International Workshop on Developmental and Regenerative Biology in Yamagata

Sep 10 / 2014 

S401 Lecture Room

Faculty of Science

Yamagata University

 

1. 14:00 – 14:40  Epigenetic regulations in the vertebrate heart regeneration

Kazuko Koshiba-Takeuchi1, Ryo Nakamura1, Tetsuo Sasano2, Tetsushi Furukawa2, Hesham A Sadek3, Jun K Takeuchi1

1: IMCB, The University of Tokyo; 2: Tokyo Medical and Dental University; 3: Southwestern  Medical Center, University of Texas

 

2. 14:40 – 15:20  Transcriptomics and proteomics-guided analysis of tissue regeneration in adult salamanders

Thomas Braun, Tania Piatkowski, Mario Looso

Max Planck Institute for Heart and Lung Research                                                    

 

        <coffee break>

 

3. 15:35 – 16:15  Can we learn the cause of differences in regeneration-competency from the newt?

Chikafumi Chiba1, Fubito Toyama2, Fumiaki Maruo1, Md. Rafiqul Islam3, Martin Miguel Casco-Robles4, Roman Miguel Casco-Robles3

1: Faculty of Life and Environmenta,l Sciences, University of Tsukuba; 2: Information Science,Faculty of Engineering, Utsunomiya University; 3: Graduate School of Life and Environmental Sciences, University of Tsukuba; 4: JSPS Postdoctoral fellow

 

4. 16:15 – 16:30  Identification of a cis-regulatory module for glomerular regeneration using  comparative genomics and transgenesis techniques for amphibian

Nanoka Suzuki1, Kazuyuki Hoshijima2, Hajime Ogino3, Haruki Ochi1

1: School of Medicine, Yamagata University; 2: Human Genetics, University of Utah; 3: Department of Animal Bioscience, Nagahama Institute of Bio-Science and Technology

 

5. 16:30 – 16:40  Tyrosinase gene knockout by TALEN technology in the red-bellied newt, Cynops pyrrhogaster

Kodai Hirano1, Haruki Ochi2, Kazuyuki Hoshijima3, Kazuhito Takeshima4, Eriko Takayama-Watanabe4, Akihiko Watanabe1

1: Department of Biology, Faculty of Science; 2: School of Medicine, Yamagata University; 3: Human Genetics, University of Utah; 4: Radioisotope Research Center, Nagoya University; 5: Institute of Arts and Sciences, Yamagata University

 

6. 16:40-17:10  Global survey of the genes involving in the mechanism of newt fertilization using de novo assembled RNAseq

Akihiko Watanabe1, Eriko Takayama-Watanabe2  

1: Department of Biology, Faculty of Science; 2: Institute of Arts and Sciences, Yamagata University

 

This workshop is presented by Yamagata University (Faculty of Science), YU-COE (E): Innovation of Advanced Reproductive Technologies, and Japan Newt Research Community, and financially supported by Center for International Business Planning Research in Yamagata University.

 

 

 

 

 

Abstracts:

 

Epigenetic regulations in the vertebrate heart regeneration

 

Kazuko Koshiba-Takeuchi1, Ryo Nakamura1, Tetsuo Sasano2, Tetsushi Furukawa2, Hesham A Sadek3, Jun K Takeuchi1

1: IMCB, The University of Tokyo; 2: Tokyo Medical and Dental University; 3: University of Texas Southwestern Medical Center

 

    Fish and amphibians can regenerate their hearts throughout their life, while mice can regenerate the heart in a couple days after birth and lose the capacity by 7 days. The reduction of regenerative capacity in mammals would be caused by epigenetic modification associating with cardiac maturation. However, any remarkable epigenetic modifiers have not been elucidated. We found that Baf60c, a component of activated-type SWI/SNF chromatin-remodeling factors, was strongly upregulated in ventricular cardiomyocytes after heart resection in both neonatal mice and axolotls. To address the function of Baf60c in heart regeneration, we generated two types of Baf60c-overepression mice; one could stably express Baf60c under a MHC promoter and another could transiently express Baf60c by lentiviral infection. Histological analyses showed that the Baf60c-overexpression promoted cardiac proliferation with protection of fibrosis. Interestingly, in the Baf60c-viral infected hearts, the specific promoter/enhancer regions on several cardiac genes encoding transcriptional regulators, contraction factors and angiogenesis factors were activated with recruitment of PolII via histone modification. In contrast, inflammation and hypoxia-induced cell death associated genes were repressed. These data indicate that the Baf60c-dependent chromatin remodeling should be critical for heart maturation and regeneration.   

 

 

Transcriptomics and proteomics-guided analysis of tissue regeneration in adult salamanders

 

Thomas Braun, Tania Piatkowski, Mario Looso

Max-Planck-Institute for Heart and Lung Research

 

  The newt Notophthalmus viridescens , which belongs to the family of salamanders (Urodela), owns remarkable regenerative capacities allowing efficient scar-free repair of various organs including the heart. Unfortunately, very little is known about the molecules and the regulatory circuits facilitating efficient tissue regeneration in salamanders. Moreover, the long reproduction cycle time of salamanders renders traditional in vivo transgenesis approaches for mechanistic studies difficult. We have developed a number of approaches to overcome these limitations taking advantages of high-end mass spectrometry and next-generation sequencing. In total we identified ca. 121,00 non-redundant transcripts of which ca. 39,000 were annotated. Around 15,000 transcripts were experimentally validated as protein coding by mass spectrometry-based proteomics. In addition, we determined the miRNAome of Notophthalmus viridescens and identified miRNAs, which were dynamically regulated during heart regeneration. Pulsed SILAC-based mass spectrometry enabled us to characterized new salamander-specific proteins, which might be part of the regulatory network driving organ regeneration. Specifically, we identified a new member of the CCN protein family, showing a wound specific activation in the damaged heart. Systematic analysis of kinase-dependent signaling cascades in regenerating newt hearts combined with the use of small-molecule pathway inhibitors allowed us to determine the functional relevance of distinct regulatory circuits for the regulation of heart regeneration. Finally, we developed a new ex-vivo heart explant model to monitor and manipulate organ regeneration of salamanders in vivo. Preliminary evidence suggests that initiation of cardiomyocyte proliferation in regenerating hearts requires signals derived from circulating myeloid cells, which accumulate in the heart following damage.

 

 

Can we learn the cause of differences in regeneration-competency from the newt?

 

Chikafumi Chiba1, Fubito Toyama2, Fumiaki Maruo1, Md. Rafiqul Islam3, Martin Miguel Casco-Robles4, Roman Miguel Casco-Robles3

1: Faculty of Life and Environmenta,l Sciences, University of Tsukuba; 2: Information Science, Faculty of Engineering, Utsunomiya University; 3: Graduate School of Life and Environmental Sciences, University of Tsukuba; 4: JSPS Postdoctoral fellow

 

   Our talk focuses on the recent findings in the study of retinal disorders and regeneration, which has been pursued on the basis of resources, techniques and information for the newt Cynops pyrrhogaster.

 

 

Identification of a cis-regulatory module for glomerular regeneration using comparative genomics and transgenesis techniques for amphibian

 

Nanoka Suzuki1, Kazuyuki Hoshijima2, Hajime Ogino3, Haruki Ochi1

1: School of Medicine, Yamagata University; 2: Human Genetics, University of Utah; 3: Department of Animal Bioscience, Nagahama Institute of Bio-Science and Technology

 

   We are studying the cis-regulatory logic of glomerular regeneration. To date, we have identified several nephron enhancers for Lhx1, Six2, Pax2, and Pax8 using the X. laevis transgenic system. We are in the process of identifying stem cell enhancers for Lhx1 and Six2 and regeneration-responding enhancers for Pax2 and Pax8. TALEN-mediated enhancer disruption experiments are also being conducted to examine the significance of the identified enhancers of glomerular regeneration.

 

 

Tyrosinase gene disruption by TALEN in the red-bellied newt, Cynops pyrrhogaster

 

Kodai Hirano1, Haruki Ochi2, Kazuyuki Hoshijima3, Kazuhito Takeshima4, Eriko Takayama-Watanabe4, Akihiko Watanabe1

1: Department of Biology, Faculty of Science; 2: School of Medicine, Yamagata University; 3: Human Genetics, University of Utah, 4: Radioisotope Research Center, Nagoya University; 5: Institute of Arts and Sciences, Yamagata University

 

   Genome editing by the transcription activator-like nuclease (TALEN) is a powerful tool to analyze in vivo function of a single gene in the process of embryonic development and regeneration of non-model animals. In urodele amphibians, the availability of the TALEN has been reported in Preurodeles Waltli, in which albino phenotype was developed by tyrosinase gene disruption (Hayashi et al., 2014). In order to optimize the procedure for gene disruption by TALEN in the red-bellied newt, Cynops pyrrhogaster, in which many studies were accumulated in correlation with the mechanism of embryonic development and regeneration, we identified a base sequence for tyrosinase in de novo assembled RNAseq data and designed the TALENs at the translation initiation site and putative active site. By injecting 150-400 pg of the mRNA for the TALENs into cytoplasm of fertilized eggs, we successfully obtained the less-pigmented larvae compared to wild type. We also developed the protocol for evaluation of the deletion in the targeted site using a small tail tissue of St.50 larva without sacrificing. We are now determining the experimental conditions that provide better efficiency of TALEN-driven gene disruption.

 

 

Global survey of the genes involving in the mechanism of newt fertilization using de novo assembled RNAseq

 

Akihiko Watanabe1, Eriko Takayama-Watanabe2  

1: Department of Biology, Faculty of Science; 2: Institute of Arts and Sciences, Yamagata University

 

   Diversification of reproductive modes based on the modification of molecular mechanisms for sperm-egg interaction is a primary nature in animals to adapt to specific reproductive environments. The highly diversified modes of amphibian reproduction provide a potential model for investigating the molecular mechanism to modify the sperm-egg interaction in adaptation to the fertilization environment, although poor gene information in non-model amphibians has presented a large difficulty in such investigations. In the present study, to estimate the availability of de novo assembled RNAseq from ovary, testis and oviduct samples of the red-bellied newt, Cynops pyrrhogaster, we screened molecules to identify base sequences encoding multiple proteins involving the sperm-egg interaction such as zona pellucida (ZP) proteins, voltage-dependent Ca2+ channels, and cysteine-rich secretory proteins (CRISPs), which are the sperm receptor for the egg envelope, major mediators of sperm intracellular signaling, and the expected extracellular modulators for sperm function in the female reproductive tract, respectively. The RNAseq from the ovary samples included a ZP1 homologue that has not been found in amphibians and 6 identical ZPC homologues, which may be involved in the specific machinery of sperm binding to egg envelopes. CaV1.1, 1.2, and 3.2 was expressed in the testis, making them candidate mediators for sperm-egg interaction specific to the internal fertilization of C. pyrrhogaster. In addition, we found a novel CRISP in the oviduct. These results indicate that de novo assembled RNAseq is a powerful tool for obtaining global gene information involved in the specific sperm-egg interactions of non-model amphibians.