Masaki Takechi

Temporal and spatial changes in the expression pattern of multiple red and green subtype opsin genes during zebrafish development

SUMMARY Zebrafish have two red, LWS-1 and LWS-2, and four green, RH2-1, RH2-2, RH2-3 and RH2-4, opsin genes encoding photopigments with distinct absorption spectra. Occurrence of opsin subtypes by gene duplication is characteristic of fish but little is known whether the subtypes are expressed differently in the retina, either spatially or temporally. Here we show by in situ hybridization the dynamic expression patterns of the opsin subtypes in the zebrafish retina. Expression of red type opsins is initiated with the shorter-wavelength subtype LWS-2, followed by the longer-wavelength subtype LWS-1. In the adult retina, LWS-2 was expressed in the central to dorsal area and LWS-1 in the ventral and peripheral areas. Expression patterns of green type opsins were similar to those of the red type opsins. The expression started with the shortest wavelength subtype RH2-1 followed by the longer wavelength ones, and in the adult retina, the shorter wavelength subtypes (RH2-1 and RH2-2) were expressed in the central to dorsal area and longer wavelength subtypes (RH2-3 and RH2-4) in the ventral and peripheral areas. These results provide the framework for subsequent studies of opsin gene regulation and for probing functional rationale of the developmental changes by using the power of zebrafish genetics.

Evolution of the Turtle Body Plan by the Folding and Creation of New Muscle Connections

Shelling Turtles In almost all vertebrates, the shoulder girdle (scapula) lies outside the ribs. The turtle is unique in that the carapace, the dorsal part of the shell, which is formed from the ribs, encapsulates the scapula. To understand the origin of the turtle-specific body plan, Nagashima et al. (p. 193; see the cover; see the Perspective by Rieppel) compared chicken, mouse, and the Chinese soft shelled-turtle, Pelodiscus sinensis. Modern embryos were studied via whole-mount immunostaining, three-dimensional reconstructions, and with markers for early skeletal precursors and compared with previously reported fossils. Initially, embryos of the three animals share a common developmental pattern, one that is likely to have been shared with their last common ancestor. This pattern, however, is modified in the turtle by a specific folding of its body wall during embryogenesis. This folding preserves some of the connectivity between skeletal and muscle elements but also produces new connections. The turtle body plan, unique among amniotes, is based on the folding of an ancestral pattern during embryogenesis. The turtle shell offers a fascinating case study of vertebrate evolution, based on the modification of a common body plan. The carapace is formed from ribs, which encapsulate the scapula; this stands in contrast to the typical amniote body plan and serves as a key to understanding turtle evolution. Comparative analyses of musculoskeletal development between the Chinese soft-shelled turtle and other amniotes revealed that initial turtle development conforms to the amniote pattern; however, during embryogenesis, lateral rib growth results in a shift of elements. In addition, some limb muscles establish new turtle-specific attachments associated with carapace formation. We propose that the evolutionary origin of the turtle body plan results from heterotopy based on folding and novel connectivities.

Cone photoreceptor types in zebrafish are generated by symmetric terminal divisions of dedicated precursors

Significance Color vision requires multiple types of cone photoreceptors, each with peak sensitivity to a specific wavelength. How different cone types are generated in vivo is not clear. We show that there are precursor cells individually dedicated to producing a single cone type. We tracked cone genesis in vivo in transgenic zebrafish in which red cones and their progenitors express fluorescent protein driven by the thyroid hormone receptor β2 promoter. We discovered that red cones are generated by symmetric terminal divisions of a red-cone precursor. Moreover, UV, blue, and green cones also have their own dedicated precursors. Thyroid hormone receptor β2 expression in cone precursors is required to produce pure red cones, whereas expression after cell division results in cones with mixed opsins. Proper functioning of sensory systems requires the generation of appropriate numbers and proportions of neuronal subtypes that encode distinct information. Perception of color relies on signals from multiple cone photoreceptor types. In cone-dominated retinas, each cone expresses a single opsin type with peak sensitivity to UV, long (L) (red), medium (M) (green), or short (S) (blue) wavelengths. The modes of cell division generating distinct cone types are unknown. We report here a mechanism whereby zebrafish cone photoreceptors of the same type are produced by symmetric division of dedicated precursors. Transgenic fish in which the thyroid hormone receptor β2 (trβ2) promoter drives fluorescent protein expression before L-cone precursors themselves are produced permitted tracking of their division in vivo. Every L cone in a local region resulted from the terminal division of an L-cone precursor, suggesting that such divisions contribute significantly to L-cone production. Analysis of the fate of isolated pairs of cones and time-lapse observations suggest that other cone types can also arise by symmetric terminal divisions. Such divisions of dedicated precursors may help to rapidly attain the final numbers and proportions of cone types (L > M, UV > S) in zebrafish larvae. Loss- and gain-of-function experiments show that L-opsin expression requires trβ2 activity before cone differentiation. Ectopic expression of trβ2 after cone differentiation produces cones with mixed opsins. Temporal differences in the onset of trβ2 expression could explain why some species have mixed, and others have pure, cone types.

Fluorescence visualization of ultraviolet‐sensitive cone photoreceptor development in living zebrafish

Cone photoreceptor cells of fish retinae are arranged in a highly organized fashion. However, the molecular mechanisms underlying photoreceptor development and retinal pattern formation are largely unknown. Here we established transgenic lines of zebrafish carrying green fluorescent protein (GFP) cDNA with the 5.5‐kb upstream region of the ultraviolet‐sensitive cone opsin gene (SWS1). In the transgenic fish, GFP gene expression proceeded in the same spatiotemporal pattern as SWS1 in the retinae of embryos. In the adult retina, GFP expression was observed throughout the short single cone (SSC) layer where SWS1 is specifically expressed. Therefore, the transgenic fish provides an excellent genetic background to study retinal pattern formation, photoreceptor determination and differentiation, and factors regulating these processes and SSC‐specific expression of SWS1.

Ontogeny of cone photoreceptor mosaics in zebrafish.

Cone photoreceptors in fish are typically arranged into a precise, reiterated pattern known as a “cone mosaic.” Cone mosaic patterns can vary in different fish species and in response to changes in habitat, yet their function and the mechanisms of their development remain speculative. Zebrafish (Danio rerio) have four cone subtypes arranged into precise rows in the adult retina. Here we describe larval zebrafish cone patterns and investigate a previously unrecognized transition between larval and adult cone mosaic patterns. Cone positions were determined in transgenic zebrafish expressing green fluorescent protein (GFP) in their UV‐sensitive cones, by the use of multiplex in situ hybridization labelling of various cone opsins. We developed a “mosaic metric” statistical tool to measure local cone order. We found that ratios of the various cone subtypes in larval and adult zebrafish were statistically different. The cone photoreceptors in larvae form a regular heterotypic mosaic array; i.e., the position of any one cone spectral subtype relative to the other cone subtypes is statistically different from random. However, the cone spectral subtypes in larval zebrafish are not arranged in continuous rows as in the adult. We used cell birth dating to show that the larval cone mosaic pattern remains as a distinct region within the adult retina and does not reorganize into the adult row pattern. In addition, the abundance of cone subtypes relative to other subtypes is different in this larval remnant compared with that of larvae or canonical adult zebrafish retina. These observations provide baseline data for understanding the development of cone mosaics via comparative analysis of larval and adult cone development in a model species. J. Comp. Neurol. 518:4182–4195, 2010.

Evolution of oropharyngeal patterning mechanisms involving Dlx and endothelins in vertebrates.

In jawed vertebrates, the Dlx code, or nested expression patterns of Dlx genes, specify the dorsoventral polarity of pharyngeal arches, downstream of endothelin-1 (Edn-1) and its effectors, Bapx1 (Nkx3.2) and dHand (Hand2). To elucidate the evolution of the specification mechanism of the oropharyngeal skeletal system, lamprey homologs of Dlx, Edn, endothelin receptor (Ednr), Bapx1, and dHand were identified. Our analysis suggested that the Edn gene family emerged at the advent of vertebrates, and that gene duplications leading to the different Edn gnathostome subtypes (Edn1-3) occurred before the cyclostome-gnathostome split. This timing of gene duplications, giving rise to multiple subtypes, was also implied for Dlx, Ednr, Hand, and Bapx. In lamprey embryos, nested expressions of Dlx genes were not observed in pharyngeal arches, nor was any focal expression of Bapx1, known in gnathostomes to specify the jaw joint. The dHand homolog, however, was expressed more intensively ventrally, as in gnathostomes. Lamprey homologs of Edn-1 and EdnrA were also shown to be expressed as described in mice, indicating involvement of this signaling pathway in the craniofacial patterning early in vertebrate evolution. These results suggest that the last common ancestor of all the extant vertebrates would have possessed basic gene repertoires involved in oropharyngeal patterning in gnathostomes, but the elaborate genetic program leading to the Dlx code is likely to have been acquired uniquely in gnathostomes.

History of Studies on Mammalian Middle Ear Evolution: A Comparative Morphological and Developmental Biology Perspective

The mammalian middle ear represents one of the most fundamental morphological features that define this class of vertebrates. Its skeletal pattern differs conspicuously from those of other amniotes and has attracted the attention of comparative zoologists for about 200 years. To reconcile this morphological inconsistency, early comparative morphologists suggested that the mammalian middle ear was derived from elements of the jaw joint of nonmammalian amniotes. Fossils of mammalian ancestors also implied a transition in skeletal morphology that resulted in the mammalian state. During the latter half of the 20th century, developmental mechanisms controlling the formation of the jaw skeleton became the subject of studies in developmental biology and molecular genetics. Mammalian middle ear evolution can now be interpreted as a series of changes in the developmental program of the pharyngeal arches. In this review, we summarize the history of middle ear research, highlight some of the remaining problems, and suggest possible future directions. We propose that to understand mammalian middle ear evolution, it is essential to identify the critical developmental events underlying the particular mammalian anatomy and to describe the evolutionary sequence of changes in developmental and molecular terms. We also discuss the degree of consistency between the developmental explanation of the mammalian middle ear based on molecular biology and morphological changes in the fossil record.

Identification of cis-Acting Elements Repressing Blue Opsin Expression in Zebrafish UV Cones and Pineal Cells

Opsin genes are expressed in a cell type-specific manner in the retina and the pineal organ for visual and nonvisual photoreceptive purposes, but the regulatory mechanism behind the tissue and cell selectivity is not well understood. In this study, we focus on the expression regulation of the blue-sensitive opsin gene SWS2 of zebrafish by taking a transgenic approach using the green fluorescence protein as an expression reporter. The zebrafish SWS2 is a single-copy gene and is expressed specifically in the “long single cones” in the retina. We found the following. 1) A 0.3-kb region between 0.6 and 0.3 kb 5′ of the SWS2 initiation codon, encompassing four cone-rod homeobox-binding sites (OTX sequences), contains the region necessary and sufficient to drive gene expression in long single cones. 2) A 15-bp portion (-341 to -327) in the 0.3-kb region represses the gene expression in the “short single cones,” which are dedicated to the UV-sensitive opsin gene SWS1. 3) An 11-bp sequence TAACTGCCAGT (-441 to -431) in the 0.3-kb region, with its adjacent OTX element, also works as a repressor for gene expression in the pineal cells. 4) Finally, this OTX site is necessary for expression repression in the bipolar cells in the retina. These findings open a way for understanding the complex interaction of positive and negative regulatory factors that govern the cell type specificity of the opsin gene expression in the photoreceptive cells in the retina and the pineal organ. We termed the novel 11-bp sequence as the pineal negative regulatory element, PINE.

Development of the head and trunk mesoderm in the dogfish, Scyliorhinus torazame: II. Comparison of gene expression between the head mesoderm and somites with reference to the origin of the vertebrate head

The vertebrate mesoderm differs distinctly between the head and trunk, and the evolutionary origin of the head mesoderm remains enigmatic. Although the presence of somite‐like segmentation in the head mesoderm of model animals is generally denied at molecular developmental levels, the appearance of head cavities in elasmobranch embryos has not been explained, and the possibility that they may represent vestigial head somites once present in an amphioxus‐like ancestor has not been ruled out entirely. To examine whether the head cavities in the shark embryo exhibit any molecular signatures reminiscent of trunk somites, we isolated several developmentally key genes, including Pax1, Pax3, Pax7, Pax9, Myf5, Sonic hedgehog, and Patched2, which are involved in myogenic and chondrogenic differentiation in somites, and Pitx2, Tbx1, and Engrailed2, which are related to the patterning of the head mesoderm, from an elasmobranch species, Scyliorhinus torazame. Observation of the expression patterns of these genes revealed that most were expressed in patterns that resembled those found in amniote embryos. In addition, the head cavities did not exhibit an overt similarity to somites; that is, the similarity was no greater than that of the unsegmented head mesoderm in other vertebrates. Moreover, the shark head mesoderm showed an amniote‐like somatic/visceral distinction according to the expression of Pitx2, Tbx1, and Engrailed2. We conclude that the head cavities do not represent a manifestation of ancestral head somites; rather, they are more likely to represent a derived trait obtained in the lineage of gnathostomes.

Developmental genetic bases behind the independent origin of the tympanic membrane in mammals and diapsids

The amniote middle ear is a classical example of the evolutionary novelty. Although paleontological evidence supports the view that mammals and diapsids (modern reptiles and birds) independently acquired the middle ear after divergence from their common ancestor, the developmental bases of these transformations remain unknown. Here we show that lower-to-upper jaw transformation induced by inactivation of the Endothelin1-Dlx5/6 cascade involving Goosecoid results in loss of the tympanic membrane in mouse, but causes duplication of the tympanic membrane in chicken. Detailed anatomical analysis indicates that the relative positions of the primary jaw joint and first pharyngeal pouch led to the coupling of tympanic membrane formation with the lower jaw in mammals, but with the upper jaw in diapsids. We propose that differences in connection and release by various pharyngeal skeletal elements resulted in structural diversity, leading to the acquisition of the tympanic membrane in two distinct manners during amniote evolution.