Evolution of touch and proprioception of the limbs: Insights from fish and humans

The function of the hands is inextricably linked to cutaneous mechanosensation, both in touch and in how hand movement and posture (proprioception) are controlled. The structure and behavior of hands and distal forelimbs of other vertebrates have been evolutionarily shaped by these mechanosensory functions. The distal forelimb of tetrapod vertebrates is homologous to the pectoral fin rays and membrane of fishes. Fish fins demonstrate similar mechanosensory abilities to hands and other distal tetrapod forelimbs in touch and proprioception. These results indicate that vertebrates were using the core mechanosensory inputs, such as fast adapting and slow adapting nerve responses, to inform fin and limb function and behavior before their diversification in fish and tetrapod lineages.     

The tetrapod limb: a hypothesis on its origin

A wrist joint and structures typical of the hand, such as digits, however, are absent in [Eustenopteron] (Andrews and Westoll, '68, p 240). Great changes must have been undergone during evolution of the ankle joint; the small number of large bones in the fin must somehow have developed into a large number of small bones, and it is very difficult to draw homologies in this region, or even be certain of what is being compared (Andrews and Westoll, '68, p 268). The tetrapod limb is one of the major morphological adaptations that facilitated the transition from an aquatic to a terrestrial lifestyle in vertebrate evolution. We review the paleontological evidence for the fin-limb transition and conclude that the innovation associated with evolution of the tetrapod limb is the zeugopodial-mesopodial transition, i.e., the evolution of the developmental mechanism that differentiates the distal parts of the limb (the autopodium, i.e., hand or foot) from the proximal parts. Based on a review of tetrapod limb and fish fin development, we propose a genetic hypothesis for the origin of the autopodium. In tetrapods the genes Hoxa-11 and Hoxa-13 have locally exclusive expression domains along the proximal-distal axis of the limb bud. The junction between the distal limit of Hoxa-11 expression and of the proximal limit of Hoxa-13 expression is involved in establishing the border between the zeugopodial and autopodial anlagen. In zebrafish, the expression domains of these genes are overlapping and there is no evidence for an autopodial equivalent in the fin skeleton. We propose that the evolution of the derived expression patterns of Hoxa-11 and Hoxa-13 may be causally involved in the origin of the tetrapod limb.     

Expression of Hoxa-11 and Hoxa-13 in the pectoral fin of a basal ray-finned fish, Polyodon spathula: implications for the origin of tetrapod limbs

Summary Paleontological and anatomical evidence suggests that the autopodium (hand or foot) is a novel feature that distinguishes limbs from fins, while the upper and lower limb (stylopod and zeugopod) are homologous to parts of the sarcopterygian paired fins. In tetrapod limb development Hoxa-11 plays a key role in differentiating the lower limb and Hoxa-13 plays a key role in differentiating the autopodium. It is thus important to determine the ancestral functions of these genes in order to understand the developmental genetic changes that led to the origin of the tetrapod autopodium. In particular it is important to understand which features of gene expression are derived in tetrapods and which are ancestral in bony fishes. To address these questions we cloned and sequenced the Hoxa-11 and Hoxa-13 genes from the North American paddlefish, Polyodon spathula, a basal ray-finned fish that has a pectoral fin morphology resembling that of primitive bony fishes ancestral to the tetrapod lineage. Sequence analysis of these genes shows that they are not orthologous to the duplicated zebrafish and fugu genes. This implies that the paddlefish has not duplicated its HoxA cluster, unlike zebrafish and fugu. The expression of Hoxa-11 and Hoxa-13 in the pectoral fins shows two main phases: an early phase in which Hoxa-11 is expressed proximally and Hoxa-13 is expressed distally, and a later phase in which Hoxa-11 and Hoxa-13 broadly overlap in the distal mesenchyme of the fin bud but are absent in the proximal fin bud. Hence the distal polarity of Hoxa-13 expression seen in tetrapods is likely to be an ancestral feature of paired appendage development. The main difference in HoxA gene expression between fin and limb development is that in tetrapods (with the exception of newts) Hoxa-11 expression is suppressed by Hoxa-13 in the distal limb bud mesenchyme. There is, however, a short period of limb bud development where Hoxa-11 and Hoxa-13 overlap similarly to the late expression seen in zebrafish and paddlefish. We conclude that the early expression pattern in tetrapods is similar to that seen in late fin development and that the local exclusion by Hoxa-13 of Hoxa-11 from the distal limb bud is a derived feature of limb developmental regulation.     

Functional analysis of limb enhancers in the developing fin

Despite diverging ～365 million years ago, tetrapod limbs and pectoral fins express similar genes that could be regulated by shared regulatory elements. In this study, we set out to analyze the ability of enhancers to maintain tissue specificity in these two divergent structures. We tested 22 human sequences that were previously reported as mouse limb enhancers for their enhancer activity in zebrafish (Danio rerio). Using a zebrafish enhancer assay, we found that 10/22 (45 %) were positive for pectoral fin activity. Analysis of the various criteria that correlated with positive fin activity found that both spatial limb activity and evolutionary conservation are not good predictors of fin enhancer activity. These results suggest that zebrafish enhancer assays may be limited in detecting human limb enhancers, and this limitation does not improve by the use of limb spatial expression or evolutionary conservation.     

Pectoral fins of rhizodontids and the evolution of pectoral appendages in the tetrapod stem-group

The Rhizodontida are an extinct order of large, predatory, lobe-finned fishes, found world-wide in Devonian and Carboniferous freshwater deposits. They are thought to be basal members of the tetrapod lineage. In this paper the pectoral fin skeleton of Rhizodus hibberti , a derived member of the group, is described in detail for the first time. It shows that muscular processes of the humerus (the deltoid and supinator processes) may have appeared later in tetrapod evolution than previously thought. The rhizodontids share an unusual fin-ray morphology, with highly elongated basal hemisegments, overlapping extensively with the endoskeleton. This morphology raises the possibility that segmentation and endoskeletal overlap share some common upstream genetic control during lepidotrichial development. The relative size of the lepidotrichia and the complexity of the endoskeleton does not fit with a 'clockface' model of limb developmental evolution.     

Fish fingers: digit homologues in sarcopterygian fish fins

A defining feature of tetrapod evolutionary origins is the transition from fish fins to tetrapod limbs. A major change during this transition is the appearance of the autopod (hands, feet), which comprises two distinct regions, the wrist/ankle and the digits. When the autopod first appeared in Late Devonian fossil tetrapods, it was incomplete: digits evolved before the full complement of wrist/ankle bones. Early tetrapod wrists/ankles, including those with a full complement of bones, also show a sharp pattern discontinuity between proximal elements and distal elements. This suggests the presence of a discontinuity in the proximal-distal sequence of development. Such a discontinuity occurs in living urodeles, where digits form before completion of the wrist/ankle, implying developmental independence of the digits from wrist/ankle elements. We have observed comparable independent development of pectoral fin radials in the lungfish Neoceratodus (Osteichthyes: Sarcopterygii), relative to homologues of the tetrapod limb and proximal wrist elements in the main fin axis. Moreover, in the Neoceratodus fin, expression of Hoxd13 closely matches late expression patterns observed in the tetrapod autopod. This evidence suggests that Neoceratodus fin radials and tetrapod digits may be patterned by shared mechanisms distinct from those patterning the proximal fin/limb elements, and in that sense are homologous. The presence of independently developing radials in the distal part of the pectoral (and pelvic) fin may be a general feature of the Sarcopterygii.     

Why we have (only) five fingers per hand: hox genes and the evolution of paired limbs.

Limb development has long been a model system for studying vertebrate pattern formation. The advent of molecular biology has allowed the identification of some of the key genes that regulate limb morphogenesis. One important class of such genes are the homeobox-containing, or Hox genes. Understanding of the roles these genes play in development additionally provides insights into the evolution of limb pattern. Hox gene expression patterns divide the embryonic limb bud into five sectors along the anterior/posterior axis. The expression of specific Hox genes in each domain specifies the developmental fate of that region. Because there are only five distinct Hox-encoded domains across the limb bud there is a developmental constraint prohibiting the evolution of more than five different types of digits. The expression patterns of Hox genes in modern embryonic limb buds also gives clues to the shape of the ancestral fin field from which the limb evolved, hence elucidating the evolution of the tetrapod limb.     

Early steps of paired fin development in zebrafish compared with tetrapod limb development

The development of zebrafish paired fins and tetrapod forelimbs and hindlimbs show striking similarities at the molecular level. In recent years, the zebrafish, Danio rerio has become a valuable model for the study of the development of vertebrate paired appendages and several large-scale mutagenesis screens have identified novel fin mutants. This review summarizes recent advances in research into zebrafish paired fin development and highlights features that are shared with and distinct from limb development in other main animal models.     

Development and evolution of the muscles of the pelvic fin

Locomotor strategies in terrestrial tetrapods have evolved from the utilisation of sinusoidal contractions of axial musculature, evident in ancestral fish species, to the reliance on powerful and complex limb muscles to provide propulsive force. Within tetrapods, a hindlimb-dominant locomotor strategy predominates, and its evolution is considered critical for the evident success of the tetrapod transition onto land. Here, we determine the developmental mechanisms of pelvic fin muscle formation in living fish species at critical points within the vertebrate phylogeny and reveal a stepwise modification from a primitive to a more derived mode of pelvic fin muscle formation. A distinct process generates pelvic fin muscle in bony fishes that incorporates both primitive and derived characteristics of vertebrate appendicular muscle formation. We propose that the adoption of the fully derived mode of hindlimb muscle formation from this bimodal character state is an evolutionary innovation that was critical to the success of the tetrapod transition.     

Development of limbs in urodeles and the origin of tetrapod limbs

On the basis of studies on serial sections of larval Ranodon sibiricus limbs and published data, the hypothesis of the origin of tetrapod limbs from the biserial archipterygium is proposed. The mesomeres of the central axis of the biserial fin correspond (in proximodistal direction) to the humerus, ulna, ulnare, all carpalia distalia, metacarpale 1, and phalanges of the first digit in the forelimb of caudate amphibians and to the femur, fibula, fibulare, tarsalia distalia, metatarsale 1, and phalanges of the first digit in the hind limb. The preaxial elements of the zygopodium and autopodium, which are positioned proximal to the digital arch, correspond to the preaxial rays of the biserial fin, and digits 2–5 correspond to its postaxial rays. As the fin transformed into the limb, the central axis curved preaxially, forming the digital arch and resulting in partial reduction and fusion of preaxial rays.     

Cloning and embryonic expression of six wnt genes in the medaka (Oryzias latipes) with special reference to expression of wnt5a in the pectoral fin buds

WNTs are secreted signaling molecules which control cell differentiation and proliferation. They are known to play essential roles in various developmental processes. Wnt genes have been identified in a variety of animals, and it has been shown that their amino acid sequences are highly conserved throughout evolution. To investigate the role of wnt genes during fish development from the evolutionary viewpoint, six medaka wnt genes (wnt4, wnt5a, wnt6, wnt7b, wnt8b and wnt8-like) were isolated and their embryonic expression was examined. These wnt genes were expressed in various tissues during embryonic development, and most of their expression patterns were conserved or comparable to those of other vertebrates. Thus, these wnt genes may be useful as molecular markers to investigate development and organogenesis using the medaka. Focus was on wnt5a, which was expressed in the pectoral fin buds, because its expression pattern was particularly comparable to that in tetrapod limbs. Its detailed expression pattern was further examined during pectoral fin bud development. The conservation and diversification of Wnt5a expression through the evolutionary transition from fish fins to tetrapod limbs is discussed.     

Mechanism of pectoral fin outgrowth in zebrafish development

Fins and limbs, which are considered to be homologous paired vertebrate appendages, have obvious morphological differences that arise during development. One major difference in their development is that the AER (apical ectodermal ridge), which organizes fin/limb development, transitions into a different, elongated organizing structure in the fin bud, the AF (apical fold). Although the role of AER in limb development has been clarified in many studies, little is known about the role of AF in fin development. Here, we investigated AF-driven morphogenesis in the pectoral fin of zebrafish. After the AER-AF transition at ～36 hours post-fertilization, the AF was identifiable distal to the circumferential blood vessel of the fin bud. Moreover, the AF was divisible into two regions: the proximal AF (pAF) and the distal AF (dAF). Removing the AF caused the AER and a new AF to re-form. Interestingly, repeatedly removing the AF led to excessive elongation of the fin mesenchyme, suggesting that prolonged exposure to AER signals results in elongation of mesenchyme region for endoskeleton. Removal of the dAF affected outgrowth of the pAF region, suggesting that dAF signals act on the pAF. We also found that the elongation of the AF was caused by morphological changes in ectodermal cells. Our results suggest that the timing of the AER-AF transition mediates the differences between fins and limbs, and that the acquisition of a mechanism to maintain the AER was a crucial evolutionary step in the development of tetrapod limbs.     

First discovery of a primitive coelacanth fin fills a major gap in the evolution of lobed fins and limbs

The fossil record provides unique clues about the primitive pattern of lobed fins, the precursors of digit-bearing limbs. Such information is vital for understanding the evolutionary transition from fish fins to tetrapod limbs, and it guides the choice of model systems for investigating the developmental changes underpinning this event. However, the evolutionary preconditions for tetrapod limbs remain unclear. This uncertainty arises from an outstanding gap in our knowledge of early lobed fins: there are no fossil data that record primitive pectoral fin conditions in coelacanths, one of the three major groups of sarcopterygian (lobe-finned) fishes. A new fossil from the Middle-Late Devonian of Wyoming preserves the first and only example of a primitive coelacanth pectoral fin endoskeleton. The strongly asymmetrical skeleton of this fin corroborates the hypothesis that this is the primitive sarcopterygian pattern, and that this pattern persisted in the closest fish-like relatives of land vertebrates. The new material reveals the specializations of paired fins in the modern coelacanth, as well as in living lungfishes. Consequently, the context in which these might be used to investigate evolutionary and developmental relationships between vertebrate fins and limbs is changed. Our data suggest that primitive actinopterygians, rather than living sarcopterygian fishes and their derived appendages, are the most informative comparators for developmental studies seeking to understand the origin of tetrapod limbs.     

Musculoskeletal morphology of the pelvis and pelvic fins in the lungfish Protopterus annectens

The West African lungfish (Protopterus annectens) performs benthic, pelvic fin‐driven locomotion with gaits common to tetrapods, the sister group of the lungfishes. Features of P. annectens movement are similar to those of modern tetrapods and include use of the distal region of the pelvic fin as a “foot,” use of the fin to lift the body above the substrate and rotation of the fin around the joint with the pelvis. In contrast to these similarities in movement, the pelvic fins of P. annectens are long, slender structures that are superficially very different from tetrapod limbs. Here, we describe the musculoskeletal anatomy of the pelvis and pelvic fins of P. annectens with dissection, magnetic resonance imaging, histology and 3D‐reconstruction methods. We found that the pelvis is embedded in the hypaxial muscle by a median rostral and two dorsolateral skeletal projections. The protractor and retractor muscles at the base of the pelvic fin are fan‐shaped muscles that cup the femur. The skeletal elements of the fin are serially repeating cartilage cylinders. Along the length of the fin, repeating truncated cones of muscles, the musculus circumradialis pelvici, are separated by connective tissue sheets that connect the skeletal elements to the skin. The simplicity of the protractor and retractor muscles at the base of the fin is surprising, given the complex rotational movement those muscles generate. In contrast, the series of many repeating segmental muscles along the length of the fin is consistent with the dexterity of bending of the distal limb. P. annectens can provide a window into soft‐tissue anatomy and sarcopterygian fish fin function that complements the fossil data from related taxa. This work, combined with previous behavioral examination of P. annectens, illustrates that fin morphologies that do not appear to be capable of walking can accomplish that function, and may inform the interpretation of fossil anatomical evidence. J. Morphol. 275:431–441, 2014. © 2013 Wiley Periodicals, Inc.     

Evolutionary history of vertebrate appendicular muscle

The evolutionary history of muscle development in the paired fins of teleost fish and the limbs of tetrapod vertebrates is still, to a large extent, uncertain. There has been a consensus, however, that in the vertebrate clade the ancestral mechanism of fin and limb muscle development involves the extension of epithelial tissues from the somite into the fin/limb bud. This mechanism has been documented in chondrichthyan, dipnoan, chondrostean and teleost fishes. It has also been assumed that in amniotes, in contrast, individual progenitor cells of muscles migrate from the somites into the limb buds. Neyt et al. now present the exciting finding that in zebrafishes this presumably derived mechanism involving individual cell migration, is present. They conclude, based on data on sharks, zebrafishes, chickens, quails and mice that the derived mechanism was present in the sarcopterygians. This conclusion, however, may be premature in the light of further data available in the literature, which show a highly mosaic distribution of this character in the vertebrate clade. Furthermore, a developmental mode exists that is intermediate between the supposed ancestral and derived modes in teleosts, reptiles and possibly amphibians.     

The evolution of paired appendages in vertebrates: T-box genes in the zebrafish

The presence of two sets of paired appendages is one of the defining features of jawed vertebrates. We are interested in identifying genetic systems that could have been responsible for the origin of the first set of such appendages, for their subsequent duplication at a different axial level, and/or for the generation of their distinct identities. It has been hypothesized that four genes of the T-box gene family (Tbx2–Tbx5) played important roles in the course of vertebrate limb evolution. To test this idea, we characterized the orthologs of tetrapod limb-expressed T-box genes from a teleost, Danio rerio. Here we report isolation of three of these genes, tbx2, tbx4, and tbx5. We found that their expression patterns are remarkably similar to those of their tetrapod counterparts. In particular, expression of tbx5 and tbx4 is restricted to pectoral and pelvic fin buds, respectively, while tbx2 can be detected at the anterior and posterior margins of the outgrowing fin buds. This, in combination with conserved expression patterns in other tissues, suggests that the last common ancestor of teleosts and tetrapods possessed all four of these limb-expressed T-box genes (Tbx2–Tbx5), and that these genes had already acquired, and have subsequently maintained, their gene-specific functions. Furthermore, this evidence provides molecular support for the notion that teleost pectoral and pelvic fins and tetrapod fore- and hindlimbs, respectively, are homologous structures, as suggested by comparative morphological analyses. Received: 14 July 1999 / Accepted: 4 September 1999     

Limb ossification in the Paleozoic branchiosaurid Apateon (Temnospondyli) and the early evolution of preaxial dominance in tetrapod limb development

Despite the wide range of shapes and sizes that accompany a vast variety of functions, the development of tetrapod limbs follows a conservative pattern of de novo condensation, branching, and segmentation. Development of the zeugopodium and digital arch typically occurs in a posterior to anterior sequence, referred to as postaxial dominance, with a digital sequence of 4-3-5-2-1. The only exception to this pattern in all of living Tetrapoda can be found in salamanders, which display a preaxial dominance in limb development, a de novo condensation of a basale commune (distal carpal/tarsal 1+2) and a precoccial development of digits I and II. These divergent patterns have puzzled researchers for over a century leading to various explanatory hypotheses. Despite many advances in research on tetrapod limb development, the divergent evolution of these two pathways and its causes are still not understood. Based on an extensive ontogenetic series we investigated the pattern of limb development of the 300 Ma old branchiosaurid amphibian Apateon. This revealed a preaxial dominance in limb development that was previously believed to be unique and derived for modern salamanders. The Branchiosauridae are favored as close relatives of extant salamanders in most phylogenetic hypotheses of the highly controversial origins and relationships of extant amphibians. The findings provide new insights into the evolution of developmental pathways in tetrapod limb development, the relationships of modern amphibians with possible Paleozoic antecedents, and their initial timing of divergence.