Tameness can be divided into two components: active tameness and passive tameness. We conducted selective breeding for higher scores of active tameness using a wild-derived heterogeneous stock. In the analyses of nine behavioral traits related to tameness, we found that five traits showed changes in the selective groups compared to the control groups through the generations. We conducted cluster analyses to evaluate the relationship among the nine traits and RNA-Seq analysis to characterize the molecular network related to tameness. The results suggested that active tameness was hidden in the control groups but became apparent in the selected populations by selective breeding, potentially driven by changes in gene expression networks.

Figure: The wild-derived heterogeneous stock was divided into four groups, two groups were selected for active tameness, and the other two groups were unselected controls. A: As the generation progressed, active tameness increased in the two selection groups, whereas there was no significant increase in the two control groups. B: RNA-seq analysis was performed using samples collected from the brains (hippocampus) of mice in the selection groups and the control groups. As a result, genes with different expression levels were found in the selection groups and the control groups, and differences in the gene network were also found.

When DNA double-strand breaks occur, four-stranded DNA structures called Holliday junctions (HJs) form during homologous recombination (HR). Because HJs connect homologous DNA by a covalent link, resolution of HJ is crucial to terminate HR and segregate the pair of DNA molecules faithfully. We recently identified Monokaryotic Chloroplast 1 (MOC1) as a plastid DNA (ptDNA) HJ resolvase in algae and plants. Although Cruciform cutting endonuclease 1 (CCE1) was identified

as a mitochondrial DNA (mtDNA) HJ resolvase in yeasts, homologs or other mitochondrial HJ resolvases have not been identified in other eukaryotes. Here, we demonstrated that MOC1 depletion in the green alga Chlamydomonas reinhardtii and the moss Physcomitrella patens induced ectopic recombination between short dispersed repeats (SDRs) in ptDNA. In addition, MOC1 depletion disorganized thylakoid membranes in plastids. In some land plant lineages, such as the moss P. patens, a liverwort and a fern, MOC1 dually targeted to plastids and mitochondria. Moreover, mitochondrial targeting of MOC1 was also predicted in charophyte algae and some land plant species. Besides causing instability of ptDNA, MOC1 depletion in P. patens induced SDR-mediated ectopic recombination in mtDNA and disorganized cristae in mitochondria. Similar phenotypes in plastids and mitochondria were previously observed in mutants of plastid-targeted (RECA2) and mitochondrion-targeted (RECA1) recombinases, respectively. These results suggest that MOC1 functions in the double-strand break repair in which a recombinase generates HJs and MOC1 resolves HJs in mitochondria of some lineages of algae and plants in addition to plastids in algae and plants.

Figure: (A) Schematic diagram of double strand break repair that is mediated by a recombinase RECA and a Holliday junction resolvase MOC1.
(B) Evolutionary transition of MOC1-mediated organelle DNA maintenance in plants.

Limbed vertebrates exhibit the basic form of locomotion, consisting of alternating activities of the flexor and extensor muscles within each limb coupled with left/right limb alternation. Zebrafish pectoral fin movements show the fundamental aspects of this basic movement: abductor/adductor alternation (corresponding to flexor/extensor alternation) and left/right fin alternation. Thus, zebrafish can serve as a good model to identify the neuronal networks of the central pattern generator (CPG) that controls rhythmic appendage movements. Here, we investigate neuronal circuits underlying rhythmic pectoral fin movements in larval zebrafish, using transgenic fish that specifically express GFP in abductor or adductor motor neurons (MNs) and candidate CPG neurons. First, we showed that spiking activities of abductor and adductor MNs are essentially alternating. Second, both abductor and adductor MNs receive rhythmic excitatory and inhibitory synaptic inputs in their active and inactive phases, respectively, indicating that the MN activities are controlled in a push-pull manner. Further, we obtained the evidence that dmrt3a-expressing commissural inhibitory neurons are involved in regulating the activities of abductor MNs: (1) strong inhibitory synaptic connections were found from dmrt3a neurons to abductor MNs; and (2) ablation of dmrt3a neurons shifted the spike timing of abductor MNs. Thus, in this simple system of abductor/adductor alternation, the last-order inhibitory inputs originating from the contralaterally located neurons play an important role in controlling the firing timings of MNs.

This study was conducted as collaboration with Professor Shin-ichi Higashijima at National Institute for Basic Biology. This study was partially supported by NBRP and NBRP/Fundamental Technologies Upgrading Program from the Japan Agency for Medical Research and Development.

Figure: Rhythmic pectoral fin movements in 3 dpf larvae.

Figure: A1, A2 Transgenic fish that labeled abductor motor neurons.
B1, B2 Transgenic fish that labeled abductor motor neurons.

DNA methylation is important for silencing transposable elements (TEs) in diverse eukaryotes including plants. In plant genomes, TEs are silenced by methylation of histone H3 lysine 9 (H3K9) and cytosines in both CG and non-CG contexts. The role of RNA interference (RNAi) in establishing TE-specific silent marks has been extensively studied, but the significance of RNAi-independent pathways remains largely unexplored. Here, we directly investigated transgenerational de novo DNA methylation of TEs after the loss of silent marks. Our analyses uncovered potent and precise RNAi-independent pathways for recovering non-CG methylation and H3K9 methylation in most TE genes (i.e. coding regions within TEs). Characterization of a subset of TE genes without the recovery revealed the impacts of H3K9 demethylation, replacement of histone H2A variants, and their interaction with CG methylation, together with feedback from transcription. These chromatin components are conserved among eukaryotes and may contribute to chromatin reprograming in a conserved manner.

Supported by Systems Functional Genetics Project of the Transdisciplinary Research Integration Center, ROIS.

Figure: H3K9 and non-CG methylation can be maintained and inherited by mutual reinforcement. These marks are established by RNAi-dependent and -independent pathways. The RNAi-independent pathway mainly works in coding regions of TEs.