Paired fins in vertebrate evolution and ontogeny.

The origin of paired appendages became one of the most important adaptations of vertebrates, allowing them to lead active lifestyles and explore a wide range of ecological niches. The basic form of paired appendages in evolution is the fins of fishes. The problem of paired appendages has attracted the attention of researchers for more than 150 years. During this time, a number of theories have been proposed, mainly based on morphological data, two of which, the Balfour-Thacher-Mivart lateral fold theory and Gegenbaur's gill arch theory, have not lost their relevance. So far, however, none of the proposed ideas has been supported by decisive evidence. The study of the evolutionary history of the appearance and development of paired appendages lies at the intersection of several disciplines and involves the synthesis of paleontological, morphological, embryological, and genetic data. In this review, we attempt to summarize and discuss the results accumulated in these fields and to analyze the theories put forward regarding the prerequisites and mechanisms that gave rise to paired fins and limbs in vertebrates.

The Molecular Mechanism of Body Axis Induction in Lampreys May Differ from That in Amphibians.

Lamprey homologues of the classic embryonic inducer Noggin are similar in expression pattern and functional properties to Noggin homologues of jawed vertebrates. All noggin genes of vertebrates apparently originated from a single ancestral gene as a result of genome duplications. nogginA, nogginB and nogginC of lampreys, like noggin1 and noggin2 of gnathostomes, demonstrate the ability to induce complete secondary axes with forebrain and eye structures when overexpressed in Xenopus laevis embryos. According to current views, this finding indicates the ability of lamprey Noggin proteins to suppress the activity of the BMP, Nodal/Activin and Wnt/beta-catenin signaling pathways, as shown for Noggin proteins of gnathostomes. In this work, by analogy with experiments in Xenopus embryos, we attempted to induce secondary axes in the European river lamprey Lampetra fluviatilis by injecting noggin mRNAs into lamprey eggs in vivo. Surprisingly, unlike what occurs in amphibians, secondary axis induction in the lampreys either by noggin mRNAs or by chordin and cerberus mRNAs, the inductive properties of which have been described, was not observed. Only wnt8a mRNA demonstrated the ability to induce secondary axes in the lampreys. Such results may indicate that the mechanism of axial specification in lampreys, which represent jawless vertebrates, may differ in detail from that in the jawed clade.

Loss of noggin1, a classic embryonic inducer gene, in elasmobranchs.

Secreted proteins of the Noggin family serve as pivotal regulators of early development and cell differentiation in all multicellular animals, including vertebrates. Noggin1 was identified first among all Noggins. Moreover, it was described as the first known embryonic inducer specifically secreted by the Spemann organizer and capable of inducing a secondary body axis when expressed ectopically. In the classical default model of neural induction, Noggin1 is presented as an antagonist of BMP signalling, playing a role as a neural inducer. Additionally, Noggin1 is involved in the dorsalization of embryonic mesoderm and later controls the differentiation of various tissues, including muscles, bones, and neural crest derivatives. Hitherto, noggin1 was found in all studied vertebrates. Here, we report the loss of noggin1 in elasmobranchs (sharks, rays and skates), which is a unique case among vertebrates. noggin2 and noggin4 retained in this group and studied in the embryos of the grey bamboo shark Chiloscyllium griseum revealed similarities in expression patterns and functional properties with their orthologues described in other vertebrates. The loss of noggin1 in elasmobranchs may be associated with histological features of the formation of their unique internal cartilaginous skeleton, although additional research is required to establish functional connections between these events.

Three foxg1 paralogues in lampreys and gnathostomes-brothers or cousins?

Foxg1 is a key regulator of the early development of the vertebrate forebrain and sensory organs. In this study, we describe for the first time three foxg1 paralogues in lamprey, representative of one of two basally diverged lineages of vertebrates-the agnathans. We also first describe three foxg1 genes in sterlet-representative of one of the evolutionarily ancient clades of gnathostomes. According to the analysis of local genomic synteny, three foxg1 genes of agnathans and gnathostomes have a common origin as a result of two rounds of genomic duplications in the early evolution of vertebrates. At the same time, it is difficult to reliably establish pairwise orthology between foxg1 genes of agnathans and gnathostomes based on the analysis of phylogeny and local genomic synteny, as well as our studies of the spatiotemporal expression of foxg1 genes in the river lamprey Lampetra fluviatilis and the sterlet Acipenser ruthenus. Thus, the appearance of three foxg1 paralogues in agnathans and gnathostomes could have occurred either as a result of two rounds of duplication of the vertebrate common ancestor genome (2R hypothesis) or as a result of the first common round followed by subsequent independent polyploidizations in two evolutionary lineages (1R hypothesis).

Publisher Correction: Discovery of four Noggin genes in lampreys suggests two rounds of ancient genome duplication.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Discovery of four Noggin genes in lampreys suggests two rounds of ancient genome duplication.

The secreted protein Noggin1 was the first discovered natural embryonic inducer produced by cells of the Spemann organizer. Thereafter, it was shown that vertebrates have a whole family of Noggin genes with different expression patterns and functional properties. For example, Noggin1 and Noggin2 inhibit the activity of BMP, Nodal/Activin and Wnt-beta-catenin signalling, while Noggin4 cannot suppress BMP but specifically modulates Wnt signalling. In this work, we described and investigated phylogeny and expression patterns of four Noggin genes in lampreys, which represent the most basally divergent group of extant vertebrates, the cyclostomes, belonging to the superclass Agnatha. Assuming that lampreys have Noggin homologues in all representatives of another superclass of vertebrates, the Gnathostomata, we propose a model for Noggin family evolution in vertebrates. This model is in agreement with the hypotheses suggesting two rounds of genome duplication in the ancestor of vertebrates before the divergence of Agnatha and Gnathostomata.

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.

Agr2-interacting Prod1-like protein Tfp4 from Xenopus laevis is necessary for early forebrain and eye development as well as for the tadpole appendage regeneration.

The Agr family genes, Ag1, Agr2, and Agr3, encode for the thioredoxin domain containing secreted proteins and are specific only for vertebrates. These proteins are attracting increasing attention due to their involvement in many physiological and pathological processes, including exocrine secretion, cancer, regeneration of the body appendages, and the early brain development. At the same time, the mode by which Agrs regulate intracellular processes are poorly understood. Despite that the receptor to Agr2, the membrane anchored protein Prod1, has been firstly discovered in Urodeles, and it has been shown to interact with Agr2 in the regenerating limb, no functional homologs of Prod1 were identified in other vertebrates. This raises the question of the mechanisms by which Agrs can regulate regeneration in other lower vertebrates. Recently, we have identified that Tfp4 (three-fingers Protein 4), the structural and functional homolog of Prod1 in Anurans, interacts with Agr2 in Xenopus laevis embryos. In the present work we show by several methods that the activity of Tfp4 is essential for the tadpole tail regeneration as well as for the early eye and forebrain development during embryogenesis. These data show for the first time the common molecular mechanism of regeneration regulation in amphibians by interaction of Prod1 and Agr2 proteins.

The presence of Anf/Hesx1 homeobox gene in lampreys suggests that it could play an important role in emergence of telencephalon.

Accumulated evidence indicates that the core genetic mechanisms regulating early patterning of the brain rudiment in vertebrates are very similar to those operating during development of the anterior region of invertebrate embryos. However, the mechanisms underlying the morphological differences between the elaborate vertebrate brain and its simpler invertebrate counterpart remain poorly understood. Recently, we hypothesized that the emergence of the most anterior unit of the vertebrate brain, the telencephalon, could be related to the appearance in vertebrates' ancestors of a unique homeobox gene, Anf/Hesx1(further Anf), which is absent from all invertebrates and regulates the earliest steps of telencephalon development in vertebrates. However, the failure of Anf to be detected in one of the most basal extant vertebrate species, the lamprey, seriously compromises this hypothesis. Here, we report the cloning of Anf in three lamprey species and demonstrate that this gene is indeed expressed in embryos in the same pattern as in other vertebrates and executes the same functions by inhibiting the expression of the anterior general regulator Otx2 in favour of the telencephalic regulator FoxG1. These results are consistent with the hypothesis that the Anf homeobox gene may have been important in the evolution of the telencephalon.

Noggin4 is a long-range inhibitor of Wnt8 signalling that regulates head development in Xenopus laevis.

Noggin4 is a Noggin family secreted protein whose molecular and physiological functions remain unknown. In this study, we demonstrate that in contrast to other Noggins, Xenopus laevis Noggin4 cannot antagonise BMP signalling; instead, it specifically binds to Wnt8 and inhibits the Wnt/ß -catenin pathway. Live imaging demonstrated that Noggin4 diffusivity in embryonic tissues significantly exceeded that of other Noggins. Using the Fluorescence Recovery After Photobleaching (FRAP) assay and mathematical modelling, we directly estimated the affinity of Noggin4 for Wnt8 in living embryos and determined that Noggin4 fine-tune the Wnt8 posterior-to-anterior gradient. Our results suggest a role for Noggin4 as a unique, freely diffusing, long-range inhibitor of canonical Wnt signalling, thus explaining its ability to promote head development.

Noggin4 expression during chick embryonic development.

We describe here the expression pattern of Noggin4 during the early development of the chick embryo (Gallus gallus). The expression of this gene starts with the onset of gastrulation (stage HH4), in two bilateral bands along the primitive streak, with a local maximum around Hensen's node. By the end of gastrulation, Noggin4 transcripts are distributed diffusely throughout the epiblast, with the highest concentration in the head ectoderm. Interestingly, the expression of Noggin4 during the first half of gastrulation demonstrates a clear left-right asymmetry in Hensen's node, being much more intensive in its right anterior portion. During neurulation, Noggin4 is expressed mainly in the neuroectoderm, with the most intensive expression in the head and lateral neural folds. In mesoderm derivatives, expression is seen in somites but not in the notochord. In general, primarily ectodermal and diffusive expression of Noggin4 in chick embryo, with a maximum in the anterior neurectoderm, resembles that of its ortholog in Xenopus, which indicates a conservative function of this gene in evolution.

Novel functions of Noggin proteins: inhibition of Activin/Nodal and Wnt signaling.

The secreted protein Noggin1 is an embryonic inducer that can sequester TGFß cytokines of the BMP family with extremely high affinity. Owing to this function, ectopic Noggin1 can induce formation of the headless secondary body axis in Xenopus embryos. Here, we show that Noggin1 and its homolog Noggin2 can also bind, albeit less effectively, to ActivinB, Nodal/Xnrs and XWnt8, inactivation of which, together with BMP, is essential for the head induction. In support of this, we show that both Noggin proteins, if ectopically produced in sufficient concentrations in Xenopus embryo, can induce a secondary head, including the forebrain. During normal development, however, Noggin1 mRNA is translated in the presumptive forebrain with low efficiency, which provides the sufficient protein concentration for only its BMP-antagonizing function. By contrast, Noggin2, which is produced in cells of the anterior margin of the neural plate at a higher concentration, also protects the developing forebrain from inhibition by ActivinB and XWnt8 signaling. Thus, besides revealing of novel functions of Noggin proteins, our findings demonstrate that specification of the forebrain requires isolation of its cells from BMP, Activin/Nodal and Wnt signaling not only during gastrulation but also at post-gastrulation stages.

Expression patterns of genes encoding small GTPases Ras-dva-1 and Ras-dva-2 in the Xenopus laevis tadpoles.

Small GTPases of the recently discovered Ras-dva family are specific to the Vertebrate phylum. In Xenopus laevis, Ras-dva-1 is expressed during gastrulation and neurulation in the anterior ectoderm where it regulates the early development of the forebrain and cranial placodes (Tereshina et al., 2006). In the present work, we studied the expression of Ras-dva-1 at later developmental stages. As a result, the Ras-dva-1 expression was revealed in the eye retina, epiphysis (pineal gland), hypophysis (pituitary), branchial arches, pharynx, oesophagus, stomach and gall bladder of swimming tadpoles. Additionally, we investigated for the first time the expression pattern of Ras-dva-2. This gene encodes a protein belonging to a novel sub-group of Ras-dva GTPases that we identified by phylogenetic analysis within Ras-dva family. In contrast to Ras-dva-1, Ras-dva-2 is not expressed before the swimming tadpole stage. At the swimming tadpole stage, however, Ras-dva-2 transcripts can be detected in the eye retina and brain. Later in development, the expression of Ras-dva-2 can also be revealed in the mesonephros and stomach.

Multiple noggins in vertebrate genome: cloning and expression of noggin2 and noggin4 in Xenopus laevis.

Noggin is a neural inducer secreted by cells of the Spemann organizer. A single noggin gene was identified until very recently in all tested vertebrates. The only exception was zebrafish, in which two close homologs of noggin, named noggin1 and noggin3, and one gene more diverged from them, noggin2, were cloned. Nevertheless, finding of three zebrafish noggins was attributed exclusively to specific genomic duplications in the fish evolutionary branch. However, very recently it was shown that Xenopus tropicalis have additional noggin homolog, called noggin2 [Fletcher, R.B., Watson, A.L., Harland, R.M. (2004). Expression of Xenopus tropicalis noggin1 and noggin2 in early development: two noggin genes in a tetrapod. Gene Expr. Patterns 5, 225-230], which indicates at least two independent noggin genes in vertebrate phylum. Now we report identification of two novel noggin homologs in each of so evolutionary distant species as Xenopus laevis, chicken and fugu. One of these noggins is ortholog of the X. tropicalis and zebrafish noggin2, whereas another, named noggin4, was not known previously. In the X. laevis embryos, the expression of noggin2 very resembles that of its counterpart in X. tropicalis: it begins with neurulation at the anterior margin of the neural plate and, afterward, continues mainly in the forebrain and dorsal hindbrain. At the same time, noggin4 is expressed starting from the beginning of gastrulation, throughout the ectoderm, with a local expression maximum in the prospective anterior neurectoderm. Later, it is widely expressed on the dorsal side of embryo, including neural tube, eyes, otic vesicles, cranial placodes, branchial arches, and somites. The data presented here demonstrate that the vertebrate phylum contains at least three distinct noggin genes.

The homeodomain-containing transcription factor X-nkx-5.1 inhibits expression of the homeobox gene Xanf-1 during the Xenopus laevis forebrain development.

Expression of the homeobox gene Xanf-1 starts within the presumptive forebrain primordium of the Xenopus embryo at the midgastrula stage and is inhibited by the late neurula. Such stage-specific inhibition is essential for the normal development as the experimental prolongation of the Xanf-1 expression elicits severe brain abnormalities. To identify transcriptional regulators that are responsible for the Xanf-1 inhibition, we have used the yeast one-hybrid system and identified a novel Xenopus homeobox gene X-nkx-5.1 that belongs to a family of Nkx-5.1 transcription factors. In terms of gene expression, X-nkx-5.1 shares many common features with its orthologs in other species, including expression in the embryonic brain and in the ciliated cells of the otic and lateral line placodes. However, we have also observed several features specific for X-nkx-5.1, such as expression in precursors of the epidermal ciliated cells that may indicate a possible common evolutionary origin of all ciliated cells derived from the embryonic ectoderm. Another specific feature is that the X-nkx-5.1 expression in the anterior neural plate starts early, within the area overlapping the Xanf-1 expression territory at the midneurula stage, and it correlates with the beginning of the Xanf-1 inhibition. Using various loss and gain-of-function techniques, including microinjections of antisense morpholino oligonucleotides and mRNA encoding for the X-nkx-5.1 and its dominant repressor and activator versions, we have shown that X-nkx-5.1 can indeed play a role of stage-specific inhibitor of Xanf-1 in the anterior neural plate during the Xenopus development.