伊藤 素行

We have reported that an environmental pollutant, cadmium, promotes cell death in the human renal tubular cells (RTCs) through hyperactivation of a serine/threonine kinase Akt. However, the molecular mechanisms downstream of Akt in this process have not been elucidated. Cadmium has a potential to accumulate misfolded proteins, and proteotoxicity is involved in cadmium toxicity. To clear the roles of Akt in cadmium exposure-induced RTCs death, we investigated the possibility that Akt could regulate proteotoxicity through autophagy in cadmium chloride (CdCl2)-exposed HK-2 human renal proximal tubular cells. CdCl2 exposure promoted the accumulation of misfolded or damaged proteins, the formation of aggresomes (pericentriolar cytoplasmic inclusions), and aggrephagy (selective autophagy to degrade aggresome). Pharmacological inhibition of Akt using MK2206 or Akti-1/2 enhanced aggrephagy by promoting dephosphorylation and nuclear translocation of transcription factor EB (TFEB)/transcription factor E3 (TFE3), lysosomal transcription factors. TFEB or TFE3 knockdown by siRNAs attenuated the protective effects of MK2206 against cadmium toxicity. These results suggested that aberrant activation of Akt attenuates aggrephagy via TFEB or TFE3 to facilitate CdCl2-induced cell death. Furthermore, these roles of Akt/TFEB/TFE3 were conserved in CdCl2-exposed primary human RTCs. The present study shows the molecular mechanisms underlying Akt activation that promotes cadmium-induced RTCs death.

Many age-related neurological diseases still lack effective treatments, making their understanding a critical and urgent issue in the globally aging society. To overcome this challenge, an animal model that accurately mimics these diseases is essential. To date, many mouse models have been developed to induce age-related neurological diseases through genetic manipulation or drug administration. These models help in understanding disease mechanisms and finding potential therapeutic targets. However, some age-related neurological diseases cannot be fully replicated in human pathology due to the different aspects between humans and mice. Although zebrafish has recently come into focus as a promising model for studying aging, there are few genetic zebrafish models of the age-related neurological disease. This review compares the aging phenotypes of humans, mice, and zebrafish, and provides an overview of age-related neurological diseases that can be mimicked in mouse models and those that cannot. We presented the possibility that reproducing human cerebral small vessel diseases during aging might be difficult in mice, and zebrafish has potential to be another animal model of such diseases due to their similarity of aging phenotype to humans.

The brain is an essential organ that controls various biological activities via the nervous system. The cerebral blood vessels supply oxygen and nutrients to neuronal cells and carry away waste products, which is essential in maintaining brain functions. Aging affects cerebral vascular function and decreases brain function. However, the physiological process of age-dependent cerebral vascular dysfunction is not fully understood. In this study, we examined aging effects on cerebral vascular patterning, vascular function, and learning ability in adult zebrafish. We found that the tortuosity of the blood vessels was increased, and the blood flow rate was reduced with aging in the zebrafish dorsal telencephalon. Moreover, we found cerebral blood flow positively correlated with learning ability in middle-old-aged zebrafish, as in aged humans. In addition, we also found that the elastin fiber decreased in the middle-old-aged fish brain vessel, suggesting a possible molecular mechanism underlying vessel dysfunction. Therefore, adult zebrafish may serve as a useful model for studying the aging-dependent decline in vascular function and human diseases such as vascular dementia.

Memory formation and sleep regulation are critical for brain functions in animals from invertebrates to humans. Neuropeptides play a pivotal role in regulating physiological behaviors, including memory formation and sleep. However, the detailed mechanisms by which neuropeptides regulate these physiological behaviors remains unclear. Herein, we report that neuropeptide diuretic hormone 31 (DH31) positively regulates memory formation and sleep in Drosophila melanogaster. The expression of DH31 in the dorsal and ventral fan-shaped body (dFB and vFB) neurons of the central complex and ventral lateral clock neurons (LNvs) in the brain was responsive to sleep regulation. In addition, the expression of membrane-tethered DH31 in dFB neurons rescued sleep defects in Dh31 mutants, suggesting that DH31 secreted from dFB, vFB, and LNvs acts on the DH31 receptor in the dFB to regulate sleep partly in an autoregulatory feedback loop. Moreover, the expression of DH31 in octopaminergic neurons, but not in the dFB neurons, is involved in forming intermediate-term memory. Our results suggest that DH31 regulates memory formation and sleep through distinct neural pathways.

As a normal physiological phenomenon, aging has a significant impact on sleep. Aging leads to sleep impairment, including sleep loss, fragmented sleep, and a lower arousal threshold, leading to various diseases. Because sleep regulates memory consolidation, age-dependent sleep impairment also affects memory. However, the mechanisms underlying age-related sleep dysregulation and its impact on memory remain unclear. Using male and female Drosophila as a model, which possesses sleep characteristics similar to those of mammals and exhibits age-dependent sleep impairment, we performed small-molecule screening to identify novel regulators of age-dependent decline in sleep. The screening identified 3,3'-difluorobenzaldazine (DFB), a positive allosteric modulator of the metabotropic glutamate receptor (mGluR) 5, as a novel sleep-promoting compound in aged flies. We found that mutant flies of mGluR, a single mGluR gene in Drosophila, and decreased mGluR expression had significant impairment in sleep and memory due to olfactory conditioning. The decreased sleep phenotype in the mGluR mutants was not promoted by DFB, suggesting that the effects of DFB on age-dependent sleep impairment are dependent on mGluR. Although aging decreases the expression of mGluR and the binding scaffold proteins Homer and Shank, the transient overexpression of mGluR in neurons improves sleep in both young and aged flies. Overall, these findings indicate that age-dependent decreased expression or function of mGluR impairs sleep and memory in flies, which could lead to age-related sleep and memory impairment.

BMP signaling is critical for many biological processes. Therefore, small molecules that modulate BMP signaling are useful for elucidating the function of BMP signaling and treating BMP signaling-related diseases. Here, we performed a phenotypic screening in zebrafish to examine the in vivo effects of N-substituted-2-amino-benzoic acid analogs NPL1010 and NPL3008 and found that they affect BMP signaling-dependent dorsal-ventral (D-V) patterning and bone formation in zebrafish embryos. Furthermore, NPL1010 and NPL3008 suppressed BMP signaling upstream of BMP receptors. BMP1 cleaves Chordin, an antagonist of BMP, and negatively regulates BMP signaling. Docking simulations demonstrated that NPL1010 and NPL3008 bind BMP1. We found that NPL1010 and NPL3008 partially rescued the disruptions in the D-V phenotype caused by bmp1 overexpression and selectively inhibited BMP1-dependent Chordin cleavage. Therefore, NPL1010 and NPL3008 are potentially valuable inhibitors of BMP signaling that act through selective inhibition of Chordin cleavage.

Age-related changes in the transcriptome lead to memory impairment. Several genes have been identified to cause age-dependent memory impairment (AMI) by changes in their expression, but genetic screens to identify genes critical for AMI have not been performed. The fruit fly is a useful model for studying AMI due to its short lifespan and the availability of consistent techniques and environments to assess its memory ability. We generated a list of candidate genes that act as AMI regulators by performing a comprehensive analysis of RNAsequencing data from young and aged fly heads and genome-wide RNAi screening data to identify memory-regulating genes. A candidate screen using temporal and panneuronal RNAi expression was performed to identify genes critical for AMI. We identified the guanylyl cyclase β-subunit at 100B (gycβ) gene, which encodes a subunit of soluble guanylyl cyclase (sGC), the only intracellular nitric oxide (NO) receptor in fruit flies, as a negative regulator of AMI. RNAi knockdown of gycβ in neurons and NO synthase (NOS) in glia or neurons enhanced the performance of intermediate-term memory (ITM) without apparent effects on memory acquisition. We also showed that pharmacological inhibition of sGC and NOS enhanced ITM in aged individuals, suggesting the possibility that age-related enhancement of the NO-sGC pathway causes memory impairment.

Notch signaling, which is essential for tissue development and homeostasis, has received attention as an attractive target for cancer therapy, tissue engineering and regenerative medicine. For signal activation, the Notch receptor undergoes proteolysis after binding to its ligand. This process is mediated by a mechanical pulling force, and receptor trans-endocytosis is known to play a central role in supplying the force. On the other hand, Notch ligands immobilized on carrier materials also induce artificial Notch activation. However, the mechanism of signal activation by immobilized ligand proteins is not fully understood. Here, we found that the actin cytoskeleton in Notch1-expressing cells contributes to signal activation induced by immobilized DLL4 (Delta-like ligand 4), and the results showed that pharmacological inhibition of actin dynamics impaired Notch signaling induced by DLL4-coated beads. Moreover, inhibition of actin dynamics remarkably impaired cell migration and was correlated with Notch signaling activity. We also investigated the contribution of Notch cis-endocytosis (the endocytosis of Notch receptor into signal-receiving cells) as an actin-mediated cell biological process to further explore the mechanism of Notch activation by DLL4-coated beads. Compromising the receptor cis-endocytosis pathway with the dynamin inhibitor did not alter DLL4-coated bead-induced Notch signaling, indicating that signal activation is not mediated by dynamin-dependent receptor cis-endocytosis. These findings suggest that Notch activation by immobilized ligands is primarily driven by actin-based cell movement, which might supply a sufficient mechanical force for receptor cleavage, but not by receptor cis-endocytosis.

Aging is a major risk factor for many chronic diseases, causing a general decline in physiological function and loss of homeostasis. Recently, small teleost fish have been used as animal models of aging research because their genetic structures and organs closely resemble those of humans. Guppy (Poecilia reticulata), a small teleost fish, has a shorter lifespan than zebrafish. However, the age-dependent changes in physiology and genetics in guppies are not well understood. Here, we investigated the age-associated changes in metabolic rate, physical activity, and gene expression in guppies. Our results indicated that the resting metabolic rate and spontaneous motor activity in guppies decreased from an earlier age than those in mice. Moreover, the mRNA expression level of ppargc1a and the accumulation of lipofuscin were affected by age in the guppy livers; however, these changes were species-specific. On the other hand, in aged guppy brains, the mRNA expression changes of some genes were partly consistent with aged mammals. Although the process of senescence of the liver in guppies might vary from mammals, our findings suggest that guppy could be a useful animal model for age-related changes in physiological functions.

Activation of Notch signaling requires physical interaction between ligand- and receptor-expressing cells and pulling force to release the Notch intracellular domain. Therefore, the soluble recombinant ligand protein is not suitable for the activation of Notch signaling in a cell culture system. Here, we describe an efficient method for transient activation of Notch signaling using immobilized ligand beads. Using this method, the timing of Notch signaling can be efficiently controlled.

In this work, we aim to construct a new behavior analysis method by using machine learning. We used two cameras to capture three-dimensional (3D) tracking data of zebrafish, which were analyzed using fuzzy adaptive resonance theory (FuzzyART), a type of machine learning algorithm, to identify specific behavioral features. The method was tested based on an experiment in which electric shocks were delivered to zebrafish and zebrafish swimming was tracked in 3D simultaneously to find electric shock-associated behaviors. By processing the obtained data with FuzzyART, we discovered that distinguishing behaviors were statistically linked to the electric shock based on the machine learning algorithm. Moreover, our system could accept user-supplied data for detection and quantitative analysis of the behavior features, such as the behavior features defined by the 3D tracking analysis above. This system could be applied to discover new distinct behavior features in mutant zebrafish and used for drug administration screening and cognitive ability tests of zebrafish in the future.

Glucocorticoid-induced osteoporosis (GIOP) is a major cause of secondary osteoporosis, and the pathogenic mechanisms of GIOP remain to be elucidated. Here, we show a rapid dexamethasone-induced osteoporosis animal model using zebrafish scales. Intraperitoneal injection of dexamethasone over a 5-day period suppressed the regeneration of scales. Furthermore, the circularity of the newly formed regenerated scales was also slightly reduced compared to that of the control group on day 5. The changes in bone-related enzymes, such as cathepsin K, tartrate-resistant acid phosphatase (TRAP) for bone resorption, and alkaline phosphatase (ALP) for bone formation, provide insight into the progression of bone diseases; therefore, we further developed a method to measure the activities of cathepsin K, TRAP, and ALP using zebrafish scales. We found that a lysis buffer with detergent at neutral pH under sonication efficiently helped extract these three enzymes with high activity levels. Interestingly, treatment with a dexamethasone injection produced considerably higher levels of cathepsin K activity and a lower Ca/P ratio than those in the control group, suggesting that dexamethasone increased osteoclast activity, with no significant changes in the activities of TRAP and ALP. Our GIOP model and enzyme assay method could help to design better treatments for GIOP.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetic small vessel disease characterized by NOTCH3 mutation and abnormal aggregation of NOTCH3 mutant proteins around vessel walls. NOTCH3 is a transmembrane receptor that is degraded by JAGGED1 (JAG1) through a process called trans-endocytosis. There are two types of CADASIL-associated NOTCH3 mutations: signal-active (SA) and signal-deficient (SD) mutations. However, the conditions that lead to abnormal aggregation of NOTCH3 mutant proteins remain poorly understood. Performing a coculture assay, we found that the SA NOTCH3 mutants (C49Y, R90C, R141C, and C185R) were degraded and trans-endocytosed by JAG1 similar to wild-type (WT) NOTCH3, but the SD NOTCH3 mutant (C428S) was not degraded or endocytosed by JAG1, suggesting that other environmental factors may be necessary for the aggregation of SA NOTCH3 mutants. Lunatic fringe (LFNG) is a glycosyltransferase of NOTCH3, but whether LFNG affects the aggregation of NOTCH3 mutants remains unknown. Performing a sucrose gradient ultracentrifugation assay, we found that LFNG might decrease the aggregation propensity of WT NOTCH3 but increase that of C185R NOTCH3. In conclusion, the SD NOTCH3 mutant may be more likely to accumulate than the SA NOTCH3 mutants upon interaction with JAG1. Moreover, LFNG may play an important role in promoting the aggregation of SA NOTCH3 mutants.

Polypyrrole-based polyamides are used as sequence-specific DNA probes. However, their cellular uptake and distribution are affected by several factors and have not been extensively studied<italic>in vivo</italic>. Here, we generated a series of fluorescence-conjugated polypyrrole compounds and examined their cellular distribution using live zebrafish and cultured human cells. Among the evaluated compounds, Py₃-FITC was able to visualize collagen-rich tissues, such as the jaw cartilage, opercle and bulbus arteriosus, in early-stage living zebrafish embryos. Then, we stained cultured human cells with Py₃-FITC and found that the staining became more intense as the amount of collagen was increased. In addition, Py₃-FITC-stained HR cells, which represent a type of ionocyte on the body surface of living zebrafish embryos. Py₃-FITC has low toxicity, and collagen-rich tissues and ionocytes can be visualized when soaked in Py₃-FITC solution. Therefore, Py₃-FITC may be a useful live imaging tool for detecting changes in collagen-rich tissue and ionocytes, including their mammalian analogues, during both normal development and disease progression.

Upstream open reading frames (uORFs) are present in the 5'-untranslated regions of many eukaryotic mRNAs, and some peptides encoded by these regions play important regulatory roles in controlling main ORF (mORF) translation. We previously developed a novel pipeline, ESUCA, to comprehensively identify plant uORFs encoding functional peptides, based on genome-wide identification of uORFs with conserved peptide sequences (CPuORFs). Here, we applied ESUCA to diverse animal genomes, because animal CPuORFs have been identified only by comparing uORF sequences between a limited number of species, and how many previously identified CPuORFs encode regulatory peptides is unclear. By using ESUCA, 1517 (1373 novel and 144 known) CPuORFs were extracted from four evolutionarily divergent animal genomes. We examined the effects of 17 human CPuORFs on mORF translation using transient expression assays. Through these analyses, we identified seven novel regulatory CPuORFs that repressed mORF translation in a sequence-dependent manner, including one conserved only among Eutheria. We discovered a much higher number of animal CPuORFs than previously identified. Since most human CPuORFs identified in this study are conserved across a wide range of Eutheria or a wider taxonomic range, many CPuORFs encoding regulatory peptides are expected to be found in the identified CPuORFs.

In the vertebrate ventral spinal cord, p2 progenitors give rise to two interneuron subtypes: excitatory V2a interneurons and inhibitory V2b interneurons. In the differentiation of V2a and V2b cells, Notch signaling promotes V2b fate at the expense of V2a fate. Later, V2b cells extend axons along the ipsilateral side of the spinal cord and express the inhibitory transmitter GABA. Notch signaling has been reported to inhibit the axonal outgrowth of mature neurons of the central nervous system; however, it remains unknown how Notch signaling modulates V2b neurite outgrowth and maturation into GABAergic neurons. Here, we have investigated neuron-specific Notch functions regarding V2b axon growth and maturation into zebrafish GABAergic neurons. We found that continuous neuron-specific Notch activation enhanced V2b fate determination but inhibited V2b axonal outgrowth and maturation into GABAergic neurons. These results suggest that Notch signaling activation is required for V2b fate determination, whereas its downregulation at a later stage is essential for V2b maturation. Accordingly, we found that a Notch signaling downstream gene, her15.1, showed biased expression in V2 linage cells and downregulated expression during the maturation of V2b cells, and continuous expression of her15.1 repressed V2b axogenesis. Our data suggest that spatiotemporal control of Notch signaling activity is required for V2b fate determination, maturation and axogenesis.

The formation of memory declines with advancing age. However, susceptibility to memory impairments depends on several factors, including the robustness of memory, the responsible neural circuits, and the internal state of aged individuals. How age-dependent changes in internal states and neural circuits affect memory formation remains unclear. Here, we show in Drosophila melanogaster that aged flies of both sexes form robust appetitive memory conditioned with nutritious sugar, which suppresses their high mortality rates during starvation. In contrast, aging impairs the formation of appetitive memory conditioned with non-nutritious sugar that lacks survival benefits for the flies. We found that aging enhanced the preference for nutritious sugar over non-nutritious sugar correlated with an age-dependent increase in the expression of Drosophila neuropeptide F, an ortholog of mammalian neuropeptide Y. Furthermore, a subset of dopaminergic neurons that signal the sweet taste of sugar decreases its function with aging, while a subset of dopaminergic neurons that signal the nutritional value of sugar maintains its function with age. Our results suggest that aging impairs the ability to form memories without survival benefits; however, the ability to form memories with survival benefits is maintained through age-dependent changes in the neural circuits and neuropeptides.SIGNIFICANCE STATEMENT The susceptibility to age-dependent memory impairments depends on the strength of the memory, changes in the responsible neurons, and internal states of aged individuals. How age-dependent changes in such internal states affect neural activity and memory formation remains unclear. We show in Drosophila melanogaster that aged flies of both sexes form robust appetitive memory conditioned with nutritious sugar, which has survival benefits for aged flies. In contrast, aging impairs the formation of appetitive memory conditioned with non-nutritious sugar that lacks survival benefits for the flies. Aging changes the neural circuits including dopamine neurons and neuropeptide F-expressing neurons, leading to the age-dependent impairment in memory with insufficient survival benefits and the preservation of the ability to form memory with survival benefits.

Metabolic alterations are relevant for the aging process. Declining metabolic rate with age is a common future of many animals, but it is not well understood how it does so. Here, we used zebrafish as a model for understanding how metabolic changes occur during aging and the interaction between aging and obesity on the metabolic rate. The oxygen consumption rate (OCR) has been used as an index of metabolic processes; however, it is difficult to accurately evaluate OCR with movement being considered because zebrafish need to move freely during the OCR measurement. To measure metabolic rate with high accuracy and efficiency, we developed a method for simultaneously collecting data on sequential oxygen consumption and distance moved by zebrafish using optical dissolved-oxygen sensors and the EthoVision video-tracking system as well as an automatic feeding system for zebrafish whereby obese zebrafish were produced by short-term overfeeding treatment. Using these systems, we examined metabolic changes during aging and overfeeding. First, we used 1- to 22-month-old zebrafish to evaluate changes in metabolism during the aging process. Measurements of body mass and length showed that the growth of the body rarely continued beyond 6 months, at which point zebrafish reach adulthood. Spontaneous swimming activity peaked at approximately 6-10 months and declined thereafter. Metabolic rates at low movement dramatically dropped during the first 4 months and gradually decreased with age after 10 months. These data suggest that metabolic aging becomes evident at approximately 10-14 months and that the metabolic rate (low movement) is useful for the detection of age-related metabolic changes in zebrafish. Second, by short-term overfeeding treatment using the automatic feeding system, we found that overweight is a strong risk factor for the development of metabolic disorders in zebrafish, but there was no interaction between obesity and aging on the metabolic rate. Therefore, our data suggest that the aging-related decline in metabolic-rate may be mostly programmed rather than being affected by energy balance disorder.

Glycolysis, the classic pathway for producing energy, has been known to be involved in neural development. Notch signaling also contributes to neural development and regulation of glycolysis in various tissues. However, the role of Notch signaling in glycolysis-related gene regulation during neural development is poorly understood. Here, we analyzed mRNA expression patterns and levels of glucose transporters (GLUT) as well as rate-limiting enzymes in glycolysis using zebrafish mib1ta52b mutants, in which Notch signaling was deficient at the early embryonic and larval stages. Our results indicated that in neural tissues, Notch signaling positively regulates glut1a and glut3a expression and negatively regulates hk2 expression at the larval stage but may not regulate them during early embryonic stages. Therefore, these results suggest that Notch signaling regulates glycolysis-related gene expression in a context-dependent manner in neural tissues at different developmental stages.

While pericytes wrap around microvascular endothelial cells throughout the human body, their highest coverage rate is found in the brain. Brain pericytes actively contribute to various brain functions, including the development and stabilization of the blood-brain barrier (BBB), tissue regeneration, and brain inflammation. Accordingly, detailed characterization of the functional nature of brain pericytes is important for understanding the mechanistic basis of brain physiology and pathophysiology. Herein, we report on the development of a new human brain pericyte cell line, hereafter referred to as the human brain pericyte/conditionally immortalized clone 37 (HBPC/ci37). Developed via the cell conditionally immortalization method, these cells exhibited excellent proliferative ability at 33 °C. However, when cultured at 37 °C, HBPC/ci37 cells showed a differentiated phenotype that was marked by morphological alterations and increases in several pericyte-enriched marker mRNA levels, such as platelet-derived growth factor receptor β. It was also found that HBPC/ci37 cells possessed the facilitative ability of in vitro BBB formation and differentiation into a neuronal lineage. Furthermore, HBPC/ci37 cells exhibited the typical "reactive" features of brain pericytes in response to pro-inflammatory cytokines. To summarize, our results clearly demonstrate that HBPC/ci37 cells possess the ability to perform several key brain pericyte functions while also showing the capacity for extensive and continuous proliferation. Based on these findings, it can be expected that, as a unique human brain pericyte model, HBPC/ci37 cells have the potential to contribute to significant advances in the understanding of human brain pericyte physiology and pathophysiology.

We designed an automated device to study active avoidance learning abilities of zebrafish. Open source tools were used for the device control, statistical computing, and graphic outputs of data. Using the system, we developed active avoidance tests to examine the effects of trial spacing and aging on learning. Seven-month-old fish showed stronger avoidance behavior as measured by color preference index with discrete spaced training as compared to successive spaced training. Fifteen-month-old fish showed a similar trend, but with reduced cognitive abilities compared with 7-month-old fish. Further, in 7-month-old fish, an increase in learning ability during trials was observed with discrete, but not successive, spaced training. In contrast, 15-month-old fish did not show increase in learning ability during trials. Therefore, these data suggest that discrete spacing is more effective for learning than successive spacing, with the zebrafish active avoidance paradigm, and that the time course analysis of active avoidance using discrete spaced training is useful to detect age-related learning impairment.

Persistent directional cell migration is involved in animal development and diseases. The small GTPase Rac1 is involved in F-actin and focal adhesion dynamics. Local Rac1 activity is required for persistent directional migration, whereas global, hyperactivated Rac1 enhances random cell migration. Therefore, precise control of Rac1 activity is important for proper directional cell migration. However, the molecular mechanism underlying the regulation of Rac1 activity in persistent directional cell migration is not fully understood. Here, we show that the ubiquitin ligase mind bomb 1 (Mib1) is involved in persistent directional cell migration. We found that knockdown of MIB1 led to an increase in random cell migration in HeLa cells in a wound-closure assay. Furthermore, we explored novel Mib1 substrates for cell migration and found that Mib1 ubiquitinates Ctnnd1. Mib1-mediated ubiquitination of Ctnnd1 K547 attenuated Rac1 activation in cultured cells. In addition, we found that posterior lateral line primordium cells in the zebrafish mib1 ta52b mutant showed increased random migration and loss of directional F-actin-based protrusion formation. Knockdown of Ctnnd1 partially rescued posterior lateral line primordium cell migration defects in the mib1 ta52b mutant. Taken together, our data suggest that Mib1 plays an important role in cell migration and that persistent directional cell migration is regulated, at least in part, by the Mib1-Ctnnd1-Rac1 pathway.

Notch is a critical signaling pathway that controls cell fate and tissue homeostasis, but the functional characterization of Notch ligand domains that activate Notch receptors remains incomplete. Here, we established a method for immobilizing Notch ligand proteins onto beads to measure time-dependent Notch activity after the addition of Notch ligand-coated beads. A comparison between activities by the Notch ligand found on the cell surface to that of the ligand immobilized on beads showed that immobilized Notch ligand protein produces comparable signal activity during the first 10 h. Follow-up truncation studies showed that the N-terminal epidermal growth factor (EGF) repeat three region of delta like canonical Notch ligand 4 (DLL4) or jagged 1 (JAG1) is the minimum region for activating Notch signaling, and the DLL4 EGF repeat three domain may have a role in activation through a mechanism other than by increasing binding affinity. In addition, we found that reconstruction of the DLL4 delta and OSM-11 (DOS) motif (N257P) resulted in an increase in both binding affinity and signaling activity, which suggests that the role of the DOS motif is conserved among Notch ligands. Furthermore, active DLL4 protein on beads promoted T cell differentiation or inhibited B cell differentiation in vitro, whereas JAG1 proteins on beads did not have any effect. Taken together, our findings provide unambiguous evidence for the role of different Notch ligands and their domains in Notch signal activation, and may be potential tools for controlling Notch signaling activation. J. Cell. Biochem. 118: 785–796, 2017. © 2016 Wiley Periodicals, Inc.

Insulin and insulin-growth-factor-like signaling (IIS) plays important roles in the regulation of development, growth, metabolic homeostasis, and aging, as well as in brain functions such as learning and memory. The temporal-spatial role of IIS in learning and memory and its effect on age-dependent memory impairment remain unclear. Here, we report that intermediate-term memory (ITM), but not short-term memory (STM), in Drosophila aversive olfactory memory requires transient IIS during adulthood. The expression of Drosophila insulin-like peptide 3 (Dilp3) in insulin-producing cells and insulin receptor function in the fat body are essential for ITM. Although the expression of dilp3 decreases with aging, which is unique among dilp genes, the transient expression of dilp3 in aged flies enhances ITM. These findings indicate that ITM is systemically regulated by communication between insulin-producing cells and fat body and that age-dependent changes in IIS contribute to age-related memory impairment.

We identified Erythrocyte membrane protein band 4.1-like 5 (Epb41l5) as a substrate for the E3 ubiquitin ligase Mind bomb 1 (Mib1), which is essential for activation of Notch signaling. Although loss of Epb41l5 does not significantly alter the pattern of neural progenitor cells (NPCs) specified as neurons at the neural plate stage, it delays their delamination and differentiation after neurulation when NPCs normally acquire organized apical junctional complexes (AJCs) in the zebrafish hindbrain. Delays in differentiation are reduced by knocking down N-cadherin, a manipulation expected to help destabilize adherens junctions (AJs). This suggested that delays in neuronal differentiation in epb41l5-deficient embryos are related to a previously described role for Epb41l5 in facilitating disassembly of cadherin-dependent AJCs. Mib1 ubiquitylates Epb41l5 to promote its degradation. DeltaD can compete with Epb41l5 to reduce Mib1-dependent Epb41l5 degradation. In this context, increasing the number of NPCs specified to become neurons, i.e. cells expressing high levels of DeltaD, stabilizes Epb41l5 in the embryo. Together, these observations suggest that relatively high levels of Delta stabilize Epb41l5 in NPCs specified as neurons. This, we suggest, helps coordinate NPC specification with Epb41l5-dependent delamination and differentiation as neurons.

Notch signaling regulates normal development and tissue homeostasis. Ligand endocytosis plays critical roles in Notch signaling activation. Endocytic proteins such as epsin and dynamin participate in Notch ligand activity by mediating Notch ligand endocytosis. The ubiquitin ligase Mib1 also plays essential roles in Notch signaling via Notch ligand ubiquitination. However, the molecular links between Mib1 and endocytic proteins have not been fully defined. Here, we show that Mib1 is involved in dynamin 2 recruitment to Dll1 and that Snx18, which interacts with dynamin 2, modestly regulates Dll1 endocytosis. Furthermore, the ubiquitin ligase activity of Mib1 is induced by Notch ligand-receptor interactions. Mib1 promotes the interaction between dynamin 2 and Snx18 in an ubiquitin ligase activity-dependent manner. These results suggest that Mib1 modulates dynamin recruitment by regulating the interaction between Snx18 and dynamin 2, thereby helping to ensure the efficient signaling activity of Notch ligands.

The Mindbomb E3 ubiquitin protein ligase (Mib) family of proteins, Mib1 and Mib2, are RING finger ubiquitin ligases that share specific substrates. Mib1 is known to play essential roles in Notch signalling by ubiquitinating Notch ligands in vivo. Conversely, the functions of Mib2 in vivo are not fully understood, although Mib2 ubiquitinates multiple substrates, including Notch ligands, in vitro. To determine the Notch-dependent and Notch-independent functions of Mib2 in vivo, we generated mutant alleles of zebrafish mib2 using transcription activator-like effector nucleases (TALENs). We found that mib2 homozygous mutants were viable and fertile. Notch-mediated functions, such as early neurogenesis, somitogenesis, and pigment cell development, were not affected in mib2 mutant embryos. The lack of Notch-deficient phenotypes in mib2 mutants was not due to compensation by a mib2 maternal gene product because mib2 maternal-zygotic mutants also did not exhibit a distinct phenotype. We also showed that Mib2 does not redundantly act with Mib1 because the genetic ablation of mib2 neither enhanced mib(tfi91)-null phenotypes nor did it alleviate antimorphic mib(ta52b) phenotypes. Furthermore, the postulated Notch-independent roles of Mib2 in maintaining muscular integrity and N-methyl-D-aspartate receptor (NMDAR) activity were not evident: mib2 mutants did not show phenotypes different from that of the control embryos. These observations suggest that Mib2 is dispensable for embryonic development and does not have redundant functions with Mib1 in Notch signalling at least during early development stages in zebrafish.

Wnt signaling plays important roles in proliferation, differentiation, development of cells, and various diseases. Activity-guided fractionation of the MeOH extract of the Ricinus communis stem led to the isolation of four compounds (1-4). The TCF/beta-catenin transcription activities of 1 and 3 were 2.2 and 2.5 fold higher at 20 and 30 mu M, respectively. Cells treated with ricinine (1) had higher beta-catenin and lower of p-beta-catenin (ser 33, 37, 45, Thr 41) protein levels, whereas glycogen synthase kinase 3 beta (GSK3 beta) and casein kinase 1 alpha (CK1 alpha) protein levels remained unchanged. Cells treated with pyrvinium, an activator of CK1 alpha, had lower beta-catenin levels. However, the combined treatment of pyrvinium and 1 led to higher beta-catenin levels than those in cells treated with pyrvinium alone, which suggested that 1 inhibited CK1 alpha activity. Furthermore, 1 increased beta-catenin protein levels in zebrafish embryos. These results indicated that 1 activated the Wnt signaling pathway by inhibiting CK1 alpha. (C) 2014 Elsevier Ltd. All rights reserved.

The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-intemeuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Here, using zebrafish embryos with altered Notch signaling, we show that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD, and three receptors, Notch1a, Notch1b, and Notch3 redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib, which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand-receptor combinations that occur concurrently during development in zebrafish. (C) 2014 Elsevier Inc. All rights reserved.

Spatially and temporally controlled gene expression, including transcription, several mRNA processing steps, and the export of mature mRNA to the cytoplasm, is essential for developmental processes. It is well known that RNA helicases of the DExD/H-box protein family are involved in these gene expression processes, including transcription, pre-mRNA splicing, and rRNA biogenesis. Although one DExD/H-box protein, Prp5, a homologue of vertebrate Ddx46, has been shown to play important roles in pre-mRNA splicing in yeast, the in vivo function of Ddx46 remains to be fully elucidated in metazoans. In this study, we isolated zebrafish morendo (mor), a mutant that shows developmental defects in the digestive organs and brain, and found that it encodes Ddx46. The Ddx46 transcript is maternally supplied, and as development proceeds in zebrafish larvae, its ubiquitous expression gradually becomes restricted to those organs. The results of whole-mount in situ hybridization showed that the expression of various molecular markers in these organs is considerably reduced in the Ddx46 mutant. Furthermore, splicing status analysis with RT-PCR revealed unspliced forms of mRNAs in the digestive organ and brain tissues of the Ddx46 mutant, suggesting that Ddx46 may be required for pre-mRNA splicing during zebrafish development. Therefore, our results suggest a model in which zebrafish Ddx46 is required for the development of the digestive organs and brain, possibly through the control of pre-mRNA splicing.

Although recent findings showed that some Drosophila doublesex and Caenorhabditis elegans mab-3 related genes are expressed in neural tissues during development, their functions have not been fully elucidated. Here, we isolated a zebrafish mutant, ha2, that shows defects in telencephalic neurogenesis and found that ha2 encodes Doublesex and MAB-3 related transcription factor like family A2 (Dmrta2). dmrta2 expression is restricted to the telencephalon, diencephalon and olfactory placode during somitogenesis. We found that the expression of the proneural gene, neurogenin1, in the posterior and dorsal region of telencephalon (posterior-dorsal telencephalon) is markedly reduced in this mutant at the 14-somite stage without any defects in cell proliferation or cell death. In contrast, the telencephalic expression of her6, a Hes-related gene that is known to encode a negative regulator of neurogenin1, expands dramatically in the ha2 mutant. Based on over-expression experiments and epistatic analyses, we propose that zebrafish Dmrta2 controls neurogenin1 expression by repressing her6 in the posterior-dorsal telencephalon. Furthermore, the expression domains of the telencephalic marker genes, foxg1 and emx3, and the neuronal differentiation gene, neurod, are downregulated in the ha2 posterior-dorsal telencephalon during somitogenesis. These results suggest that Dmrta2 plays important roles in the specification of the posterior-dorsal telencephalic cell fate during somitogenesis. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Sensory systems are specialized to recognize environmental changes. Sensory organs are complex structures composed of different cell types, including neurons and sensory receptor cells, and how these organs are generated is an important question in developmental neurobiology. The posterior lateral line (pLL) is a simple sensory system in fish and amphibians that detects changes in water motion. It consists of neurons and sensory receptor hair cells, both of which are derived from the cranial ectoderm preplacodal region. However, it is not clearly understood how neurons and the sensory epithelium develop separately from the same preplacodal progenitors. We found that the numbers of posterior lateral line ganglion (pLLG) neurons, which are marked by neurod expression, increased in embryos with reduced Notch activity, but the forced activation of Notch reduced their number, suggesting that Notch-mediated lateral inhibition regulates the pLLG cell fate in zebrafish. By fate-mapping analysis, we found that cells adjacent to the pLLG neurons in the pre-pLL placodal region gave rise to the anterior part of the pLL primordium (i.e., sensory epithelial progenitor cells), and that the choice of cell fate between pLLG neuron or pLL primordium was regulated by Notch signaling. Since Notch signaling also affects hair cell fate determination at a later stage, our study suggests that Notch signaling has dual, time-dependent roles in specifying multiple cell types during pLL development. © 2011 the authors.

In the developing embryo, cell-cell signalling is necessary for tissue patterning and structural organization. During midline development, the notochord plays roles in the patterning of its surrounding tissues while forming the axial structure however, how these patterning and structural roles are coordinated remains elusive. Here, we identify a mechanism by which Notch signalling regulates the patterning activities and structural integrity of the notochord. We found that Mind bomb (Mib) ubiquitylates Jagged 1 (Jag1) and is essential in the signal-emitting cells for Jag1 to activate Notch signalling. In zebrafish, loss- and gain-of-function analyses showed that Mib-Jag1-Notch signalling favours the development of non-vacuolated cells at the expense of vacuolated cells in the notochord. This leads to changes in the peri-notochordal basement membrane formation and patterning surrounding the muscle pioneer cells. These data reveal a previously unrecognized mechanism regulating the patterning and structural roles of the notochord by Mib-Jag1-Notch signalling-mediated cell-fate determination.

Failure of Notch signaling in zebrafish mind bomb (mib) mutants results in a neurogenic phenotype where an overproduction of early differentiating neurons is accompanied by the loss of later-differentiating cell types. We have characterized in detail the hindbrain phenotype of mib mutants. Hindbrain branchiomotor neurons (BMNs) are reduced in number but not missing in mib mutants. In addition, BMN clusters are frequently fused across the midline in mutants. Mosaic analysis indicates that the BMN patterning and fusion defects in the mib hindbrain arise non-cell autonomously. Ventral midline signaling is defective in the mutant hindbrain, in part due to the differentiation of some midline cells into neural cells. Interestingly, while early hindbrain patterning appears normal in mib mutants, subsequent rhombomere-specific gene expression is completely lost. The defects in ventral midline signaling and rhombomere patterning are accompanied by an apparent loss of neuroepithelial cells in the mutant hindbrain. These observations suggest that, by regulating the differentiation of neuroepithelial cells into neurons, Notch signaling preserves a population of non-neuronal cells that are essential for maintaining patterning mechanisms in the developing neural tube. (C) 2003 Wiley-Liss, Inc.

Caudalizing factors operate in the context of Wnt/beta-catenin signaling to induce gene expression in discrete compartments along the rostral-caudal axis of the developing vertebrate nervous system. In zebrafish, basal repression of caudal genes is achieved through the function of Headless (Hdl), a Tcf3 homolog. In this study, we show that a second Tcf3 homolog, Tcf3b, limits caudalization caused by loss of Hdl function and although this Lef/Tcf family member can rescue hdl mutants, Lefl cannot. Wnts can antagonize repression mediated by Tcf3 and this derepression is dependent on a Tcf3 beta-catenin binding domain. Systematic changes in gene expression caused by reduced Tcf3 function help predict the shape of a caudalizing activity gradient that defines compartments along the rostral-caudal axis. In addition, Tcf3b has a second and unique role in the morphogenesis of rhombomere boundaries, indicating that it controls multiple aspects of brain development.

The midbrain-hindbrain (MH) domain of the vertebrate embryonic neural plate displays a stereotypical profile of neuronal differentiation, organized around a neuron-free zone ('intervening zone', IZ) at the midbrain-hindbrain boundary (MHB). The mechanisms establishing this early pattern of neurogenesis are unknown. We demonstrate that the MHB is globally refractory to neurogenesis, and that forced neurogenesis in this area interferes with the continued expression of genes defining MHB identity. We further show that expression of the zebrafish bHLH Hairy/E(spl)-related factor Her5 prefigures and then precisely delineates the IZ throughout embryonic development. Using morpholino knock-down and conditional gain-of-function assays, we demonstrate that Her5 is essential to prevent neuronal differentiation and promote cell proliferation in a medial compartment of the IZ. We identify one probable target of this activity, the zebrafish Cdk inhibitor p27(Xic1). Finally, although the her5 expression domain is determined by anteroposterior patterning cues, we show Her5 does not retroactively influence MH patterning. Together, our results highlight the existence of a mechanism that actively inhibits neurogenesis at the MHB, a process that shapes MH neurogenesis into a pattern of separate neuronal clusters and might ultimately be necessary to maintain MHB integrity. Her5 appears as a partially redundant component of this inhibitory process that helps translate early axial patterning information into a distinct spatiotemporal pattern of neurogenesis and cell proliferation within the MH domain.