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In this study, we comprehensively searched for fish-specific genes in gnathostomes that contribute to development of the fin, a fish-specific trait. Many previous reports suggested that animal group-specific genes are often important for group-specific traits. Clarifying the roles of fish-specific genes in fin development of gnathostomes, for example, can help elucidate the mechanisms underlying the formation of this trait. We first identified 91 fish-specific genes in gnathostomes by comparing the gene repertoire in 16 fish and 35 tetrapod species. RNA-seq analysis narrowed down the 91 candidates to 33 genes that were expressed in the developing pectoral fin. We analyzed the functions of approximately half of the candidate genes by loss-of-function analysis in zebrafish. We found that some of the fish-specific and fin development-related genes, including fgf24 and and1/and2, play roles in fin development. In particular, the newly identified fish-specific gene qkia is expressed in the developing fin muscle and contributes to muscle morphogenesis in the pectoral fin as well as body trunk. These results indicate that the strategy of identifying animal group-specific genes is functional and useful. The methods applied here could be used in future studies to identify trait-associated genes in other animal groups., (© 2024 The Authors. Development, Growth & Differentiation published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Developmental Biologists.)

In the initiation of cardiogenesis, the heart primordia transform from bilateral flat sheets of mesoderm into an elongated midline tube. Here, we discover that this rapid architectural change is driven by actomyosin-based oriented cell rearrangement and resulting dynamic tissue reshaping (convergent extension, CE). By labeling clusters of cells spanning the entire heart primordia, we show that the heart primordia converge toward the midline to form a narrow tube, while extending perpendicularly to rapidly lengthen it. Our data for the first time visualize the process of early heart tube formation from both the medial (second) and lateral (first) heart fields, revealing that both fields form the early heart tube by essentially the same mechanism. Additionally, the adjacent endoderm coordinately forms the foregut through previously unrecognized movements that parallel those of the heart mesoderm and elongates by CE. In conclusion, our data illustrate how initially two-dimensional flat primordia rapidly change their shapes and construct the three-dimensional morphology of emerging organs in coordination with neighboring morphogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

To understand the developmental mechanism that determines limb size and the consequent limb-to-trunk proportions in the tetrapod body, we investigated the role of the paraxial mesoderm in the specification of the limb and flank fields in the chick embryo. We found that the paraxial mesoderm subjacent to the limb field can affect the size of the limb bud along the anterior-posterior and proximal-distal axes. We also found that the paraxial mesoderm subjacent to the flank plays roles in suppressing the emergence and growth of the limb bud and in promoting flank-specific apoptosis in the lateral plate mesoderm. Our results suggest that signals from the paraxial mesoderm specify the limb and flank fields in the competent lateral plate mesoderm., (Copyright © 2011 Wiley-Liss, Inc.)

The identities of the digits of the avian forelimb are disputed. Whereas paleontological findings support the position that the digits correspond to digits one, two, and three, embryological evidence points to digit two, three, and four identities. By using transplantation and cell-labeling experiments, we found that the posteriormost digit in the wing does not correspond to digit four in the hindlimb; its progenitor segregates early from the zone of polarizing activity, placing it in the domain of digit three specification. We suggest that an avian-specific shift uncouples the digit anlagen from the molecular mechanisms that pattern them, resulting in the imposition of digit one, two, and three identities on the second, third, and fourth anlagens.

Every vertebrate species has its own unique morphology adapted to a particular lifestyle and habitat. Limbs and fins are strikingly diversified in size, shape, and position along the body axis. This diversity in morphology suggests the existence of a variety of embryonic developmental programs. However, comparisons of various embryos suggest common mechanisms underlying limb/fin formation. Here, we report the existence of continuous stripes of competency for appendage formation along the dorsal midline and the lateral trunk of all of the major jawed vertebrate (gnathostome) groups. We also show that the developing fin buds of cartilaginous fish share a mechanism of anterior-posterior axis formation as well as an shh (sonic hedgehog) expression domain in the posterior bud. We hypothesize a continuous distribution of competent stripes that represents the common developmental program at the root of appendage formation in gnathostomes. This schema would have permitted subsequent divergence into various levels of limbs/fins in each animal group.

The autopod, including the mesopodium and the acropodium, is the most distal part of the tetrapod limb, and developmental mechanisms of autopod formation serve as a model system of pattern formation during development. Cartilage rudiments of the autopod develop after proximal elements have differentiated. The autopod region is marked by a change in the expression of two homeobox genes: future autopod cells are first Hoxa11/Hoxa13-double-positive and then Hoxa13-single-positive. The change in expression of these Hox genes is controlled by upstream mechanisms, including the retinoic acid pathway, and the expression of Hoxa13 is connected to downstream mechanisms, including the autopod-specific cell surface property mediated by molecules, including cadherins and ephrins/Ephs, for cell-to-cell communication and recognition. Comparative analyses of the expression of Hox genes in fish fins and tetrapod limb buds support the notion on the origin of the autopod in vertebrates. This review will focus on the cellular and molecular regulation of the formation of the autopod during development and evolutionary developmental aspects of the origin of the autopod.

Tetrapod limbs, forelimbs and hindlimbs, emerge as limb buds during development from appropriate positions along the rostro-caudal axis of the main body. In this study, tissue interactions by which rostro-caudal level-specific limb initiation is established were analyzed. The limb bud originates from the lateral plate located laterally to the paraxial mesoderm, and we obtained evidence that level-specific tissue interactions between the paraxial mesoderm and the lateral plate mesoderm are important for the determination of the limb-type-specific gene expression and limb outgrowth. When the wing-level paraxial mesoderm was transplanted into the presumptive leg region, the wing-level paraxial mesoderm upregulated the expression of Tbx5, a wing marker gene, and down regulated the expression of Tbx4 and Pitx1, leg marker genes, in the leg-level lateral plate. The wing-level paraxial mesoderm relocated into the leg level also inhibited outgrowth of the hindlimb bud and down regulated Fgf10 and Fgf8 expression, demonstrating that the wing-level paraxial mesoderm cannot substitute for the function of the leg-level paraxial mesoderm in initiation and outgrowth of the hindlimb. The paraxial mesoderm taken from the neck- and flank-level regions also had effects on Tbx5/Tbx4 expression with different efficiencies. These findings suggest that the paraxial mesoderm has level-specific abilities along the rostro-caudal axis in the limb-type-specific mechanism for limb initiation.

Positioning of the limb is one of the important events for limb development. In the early stage of embryogenesis, the lateral plate mesoderm splits into two layers and the dorsal layer (the somatic mesoderm) gives rise to a series of distinct structures along the rostrocaudal axis, including the forelimb bud, flank body wall, and hindlimb bud. To determine whether positional information in the somatic mesoderm for regionalization along the rostrocaudal axis is also inherited by the ventral layer of the lateral plate mesoderm (the splanchnic mesoderm), experiments in which the splanchnic mesoderm was transplanted under the ectoderm in an in ovo chick system were carried out. Transplantation of the wing-, flank-, and leg-level splanchnic mesoderm resulted in the formation of wings, nothing, and legs, respectively. These results suggest that the splanchnic mesoderm possesses the ability to form limbs and that the ability differs according to the position along the rostrocaudal axis. The position-specific ability to form limbs suggests that there are some domains involved in the formation of position-specific structures in the digestive tract derived from the splanchnic mesoderm, and results of cell fate tracing supported this possibility. In contrast, analysis of shh expression suggested that the anteroposterior polarity in the limb region seems not to be inherited by the splanchnic mesoderm. We propose that the positioning of limb buds is specified and determined in the very early stage of development of the lateral plate mesoderm before splitting and that the polarity in a limb bud is established after the splitting of the mesoderm., (Copyright 2005 Wiley-Liss, Inc)

In a differential display screening for genes regulated by retinoic acid in the developing chick limb bud, we have isolated a novel gene, termed rigf, retinoic-acid induced growth factor, that encodes a protein belonging to the vascular endothelial growth factor (VEGF) family. Rigf transcripts were found in the posterior region of the limb bud in a region-specific manner as well as in other embryonic tissues and regions, including the notochord, head and trunk mesenchyme, retinal pigment epithelium, and branchial arches. Several manipulations revealed that retinoic acid and sonic hedgehog signaling pathways regulate rigf expression in the limb bud. VEGF family members, which promote the migration, differentiation and proliferation of endothelial cells in both blood and lymphatic vessels, are important factors for the formation of blood and lymphatic vasculatures during development. We demonstrated that the anterior border of the rigf expression domain in the limb bud corresponds with the position of the primary central artery (the subclavian artery in the forelimb), which is a main artery for supplying blood to the limb. These observations taken together with results from some experimental manipulations suggest that the limb tissue attracts blood vessels into the limb bud and that rigf is involved in the pattern formation of blood vessels in the limb.

Limb-type-specific expression of Tbx5/Tbx4 plays a key role in drawing distinction between a forelimb and a hindlimb. Here, we show insights into specification and determination during commitment of limb-type identity, in particular that median tissues regulate Tbx expressions. By using the RT-PCR technique on chick embryos, the onset of specific Tbx5/Tbx4 expression in the wing/leg region was estimated to be stage 13. Specification of the limb-type identity is thought to occur before stage 9, since all explants from stage 9 through 14 expressed the intrinsic Tbx gene autonomously in a simple culture medium. The results of transplantation experiments revealed that axial structures medial to the lateral plate mesoderm at the level of the wing region are capable of transforming leg identity to wing identity, suggesting that a factor(s) from the median tissues is involved in the limb-type determination. Nevertheless, the transplanted wing region was not converted to leg identity. The results of the transplantation experiments also suggested that wing-type identity is determined much earlier than is leg-type identity. Finally, we also found that inhibitory effects of median tissues mediate the specific expression of Tbx5/Tbx4 in the presumptive wing/leg region. We propose a model for limb-type identification in which inhibitory regulation is involved in restricting one Tbx gene expression by masking the other Tbx expression there.

Emerging developmental studies contribute to our understanding of vertebrate evolution because changes in the developmental process and the genes responsible for such changes provide a unique way for evaluating the evolution of morphology. Endoskeletal limbs, the locomotor organs that are unique to vertebrates, are a popular model system in the fields of palaeontology and phylogeny because their structure is highly visible and their bony pattern is easily preserved in the fossil records. Similarly, limb development has long served as an excellent model system for studying vertebrate pattern formation. In this review, the evolution of vertebrate limb development is examined in the light of the latest knowledge, viewpoints and hypotheses.

To clarify the roles of fibroblast growth factors (FGF) in limb cartilage pattern formation, the effects of various FGF on recombinant limbs that were composed of dissociated and reaggregated mesoderm and ectodermal jackets were examined. Fibroblast growth factor-soaked beads were inserted just under the apical ectodermal ridge (AER) of recombinant limbs and the recombinant limbs were grafted and allowed to develop. Control recombinant limbs without FGF beads formed one or two cartilage elements. Recombinants with FGF-4 beads formed up to five cartilage elements, which were aligned along the anteroposterior (AP) axis. Each cartilage element showed digit-like segmentation. In contrast, recombinants with FGF-2 beads showed formation of multiple thick and unsegmented cartilage rods, which elongated inside and outside the AP plane from the distal end of the recombinants. Recombinants with FGF-8 beads formed a truncated cartilage pattern and recombinants with FGF-10 beads formed a cartilage pattern similar to that of the control recombinants. The expression of the Fgf-8, Msx-1 and Hoxa-13 genes in the developing recombinant limbs were examined. FGF-4 induced extension of the length of the Fgf-8-positive epidermis, or AER, along the AP axis 5 days after grafting, at which time the digits are specified. FGF-2 induced expansion of the Msx-1-positive area, first in the proximal direction and then along the dorsoventral axis. The functions of these FGF in recombinant and normal limb patterning are discussed in this paper.

A young tadpole of an anuran amphibian can completely regenerate an amputated limb, and it exhibits an ontogenetic decline in the ability to regenerate its limbs. However, whether mesenchymal or epidermal tissue is responsible for this decrease of the capacity remains unclear. Moreover, little is known about the molecular interactions between these two tissues during regeneration. The results of this study showed that fgf-10 expression in the limb mesenchymal cells clearly corresponds to the regenerative capacity and that fgf-10 and fgf-8 are synergistically reexpressed in regenerating blastemas. However, neither fgf-10 nor fgf-8 is reexpressed after amputation of a nonregenerative limb. Nevertheless, nonregenerative epidermal tissue can reexpress fgf-8 under the influence of regenerative mesenchyme, as was demonstrated by experiments using a recombinant limb composed of regenerative limb mesenchyme and nonregenerative limb epidermis. Taken together, our data demonstrate that the regenerative capacity depends on mesenchymal tissue and suggest that fgf-10 is likely to be involved in this capacity., (Copyright 2000 Academic Press.)

In vertebrates visceral asymmetry is conserved along the left-right axis within the body. Only a small percentage of randomization (situs ambiguus), or complete reversal (situs inversus) of normal internal organ position and structural asymmetry is found in humans. A breakdown in left-right asymmetry is occasionally associated with severe malformations of the organs, clearly indicating that the regulated asymmetric patterning could have an evolutionary advantage over allowing random placement of visceral organs. Genetic, molecular and cell transplantation experiments in humans, mice, zebrafish, chick and Xenopus have advanced our understanding of how initiation and establishment of left-right asymmetry occurs in the vertebrate embryo. In particular, the chick embryo has served as an extraordinary animal model to manipulate genes, cells and tissues. This chick model system has enabled us to reveal the genetic pathways that occur during left-right development. Indeed, genes with asymmetric expression domains have been identified and well characterized using the chick as a model system. The present review summarizes the molecular and experimental studies employed to gain a better understanding of left-right asymmetry pattern formation from the first split of symmetry in embryos, to the exhibition of asymmetric morphologies in organs.

Asymmetric expression of Sonic hedgehog (Shh) in Hensen's node of the chicken embryo plays a key role in the genetic cascade that controls left-right asymmetry, but its involvement in left-right specification in other vertebrates remains unclear. We show that mouse embryos lacking Shh display a variety of laterality defects, including pulmonary left isomerism, alterations of heart looping, and randomization of axial turning. Expression of the left-specific gene Lefty-1 is absent in Shh(-/-) embryos, suggesting that the observed laterality defects could be the result of the lack of Lefty-1. We also demonstrate that retinoic acid (RA) controls Lefty-1 expression in a pathway downstream or parallel to Shh. Further, we provide evidence that RA controls left-right development across vertebrate species. Thus, the roles of Shh and RA in left-right specification indeed are conserved among vertebrates, and the Shh and RA pathways converge in the control of Lefty-1.

During vertebrate limb development, the apical ectodermal ridge (AER) plays a vital role in both limb initiation and distal outgrowth of the limb bud. In the early chick embryo the prelimb bud mesoderm induces the AER in the overlying ectoderm. However, the direct inducer of the AER remains unknown. Here we report that FGF7 and FGF10, members of the fibroblast growth factor family, are the best candidates for the direct inducer of the AER. FGF7 induces an ectopic AER in the flank ectoderm of the chick embryo in a different manner from FGF1, -2, and -4 and activates the expression of Fgf8, an AER marker gene, in a cultured flank ectoderm without the mesoderm. Remarkably, FGF7 and FGF10 applied in the back induced an ectopic AER in the dorsal median ectoderm. Our results suggest that FGF7 and FGF10 directly induce the AER in the ectoderm both of the flank and of the dorsal midline and that these two regions have the competence for AER induction. Formation of the AER of the dorsal median ectoderm in the chick embryo is likely to appear as a vestige of the dorsal fin of the ancestors., (Copyright 1999 Academic Press.)

T-box genes are conserved in all animal species. We have identified two members of the T-box gene family from the zebrafish, Danio rerio. Zf-tbr1 and zf-tbx3 share high amino acid identity with human, murine, chick and Xenopus orthologs and are expressed in specific regions during zebrafish development., (Copyright 1998 Elsevier Science Ireland Ltd.)

The handedness of visceral organs is conserved among vertebrates and is regulated by asymmetric signals relayed by molecules such as Shh, Nodal and activin. The gene Pitx2 is expressed in the left lateral plate mesoderm and, subsequently, in the left heart and gut of mouse, chick and Xenopus embryos. Misexpression of Shh and Nodal induces Pitx2 expression, whereas inhibition of activin signalling blocks it. Misexpression of Pitx2 alters the relative position of organs and the direction of body rotation in chick and Xenopus embryos. Changes in Pitx2 expression are evident in mouse mutants with laterality defects. Thus, Pitx2 seems to serve as a critical downstream transcription target that mediates left-right asymmetry in vertebrates.