Sakiko Matsumoto

Expression analysis of the regenerating gene (Reg) family members Reg-IIIβ and Reg-IIIγ in the mouse during development

The regenerating gene/regenerating islet‐derived (Reg) family is a group of small secretory proteins. Within this family, Reg type‐III (Reg‐III) consists of: Reg‐IIIα, ‐β, ‐γ, and ‐δ. To elucidate the physiological relevance of Reg‐III, we examined the localization and ontogeny of Reg‐IIIβ and Reg‐IIIγ in mice at different time points spanning from embryonic day 13.5 to 7 weeks old, using in situ hybridization and immunohistochemistry. Our results showed that Reg‐IIIβ was expressed in specific subsets of primary sensory neurons and motor neurons, and that expression was transient during the embryonic and perinatal periods. Reg‐IIIβ expression was also observed in absorptive epithelial cells of the intestine. In contrast, Reg‐IIIγ expression was mainly observed in epithelial cells of the airways and intestine, but not in the nervous system, and expression levels showed a gradually increasing pattern along with development. In the airways Reg‐IIIγ was expressed in goblet and Clara‐like cells, whereas in the intestine Reg‐IIIγ was expressed in the absorptive epithelial cells and Paneth cells, and was found to be expressed in development before these organs had been exposed to the outside world. The present findings imply that Reg‐IIIβ and Reg‐IIIγ expression is regulated along divergent pathways. Furthermore, we also suggest that expression of Reg‐IIIγ in the airway and intestinal epithelia may occur to protect these organs from exposure to antigens or other factors (e.g., microbes) in the outer world, whereas the transient expression of Reg‐IIIβ in the nervous system may be associated with the development of the peripheral nervous system including such processes as myelination. J. Comp. Neurol., 2012;520:479–494.

Motor Nerve Arborization Requires Proteolytic Domain of Damage-Induced Neuronal Endopeptidase (DINE) during Development

Damage-induced neuronal endopeptidase (DINE)/endothelin-converting enzyme-like 1 (ECEL1) is a membrane-bound metalloprotease, which we originally identified as a nerve regeneration-associated molecule. Abundant expression of DINE is observed in regenerating neurons, as well as in developing spinal motor neurons. In line with this, DINE-deficient (DINE KO) embryos fail to arborize phrenic motor nerves in the diaphragm and to form proper neuromuscular junctions (NMJ), which lead to death shortly after birth. However, it is unclear whether protease activity of DINE is involved in motor nerve terminal arborization and how DINE participates in the process. To address these issues, we performed an in vivo rescue experiment in which three types of motor-neuron specific DINE transgenic mice were crossed with DINE KO mice. The DINE KO mice, which overexpressed wild-type DINE in motor neurons, succeeded in rescuing the aberrant nerve terminal arborization and lethality after birth, while those overexpressing two types of protease domain-mutated DINE failed. Further histochemical analysis showed abnormal behavior of immature Schwann cells along the DINE-deficient axons. Coculture experiments of motor neurons and Schwann cells ensured that the protease domain of neuronal DINE was required for proper alignment of immature Schwann cells along the axon. These findings suggest that protease activity of DINE is crucial for intramuscular innervation of motor nerves and subsequent NMJ formation, as well as proper control of interactions between axons and immature Schwann cells. SIGNIFICANCE STATEMENT Damage-induced neuronal endopeptidase (DINE) is a membrane-bound metalloprotease; expression is abundant in developing spinal motor neurons, as well as in nerve-injured neurons. DINE-deficient (KO) embryos fail to arborize phrenic motor nerves in the diaphragm and to form a neuromuscular junction, leading to death immediately after birth. To address whether proteolytic activity of DINE is involved in this process, we performed in vivo rescue experiments with DINE KO mice. Transgenic rescue of DINE KO mice was accomplished by overexpression of wild-type DINE, but not by protease domain-mutated DINE. Immature Schwann cells were abnormally aligned along the DINE protease-deficient axons. Thus, the protease activity of DINE is crucial for motor axon arborization, as well as the interaction between axons and immature Schwann cells.

Sex-Related Differences in the Foraging Movement of Streaked Shearwaters Calonectris leucomelas Breeding on Awashima Island in the Sea of Japan

Abstract Sex-related differences in foraging habitat are common among seabirds. Streaked Shearwaters Calonectris leucomelas breeding on Awashima Island in the Sea of Japan are considered to exhibit gender differences in foraging habitat: only males cross the windy Tsugaru Strait into the Pacific Ocean. Since males are larger, with greater wing loading than females, winds are expected to increase the effect of sexual size dimorphism on their flight performance, which may determine accessibility to foraging habitats. To assess the sex-related differences in foraging movements among years in which environmental and wind conditions differed, we analyzed foraging trips of male (N=243) and female (N=241) Streaked Shearwaters during the chick-rearing period by using GPS loggers in 2011, 2012, and 2013. Both males and females were found to travel through the Tsugaru Strait into the Pacific Ocean, but the frequency was higher for males than for females. Nevertheless, we found that wind velocities had no effect on the probability of transiting the Tsugaru Strait. Greater wing loading requires higher energy demands for flight; therefore, males possibly needed to travel into the Pacific Ocean to feed on the energy-rich Pacific Saury Cololabis saira. In 2012, when the sea surface temperature (SST) in the Sea of Japan was the highest among the three study years, the frequency of foraging in the Pacific Ocean was similar for males and females. Shearwaters are considered to forage in association with predatory fish, the distribution of which is largely influenced by the Tsushima Warm Current migrating partially into the Tsugaru Strait. Hence, both males and females were more likely to travel into the Pacific Ocean when the Sea of Japan SST was high, generating conditional sex-related differences in foraging habitat.

Damage-induced neuronal endopeptidase (DINE) enhances axonal regeneration potential of retinal ganglion cells after optic nerve injury

Damage-induced neuronal endopeptidase (DINE)/endothelin-converting enzyme-like 1 (ECEL1) is a membrane-bound metalloprotease that we identified as a nerve regeneration-associated molecule. The expression of DINE is upregulated in response to nerve injury in both the peripheral and central nervous systems, while its transcription is regulated by the activating transcription factor 3 (ATF3), a potent hub-transcription factor for nerve regeneration. Despite its unique hallmark of injury-induced upregulation, the physiological relevance of DINE in injured neurons has been unclear. In this study, we have demonstrated that the expression of DINE is upregulated in injured retinal ganglion cells (RGCs) in a coordinated manner with that of ATF3 after optic nerve injury, whereas DINE and ATF3 are not observed in any normal retinal cells. Recently, we have generated a mature DINE-deficient (KOTg) mouse, in which exogenous DINE is overexpressed specifically in embryonic motor neurons to avoid aberrant arborization of motor nerves and lethality after birth that occurs in the conventional DINE KO mouse. The DINE KOTg mice did not show any difference in retinal structure and the projection to brain from that of wild–type (wild type) mice under normal conditions. However, injured RGCs of DINE KOTg mice failed to regenerate even after the zymosan treatment, which is a well-known regeneration-promoting reagent. Furthermore, a DINE KOTg mouse crossed with a Atf3:BAC Tg mouse, in which green fluorescent protein (GFP) is visualized specifically in injured RGCs and optic nerves, has verified that DINE deficiency leads to regeneration failure. These findings suggest that injury-induced DINE is a crucial endopeptidase for injured RGCs to promote axonal regeneration after optic nerve injury. Thus, a DINE-mediated proteolytic mechanism would provide us with a new therapeutic strategy for nerve regeneration.

N-terminal Cleaved Pancreatitis-associated Protein-III (PAP-III) Serves as a Scaffold for Neurites and Promotes Neurite Outgrowth

Background: PAP-III is a secretory protein expressed in injured nerves. Results: Extracellular PAP-III formed fibrillar structures upon proteolytic N-terminal processing, interacted with neuronal surfaces, and enhanced neurite extension. Conclusion: Fibrillar PAP-III formed around injury sites may serve as a scaffold for the growth of regenerating axons. Significance: Clarifying fibrillar PAP-III functions would provide a novel strategy for nerve regeneration. Pancreatitis-associated protein (PAP)-III, also known as regenerating gene/regenerating islet-derived (Reg)-IIIγ, is a small secretory protein whose expression is substantially induced in injured nerves. Here, we found that PAP-III protein underwent proteolytic N-terminal processing by trypsin-like protease(s) in injured sciatic nerves after axotomy. In vitro studies demonstrated that the N terminus-truncated PAP-III (ΔN-PAP-III) polymerized into a filament with a relatively uniform diameter of 10–20 nm, and the filaments formed higher order structures in a Na+ concentration-dependent manner. When the ΔN-PAP-III fibers were added to the culture media, the ΔN-PAP-III fibers were tightly attached to neurites and somata of primary cortical neurons in vitro. In contrast, little association with glial cells was observed. When dense matrices of ΔN-PAP-III fibers were sheeted on a culture dish, neurites preferentially adhered to the fibers, and neurite extension was enhanced. This neurite outgrowth activity was significantly suppressed by preincubation with antibodies against PAP-III. These results imply that the released PAP-III might be cleaved and forms ΔN-PAP-III fibers at the nerve injury sites. Consequently, these resulting fibers would provide regenerating axons with a platform for extension.

Continuous stress-induced dopamine dysregulation augments PAP-I and PAP-II expression in melanotrophs of the pituitary gland

Under continuous stress (CS) in rats, melanotrophs, the predominant cell-type in the intermediate lobe (IL) of the pituitary, are hyperactivated to secrete α-melanocyte-stimulating hormone and thereafter degenerate. Although these phenomena are drastic, the molecular mechanisms underlying the cellular changes are mostly unknown. In this study, we focused on the pancreatitis-associated protein (PAP) family members of the secretory lectins and characterized their expression in the IL of CS model rats because we had identified two members of this family as up-regulated genes in our previous microarray analysis. RT-PCR and histological studies demonstrated that prominent PAP-I and PAP-II expression was induced in melanotrophs in the early stages of CS, while another family member, PAP-III, was not expressed. We further examined the regulatory mechanisms of PAP-I and PAP-II expression and revealed that both were induced by the decreased dopamine levels in the IL under CS. Because the PAP family members are implicated in cell survival and proliferation, PAP-I and PAP-II secreted from melanotrophs may function to sustain homeostasis of the IL under CS conditions in an autocrine or a paracrine manner.

Compass orientation drives naïve pelagic seabirds to cross mountain ranges

Wildlife migration is a spectacular phenomenon [1]. Studies using telemetry - tracking devices attached on free-living animals - have shown that large topographic barriers and obstacles, such as oceans and deserts, elicit extreme feats of migration [2]. Overcoming the challenges of these obstacles might require experience and skill that young individuals lack [2-5]. Further, younger, inexperienced animals might determine their migration routes using navigation strategies different from those of older animals [6-9], but our knowledge of how orientation mechanisms and experience drive migration strategy is limited. We have studied how experienced (adults) and inexperienced (first-time migrating fledglings) streaked shearwaters (Calonectris leucomelas) approach the challenge of migration using animal-borne tracking devices. The study birds migrate from a colony on the north of a large topographic barrier, Honshu Island, Japan. Shearwaters use a wind- and wave-based flight pattern-dynamic soaring-to extract energy for highly efficient travel over oceans [10]. We therefore expected that shearwaters migrating southward from the colony would make substantial detours to avoid any landmasses. We found that migrating adults followed one of two paths that detour around landmasses that hinder direct southerly migration. In contrast, inexperienced fledglings followed a straight course in a south-oriented direction that forced them to complete a trans-mountain journey, suggesting that the birds rely on an innate compass. Thus, we suggest that fledglings would eventually override the simple compass navigation, which appears to be the primary driver for their extreme migration, before being able to interact appropriately with the marine environment.

Finding Discriminative Animal Behaviors from Sequential Bio-Logging Trajectory Data

Recent advancement of bio-logging devices such as GPS sensor enables researchers in ecology to quantitatively measure animal trajectories. These animal trajectory data are often represented in the form of multi-dimensional time-series. In this paper, we develop a method for extracting interesting animal behaviors from these multi-dimensional time-series. To this end, we represent a multi-dimensional time-series as a discrete symbol sequence, and introduce some techniques developed in the context of sequential pattern mining, which has been actively studied in the literature of knowledge discovery and data mining. In animal behavior studies, it is often desired to conduct comparative studies for finding different animal behaviors in different groups, e.g, different behaviors between male and female animals etc. We use a sequential pattern mining method designed for finding so-called discriminative sequential patterns, i.e., sequential patterns that are useful for discriminating different group of animals. We apply the method to several animal trajectory datasets for demonstrating its effectiveness.