Takehiro G. Kusakabe

Individuals with mutations in XPNPEP3 , which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy

The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1-NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are ciliopathies. Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.

Glutamatergic networks in the Ciona intestinalis larva

Glutamate is a major neurotransmitter in the excitatory synapses of both vertebrate and invertebrate nervous systems and is involved in many neural processes including photo‐, mechano‐, and chemosensations, neural development, motor control, learning, and memory. We identified and characterized the gene (Ci‐VGLUT) encoding a member of the vesicular glutamate transporter subfamily, a specific marker of glutamatergic neurons, in the ascidian Ciona intestinalis. The Ci‐VGLUT gene is expressed in the adhesive organ, the epidermal neurons, and the brain vesicle, but not in the visceral ganglion. The Ci‐VGLUT promoter and an anti‐Ci‐VGLUT antibody were used to analyze the distribution and axonal connections of prospective glutamatergic neurons in the C. intestinalis larva. The green fluorescent protein (GFP) reporter driven by the 4.6‐kb upstream region of Ci‐VGLUT recapitulated the endogenous gene expression patterns and visualized both the cell bodies and neurites of glutamatergic neurons. Papillar neurons of the adhesive organs, almost all epidermal neurons, the otolith cell, and ocellus photoreceptor cells were shown to be glutamatergic. Each papillar neuron connects with a rostral epidermal neuron. Axons from rostral epidermal neurons, ocellus photoreceptor cells, and neurons underlying the otolith terminate in the posterior brain vesicle. Some caudal epidermal neurons also send long axons toward the brain vesicle. The posterior brain vesicle contains a group of Ci‐VGLUT‐positive neurons that send axons posteriorly to the visceral ganglion. Our results suggest that glutamatergic neurotransmission plays a major role in sensory systems and in the integration of the sensory inputs of the ascidian larva. J. Comp. Neurol. 508:249–263, 2008.

Tube formation by complex cellular processes in Ciona intestinalis notochord

In the course of embryogenesis multicellular structures and organs are assembled from constituent cells. One structural component common to many organs is the tube, which consists most simply of a luminal space surrounded by a single layer of epithelial cells. The notochord of ascidian Ciona forms a tube consisting of only 40 cells, and serves as a hydrostatic skeleton essential for swimming. While the early processes of convergent extension in ascidian notochord development have been extensively studied, the later phases of development, which include lumen formation, have not been well characterized. Here we used molecular markers and confocal imaging to describe tubulogenesis in the developing Ciona notochord. We found that during tubulogenesis each notochord cell established de novo apical domains, and underwent a mesenchymal-epithelial transition to become an unusual epithelial cell with two opposing apical domains. Concomitantly, extracellular luminal matrix was produced and deposited between notochord cells. Subsequently, each notochord cell simultaneously executed two types of crawling movements bi-directionally along the anterior/posterior axis on the inner surface of notochordal sheath. Lamellipodia-like protrusions resulted in cell lengthening along the anterior/posterior axis, while the retraction of trailing edges of the same cell led to the merging of the two apical domains. As a result, the notochord cells acquired endothelial-like shape and formed the wall of the central lumen. Inhibition of actin polymerization prevented the cell movement and tube formation. Ciona notochord tube formation utilized an assortment of common and fundamental cellular processes including cell shape change, apical membrane biogenesis, cell/cell adhesion remodeling, dynamic cell crawling, and lumen matrix secretion.

Pigmented and nonpigmented ocelli in the brain vesicle of the ascidian larva

The vertebrate‐type opsin, Ci‐opsin1, is localized in the outer segments of the photoreceptor cells of larvae of the ascidian Ciona intestinalis. The absorption spectrum of the photopigment reconstituted from Ci‐opsin1 and 11‐cis‐retinal suggested that the photopigment is responsible for photic behavior of the larvae. The structure and function of Ci‐opsin1‐positive photoreceptor cells were examined by immunohistochemistry, confocal microscopy, electron microscopy, laser ablation, and behavioral analysis. Ciona larvae have three morphologically distinct groups of photoreceptor cells in the brain vesicle. Group I and group II photoreceptor cells are associated with the ocellus pigment cell on the right side of the brain vesicle. The outer segments of the group I photoreceptor cells are regularly arranged inside the small cavity encircled by the cup‐shaped pigment cell. The outer segments of the group II photoreceptor cells are located outside the pigment cavity and exposed to the lumen of the brain vesicle. The outer segments of the group III photoreceptor cells are located near the otolith on the left ventral side of the brain vesicle. Thus, the brain vesicle of the ascidian larva has two ocelli: a ‘conventional’ pigmented ocellus containing the group I and group II photoreceptor cells and a novel nonpigmented ocellus solely consisting of the group III photoreceptor cells. Laser ablation experiments suggest that the pigmented ocellus is responsible for the photic swimming behavior. The nonpigmented ocellus might relate to later developmental or physiological events, such as metamorphosis, because Ci‐opsin1 immunoreactivity appears in the late larval stage and becomes intense just before the onset of metamorphosis. J. Comp. Neurol. 509:88–102, 2008.

The pre-vertebrate origins of neurogenic placodes

The sudden appearance of the neural crest and neurogenic placodes in early branching vertebrates has puzzled biologists for over a century. These embryonic tissues contribute to the development of the cranium and associated sensory organs, which were crucial for the evolution of the vertebrate “new head”. A previous study suggests that rudimentary neural crest cells existed in ancestral chordates. However, the evolutionary origins of neurogenic placodes have remained obscure owing to a paucity of embryonic data from tunicates, the closest living relatives to those early vertebrates. Here we show that the tunicate Ciona intestinalis exhibits a proto-placodal ectoderm (PPE) that requires inhibition of bone morphogenetic protein (BMP) and expresses the key regulatory determinant Six1/2 and its co-factor Eya, a developmental process conserved across vertebrates. The Ciona PPE is shown to produce ciliated neurons that express genes for gonadotropin-releasing hormone (GnRH), a G-protein-coupled receptor for relaxin-3 (RXFP3) and a functional cyclic nucleotide-gated channel (CNGA), which suggests dual chemosensory and neurosecretory activities. These observations provide evidence that Ciona has a neurogenic proto-placode, which forms neurons that appear to be related to those derived from the olfactory placode and hypothalamic neurons of vertebrates. We discuss the possibility that the PPE-derived GnRH neurons of Ciona resemble an ancestral cell type, a progenitor to the complex neuronal circuit that integrates sensory information and neuroendocrine functions in vertebrates.

Simple Motor System of the Ascidian Larva: Neuronal Complex Comprising Putative Cholinergic and GABAergic/Glycinergic Neurons

The ascidian larva is an excellent model for studies of the functional organization and neuronal circuits of chordates due to its remarkably simple central nervous system (CNS), comprised of about 100 neurons. To date, however, the identities of the various neurons in the ascidian larva, particularly their neurotransmitter phenotypes, are not well established. Acetylcholine, GABA, and glycine are critical neurotransmitters for locomotion in many animals. We visualized putative cholinergic neurons and GABAergic/glycinergic neurons in the ascidian larva by immunofluorescent staining using antibodies against vesicular acetylcholine transporter (VACHT) and vesicular GABA/glycine transporter (VGAT), respectively. Neurons expressing a cholinergic phenotype were found in the brain vesicle and the visceral ganglion. Five pairs of VACHT-positive neurons were located in the visceral ganglion. These putative cholinergic neurons extended their axons posteriorly and formed nerve terminals proximal to the most anterior muscle cells in the tail. VGAT-positive neurons were located in the brain vesicle, the visceral ganglion, and the anterior nerve cord. Two distinct pairs of VGAT-positive neurons, bilaterally aligned along the anterior nerve cord, extended axons anteriorly, near to the axons of the contralateral VACHT-positive neurons. Cell bodies of the VGAT-positive neurons lay on these nerve tracts. The neuronal complex, comprising motor neurons with a cholinergic phenotype and some of the GABA/glycinergic interneurons, has structural features that are compatible with a central pattern generator (CPG) producing a rhythmic movement of the tail. The simple CPG of the ascidian larva may represent the ancestral state of the vertebrate motor system.

Photoreceptive Systems in Ascidians

The brain vesicle of the tadpole larva of ascidians, simple basal chordates, contains an eye‐spot (ocellus), which is responsible for the photic swimming behavior. Ascidian adults also exhibit several types of light‐responsive behaviors. Molecular phylogenetic studies have suggested that ascidians are the closest living relatives of vertebrates, and therefore, understanding the photoreceptive systems in ascidians is a key to uncover the origin and evolution of the vertebrate eyes. The ocellus of the ascidian larva has ciliary photoreceptors resembling those of the retina and pineal eye of vertebrates. Recent studies have indicated that the ascidian larva has phototransduction and visual cycle systems similar to those of vertebrate eyes. Comparative studies on photoreceptor systems between ascidians and vertebrates provide us clues to reconstructing the evolutionary pathway leading to the lateral and median eyes of vertebrates.

Functional Diversity of Signaling Pathways through G Protein–Coupled Receptor Heterodimerization with a Species-Specific Orphan Receptor Subtype

Gonadotropin-releasing hormones (GnRHs) play pivotal roles in control of reproduction via a hypothalamic-pituitary-periphery endocrine system and nervous systems of not only vertebrates but also invertebrates. GnRHs trigger several signal transduction cascades via GnRH receptors (GnRHRs), members of the G protein-coupled receptor (GPCR) family. Recently, six GnRHs (tunicate GnRH [tGnRH]-3 to tGnRH-8) and four GnRHRs (Ciona intestinalis [Ci]-GnRHR1 to GnRHR-4), including a species-specific paralog, Ci-GnRHR4 (R4) regarded as an orphan receptor or nonfunctional receptor, were identified in the protochordate, C. intestinalis, which lacks the hypothalamic-pituitary system. Here, we show novel functional modulation of GnRH signaling pathways via GPCR heterodimerization. Immunohistochemical analysis showed colocalization of R1 and R4 in test cells of the ascidian ovary. The native R1-R4 heterodimerization was detected in the Ciona ovary by coimmunoprecipitation analysis. The heterodimerization in HEK293 cells cotransfected with R1 and R4 was also observed by coimmunoprecipitation and fluorescent energy transfer analyses. Binding assay revealed that R4 had no affinity for tGnRHs, and the heterodimerization did not alter the binding affinity of R1 to the ligands. The R1-R4 elicited 10-fold more potent Ca2+ mobilization than R1 exclusively by tGnRH-6, although R1-mediated cyclic AMP production was not affected by any of tGnRHs via the R1-R4 heterodimer. Moreover, the R1-R4 heterodimer potentiated translocation of both Ca2+-dependent protein kinase C-alpha (PKCalpha) by tGnRH-6 and Ca2+-independent PKCzeta by tGnRH-5 and tGnRH-6, eventually leading to the upregulation of extracellular signal-regulated kinase (ERK) phosphorylation compared with R1 alone. These results provide evidence that the species-specific GnRHR orphan paralog, R4, serves as an endogenous modulator for the fine-tuning of activation of PKC subtype-selective signal transduction via heterodimerization with R1 and that the species-specific GPCR heterodimerization, in concert with multiplication of tGnRHs and Ci-GnRHRs, participates in functional evolution of neuropeptidergic GnRH signaling pathways highly conserved throughout the animal kingdom.

Evolution and the origin of the visual retinoid cycle in vertebrates

Absorption of a photon by visual pigments induces isomerization of 11-cis-retinaldehyde (RAL) chromophore to all-trans-RAL. Since the opsins lacking 11-cis-RAL lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called ‘visual cycle’. Protostomes and vertebrates use essentially different machinery of visual pigment regeneration, and the origin and early evolution of the vertebrate visual cycle is an unsolved mystery. Here we compare visual retinoid cycles between different photoreceptors of vertebrates, including rods, cones and non-visual photoreceptors, as well as between vertebrates and invertebrates. The visual cycle systems in ascidians, the closest living relatives of vertebrates, show an intermediate state between vertebrates and non-chordate invertebrates. The ascidian larva may use retinochrome-like opsin as the major isomerase. The entire process of the visual cycle can occur inside the photoreceptor cells with distinct subcellular compartmentalization, although the visual cycle components are also present in surrounding non-photoreceptor cells. The adult ascidian probably uses RPE65 isomerase, and trans-to-cis isomerization may occur in distinct cellular compartments, which is similar to the vertebrate situation. The complete transition to the sophisticated retinoid cycle of vertebrates may have required acquisition of new genes, such as interphotoreceptor retinoid-binding protein, and functional evolution of the visual cycle genes.

Cell type and function of neurons in the ascidian nervous system

Ascidians, or sea squirts, are primitive chordates, and their tadpole larvae share a basic body plan with vertebrates, including a notochord and a dorsal tubular central nervous system (CNS). The CNS of the ascidian larva is formed through a process similar to vertebrate neurulation, while the ascidian CNS is remarkably simple, consisting of about 100 neurons. Recent identification of genes that are specifically expressed in a particular subtype of neurons has enabled us to reveal neuronal networks at single‐cell resolution. Based on the information on neuron subtype‐specific genes, different populations of neurons have been visualized by whole‐mount in situ hybridization, immunohistochemical staining using specific antibodies, and fluorescence labeling of cell bodies and neurites by a fluorescence protein reporter driven by neuron‐specific promoters. Neuronal populations that have been successfully visualized include glutamatergic, cholinergic, γ‐aminobutyric acid/glycinergic, and dopaminergic neurons, which have allowed us to propose functional regionalization of the CNS and a neural circuit for locomotion. Thus, the simple nervous system of the ascidian larva can serve as an attractive model system for studying the development, function, and evolution of the chordate nervous system.