Koichi Kawakami

National Institute of Genetics · Division of Molecular and Developmental Biology

Mutations in eya1 cause branchio-oto-renal syndrome (BOR) in humans and the equivalent condition in animal models. BOR is characterized by multi-organ malformations. To better understand the role of Eya1 in organogenesis we used the zebrafish posterior lateral-line primordium. This multicellular tissue moves from head-to-tail at a constant velocity...

Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells...

A critical step for functional recovery from peripheral nerve injury is for regenerating axons to connect with their pre-injury targets. Reestablishing pre-injury target specificity is particularly challenging for limb-innervating axons as they encounter a plexus, a network where peripheral nerves converge, axons from different nerves intermingle,...

A critical step for functional recovery from peripheral nerve injury is for regenerating axons to connect with their pre-injury targets. Reestablishing pre-injury target specificity is particularly challenging for limb-innervating axons as they encounter a plexus, a network where peripheral nerves converge, axons from different nerves intermingle,...

The vertebrate skeleton changes its shape during development through the activities of chondrocytes, osteoblasts and osteoclasts. Although much is known about the mechanisms for differentiation in these cells, it is less understood how they behave in a region-specific manner to acquire unique bone shapes. To address this question, we investigated t...

The mammalian amygdala is a complex forebrain structure consisting of a heterogeneous group of nuclei derived from the pallial and subpallial telencephalon. It plays a critical role in a broad range of behaviors such as emotion, cognition, and social behavior; within the amygdala each nucleus has a distinct role in these behavioral processes. Topol...

When animals repeatedly receive a combination of neutral conditional stimulus (CS) and aversive unconditional stimulus (US), they learn the relationship between CS and US, and show conditioned fear responses after CS. They show passive responses such as freezing or panic movements (classical or Pavlovian fear conditioning), or active behavioral res...

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord. Spinal motor neurons align along the spinal cord length within the vertebral column, and extend long axons to connect with skeletal muscles covering the body surface. Due to this anatomy, spin...

Transactive response DNA-binding protein 43 kDa (TDP-43) encoded by the TARDBP gene is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) that regulates multiple steps of RNA metabolism, and its cytoplasmic aggregation characterizes degenerating motor neurons in amyotrophic lateral sclerosis (ALS). In most ALS cases, cytopl...

Tissue remodeling presents an enormous challenge to the stability of intercellular signaling domains. Here we investigate this issue during the development of the posterior lateral line in zebrafish. We find that the transcriptional co-activator and phosphatase Eya1, mutated in the branchio-oto-renal syndrome in humans, is essential for the homeost...

Neurons in the inferior olivary nuclei (IO neurons) send climbing fibers to Purkinje cells to elicit functions of the cerebellum. IO neurons and Purkinje cells are derived from neural progenitors expressing the proneural gene ptf1a In this study, we found that the homeobox gene gsx2 was co-expressed with ptf1a in IO progenitors in zebrafish. Both g...

Background The distribution of sensory organs is important for detecting environmental signals efficiently. The mechanosensory receptors of the lateral line system, neuromasts, are stereotypically distributed over the head and body surface of fish, although how neuromasts arise in these predetermined positions during development remains unclear. R...

Although domesticated goldfish strains exhibit highly diversified phenotypes in morphology, the genetic basis underlying these phenotypes is poorly understood. Here, based on analysis of transposable elements in the allotetraploid goldfish genome, we found that its two subgenomes have evolved asymmetrically since a whole-genome duplication event in...

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal...

Cytoplasmic aggregation of TDP-43 characterizes degenerating neurons in most cases of amyotrophic lateral sclerosis (ALS). Here, we develop an optogenetic TDP-43 variant (opTDP-43), whose multimerization status can be modulated in vivo through external light illumination. Using the translucent zebrafish neuromuscular system, we demonstrate that sho...

Cytoplasmic aggregation of TDP-43 characterizes degenerating neurons in most cases of amyotrophic lateral sclerosis (ALS), yet the mechanisms and cellular outcomes of TDP-43 pathology remain largely elusive. Here, we develop an optogenetic TDP-43 variant (opTDP-43), whose multimerization status can be modulated in vivo through external light illumi...

Brain activity and connectivity alter drastically during epileptic seizures. The brain networks shift from a balanced resting state to a hyperactive and hypersynchronous state. It is, however, less clear which mechanisms underlie the state transitions. By studying neural and glial activity in zebrafish models of epileptic seizures, we observe strik...

Background: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the d...

Brain activity and connectivity alters drastically during epileptic seizures. Throughout this transition, brain networks shift from a balanced resting state to a hyperactive and hypersynchronous state, spreading across the brain. It is however less clear which mechanisms underlie these state transitions. By studying neuronal and glia activity acros...

Background: Fear conditioning is a form of learning essential for animal survival and used as a behavioral paradigm to study the mechanisms of learning and memory. In mammals, the amygdala plays a crucial role in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amyg...

The nature of host organs and genes that underlie tumor-induced physiological disruption on host remains ill-defined. Here, we establish a novel zebrafish intestinal tumor model that is suitable for addressing this issue, and find that hepaticcyp7a1, the rate-limiting factor for synthesizing bile acids, in the case of zebrafish bile alcohol (BA), i...

Bone is a connective tissue composed of many cell types, including osteoblasts. How bones acquire their unique size and shape during development remains poorly understood. Herein we investigated the molecular and cellular mechanisms of bone morphogenesis in the zebrafish scale by using transgenic lines to enable visualization of specific types of o...

Background The most frequent genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) is the expansion of a GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 (C9orf72) locus. The pathological hallmarks observed in C9orf72 repeat expansion carriers are the formation...

The nature of host organs and genes that underlie tumor-induced physiological disruption on host remains ill-defined. Here, we establish a novel zebrafish intestinal tumor model that is optimized for addressing this issue, and find that hepatic cyp7a1, the rate-limiting factor for synthesizing bile acids (BAs), is such a host gene. Inducing krasG12...

Although previous studies show that the cerebellum is involved in classical fear conditioning, it is not clear which components in the cerebellum control it or how. We addressed this issue using a delayed fear-conditioning paradigm with late-stage zebrafish larvae, with the light extinguishment as the conditioned stimulus (CS) and an electric shock...

Genetic tools and mutagenesis strategies based on transposable elements are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of their inherent capacity to insert into DNA, transposons can be developed into powerful tools for chromosomal manipulations. Transposon-bas...

Limb bud patterning, outgrowth, and differentiation are precisely regulated in a spatio-temporal manner through integrated networks of transcription factors, signaling molecules, and downstream genes. However, the exact mechanisms that orchestrate morphogenesis of the limb remain to be elucidated. Previously, we have established EMBRYS, a whole-mou...

The visual system plays a major role in food/prey recognition in diurnal animals, and food intake is regulated by the hypothalamus. However, whether and how visual information about prey is conveyed to the hypothalamic feeding centre is largely unknown. Here we perform real-time imaging of neuronal activity in freely behaving or constrained zebrafi...

Pretectal response to prey in a 4 dpf zebrafish larva (Supplement to Figure 2b)

Correlated calcium signals in the pretectal areas and the inferior lobes of the hypothalamus in response to prey in an agarose-embedded larva (Supplement to Figure 3b)

Hypothalamic response to prey in an agarose-embedded larva (Supplement to Figure 1d)

Supplementary Figures

Pretectal activity in the presence of a paramecium in a 6 dpf zebrafish larva embedded in agarose (Supplement to Figure 2d)

Pretectal activity in the presence of a paramecium in a 6 dpf larva embedded in agarose (no eye convergence) (Supplement to Figure 2d)

Activity of the inferior lobes of the hypothalamus (ILH) during prey capture behaviour (Supplement to Figure 1b-c)

Pretectal response to a moving spot (Supplement to Figure 2e and f)

Prey capture is abolished in pretectum-ablated zebrafish (Supplement to Figure 2m)

gSAIzGFFM119B-labelled pretectal cells project to the inferior lobe of the hypothalamus (Supplement to Figure 3a)

The blood-brain barrier is essential for the proper homeostasis and function of the CNS, but its mechanism of function is poorly understood. Perivascular cells surrounding brain blood vessels are thought to be important for blood-brain barrier establishment, but their roles are not well defined. Here, we describe a novel perivascular cell populatio...

Scientific Reports 6 : Article number: 28873 10.1038/srep28873 ; published online: 01 July 2016 ; updated: 20 April 2017 The Acknowledgements section in this Article is incomplete. “We are grateful to Drs K. Shimamura, J.

The visual system plays a major role in food/prey recognition in diurnal animals, and food intake is regulated by the hypothalamus. However, whether and how visual information about prey is conveyed to the hypothalamic feeding centre is largely unknown. Here we perform real-time imaging of neuronal activity in freely behaving or constrained zebrafi...

il1b expression in larvae injected with std, spi1b, csf3r, or irf8 MOs.The table shows the numbers of larvae used for evaluating the il1b expression levels in larvae that were injected with respective MOs.DOI: http://dx.doi.org/10.7554/eLife.22716.013

il1b expression at respective time points after fin fold amputation.The table shows the numbers of larvae used for evaluating the il1b expression levels in WT or clo mutant at respective time points after fin fold amputation.DOI: http://dx.doi.org/10.7554/eLife.22716.004

The list of transcripts that are upregulated or downregulated in the amputated fin fold of the clo mutant. The table of upregulated genes shows the list of transcripts whose final RPKMs of the amputated clo mutant at 6 hpa are more than two times than those of the amputated WT. The table of downregulated genes shows the list of top 100 transcripts...

Cellular responses to injury are crucial for complete tissue regeneration, but their underlying processes remain incompletely elucidated. We have previously reported that myeloiddefective zebrafish mutants display apoptosis of regenerative cells during fin fold regeneration. Here, we found that the apoptosis phenotype is induced by prolonged expres...

Tissue integrity is critical for organ formation and function. During heart development, cardiomyocytes differentiate and integrate to form a coherent tissue that contracts synchronously. However, the molecular mechanisms regulating cardiac tissue integrity are poorly understood. Here we show that proteolysis, via the E3 ubiquitin ligase ASB2, regu...

Calcium imaging of Tg(myl7:gCaMP) WT heart. One-cell stage Tg(myl7:gCaMP) WT embryos were injected with tnnt2a MO and imaged at 50 hpf.

Calcium imaging of Tg(myl7:gCaMP) asb2b mutant heart shows no obvious changes in cardiac conduction. One-cell stage Tg(myl7:gCaMP) asb2b mutant embryos were injected with tnnt2a MO and imaged at 50 hpf.

Supplementary Figures and Supplementary Tables.

Time-lapse imaging of a WT heart transplanted with WT cells. Tg(myl7:LIFEACT-GFP) WT donor cells (green) were transplanted to the margin of Tg(myl7:LIFEACT-tdTomato) WT host embryos (red) at the blastula stage and imaged at 50 hpf.

Time-lapse imaging of a WT heart transplanted with asb2b mutant cells. Tg(myl7:LIFEACT-GFP) asb2b mutant donor cells (green) were transplanted to the margin of Tg(myl7:LIFEACT-tdTomato) WT host embryos (red) at the blastula stage and imaged at 50 hpf.

Habenula (Hb) plays critical roles in emotion-related behaviors through integrating inputs mainly from the limbic system and basal ganglia. However, Hb also receives inputs from multiple sensory modalities. The function and underlying neural circuit of Hb sensory inputs remain unknown. Using larval zebrafish, we found that left dorsal Hb (dHb, a ho...

Motile cilia are actively beating hair-like structures that cover the surface of multiple epithelia. The flow that ciliary beating generates is utilized for diverse functions and depends on the spatial location and biophysical properties of cilia. Here we show that the motile cilia in the nose of aquatic vertebrates are spatially organized and stab...

Hirschsprung disease (HSCR) is characterized by absence of enteric neurons from the distal colon and severe intestinal dysmotility. To understand the pathophysiology and genetics of HSCR we developed a unique zebrafish model that allows combined genetic, developmental and in vivo physiological studies. We show that ret mutant zebrafish exhibit cell...

Intestinal Motility in WT larvae. Recording (at 2.5 frames per second) of spontaneous intestinal motility of a WT larvae at 7dpf. The spatiotemporal map constructed from this original movie is shown in Fig 3. Movie is sped up 20 times. (AVI)

Intestinal Motility in rethu2846/+ larvae. Recording (at 2.5 frames per second) of spontaneous intestinal motility of a rethu2846/+ larvae at 7dpf. The spatiotemporal map constructed from this original movie is shown in Fig 3. Movie is sped up 20 times. (AVI)

Proliferation, cell death and differentiation are all unaffected in rethu2846/+ larvae. (A-C) Proliferation of ENS progenitor cells is equivalent in 48hpf WT (A) and rethu2846/+ (B) larvae, shown using GFP from SAGFF234A;UAS:GFP to label pixel area of ENS progenitors and BrdU (following 30 minute BrdU pulse) to quantify proliferating cells per 220u...

Tools used for mapk10 loss of function experiments. (A) Morpholino oligo (MO) gene knockdown of mapk10. Genomic organization of mapk10 (mapk10-001 from Zv9:CU651624.3:8583743:8720716:1) showing the position of the mapk10 splice blocking MO (mapk10 MO) at the intron3/4-exon4 boundary, which leads to excision of exon 4 (underlined). RT-PCR of injecte...

Migration of the ENCC population in WT embryos from 48–56hpf. Depth coded Z-projected recording of GFP+ ENCC cells of ret+/+;SAGFF234A;UAS:GFP embryos from 48–56hpf with a frame interval 374 seconds. (AVI)

mapk10 is expressed within the SAGFF234A;UAS:GFP expressing ENS lineage. RNA in situ hybridization for mapk10 was conducted in SAGFF234A;UAS:GFP embryos at 54 (A) and 72hpf (D). Immunostaining for GFP identifies the stream of migrating ENCCs (B, both streams shown, E, one stream shown). Merged images indicate that cells containing map10 signal loca...

rethu2846/+ larvae exhibit phenotypic variation. 7dpf larvae immunostained with the HuC/D antibody to visualize ENS neurons in WT (A) and rethu2846/+ (B-D) larvae. Asterisks indicate end of the gut tube (anal pore), and arrows denote the position of the most distal HuC/D+ neuron. At 7dpf, WT (ret+/+) larvae exhibit HuC/D+ ENS neurons along the full...

Novel transgenic tools allow labeling of ENS progenitors and ENS neurons. SAGFF234A is a transgenic line with the Gal4 expressing gene trap construct integrated within the chrm2a gene on the chromosome 4 encoding the cholinergic receptor, muscarinic 2a. When crossed to transgenic fish carrying the GFP reporter gene downstream of the Gal4 recognitio...

Migration of the ENCC population in rethu2846/+ embryos from 48–56hpf. Depth coded Z-projected recording of GFP+ ENCC cells of rethu2846/+;SAGFF234A;UAS:GFP embryos from 48–56hpf with a frame interval 374 seconds. (AVI)

Intestinal Motility in rethu2846/hu2846 larvae. Recording (at 2.5 frames per second) of spontaneous intestinal motility of a rethu2846/hu2846 larvae at 7dpf. The spatiotemporal map constructed from this original movie is shown in Fig 3. Movie is sped up 20 times. (AVI)

Mutations in the gene MAPT encoding tau, a microtubules-associated protein, cause a subtype of familial neurodegenerative disorder, known as frontotemporal lobar degeneration tauopathy (FTLD-Tau), which presents with dementia and is characterized by atrophy in the frontal and temporal lobes of the brain. Although induced pluripotent stem cell (iPSC...

Glutamatergic retinal waves, the spontaneous patterned neural activities propagating among developing retinal ganglion cells (RGCs), instruct the activity-dependent refinement of visuotopic maps. However, its initiation and underlying mechanism remain largely elusive. Here using larval zebrafish and multiple in vivo approaches, we discover that bip...