Rie Kusakabe

Ci-opsin1, a vertebrate-type opsin gene, expressed in the larval ocellus of the ascidian Ciona intestinalis.

A novel gene encoding visual pigment, Ci‐opsin1, was identified in a primitive chordate, the ascidian, Ciona intestinalis. Molecular phylogenetic analysis and the exon–intron organization suggest that Ci‐opsin1 is closely related to the retinal and pineal opsins of vertebrates. During embryogenesis, Ci‐opsin1 transcripts were first detected in part of the brain of mid tailbud embryos; its expression was confined to photoreceptor cells of the ocellus (eye spot) in the larval brain as development proceeded. These results suggest a common descent of the ascidian ocellus and the vertebrate eyes. The ocellus of ascidian larvae may represent an ancestral state of the vertebrate eye.

Evolution and Developmental Patterning of the Vertebrate Skeletal Muscles: Perspectives From the Lamprey

The myotome in gnathostome vertebrates, which gives rise to the trunk skeletal muscles, consists of epaxial (dorsal) and hypaxial (ventral) portions, separated by the horizontal myoseptum. The hypaxial portion contains some highly derived musculature that is functionally as well as morphologically well differentiated in all the gnathostome species. In contrast, the trunk muscles of agnathan lampreys lack these distinctions and any semblance of limb muscles. Therefore, the lamprey myotomes probably represent a primitive condition compared with gnathostomes. In this review, we compare the patterns of expression of some muscle‐specific genes between the lamprey and gnathostomes. Although the cellular and tissue morphology of lamprey myotomes seems uniform and undifferentiated, some of the muscle‐specific genes are expressed in a spatially restricted manner. The lamprey Pax3/7 gene, a cognate of gnathostome Pax3, is expressed only at the lateral edge of the myotomes and in the hypobranchial muscle, which we presume is homologous to the gnathostome hypobranchial muscle. Thus, the emergence of some part of a hypaxial‐specific gene regulatory cascade might have evolved before the agnathan/gnathostome divergence, or before the evolutionary separation of epaxial and hypaxial muscles. Developmental Dynamics 234:824–834, 2005.

Evolutionary perspectives from development of mesodermal components in the lamprey

Lampreys, a jawless vertebrate species, lack not only jaws but also several other organs, including ventral migratory muscles shared by gnathostomes. In the lamprey embryo, the mesoderm consists primarily of unsegmented head mesoderm, segmented somites, and yet uncharacterized lateral plate mesoderm, as in gnathostomes. Although the adult lamprey possesses segmented myotomes in the head, the head mesoderm of this animal is primarily unsegmented, similar to that in gnathostomes. In the trunk, the large part of lamprey somites is destined to form myotomes, and the Pax3/7 gene expression domain in the lateral part of somites is suggested to represent a dermomyotome homologue. Lamprey myotomes are not segregated by a horizontal myoseptum, which has been regarded as consistent with the apparent absence of a migratory population of hypaxial muscles shared by gnathostomes. However, recent analysis suggests that lampreys have established the gene regulatory cascade necessary for the ventrally migrating myoblasts, which functions in part during the development of the primordial hypobranchial muscle. There have also been new insights on the developmental cascade of lamprey cartilages, in which the Sox family of transcription factors plays major roles, as in gnathostomes. Thus, mesoderm development in lampreys may represent the ancestral state of gene regulatory mechanisms required for the evolution of the complex and diverse body plan of gnathostomes. Developmental Dynamics 236:2410–2420, 2007.

Origin of the Vertebrate Visual Cycle: Genes Encoding Retinal Photoisomerase and Two Putative Visual Cycle Proteins Are Expressed in Whole Brain of a Primitive Chordate

The absorption of light by rhodopsin leads to the cis‐to‐trans isomerization of the chromophore to generate all‐trans‐retinal. In the visual cycle, the resultant all‐trans‐retinal is converted back into the 11‐cis‐retinal. In the mammalian eye, the retinal pigment epithelium (RPE) plays an essential role in the visual cycle. We have identified cDNA clones encoding three putative visual cycle proteins, homologs of mammalian retinal G‐protein‐coupled receptor (RGR), cellular retinaldehyde‐binding protein (CRALBP) and β‐carotene 15,15′‐monooxygenase (BCO)/RPE65 in a primitive chordate, ascidian Ciona intestinalis. The mRNAs for these proteins are specifically expressed in the central nervous system during embryonic development. In the larva, the transcripts were widely distributed in the brain vesicle and visceral ganglion. Since visual pigment, Ci‐opsin1, is solely expressed in photoreceptor cells, the visual cycle in this primitive chordate may take place in two compartments, which are coupled into a cycle by the direct flow of retinoids though the intercellular matrix. The Ci‐opsin3, an ascidian homolog of mammalian RGR, was expressed in HEK 293S cells and purified after binding of retinal. The chromophore of Ci‐opsin3 is in an all‐trans‐retinal and it is isomerized to an 11‐cis‐form upon absorption of light. Mammalian CRALBP and BCO/RPE65 are believed to play critical roles in the process of reisomerization of all‐trans‐retinoid to 11‐cis‐retinoid in RPE. The present data suggest that isomerization of all‐trans‐retinoid to 11‐cis‐retinoid occurs in the brain vesicle and visceral ganglion of a primitive chordate. J. Comp. Neurol. 460:180–190, 2003.

Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: Implications for conserved mechanisms of chordate development

Abstract The cephalochordate amphioxus is thought to share a common ancestor with vertebrates. To investigate the evolution of developmental mechanisms in chordates, cDNA clones for two amphioxus actin genes, BfCA1 and BfMA1, were isolated. BfCA1 encodes a cytoplasmic actin and is expressed in a variety of tissues during embryogenesis, beginning in the dorsolateral mesendoderm of the mid-gastrula. At the open neural plate stage, BfCA1 transcripts accumulate at the bases of the neuroectodermal cells adjacent the presumptive notochord. The 3’ untranslated region of BfCA1 contains a sequence that is similar to the ”zipcode” sequence of chicken β-cytoplasmic actin gene, which is thought to direct intracellular mRNA localization. BfCA1 is also expressed in the notochord through the early larval stage, in the pharynx and in the somites at the onset of muscle-cell differentiation. BfMA1 is a vertebrate-type muscle actin gene, although the deduced amino acid sequence is fairly divergent. Transcripts first appear in the early neurula in the somites as they begin to differentiate into axial muscle cells and persist into the adult stage. In young adults, transcripts are localized in the Z-discs of the muscle cells. Smooth muscle cells around the gill slits and striated muscle cells in the pterygeal muscle also express BfMA1; however, there is never any detectable expression in the notochord, which is a modified striated muscle. Together with the alkali myosin light chain gene AmphiMLC-alk, the sequence and muscle-specific expression of BfMA1 implies a conserved mechanism of muscle cell differentiation between amphioxus and vertebrates. Evolution of the chordate actin gene family is discussed based on molecular phylogenetic analysis and expression patterns of amphioxus actin genes.

Genomic organization and evolution of actin genes in the amphioxus Branchiostoma belcheri and Branchiostoma floridae

We previously described the cDNA cloning and expression patterns of actin genes from amphioxus Branchiostoma floridae (Kusakabe, R., Kusakabe, T., Satoh, N., Holland, N.D., Holland, L.Z., 1997. Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: implications for conserved mechanisms of chordate development. Dev. Genes Evol. 207, 203-215). In the present paper, we report the characterization of cDNA clones for actin genes from a closely related species, Branchiostoma belcheri, and the exon-intron organization of B. floridae actin genes. Each of these two amphioxus species has two types of actin genes, muscle and cytoplasmic. The coding and non-coding regions of each type are well-conserved between the two species. A comparison of nucleotide sequences of muscle actin genes between the two species suggests that a gene conversion may have occurred between two B. floridae muscle actin genes BfMA1 and BfMA2. From the conserved positions of introns between actin genes of amphioxus and those of other deuterostomes, the evolution of deuterostome actin genes can be inferred. Thus, the presence of an intron at codon 328/329 in vertebrate muscle and cytoplasmic actin genes but not in any known actin gene in other deuterostomes suggests that a gene conversion may have occurred between muscle and cytoplasmic actin genes during the early evolution of the vertebrates after separation from other deuterostomes. A Southern blot analysis of genomic DNA revealed that the amphioxus genome contains multiple muscle and cytoplasmic actin genes. Some of these actin genes seem to have arisen from recent duplication and gene conversion. Our findings suggest that the multiple genes encoding muscle and cytoplasmic actin isoforms arose independently in each of the three chordate lineages, and gene duplications and gene conversions established the extant actin multigene family during the evolution of chordates.

The neural crest and evolution of the head/trunk interface in vertebrates

The migration and distribution patterns of neural crest (NC) cells reflect the distinct embryonic environments of the head and trunk: cephalic NC cells migrate predominantly along the dorsolateral pathway to populate the craniofacial and pharyngeal regions, whereas trunk crest cells migrate along the ventrolateral pathways to form the dorsal root ganglia. These two patterns thus reflect the branchiomeric and somitomeric architecture, respectively, of the vertebrate body plan. The so-called vagal NC occupies a postotic, intermediate level between the head and trunk NC. This level of NC gives rise to both trunk- and cephalic-type (circumpharyngeal) NC cells. The anatomical pattern of the amphioxus, a basal chordate, suggests that somites and pharyngeal gills coexist along an extensive length of the body axis, indicating that the embryonic environment is similar to that of vertebrate vagal NC cells and may have been ancestral for vertebrates. The amniote-like condition in which the cephalic and trunk domains are distinctly separated would have been brought about, in part, by anteroposterior reduction of the pharyngeal domain.

MiR-124 is Involved in Post-transcriptional Regulation of Polypyrimidine Tract Binding Protein 1 (PTBP1) During Neural Development in the Medaka, Oryzias latipes

MicroRNAs (miRNAs) comprise a group of small noncoding RNA molecules thought to have contributed to the evolution of vertebrate brain homogeneity and diversity. The miRNA miR-124 is well conserved between invertebrates and vertebrates and is expressed abundantly in the central nervous system (CNS). We identified miR-124 in the medaka, Oryzias latipes, and investigated its role in neural development. The five candidate genes for medaka precursor miR-124 are unlinked on four different chromosomes and differ in nucleotide length. Their sequences suggest that they can generate functional miRNAs through conventional miRNA biogenesis by folding into stem-loop structures. Whole-mount in situ hybridization and northern blotting revealed that mature miR-124 is specifically expressed in the CNS and the eyes starting at two days post-fertilization. We also examined the sequences and expression of medaka Polypyrimidine tract binding protein 1 (Ptbp1), a possible direct target of miR-124. The 3′UTR of medaka Ptbp1 contains predicted binding motifs (target sites) for miR-124. A GFP reporter assay for the target sites or the entire 3′UTR showed that exogenous miR-124 silences PTBP1 expression in vivo. Our study suggests that medaka miR-124 is involved in post-transcriptional regulation of target genes in neural development and that medaka miR-124 homologs may have spatiotemporal roles different from those in other vertebrates.

Developmental mechanisms of migratory muscle precursors in medaka pectoral fin formation

Limb muscles are formed from migratory muscle precursor cells (MMPs) that delaminate from the ventral region of dermomyotomes and migrate into the limb bud. MMPs remain undifferentiated during migration, commencing differentiation into skeletal muscle after arrival in the limb. However, it is still unclear whether the developmental mechanisms of MMPs are conserved in teleost fishes. Here, we investigate the development of pectoral fin muscles in the teleost medaka Oryzias latipes. Expression of the MMP marker lbx1 is first observed in several somites prior to the appearance of fin buds. lbx1-positive cells subsequently move anteriorly and localize in the prospective fin bud region to differentiate into skeletal muscle cells. To address the developmental mechanisms underlying fin muscle formation, we knocked down tbx5, a gene that is required for fin bud formation. tbx5 morphants showed loss of fin buds, whereas lbx1 expression initiated normally in anterior somites. Unlike in normal embryos, expression of lbx1 was not maintained in migrating fin MMPs or within the fin buds. We suggest that fin MMPs appear to undergo two phases in their development, with an initial specification of MMPs occurring independent of fin buds and a second fin bud-dependent phase of MMP migration and proliferation. Our results showed that medaka fin muscle is composed of MMPs. It is suggested that the developmental mechanism of fin muscle formation is conserved in teleost fishes including medaka. Through this study, we also propose new insights into the developmental mechanisms of MMPs in fin bud formation.

Publisher Correction: Migratory appendicular muscles precursor cells in the common ancestor to all vertebrates

In Fig. 2 of this Article originally published, some erroneous lines appeared on the left side of the images in panels c, e and g. The figure should have appeared as shown below. These errors have now been corrected in all versions of the Article.