclostomes or 'agnathans', are the only surviving lineages of jawless fish. They diverged early in vertebrate evolution, before the origin of the hinged jaws that are characteristic of gnathostome (jawed) vertebrates and before the evolution of paired appendages. However, they do share numerous characteristics with jawed vertebrates. Studies of cyclostome development can thus help us to understand when, and how, key aspects of the vertebrate body evolved. Here, we summarise the development of cyclostomes, highlighting the key species studied and experimental methods available. We then discuss how studies of cyclostomes have provided important insight into the evolution of fins, jaws, skeleton and neural crest.

The lamprey: a jawless vertebrate model system for examining origin of the neural crest and other vertebrate traits.

Lampreys are a group of jawless fishes that serve as an important point of comparison for studies of vertebrate evolution. Lampreys and hagfishes are agnathan fishes, the cyclostomes, which sit at a crucial phylogenetic position as the only living sister group of the jawed vertebrates. Comparisons between cyclostomes and jawed vertebrates can help identify shared derived (i.e. synapomorphic) traits that might have been inherited from ancestral early vertebrates, if unlikely to have arisen convergently by chance. One example of a uniquely vertebrate trait is the neural crest, an embryonic tissue that produces many cell types crucial to vertebrate features, such as the craniofacial skeleton, pigmentation of the skin, and much of the peripheral nervous system (Gans and Northcutt, 1983). Invertebrate chordates arguably lack unambiguous neural crest homologs, yet have cells with some similarities, making comparisons with lampreys and jawed vertebrates essential for inferring characteristics of development in early vertebrates, and how they may have evolved from nonvertebrate chordates. Here we review recent research on cyclostome neural crest development, including research on lamprey gene regulatory networks and differentiated neural crest fates.

[Life Forms of Lampreys (Petromyzontidae) as a Manifestation of Intraspecific Diversity of Ontogenesis].

Some lamprey genera include the forms that have significantly different life cycles (the most well-known are the "anadromous" and "resident," or the "parasitic" and "nonparasitic" forms). The analysis of data on the genetic characteristics of these forms shows that, in some cases, the nonparasitic lampreys independently derived from the parasitic ones in different aqueous systems. These data, together with the results of morphological analysis as well as data on the distribution, ecology, hybridization, and observations ofjoint spawning of parasitic and nonparasitic forms show that these forms belong to the same species (except the resident forms that were isolated long ago).

Gene expression during ovarian differentiation in parasitic and non-parasitic lampreys: implications for fecundity and life history types.

Lampreys diverged from the jawed vertebrate lineage approximately 500million years ago. Lampreys undergo sex differentiation much later than most other vertebrates, and ovarian differentiation occurs several years before testicular differentiation. The genetic basis of lamprey sex differentiation is of particular interest both because of the phylogenetic importance of lampreys and because of their unusual pattern of sex differentiation. As well, differences between parasitic and non-parasitic lampreys may first become evident at ovarian differentiation. However, nothing is known about the genetic basis of ovarian differentiation in lampreys. This study examined potential differences in gene expression before, during, and after ovarian differentiation in parasitic chestnut lamprey Ichthyomyzon castaneus and non-parasitic northern brook lamprey Ichthyomyzonfossor. Eight target genes (17ß-hydroxysteroid dehydrogenase, germ cell-less, estrogen receptor ß, insulin-like growth factor 1 receptor, daz-associated protein 1, cytochrome c oxidase subunit III, Wilms' tumour suppressor protein 1, and dehydrocholesterol reductase 7) were examined. Northern brook lamprey displayed higher expression of cytochrome c oxidase subunit III, whereas chestnut lamprey displayed higher expression of insulin-like growth factor 1 receptor; these genes may be involved in apoptosis and oocyte growth, respectively. Presumptive male larvae had higher expression of Wilms' tumour suppressor protein 1, which may be involved in the undifferentiated gonad and/or later testicular development. Differentiated females had higher expression of 17ß hydroxysteroid dehydrogenase and daz-associated protein 1, which may be involved in female development. This study is the first to identify genes that may be involved in ovarian differentiation and fecundity in lampreys.

New perspectives on pharyngeal dorsoventral patterning in development and evolution of the vertebrate jaw.

Patterning of the vertebrate facial skeleton involves the progressive partitioning of neural-crest-derived skeletal precursors into distinct subpopulations along the anteroposterior (AP) and dorsoventral (DV) axes. Recent evidence suggests that complex interactions between multiple signaling pathways, in particular Endothelin-1 (Edn1), Bone Morphogenetic Protein (BMP), and Jagged-Notch, are needed to pattern skeletal precursors along the DV axis. Rather than directly determining the morphology of individual skeletal elements, these signals appear to act through several families of transcription factors, including Dlx, Msx, and Hand, to establish dynamic zones of skeletal differentiation. Provocatively, this patterning mechanism is largely conserved from mouse and zebrafish to the jawless vertebrate, lamprey. This implies that the diversification of the vertebrate facial skeleton, including the evolution of the jaw, was driven largely by modifications downstream of a conversed pharyngeal DV patterning program.