Motor Neural Circuit Laboratory • Hirata Group

Native American myopathy (NAM) is a congenital muscular disease prevalent in the southern regions of the United States. However, the cause of this intractable myopathy has not been clarified so far. We originally studied mutant zebrafish that exhibited severe muscle weakness. The responsible gene encoded for a muscle protein Stac3. We found that Stac3 associates with dihydropyridine and ryanodine receptors and regulates calcium release during muscle contraction. Stac3-deficient zebrafish is now used for identifying drugs that mitigate the muscular defect in humans.

This is a collaborative work with Dr. John Y. Kuwada (University of Michigan).

Touch evoked swimming in wild-type (control) but not stac3 mutant zebrafish embryos at 48 hours post-fertilization. Panels show superimposed frames of swimming motion with the head embedded in agarose.

Motor Neural Circuit Laboratory • Hirata Group

Arthrogryposis multiplex congenita (AMC) is caused by heterogeneous pathologies leading to multiple antenatal joint contractures through fetal akinesia. Understanding the pathophysiology of this disorder is important for clinical care of the affected individuals and genetic counseling of the families. In this study, we identified disease-causing mutations in the zinc-finger gene ZC4H2 in an AMC subtype that is associated with multiple dysmorphic features and intellectual disability (ID). In zebrafish, antisense-morpholino-mediated zc4h2 knockdown caused abnormal swimming and impaired primary motoneuron development. All missense mutations identified herein failed to rescue the swimming defect of zebrafish morphants. We conclude that ZC4H2 defects cause a clinically variable broad-spectrum neurodevelopmental disorder of the central and peripheral nervous systems. Our results highlight the importance of ZC4H2 for genetic testing of individuals presenting with ID plus muscle weakness and minor or major forms of AMC.

This is a collaborative work with Dr. Vera M. Kalscheuer (Max Planck Institute for Molecular Genetics), Dr. Markus Schuelke (Charité Universitätsmedizin Berlin) and other researchers and is supported by the NIG Collaborative Research.

In zebrafish embryos, zc4h2 is expressed by forebrain, midbrain, hindbrain and spinal cord.

Division of Agricultural Genetics • Kakutani Group

Genomes of vertebrates and plants contain a substantial number of transposable elements (TEs), which are silenced by repressive epigenetic modifications, such as cytosine methylation and methylation of lysine 9 of histone H3. These modifications are essential for formation of inactive chromatin structures called heterochromatin. A potential complication is that active cellular genes sometimes contain TEs within their transcribed regions.

In this study, we show that heterochromatic epigenetic modifications are commonly found within actively transcribed gene units in both the Arabidopsis and rice genomes. We further show that in Arabidopsis, full-length transcription of genes with intragenic heterochromatin, most of which is formed by TE insertions, requires IBM2 (Increase in Bonsai Methylation 2), a protein with a Bromo-Adjacent Homology (BAH) domain and an RNA recognition motif (RRM). Our results reveal a novel epigenetic mechanism that masks effects of genetic variations created by TE insertions, allowing evolution of complex genomes with heterochromatic domains having diverse functions. (This work is a collaboration with Saze lab in OIST.)

Full-length transcription of the gene containing heterochromatin is impaired in ibm2 mutant. mRNA reads are indicated with green and red. Reads with dotted lines are mapped across exon-exon boundaries. Note that the inserted TE (ATLINE2) is associated with heterochromatic H3K9 methylation (bottom).

Division of Molecular and Developmental Biology • Kawakami Group

The vertebrate brain is anatomically and functionally asymmetric. The left and right cerebral hemispheres harbor neural stem cell niches at the ventricular-subventricular zone (V-SVZ) of the ventricular walls, where new neurons are continuously generated throughout life. However, any interhemispheric asymmetry of neural stem cell niches remains unclear. We performed gene-trap screens in adult zebrafish to identify genes that are differentially expressed in the two hemispheres and found that adult-born neurons expressing the neural zinc-finger protein Myt1 exist predominantly in the left V-SVZ. This lateralization could be reversed by left olfactory sensory deprivation–induced inactivation of Notch signaling. The olfactory behavioral preference for attractive amino acids was also impaired by sensory deprivation of the left olfactory system, but not of the right olfactory system. Our findings suggest that olfactory input generates interhemispheric differences in the fate of adult-born neurons in the zebrafish brain.

This study has been carried out as collaboration with Drs. Kishimoto and Sawamoto at Nagoya City University.

Interhemispheric asymmetric distribution of Myt1-positive neurons in telencephalic ventricular-subventricular zone.

Division of Molecular and Developmental Biology • Kawakami Group

Heartbeat is required for normal development of the heart, and perturbation of intracardiac flow leads to morphological defects resembling congenital heart diseases. These observations implicate intracardiac haemodynamics in cardiogenesis, but the signalling cascades connecting physical forces, gene expression and morphogenesis are largely unknown. Here we use a zebrafish model to show that the microRNA, miR-21, is crucial for regulation of heart valve formation. Expression of miR-21 is rapidly switched on and off by blood flow. Vasoconstriction and increasing shear stress induce ectopic expression of miR-21 in the head vasculature and heart. Flow-dependent expression of mir-21 governs valvulogenesis by regulating the expression of the same targets as mouse/human miR-21 (sprouty, pdcd4, ptenb) and induces cell proliferation in the valve-forming endocardium at constrictions in the heart tube where shear stress is highest. We conclude that miR-21 is a central component of a flow-controlled mechanotransduction system in a physicogenetic regulatory loop.

This study has been carried out as collaboration with Dr. Ogura at Tohoku University.

A: Normal valvulogenesis in the wild type zebrafish embryo.
B: Abnormal valvulogenesis in the zebrafish embryo in which the function of miR-21 was inhibited.

Microbial Genetics Laboratory • Niki Group

RodZ is important for maintaining the rod shape of Escherichia coli. Loss of RodZ causes conversion of the rod shape to a round shape and a growth rate slower than that of wild-type cells. Suppressor mutations that simultaneously restore both the growth rates and the rod shape were isolated. Most of the suppressor mutations are found in mreB, mrdA, or mrdB. One of the mutations was in the promoter region of zipA, which encodes a crucial component of the cell division machinery. In this study, we investigated the mechanism of the suppression by this mutation. ZipA was slightly but significantly increased in the suppressor cells and led to a delay in cell division. While round-shaped mreB and mrdA mutants lose cell bipolarity, we found that round-shaped rodZ mutants retained cell bipolarity. Therefore, we concluded that a delay in the completion of septation provides extra time to elongate the cell laterally so that the zipA suppressor mutant is able to recover its ovoid or rod shape. The suppression by zipA demonstrates that the regulation of timing of septation potentially contributes to the conversion of morphology in bacterial cells.

This study has been carried out as collaboration with Dr. Ogura at Tohoku University.

Rod shaped E. coli cells grow at the central cylinder. Ovoid shaped Streptococcus cells grow at septum while round shaped Staphylococcus cells swell. Black arrows indicate the direction of increase of cell volume. The increase in cell volume of ΔrodZ resembles that of Streptococcus, while those of ΔmreB or ΔmrdA resemble that of Staphylococcus. Dark gray zones indicate regions where peptidoglycan is actively synthesized. E. coli (WT), ΔrodZ, and Streptococcus cells retain cell polarity, while E. coli ΔmreB, ΔmrdA, and Staphylococcus cells lose polarity.