Genomic transformations during early vertebrate evolution, including two rounds of whole-genome duplication, laid the groundwork for the emergence of novel morphological features in jawed vertebrates. Among these innovations, paired appendages represent a major evolutionary milestone, whose development and diversification enabled vertebrates to exploit diverse ecological niches in aquatic, terrestrial, and aerial environments. Here, we combined phylogenetic and local genomic synteny analyses to investigate the evolutionary history of chordin-like homologs in vertebrates. Our results indicate that chordin-like1 first appeared in jawed vertebrates, suggesting a possible link between its origin and the emergence of paired appendages. To explore this hypothesis, we examined chordin-like1 expression in representatives of basal jawed vertebrate lineages - cartilaginous fishes (grey catshark, Chiloscyllium griseum) and sturgeons (sterlet, Acipenser ruthenus). We further assessed the expression and functional properties of the chordin-like1 ortholog in the African clawed frog (Xenopus laevis), a representative terrestrial vertebrate with limb morphology that markedly differs from the fins of basal gnathostomes. Together with published data, our findings support a potential role for chordin-like1 in the evolution of paired appendages. In particular, chordin-like1 may have contributed to the development of the metapterygial element and its derivatives, which formed the structural basis for the evolution of tetrapod limbs.

The hypothesis that whole genome duplications may have contributed to the origin and complexity of the vertebrate body plan was put forward in the 1970s. It was based on the analysis of the genome size of vertebrates and their closest relatives, the lancelets. The subsequent development of genetic technologies has confirmed this hypothesis and raised new questions—about the timing and number of these duplications/polyploidizations, about the fate of genes after duplication when the organism returns to an equilibrium diploid state (rediploidization), about the contribution of an increase in the number of specific genes to the formation of structures and traits. In this paper, we will briefly review the key issues related to genome duplication and consider the results of our work studying two gene families in phylogenetically basal groups of vertebrates. Genes of the Noggin and Foxg1 families have been described as key regulators of early ontogeny and the formation of vertebrate forebrain structures, including the telencephalon, a unique part. Historically, functional studies in classical laboratory objects (amphibians, birds, mice) have looked at one gene from each family. At the same time, we have shown that in evolutionarily ancient groups of vertebrates, such as agnathans (lampreys), cartilaginous fish and Chondrostei, these gene families are represented by several paralogues. In this context, the hypothesis that a multiple increase in the number of these key genes regulating early development could have been an important step in the formation of the body plan of modern vertebrates and the emergence of a number of their unique features that ensured the ecological stability and evolutionary success of the group seems quite reasonable.

The genomes of most gnathostomes contain two paralogs of the shox gene, shox and shox2, both of which are implicated in the development of two key morphological innovations: the jaw apparatus derived from the branchial arches and the paired appendages, whose evolutionary origins remain debated. Here, we investigate the expression patterns of shox and shox2 paralogs in the gray bamboo shark (Chiloscyllium griseum), a representative of Chondrichthyes, a basally divergent gnathostome lineage. The paired fins of cartilaginous fishes are considered a basal model for gnathostome appendages. Our findings suggest spatial subfunctionalization of the shox and shox2 genes. Specifically, shox is expressed in the mandibular and branchial arches, as well as in paired and unpaired fins, indicating shared developmental mechanisms among these structures. In contrast, shox2 expression is predominantly restricted to paired fins, highlighting distinct developmental features that differentiate them from the evolutionarily older median fins.

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.

The foxg1 gene has been described as one of the key regulators of early differentiation and development of the vertebrate forebrain and related sensory organs. In this article, the authors describe for the first time the presence of three foxg1 paralogs in lampreys, representatives of Agnatha, one of the most evolutionarily ancient branches of vertebrates. While maintaining several common features, an expression patterns of foxg1 paralogs in lampreys demonstrate elements of spatial subfunctionalization. An assessment of the estimated timing of duplication of foxg1 lamprey genes suggests that these genes could have appeared as a result of two rounds of whole-genome duplications at the early stages of vertebrate evolution.

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 emergence of paired appendages is one of the most important adaptations of vertebrates; it provided the opportunity to lead an active lifestyle and develop a wide range of ecological niches. Fish fins are a basic form of paired appendages in evolution. The problem of the emergence of paired appendages has attracted the attention of researchers for more than 150 years. During this time, a number of theories have been proposed, based primarily on morphological data, two of which, the Balfour–Thatcher–Mivart lateral fold theory and the Gegenbaur gill arch theory, have not lost their relevance. However, to date, none of the proposed ideas has decisive evidence in its favor. In recent years, new data have been obtained in favor of each of the theories, which emphasize the search for options for their consistent synthesis. In this review article, we attempt to consider the results and analyze the hypotheses regarding the prerequisites and mechanisms that led to the emergence of paired appendages in vertebrates.

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).

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.

Gene foxg1 is one of the key regulators of the early differentiation and development of the vertebrate forebrain and associated sensory organs. In this article, the presence of five foxg1 paralogs in Acipenseriformes, one of the evolutionarily ancient branches of gnathostomes, is described. The appearance of multiple paralogs in Acipenseriformes was apparently associated with three rounds of whole-genome duplications (WGDs), two of which occurred early in the evolution of gnathostomes and are common to the entire clade, while the third is specific to sturgeons. The goal of the work was to study the orthology of the foxg1 of Acipenseriformes with the foxg1 of other groups of vertebrates and to identify their relationship with the development of individual morphological structures by studying spatial expression. The phylogenetic analysis showed that the foxg1b genes of sturgeons occurred as a result of duplication in the common ancestor of the group, while the foxg1a paralogues appeared as a result of independent rediploidizations in the Acipenser and Polyodon lineages. These data are consistent with a model of ancestral duplication followed by asynchronous rediploidization and support duplication at the level of the common ancestor of sturgeons. The studied expression patterns indicate spatial subfunctionalization of foxg1 paralogs in Acipenseriformes and confirm the connection of foxg1 with the development of the forebrain, sensory organs, and associated cranial ganglia in Acipenseriformes as one of the archaic groups of vertebrates.

The hypothesis about whole-genome duplications as the most important driver of transformation of the structure plan and lifestyle of vertebrates at the early stages of their evolution is generally accepted today. At the same time, details such as the timing and mechanisms of these duplications still remain controversial. Research into issues of periodization, number, and in which evolutionary lineages rounds of whole-genome and/or local duplications occurred in vertebrates continues as methodology and technical capabilities develop. The role of high-throughput genomic sequencing and big data analysis is increasing, which makes it possible to identify and track the history of not only individual genes or their families but of large sections of the genome, including at the chromosomal level. New opportunities allow for considering the problem at the macro level and conduct a comparative analysis of the genomic characteristics of representatives of different evolutionary groups. In this article, which is a logical continuation of an earlier review article (in 2020), the authors make an attempt to review and summarize the data of recent years, largely related to the sequencing of genomes of representatives of evolutionarily ancient (basal) groups of vertebrates and to understand the contribution of this new information to our ideas about the early evolutionary history of the vertebrate genotype. According to new data, the divergence and observed significant differences in the morphological plans of the two evolutionary lineages of vertebrates could be ensured by different scenarios of polyploidization of their genomes.

The basic knowledge about the development and formation of the body plan of vertebrates (Vertebrata) was obtained while working with traditional and generally accepted model objects, such as the embryos of mice (Muridae), chickens (Gallus), bony fish (Teleostei), clawed frogs (Xenopus). At the same time, for understanding the evolutionary history of vertebrates, studies of so-called “non-model” objects are of particular value. These animals represent important phylogenetic branches on the evolutionary tree of vertebrates, but do not possess a set of qualities that make them convenient for laboratory work. First of all, such objects include the most ancient living representatives of the taxa, basally diverged from the common evolutionary trunk. These are cyclostomes (Cyclostomata) in the case of vertebrates in general, cartilaginous fish (Chondrichthyes) in the case of gnathostomes (Gnathostomata) and bone ganoids (Holostei) as representatives of new-finned fish (Neopterygii). The research value of these ancient groups lies in the fact that morphological features that are later characteristic of the entire taxon appear in their evolution for the first time, for example, the telencephalon and neural crest cells in cyclostomes, paired fins and the jaws in cartilaginous fish. Representatives of these groups provide an opportunity to study the evolutionary premieres of individual structures and features and mechanisms that ensured their emergence. This article presents an overview of studies of whole-genome duplications at the early stages of vertebrate evolution and the emergence of a unique forebrain region, the telencephalon, which we studied on lampreys, as the most ancient representatives of vertebrates available for laboratory research today.

Vertebrates, often considered as the most sophisticatedly organized animals, have a number of unique morphological features that ensured their evolutionary stability and success. Many have argued that the genetic basis for these innovations was provided by the genome duplications occurred at the early stages of vertebrate evolution. One of the important results of genome duplication is the emergence of additional copies of regulatory genes. Having been removed from the constraining pressure of natural selection, these copies acquired an opportunity to modify their structure and functions, influencing the organism’s development and morphology. The consequence of ancient genomic duplications is, for example, that about 35% of human genes are represented by at least two homologous copies. The idea of genome duplications occurred at the early stages of vertebrate evolution was first proposed in 1970, but questions about the number, the scale (whole-genome or local) and the evolutionary timing of these duplications are still actively debated. In recent years (2018–2020), due to the rapid development of methods for processing of big data of high-throughput genome sequencing in different lineages of vertebrates and their closest relatives, cephalochordates and tunicates, a number of comparative studies aimed at identifying groups of syntheny in genomes of different evolutionary lines and reconstruction of ancestral vertebrate chromosomes were obtained. As a result, several models describing putative scenarios of genome duplications in vertebrate evolution have been proposed. In parallel, detailed laboratory studies focused on the expression and functional properties of different families of regulatory genes were performed for representatives of several vertebrate groups. In these studies, a plethora of new information on the molecular mechanisms of the embryonic development in hitherto poorly studied representatives of the evolutionarily ancient vertebrate linages (e.g. cyclostomes, cartilaginous fishes and sturgeons) have been collected. In this review, we consider modern concepts of the mechanisms and consequences of genomic duplications in the light of recent experimental data and currently proposed models of vertebrate genome evolution.

In the context of studying the mechanisms of early development of the vertebrate nervous system, agnatha (cyclostomata) is a unique group of animals. Since the branches of agnatha (jawless) and gnathostomata were separated at the earliest stages of vertebrate evolution, the genes of lampreys, as representatives of jawless, could most likely retain ancient expression patterns characteristic of the ancestors of vertebrates. Previous studies of the noggin family of gnathostomata have shown that all three vertebrate Noggin genes (Noggin1, Noggin2, and Noggin4) differ in expression pattern and functional properties. It was shown that Noggin genes are involved in a wide range of ontogenetic processes, including the early development of vertebrate anterior head structures and brain regions. In this paper, the expression patterns of four Noggin genes in the head structures of larvae of the European river lamprey Lampetra fluviatilis at the early stages of development are analyzed. The analysis finds a lot of similarities in the gene expression patterns of Noggin in lampreys with the expression of their homologues in gnathostomata. The NogginB gene, showing a high level of expression in the terminal part of the brain (telencephalon), can be used as a specific marker of this unique part of the vertebrate brain, which firstly appears in the evolution of gnathostomata.

The telencephalon, which provides the highest forms of nervous activity in humans and other animals, is one of the most important innovations of vertebrates. Although this part of the brain has been described in all living vertebrates, its evolutionary origin is still poorly understood. This article discusses one of the possible approaches to studying the expression and functional properties of genes that regulate the early development of the forebrain in cyclostomes (lampreys) as the most archaic representatives of vertebrates. The results of studies of genes such as Anf, FoxG1, and genes of the Noggin family are described.

The emergence of the telencephalon as a forebrain part with a complex structure is one of the most important aromorphoses in vertebrate evolution. The telencephalon developed and improved in evolution to allow higher nervous activity forms observed in animals and humans. A telencephalic anlage is separated at the earliest stages of vertebrate ontogenesis, when the anterior part of the neural tube differentiates into three cerebral vesicles: the prozencephalon as an anlage of the future forebrain, the mesencephalon as the future midbrain, and the rhombencephalon as the future hindbrain. The forebrain further differentiates to form the telencephalon and the diencephalon. The development of brain structures and regions is modulated by the expression of certain regulatory genes, which code for transcription factors and signaling molecules. Problems of the evolutionary origin and ontogenesis of the telencephalon are still poorly understood at the molecular level, although they are among central problems of modern developmental biology. Recent studies of the evolutionary mechanisms responsible for the emergence of the telencephalon in vertebrates have paid much attention to cyclostomes (lampreys and hagfishes) as the most evolutionarily ancient vertebrate groups and Tunicata (ascidians) and Cephalochordata (lancelets) as the closest relatives of vertebrates. Cyclostomes are of particular interest because they were the first in evolution to have the telencephalon as a separate morphological structure and because they might preserve the expression patterns and regulatory mechanisms characteristic of vertebrate ancestors. The review summarizes and analyzes the data accumulated in recent years from studies of the genetic mechanisms of early telencephalon development in lower vertebrates and searches for telencephalon homologs in two vertebrate-related chordate groups, Cephalochordata and Tunicata.

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