Organization of the body wall in lampreys informs the evolution of the vertebrate paired appendages.

Vertebrate paired appendages are one of the most important evolutionary novelties in vertebrates. During embryogenesis, the skeletal elements of paired appendages differentiate from the somatic mesoderm, which is a layer of lateral plate mesoderm. However, the presence of the somatic mesoderm in the common ancestor of vertebrates has been controversial. To address this problem, it is necessary but insufficient to understand the developmental process of lateral plate mesoderm formation in lamprey (jawless vertebrates) embryos. Here, I show the presence of the somatic mesoderm in lamprey (Lethenteron camtschaticum) embryos using plastic sectioning and transmission electron microscopy analysis. During the early pharyngeal stages, the somatic mesoderm transforms from the lateral plate mesoderm in the trunk region. Soon after, when the cardiac structures were morphologically distinct, the somatic mesoderm was recognized through the cardiac to more caudal regions. These findings indicated that the somatic mesoderm evolved before the emergence of paired appendages. I also discuss the developmental changes in the body wall organization in the common ancestor of vertebrates, which is likely related to the evolution of the paired appendages.

Evo-devo studies of cyclostomes and the origin and evolution of jawed vertebrates.

Modern vertebrates consist of two sister groups: cyclostomes and gnathostomes. Cyclostomes are a monophyletic jawless group that can be further divided into hagfishes and lampreys, which show conspicuously different developmental and morphological patterns. However, during early pharyngula development, there appears to be a stage when the embryos of hagfishes and lampreys resemble each other by showing an "ancestral" craniofacial pattern; this pattern enables morphological comparison of hagfish and lamprey craniofacial development at late stages. This cyclostome developmental pattern, or more accurately, this developmental pattern of the jawless grade of vertebrates in early pharyngula was very likely shared by the gnathostome stem before the division of the nasohypophyseal placode led to the jaw and paired nostrils. The craniofacial pattern of the modern jawed vertebrates seems to have begun in fossil ostracoderms (including galeaspids), and was completed by the early placoderm lineages. The transition from jawless to jawed vertebrates was thus driven by heterotopy of development, mainly caused by separation and shift of ectodermal placodes and resultant ectomesenchymal distribution, and shifts of the epithelial-mesenchymal interactions that underlie craniofacial differentiation. Thus, the evolution of the jaw was not a simple modification of the mandibular arch, but a heterotopic shift of the developmental interactions involving not only the mandibular arch, but also the premandibular region rostral to the mandibular arch.

Development of the lamprey velum and implications for the evolution of the vertebrate jaw.

BACKGROUND: The vertebrate jaw is thought to have evolved through developmental modification of the mandibular arch. An extant jawless vertebrate, the lamprey, possesses a structure called "velum"-a mandibular arch derivative-in addition to the oral apparatus. This leads us to assess the velum's possible contribution to the evolution of jaws. RESULTS: The velar muscles develop from progenitor cells distinct from those from which the oral muscles develop. In addition, the oral and velar regions originate from the different sub-population of the trigeminal neural crest cells (NCCs): the former region receives NCCs from the midbrain, whereas the latter region receives NCCs from the anterior hindbrain. The expression of patterning genes (eg, DlxA and MsxA) is activated at a later developmental stage in the velum compared to the oral region, and more importantly, in different cells from those in the oral region. CONCLUSION: The lamprey mandibular arch consists of two developmental units: the anterior oral unit and the posterior velar unit. Because structural elements of the lamprey velum may be homologous to the jaw, the evolution of vertebrate jaws may have occurred by the velum being released from its functional roles in feeding or respiration in jawless vertebrates.

The expression of FoxG1 in the early development of the European river lamprey Lampetra fluviatilis demonstrates significant heterochrony with that in other vertebrates.

FoxG1, a member of the Fox/Forkhead family of winged helix transcription factors, plays key roles in the induction and spatial compartmentalization of the telencephalon in vertebrates. Loss- and gain-of-function experiments have established FoxG1 as a maintenance factor for neural progenitors and a crucial player in the specification of the ventral telencephalon (subpallium). For the first time in evolution, the telencephalon appeared in the ancestors of vertebrates, including cyclostomes. However, although FoxG1 homologues are present in cyclostomes (i.e., in lampreys and hagfishes), no systematic study of the spatial-temporal expression of FoxG1 during the embryonic development of these animals has been carried out. Given these findings, we have now studied FoxG1 spatial-temporal expression patterns in the early development of the European river lamprey Lampetra fluviatilis. We show that in contrast to other vertebrates, in which the expression of FoxG1 begins during neurulation, the expression of this gene in L. fluviatilis starts after neurulation, first at stage 21 (early head protrusion) in the area of the otic placodes and then, beginning from stage 22, in the telencephalon. Such heterochrony of FoxG1 expression in the lamprey may reflect the fact that in this basally divergent representative of vertebrates, telencephalon specification occurs relatively late. This heterochrony could be related to the evolutionary history of the telencephalon, with a recent appearance in vertebrates as an extension to more ancient anterior brain regions. Another peculiarity of FoxG1 expression in lamprey, compared to other vertebrates, is that it is not expressed in the lamprey optic structures.

Evolution of the new head by gradual acquisition of neural crest regulatory circuits.

The neural crest, an embryonic stem-cell population, is a vertebrate innovation that has been proposed to be a key component of the 'new head', which imbued vertebrates with predatory behaviour1,2. Here, to investigate how the evolution of neural crest cells affected the vertebrate body plan, we examined the molecular circuits that control neural crest development along the anteroposterior axis of a jawless vertebrate, the sea lamprey. Gene expression analysis showed that the cranial subpopulation of the neural crest of the lamprey lacks most components of a transcriptional circuit that is specific to the cranial neural crest in amniotes and confers the ability to form craniofacial cartilage onto non-cranial neural crest subpopulations3. Consistent with this, hierarchical clustering analysis revealed that the transcriptional profile of the lamprey cranial neural crest is more similar to the trunk neural crest of amniotes. Notably, analysis of the cranial neural crest in little skate and zebrafish embryos demonstrated that the transcriptional circuit that is specific to the cranial neural crest emerged via the gradual addition of network components to the neural crest of gnathostomes, which subsequently became restricted to the cephalic region. Our results indicate that the ancestral neural crest at the base of the vertebrate lineage possessed a trunk-like identity. We propose that the emergence of the cranial neural crest, by progressive assembly of an axial-specific regulatory circuit, allowed the elaboration of the new head during vertebrate evolution.

Germline-Specific Repetitive Elements in Programmatically Eliminated Chromosomes of the Sea Lamprey (Petromyzon marinus).

The sea lamprey (Petromyzon marinus) is one of few vertebrate species known to reproducibly eliminate large fractions of its genome during normal embryonic development. This germline-specific DNA is lost in the form of large fragments, including entire chromosomes, and available evidence suggests that DNA elimination acts as a permanent silencing mechanism that prevents the somatic expression of a specific subset of "germline" genes. However, reconstruction of eliminated regions has proven to be challenging due to the complexity of the lamprey karyotype. We applied an integrative approach aimed at further characterization of the large-scale structure of eliminated segments, including: (1) in silico identification of germline-enriched repeats; (2) mapping the chromosomal location of specific repetitive sequences in germline metaphases; and (3) 3D DNA/DNA-hybridization to embryonic lagging anaphases, which permitted us to both verify the specificity of elements to physically eliminated chromosomes and characterize the subcellular organization of these elements during elimination. This approach resulted in the discovery of several repetitive elements that are found exclusively on the eliminated chromosomes, which subsequently permitted the identification of 12 individual chromosomes that are programmatically eliminated during early embryogenesis. The fidelity and specificity of these highly abundant sequences, their distinctive patterning in eliminated chromosomes, and subcellular localization in elimination anaphases suggest that these sequences might contribute to the specific targeting of chromosomes for elimination or possibly in molecular interactions that mediate their decelerated poleward movement in chromosome elimination anaphases, isolation into micronuclei and eventual degradation.

Evolution of Snail-mediated regulation of neural crest and placodes from an ancient role in bilaterian neurogenesis.

A major challenge in vertebrate evolution is to identify the gene regulatory mechanisms that facilitated the origin of neural crest cells and placodes from ancestral precursors in invertebrates. Here, we show in lamprey, a primitively jawless vertebrate, that the transcription factor Snail is expressed simultaneously throughout the neural plate, neural plate border, and pre-placodal ectoderm in the early embryo and is then upregulated in the CNS throughout neurogenesis. Using CRISPR/Cas9-mediated genome editing, we demonstrate that Snail plays functional roles in all of these embryonic domains or their derivatives. We first show that Snail patterns the neural plate border by repressing lateral expansion of Pax3/7 and activating nMyc and ZicA. We also present evidence that Snail is essential for DlxB-mediated establishment of the pre-placodal ectoderm but is not required for SoxB1a expression during formation of the neural plate proper. At later stages, Snail regulates formation of neural crest-derived and placode-derived PNS neurons and controls CNS neural differentiation in part by promoting cell survival. Taken together with established functions of invertebrate Snail genes, we identify a pan-bilaterian mechanism that extends to jawless vertebrates for regulating neurogenesis that is dependent on Snail transcription factors. We propose that ancestral vertebrates deployed an evolutionarily conserved Snail expression domain in the CNS and PNS for neurogenesis and then acquired derived functions in neural crest and placode development by recruitment of regulatory genes downstream of neuroectodermal Snail activity. Our results suggest that Snail regulatory mechanisms in vertebrate novelties such as the neural crest and placodes may have emerged from neurogenic roles that originated early in bilaterian evolution.

Inner ear development in cyclostomes and evolution of the vertebrate semicircular canals.

Jawed vertebrates have inner ears with three semicircular canals, the presence of which has been used as a key to understanding evolutionary relationships. Ostracoderms, the jawless stem gnathostomes, had only two canals and lacked the lateral canal1-3. Lampreys, which are modern cyclostomes, are generally thought to possess two semicircular canals whereas the hagfishes-which are also cyclostomes-have only a single canal, which used to be regarded as a more primitive trait1,4. However, recent molecular and developmental analyses have strongly supported the monophyly of cyclostomes5-7, which has left the evolutionary trajectory of the vertebrate inner ear unclear8. Here we show the differentiation of the otic vesicle of the lamprey Lethenteron camtschaticum and inshore hagfish Eptatretus burgeri. This is the first time, to our knowledge, that the development of the hagfish inner ear is reported. We found that canal development in the lamprey starts with two depressions-which is reminiscent of the early developmental pattern of the inner ear in modern gnathostomes. These cyclostome otic vesicles show a pattern of expression of regulatory genes, including OTX genes, that is comparable to that of gnathosomes. Although two depressions appear in the lamprey vesicle, they subsequently fuse to form a single canal that is similar to that of hagfishes. Complete separation of the depressions results in anterior and posterior canals in gnathostomes. The single depression of the vesicle in hagfishes thus appears to be a secondarily derived trait. Furthermore, the lateral canal in crown gnathostomes was acquired secondarily-not by de novo acquisition of an OTX expression domain, but by the evolution of a developmental program downstream of the OTX genes.

A Notch-regulated proliferative stem cell zone in the developing spinal cord is an ancestral vertebrate trait.

Vertebrates have evolved the most sophisticated nervous systems we know. These differ from the nervous systems of invertebrates in several ways, including the evolution of new cell types, and the emergence and elaboration of patterning mechanisms to organise cells in time and space. Vertebrates also generally have many more cells in their central nervous systems than invertebrates, and an increase in neural cell number may have contributed to the sophisticated anatomy of the brain and spinal cord. Here, we study how increased cell number evolved in the vertebrate central nervous system, investigating the regulation of cell proliferation in the lamprey spinal cord. Markers of proliferation show that a ventricular progenitor zone is found throughout the lamprey spinal cord. We show that inhibition of Notch signalling disrupts the maintenance of this zone. When Notch is blocked, progenitor cells differentiate precociously, the proliferative ventricular zone is lost and differentiation markers become expressed throughout the spinal cord. Comparison with other chordates suggests that the emergence of a persistent Notch-regulated proliferative progenitor zone was a crucial step for the evolution of vertebrate spinal cord complexity.

The neural crest and origin of the neurocranium in vertebrates.

Cranium of jawed vertebrates is composed of dorsal moiety that encapsulates the brain, or the neurocranium, and the is called the neurocranium, and the ventral moiety, the viscerocranium, that supports the pharynx. In modern jawed vertebrates (crown gnathostomes), the viscerocranium is predominantly of neural crest origin, and for the neurocranium, the rostral part is derived from neural crest cells, whereas the posterior part from the mesoderm. In the cyclostome cranium, the mesoderm/neural crest boundary of the neurocranium used to be enigmatic, let alone the morphological comparison of neurocranial between two cyclostome groups, lampreys and hagfishes. By examining the hagfish development it has become clear that cyclostomes share a common craniofacial embryonic pattern that is not shared by modern gnathostomes, and cyclostome cranium can be compared among the group as developmental modular units with comparable mesoderm/neural crest boundary within the neuroranium. Also, the dual origin of the jawed vertebrate neurocranium has now turned out to represent a derived condition, and ancestrally, the neurocranium would likely have been predominantly of mesodermal origin. Enlargement of the forebrain and reorganization of the oral apparatus seem to have led to the involvement of the neural crest in the rostral neurocranium.

Gliogenesis in lampreys shares gene regulatory interactions with oligodendrocyte development in jawed vertebrates.

Glial cells in the nervous system regulate and support many functions related to neuronal activity. Understanding how the vertebrate nervous system has evolved demands a greater understanding of the mechanisms controlling evolution and development of glial cells in basal vertebrates. Among vertebrate glia, oligodendrocytes form an insulating myelin layer surrounding axons of the central nervous system (CNS) in jawed vertebrates. Jawless vertebrates lack myelinated axons but it is unclear when oligodendrocytes or the regulatory mechanisms controlling their development evolved. To begin to investigate the evolution of mechanisms controlling glial development, we identified key genes required for the differentiation of oligodendrocytes in gnathostomes, including Nkx2.2, SoxE genes, and PDGFR, analyzed their expression, and used CRISPR/Cas9 genome editing to perturb their functions in a primitively jawless vertebrate, the sea lamprey. We show in lamprey that orthologs required for oligodendrocyte development in jawed vertebrates are expressed in the lamprey ventral neural tube, in similar locations where gnathostome oligodendrocyte precursor cells (OPC) originate. In addition, they appear to be under the control of conserved mechanisms that regulate OPC development in jawed vertebrates and may also function in gliogenesis. Our results suggest that although oligodendrocytes first emerged in jawed vertebrates, regulatory mechanisms required for their development predate the divergence of jawless and jawed vertebrates.

Lamprey neural crest migration is Snail-dependent and occurs without a differential shift in cadherin expression.

The acquisition of neural crest cells was a key step in the origin of the vertebrate body plan. An outstanding question is how neural crest cells acquired their ability to undergo an epithelial-mesenchymal transition (EMT) and migrate extensively throughout the vertebrate embryo. We tested if differential regulation of classical cadherins-a highly conserved feature of neural crest EMT and migration in jawed vertebrates-mediates these cellular behaviors in lamprey, a basal jawless vertebrate. Lamprey has single copies of the type I and type II classical cadherins (CadIA and CadIIA). CadIIA is expressed in premigratory neural crest, and requires the transcription factor Snail for proper expression, yet CadIA is never expressed in the neural tube during neural crest development, suggesting that differential regulation of classical cadherin expression is not required to initiate neural crest migration in basal vertebrates. We hypothesize that neural crest cells evolved by retention of regulatory programs linking distinct mesenchymal and multipotency properties, and emigrated from the neural tube without differentially regulating type I/type II cadherins. Our results point to the coupling of mesenchymal state and multipotency as a key event facilitating the origin of migratory neural crest cells.

Reconstructing the ancestral vertebrate brain.

Highly complicated morphologies and sophisticated functions of vertebrate brains have been established through evolution. However, the origin and early evolutionary history of the brain remain elusive, owing to lack of information regarding the brain architecture of extant and fossil species of jawless vertebrates (agnathans). Comparative analyses of the brain of less studied cyclostomes (only extant agnathan group, consisting of lampreys and hagfish) with the well-known sister group of jawed vertebrates (gnathostomes) are the only tools we have available to illustrate the ancestral architecture of the vertebrate brain. Previous developmental studies had shown that the lamprey lacked well-established brain compartments that are present in gnathostomes, such as the medial ganglionic eminence and the rhombic lip. The most accepted scenario suggested that cyclostomes had fewer compartments than that of the gnathostome brain and that gnathostomes thus evolved by a stepwise addition of innovations on its developmental sequence. However, recent studies have revealed that these compartments are present in hagfish embryos, indicating that these brain regions have been acquired before the split of cyclostomes and gnathostomes. By comparing two cyclostome lineages and gnathostomes, it has become possible to speculate about a more complex ancestral state of the brain, excluding derived traits in either of the lineages. In this review, we summarize recent studies on the brain development of the lamprey and hagfish. Then, we attempt to reconstruct the possible brain architecture of the last common ancestor of vertebrates. Finally, we discuss how the developmental plan of the vertebrate brain has been modified independently in different vertebrate lineages.

CRISPR: MODIFYING THE LIFE SCIENCE LANDSCAPE.

From lampreys to human stem cells, the CRISPR/Cas9 system is challenging our notions of what is possible with genome editing. Nathan Blow talks to researchers pushing the boundaries of CRISPR/Cas9 technology to expand our understanding of biology.

Functional constraints on SoxE proteins in neural crest development: The importance of differential expression for evolution of protein activity.

Vertebrate SoxE genes (Sox8, 9, and 10) are key regulators of neural crest cell (NCC) development. These genes arose by duplication from a single SoxE gene in the vertebrate ancestor. Although SoxE paralogs are coexpressed early in NCC development, later, Sox9 is restricted to skeletogenic lineages in the head, and Sox10 to non-skeletogenic NCC in the trunk and head. When this subfunctionalization evolved and its possible role in the evolution of the neural crest are unknown. Sea lampreys are basal vertebrates that also possess three SoxE genes, while only a single SoxE is present in the cephalochordate amphioxus. In order to address the functional divergence of SoxE genes, and to determine if differences in their biochemical functions may be linked to changes in neural crest developmental potential, we examined the ability of lamprey and amphioxus SoxE genes to regulate differentiation of NCC derivatives in zebrafish colourless (cls) mutants lacking expression of sox10. Our findings suggest that the proto-vertebrate SoxE gene possessed both melanogenic and neurogenic capabilities prior to SoxE gene duplication. Following the agnathan-gnathostome split, lamprey SoxE1 and SoxE3 largely lost their melanogenic and/or enteric neurogenic properties, while gnathostome SoxE paralogs have retained functional conservation. We posit that this difference in protein subfunctionalization is a direct consequence of the independent regulation of SoxE paralog expression between the two lineages. Specifically, we propose that the overlapping expression of gnathostome SoxE paralogs in early neural crest largely constrained the function of gnathostome SoxE proteins. In contrast, the largely non-overlapping expression of lamprey SoxE paralogs allowed them to specialize with regard to their DNA-binding and/or protein interaction properties. Restriction of developmental potential among cranial and trunk neural crest in lampreys may be related to constraints on SoxE activity among duplicates, but such specialization does not appear to have occurred in gnathostomes. This highlights an important difference in the evolution of SoxE activity between these two divergent vertebrate lineages and provides insights for understanding how cell fate restriction in different NCC populations may be dependent on subfunctionalization among SoxE duplicates.

Evolution of the Vertebrate Cranium: Viewed from Hagfish Developmental Studies.

Our knowledge of vertebrate cranium evolution has relied largely on the study of gnathostomes. Recent evolutionary and developmental studies of cyclostomes have shed new light on the history of the vertebrate skull. The recent ability to obtain embryos of the hagfish, Eptatretus burgeri, has enabled new studies which have suggested an embryonic morphological pattern (the "cyclostome pattern") of craniofacial development. This pattern is shared by cyclostomes, but not by modern jawed vertebrates. Because this pattern of embryonic head development is thought to be present in some stem gnathostomes (ostracoderms), it is possible that the cyclostome pattern represents the vertebrate ancestral pattern. The study of cyclostomes may thus lead to an understanding of the most ancestral basis of craniofacial development. In this review, we summarize the development of the hagfish chondrocranium in light of the cyclostome pattern, present an updated comparison of the cyclostome chondrocranium, and discuss several aspects of the evolution and development of the vertebrate skull.

Species-specific contribution of volumetric growth and tissue convergence to posterior body elongation in vertebrates.

Posterior body elongation is a widespread mechanism propelling the generation of the metazoan body plan. The posterior growth model predicts that a posterior growth zone generates sufficient tissue volume to elongate the posterior body. However, there are energy supply-related differences between vertebrates in the degree to which growth occurs concomitantly with embryogenesis. By applying a multi-scalar morphometric analysis in zebrafish embryos, we show that posterior body elongation is generated by an influx of cells from lateral regions, by convergence-extension of cells as they exit the tailbud, and finally by a late volumetric growth in the spinal cord and notochord. Importantly, the unsegmented region does not generate additional tissue volume. Fibroblast growth factor inhibition blocks tissue convergence rather than volumetric growth, showing that a conserved molecular mechanism can control convergent morphogenesis through different cell behaviours. Finally, via a comparative morphometric analysis in lamprey, dogfish, zebrafish and mouse, we propose that elongation via posterior volumetric growth is linked to increased energy supply and is associated with an overall increase in volumetric growth and elongation.

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.

Expression patterns of Eph genes in the "dual visual development" of the lamprey and their significance in the evolution of vision in vertebrates.

Image-forming vision is crucial to animals for recognizing objects in their environment. In vertebrates, this type of vision is achieved with paired camera eyes and topographic projection of the optic nerve. Topographic projection is established by an orthogonal gradient of axon guidance molecules, such as Ephs. To explore the evolution of image-forming vision in vertebrates, lampreys, which belong to the basal lineage of vertebrates, are key animals because they show unique "dual visual development." In the embryonic and pre-ammocoete larval stage (the "primary" phase), photoreceptive "ocellus-like" eyes develop, but there is no retinotectal optic nerve projection. In the late ammocoete larval stage (the "secondary" phase), the eyes grow and form into camera eyes, and retinotectal projection is newly formed. After metamorphosis, this retinotectal projection in adult lampreys is topographic, similar to that of gnathostomes. In this study, we explored the involvement of Ephs in lamprey "dual visual development" and establishment of the image-form vision. We found that gnathostome-like orthogonal gradient expression was present in the retina during the "secondary" phase; i.e., EphB showed a gradient of expression along the dorsoventral axis, while EphC was expressed along the anteroposterior axis. However, no orthogonal gradient expression was observed during the "primary" phase. These observations suggest that Ephs are likely recruited de novo for the guidance of topographical "second" optic nerve projection. Transformations during lamprey "dual visual development" may represent "recapitulation" from a protochordate-like ancestor to a gnathostome-like vertebrate ancestor.