Tohru Yano

Trunk exoskeleton in teleosts is mesodermal in origin

The vertebrate mineralized skeleton is known to have first emerged as an exoskeleton that extensively covered the fossil jawless fish. The evolutionary origin of this exoskeleton has long been attributed to the emergence of the neural crest, but experimental evaluation for this is still poor. Here we determine the embryonic origin of scales and fin rays of medaka (teleost trunk exoskeletons) by applying long-term cell labelling methods, and demonstrate that both tissues are mesodermal in origin. Neural crest cells, however, fail to contribute to these tissues. This result suggests that the trunk neural crest has no skeletogenic capability in fish, instead highlighting the dominant role of the mesoderm in the evolution of the trunk skeleton. This further implies that the role of the neural crest in skeletogenesis has been predominant in the cephalic region from the early stage of vertebrate evolution.

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.

The making of differences between fins and limbs

‘Evo‐devo’, an interdisciplinary field based on developmental biology, includes studies on the evolutionary processes leading to organ morphologies and functions. One fascinating theme in evo‐devo is how fish fins evolved into tetrapod limbs. Studies by many scientists, including geneticists, mathematical biologists, and paleontologists, have led to the idea that fins and limbs are homologous organs; now it is the job of developmental biologists to integrate these data into a reliable scenario for the mechanism of fin‐to‐limb evolution. Here, we describe the fin‐to‐limb transition based on key recent developmental studies from various research fields that describe mechanisms that may underlie the development of fins, limb‐like fins, and limbs.

The autopod : Its formation during limb development

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.

Evolution of the fish heart by sub/neofunctionalization of an elastin gene

The evolution of phenotypic traits is a key process in diversification of life. However, the mechanisms underlying the emergence of such evolutionary novelties are largely unknown. Here we address the origin of bulbus arteriosus (BA), an organ of evolutionary novelty seen in the teleost heart outflow tract (OFT), which sophisticates their circulatory system. The BA is a unique organ that is composed of smooth muscle while the OFTs in other vertebrates are composed of cardiac muscle. Here we reveal that the teleost-specific extracellular matrix (ECM) gene, elastin b, was generated by the teleost-specific whole-genome duplication and neofunctionalized to contribute to acquisition of the BA by regulating cell fate determination of cardiac precursor cells into smooth muscle. Furthermore, we show that the mechanotransducer yap is involved in this cell fate determination. Our findings reveal a mechanism of generating evolutionary novelty through alteration of cell fate determination by the ECM.

Evidence for an amphibian sixth digit

IntroductionDespite the great diversity in digit morphology reflecting the adaptation of tetrapods to their lifestyle, the number of digits in extant tetrapod species is conservatively stabilized at five or less, which is known as the pentadactyl constraint.ResultsWe found that an anuran amphibian species, Xenopus tropicalis (western clawed frog), has a clawed protrusion anteroventral to digit I on the foot. To identify the nature of the anterior-most clawed protrusion, we examined its morphology, tissue composition, development, and gene expression. We demonstrated that the protrusion in the X. tropicalis hindlimb is the sixth digit, as is evident from anatomical features, development, and molecular marker expression.ConclusionIdentification of the sixth digit in the X. tropicalis hindlimb strongly suggests that the prehallux in other Xenopus species with similar morphology and at the same position as the sixth digit is also a vestigial digit. We propose here that the prehallux seen in various species of amphibians generally represents a rudimentary sixth digit.

Molecular developmental mechanism in polypterid fish provides insight into the origin of vertebrate lungs

The lung is an important organ for air breathing in tetrapods and originated well before the terrestrialization of vertebrates. Therefore, to better understand lung evolution, we investigated lung development in the extant basal actinopterygian fish Senegal bichir (Polypterus senegalus). First, we histologically confirmed that lung development in this species is very similar to that of tetrapods. We also found that the mesenchymal expression patterns of three genes that are known to play important roles in early lung development in tetrapods (Fgf10, Tbx4, and Tbx5) were quite similar to those of tetrapods. Moreover, we found a Tbx4 core lung mesenchyme-specific enhancer (C-LME) in the genomes of bichir and coelacanth (Latimeria chalumnae) and experimentally confirmed that these were functional in tetrapods. These findings provide the first molecular evidence that the developmental program for lung was already established in the common ancestor of actinopterygians and sarcopterygians.

Evolutionary Changes in the Developmental Origin of Hatching Gland Cells in Basal Ray-Finned Fishes

Hatching gland cells (HGCs) originate from different germ layers between frogs and teleosts, although the hatching enzyme genes are orthologous. Teleostei HGCs differentiate in the mesoendodermal cells at the anterior end of the involved hypoblast layer (known as the polster) in late gastrula embryos. Conversely, frog HGCs differentiate in the epidermal cells at the neural plate border in early neurula embryos. To infer the transition in the developmental origin of HGCs, we studied two basal ray-finned fishes, bichir (Polypterus) and sturgeon. We observed expression patterns of their hatching enzyme (HE) and that of three transcription factors that are critical for HGC differentiation: KLF17 is common to both teleosts and frogs; whereas FoxA3 and Pax3 are specific to teleosts and frogs, respectively. We then inferred the transition in the developmental origin of HGCs. In sturgeon, the KLF17, FoxA3, and HE genes were expressed during the tailbud stage in the cell mass at the anterior region of the body axis, a region corresponding to the polster in teleost embryos. In contrast, the bichir was suggested to possess both teleost- and amphibian-type HGCs, i.e. the KLF17 and FoxA3 genes were expressed in the anterior cell mass corresponding to the polster, and the KLF17, Pax3 and HE genes were expressed in dorsal epidermal layer of the head. The change in developmental origin is thought to have occurred during the evolution of basal ray-finned fish, because bichir has two HGCs, while sturgeon only has the teleost-type.

Fins and Limbs: Emergence of Morphological Differences

Paired appendages (fins and limbs) are regarded as distinct morphologies by classification of skeletal patterns. On the basis of sequential orientation and articulation of tetrapod limb bones, we can understand that stylopodial/zeugopodial skeletal elements are present in an extinct and extant basal sarcopterygian (coelacanth and lungfish) fin and that only nonhomologous radial bones exist in a zebrafish fin. From these phylogenetic views, morphological differences between fins and limbs and paleontological discoveries of limb-like fins of basal sarcopterygians emphasize both the homologous skeletal elements and tetrapodomorph evolution. During embryogenesis, on the other hand, initial fin development requires apical ectodermal ridge (AER) signals as does tetrapod limb development, and then the AER itself starts to transform into a fin-specific structure, the apical fold (AF). HoxD genes are involved in fish fin development as in tetrapod limb development, but the resultant skeletal patterns of fins are very different from those of limbs as a result of differences in regulation of genes such as HoxD. From these developmental aspects, we can understand that both fins and limbs develop by common mechanisms, including fibroblast growth factors (FGFs) from the AER and Hox genes, and that alteration of basic mechanisms by heterochronic/heterometric change in expression of AER/AF signals and Hox gives rise to morphological differences among paired appendages. In this chapter, we describe homology and difference in several research fields (genome commonality/difference, developmental commonality/difference, and anatomical or paleontological correspondence/difference) and especially explain a scenario of fin-to-limb evolution.

09-P097 Apical fold morphogenesis in zebrafish fin; differences from tetrapod limb development

The inner ear develops from a simple epithelial vesicle that gives rise to the sensory hair cells, neuroblasts, secretory cells and other non-sensory tissue of the inner ear. In the zebrafish embryo, sensory hair cells begin to differentiate at the anterior and posterior ends of the otic vesicle, forming two distinct and separate sensory patches or maculae. Otic neuroblasts arise from an anteroventral region of the epithelium between the two maculae, and subsequently delaminate and coalesce to form the statoacoustic ganglion beneath the ear. We are examining the roles of various transcription factors in patterning ventral regions of the zebrafish otic epithelium. We show that eya1/six1, tbx1 and otx1 regulate the spatial extent of the neurogenic domain in the otic vesicle and are required for the correct spacing of the sensory patches. In the vgo/tbx1 mutant and otx1 morphant at 26–27 h post-fertilisation, expression of otx1-like and gsc is lost in the otic vesicle, sensory patch spacing is reduced, the neurogenic domain expands laterally, and the developing statoacoustic ganglion is enlarged. In contrast, dog/eya1 mutants and six1 morphants show a reciprocal phenotype: expression of tbx1, otx1, otx1-like and gsc in the otic vesicle is expanded, sensory patch spacing is increased, and neurogenesis is severely suppressed. We will discuss the regulatory interactions between these transcription factors and their requirements in patterning ventral regions of the zebrafish otic epithelium.