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During mammalian corticogenesis, Notch signaling is essential to maintain neural stem cells called radial glial cells (RGCs) and the cortical architecture. Because the conventional knockout of either Notch1 or Notch2 causes a neuroepithelial loss prior to neurogenesis, their functional relationship in RGCs remain elusive. Here, we investigated the impacts of single knockout of Notch1 and Notch2 genes, and their conditional double knockout (DKO) on mouse corticogenesis. We demonstrated that Notch1 single knockout affected RGC maintenance in early to mid-neurogenesis whereas Notch2 knockout caused no apparent defect. In contrast, Notch2 plays a role in the RGC maintenance as Notch1 does at the late stage. Notch1 and Notch2 DKO resulted in the complete loss of RGCs, suggesting their cooperative function. We found that Notch activity in RGCs depends on the Notch gene dosage irrespective of Notch1 or Notch2 at late neurogenic stage, and that Notch1 and Notch2 have a similar activity, most likely due to a drastic increase in Notch2 transcription. Our results revealed that Notch1 has an essential role in establishing the RGC pool during the early stage, whereas Notch1 and Notch2 subsequently exhibit a comparable function for RGC maintenance and neurogenesis in the late neurogenic period in the mouse telencephalon., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The origin of the mammalian lymphatic vasculature has been studied for more than a century; however, details regarding organ-specific lymphatic development remain unknown. A recent study reported that cardiac lymphatic endothelial cells (LECs) stem from venous and non-venous origins in mice. Here, we identified Isl1-expressing progenitors as a potential non-venous origin of cardiac LECs. Genetic lineage tracing with Isl1-Cre reporter mice suggested a possible contribution from the Isl1-expressing pharyngeal mesoderm constituting the second heart field to lymphatic vessels around the cardiac outflow tract as well as to those in the facial skin and the lymph sac. Isl1 + lineage-specific deletion of Prox1 resulted in disrupted LYVE1 + vessel structures, indicating a Prox1-dependent mechanism in this contribution. Tracing back to earlier embryonic stages revealed the presence of VEGFR3 + and/or Prox1 + cells that overlapped with the Isl1 + pharyngeal core mesoderm. These data may provide insights into the developmental basis of heart diseases involving lymphatic vasculature and improve our understanding of organ-based lymphangiogenesis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Organoids mimicking the formation of the brain cortex have been demonstrated to be powerful tools for developmental studies as well as pathological investigations of brain malformations. Here, we report an integrated approach for the quantification of temporal neural production (neurogenic rate) in organoids derived from embryonic brains. Spherical tissue fragments with polarized cytoarchitectures were incubated in multiple cavities arranged in a polymethylmethacrylate chip. The time-dependent neurogenic rate in the organoids was monitored by the level of EGFP under the promoter of Tbr2, a transcription factor that is transiently expressed in neural fate-committed progenitors during corticogenesis. Importantly, our monitoring system exhibited a quick response to DAPT, a drug that promotes neural differentiation. Furthermore, we successfully quantified the temporal neurogenic rate in a large number of organoids by applying image processing that semi-automatically recognized the positions of organoids and measured their signal intensities from sequential images. Taken together, we provide a strategy to quantitate the neurogenic rate in brain organoids in a time-dependent manner, which will also be a potent method for monitoring organoid formation and drug activity in other tissue types., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The coordinated polarization of neighboring cells within the plane of the tissue, known as planar cell polarity (PCP), is a recurring theme in biology. It is required for numerous developmental processes for the form and function of many tissues and organs across species. The genetic pathway regulating PCP was first discovered in Drosophila, and an analogous but distinct pathway is emerging in vertebrates. It consists of membrane protein complexes known as core PCP proteins that are conserved across species. Here we report that the over-expression of the murine Ankrd6 (mAnkrd6) gene that shares homology with Drosophila core PCP gene diego causes a typical PCP phenotype in Drosophila, and mAnkrd6 can rescue the loss of function of diego in Drosophila. In mice, mAnkrd6 protein is asymmetrically localized in cells of the inner ear sensory organs, characteristic of components of conserved core PCP complexes. The loss of mAnkrd6 causes PCP defects in the inner ear sensory organs. Moreover, canonical Wnt signaling is significantly increased in mouse embryonic fibroblasts from mAnkrd6 knockout mice in comparison to wild type controls. Together, these results indicated that mAnkrd6 is a functional homolog of the Drosophila diego gene for mammalian PCP regulation and act to suppress canonical Wnt signaling., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aurora-A kinase (AurA) is a key regulator of cellular processes involving microtubules. It has also been implicated in actin-dependent events, but the mechanisms that underlie the processes are not fully understood. Here we provide genetic and biochemical evidence suggesting that AurA negatively regulates Drok, the only known Rho-kinase orthologue in Drosophila. AurA directly phosphorylates Drok in vitro, and the overexpression of the nonphosphorylatable forms of Drok in vivo causes similar, but much stronger effects than that of wild-type Drok. The defects induced by the nonphosphorylatable forms of Drok are compensated by reducing the function of myosin downstream. Thus, phosphorylation of Drok by AurA normally suppresses Drok activity. We propose that AurA directly regulates actin-dependent processes by phosphorylating Rho-kinase., (Copyright © 2013 The authors. Published by Elsevier Inc. All rights reserved.)

Asymmetric cell division generates two daughter cells of differential gene expression and/or cell shape. Drosophila neuroblasts undergo typical asymmetric divisions with regard to both features; this is achieved by asymmetric segregation of cell fate determinants (such as Prospero) and also by asymmetric spindle formation. The loss of genes involved in these individual asymmetric processes has revealed the roles of each asymmetric feature in neurogenesis, yet little is known about the fate of the neuroblast progeny when asymmetric processes are blocked and the cells divide symmetrically. We genetically created such neuroblasts, and found that in embryos, they were initially mitotic and then gradually differentiated into neurons, frequently forming a clone of cells homogeneous in temporal identity. By contrast, larval neuroblasts with the same genotype continued to proliferate without differentiation. Our results indicate that asymmetric divisions govern lineage length and progeny fate, consequently generating neural diversity, while the progeny fate of symmetrically dividing neuroblasts depends on developmental stages, presumably reflecting differential activities of Prospero in the nucleus., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Drosophila neuroblasts provide an excellent model for asymmetric cell divisions, where cell-fate determinants such as Miranda localize at the basal cortex and segregate to one daughter cell. Mechanisms underlying this process, however, remain elusive. We found that Mo25 and the GC kinase Fray act in this regulation. mo25 and fray mutants show an indistinguishable defect in Miranda localization. On the other hand, Drosophila Mo25 interacts with the tumor suppressor kinase Lkb1 in vivo, as have shown in mammals. Overexpression of Lkb1, which accumulates in the cell cortex, drastically relocalizes both Mo25 and Fray from the cytoplasm to the cortex, causing the same phenotype as mo25-mutant neuroblasts. Recovery from this defect caused by Lkb1 overexpression requires simultaneous overexpression of Mo25 and Fray. We suggest from those results that Mo25 and Fray operate together or in the same pathway in Drosophila asymmetric processes, and that their function counterbalances Lkb1.

Segmentation plays crucial roles during morphogenesis. Drosophila legs are divided into segments along the proximal-distal axis by flexible structures called joints. Notch signaling is necessary and sufficient to promote leg growth and joint formation, and is activated in distal cells of each segment in everting prepupal leg discs. The homeobox gene defective proventriculus (dve) is expressed in regions both proximal and distal to the intersegmental folds at 4 h after puparium formation (APF). Dve-expressing region partly overlaps with the Notch-activated region, and they become a complementary pattern at 6 h APF. Interestingly, dve mutant legs resulted in extra joint formation at the center of each tarsal segment, and the forced expression of dve caused a jointless phenotype. We present evidence that Dve suppresses the potential joint-forming activity, and that Notch signaling represses Dve expression to form joints.

We cloned full-length cDNA (PcCYP1f) encoding one of the cytochrome P450s in the lignin-degrading basidiomycete Phanerochaete chrysosporium, which showed high homology to P450s in the CYP53 family. PcCYP1f was expressed as an active microsomal protein using the methylotrophic yeast Pichia pastoris expression system. Using the microsomal fraction containing PcCYP1f, a typical P450 CO-difference spectrum was obtained with absorption maximum at 448nm. Recombinant PcCYP1f catalyzed the hydroxylation of benzoic acid into 4-hydroxybenzoic acid in the presence of NADPH and P. chrysosporium cytochrome P450 oxidoreductase. In contrast to other CYP53 P450s, this enzyme was shown to catalyze the hydroxylation of 3-hydroxybenzoate into 3,4-dihydroxybenzoate. Furthermore, 2- and 3-methylbenzoate were also shown to be substrates of PcCYP1f. This is the first report showing the expression of a functionally active Phanerochaete P450. Finally, real-time quantitative PCR analysis revealed that PcCYP1f is induced at a transcriptional level by exogenous addition of benzoic acid.

The functional diversity of cytochrome P450s (P450s) of the white-rot basidiomycete, Phanerochaete chrysosporium, was studied. A series of compounds known to be P450 substrates of other organisms were utilized for metabolic studies of P. chrysosporium. Metabolic conversions of benzoic acid, camphor, 1,8-cineol, cinnamic acid, p-coumaric acid, coumarin, cumene, 1,12-dodecanediol, 1-dodecanol, 4-ethoxybenzoic acid, and 7-ethoxycoumarin were observed with P. chrysosporium for the first time. 1-Dodecanol was hydroxylated at seven different positions to form 1,12-, 1,11-, 1,10-, 1,9-, 1,8-, 1,7-, and 1,6-dodecandiols. The effect of piperonyl butoxide, a P450 inhibitor, on the fungal conversion of 1-dodecanol was also investigated, indicating that hydroxylation reactions of 1-dodecanol were inhibited by piperonyl butoxide in a concentration-dependent manner. With 11 substrates, 23 hydroxylation reactions and 2 deethylation reactions were determined and 6 products were new with the position of hydroxyl group incorporated. In conclusion, fungal P450s were shown to have diverse and unique functions.

A homeobox gene, defective proventriculus (dve), is expressed in various tissues including the ventral ectoderm and midgut. Here, we show the expression pattern of dve in the ventral ectoderm, in which dve expression is induced by Spitz, a ligand for Drosophila epidermal growth factor receptor (EGFR). In spitz mutants, dve expression is only lost in the ventral ectoderm and overexpression of Spitz induces ectopic dve activation in the ventral ectoderm. Dve expression in the middle midgut depends on Decapentaplegic (Dpp) signaling, while expression of a dominant-negative form of Drosophila EGFR (DER(DN)) also causes a marked decrease in dve expression in the middle midgut. Furthermore, heterozygous mutation of thick veins (tkv), a Dpp receptor, strongly enhances the effect of DER(DN). These results indicate that EGFR signaling is crucial for dve expression in the ventral ectoderm and is required in the middle midgut where it cooperates with Dpp signaling.

Pattern formation during animal development is often induced by extracellular signaling molecules, known as morphogens, which are secreted from localized sources. During wing development in Drosophila, Wingless (Wg) is activated by Notch signaling along the dorsal-ventral boundary of the wing imaginal disc and acts as a morphogen to organize gene expression and cell growth. Expression of wg is restricted to a narrow stripe by Wg itself, repressing its own expression in adjacent cells. This refinement of wg expression is essential for specification of the wing margin. Here, we show that a homeodomain protein, Defective proventriculus (Dve), mediates the refinement of wg expression in both the wing disc and embryonic proventriculus, where dve expression requires Wg signaling. Our results provide evidence for a feedback mechanism that establishes the wg-expressing domain through the action of a Wg-induced gene product.

Prostaglandins (PGs), hepatocyte growth factor (HGF), and induction of cyclooxygenase (PG synthetase, COX) play important roles in the repair process of gastric mucosa. We hypothesized that nonsteroidal anti-inflammatory drugs (NSAIDs), including indomethacin (IND), retard the healing of ulcers by suppressing these factors. In this study, we investigated the effects of cytokines, growth factors, and IND on production of PG and HGF, and induction of COX using cultured human gastric fibroblasts. Exogenous PGs significantly increased HGF production in a dose-dependent manner. Among various potential stimulants tested, interleukin-1 beta (IL-1 beta) dramatically increased PGE2 production and significantly stimulated HGF production. IL-1 beta induced COX-2 but not COX-1 protein. IND significantly reduced both basal and IL-1 beta-induced PGE2 release and HGF production. These results suggest that the IL-1 beta-PG-HGF pathway plays a role in the repair process of gastric mucosa. Further, NSAIDs may delay the healing of gastric mucosal ulcer, in part through suppression of HGF expression via inhibition of endogenous PG production.

The regulation of cyclin D-dependent kinase activity in tissue regeneration in vivo has not been fully described. In young adult rat liver after 70% partial hepatectomy, the association of cyclin D1 with cdk4 was significantly promoted during G1 phase and was maximal at 18 hr, corresponding mainly to late G1. Cyclin D1-dependent kinase activity also strongly increased during G1 phase. The timing of the induction of cyclin D1 / cdk4 complex assembly correlated well with that of cyclin D1-dependent kinase activity. At 18 hr after partial hepatectomy, the amounts of CDK inhibitors p21(CIP1) and p27(KIP1) were also maximal, while only one-tenth of p21(CIP1) and of p27(KIP1) was associated with cyclin D1. These findings suggest that cyclin D1, cdk4 and their association act as promoting factors, and that both p21(CIP1) and p27(KIP1) may have physiological functions as adaptor proteins in additions to their roles as CDK inhibitors in rat liver regeneration., (Copyright 1998 Academic Press.)

The Drosophila central nervous system comprises an enormous diversity of neurons that are originated from neuronal stem cells, neuroblasts. They generate a specific series of ganglion mother cells, each of which is once cleaved into a pair of neurons. Among genes known to control neurogenesis, prospero (pros) was recently identified as a gene required for gene expression specifying properties of some identified neurons. Here we report that pros encodes a nuclear protein containing a homeodomain-like sequence. In neuronal lineages of the central nervous system, pros protein is specifically detected in ganglion mother cells, although their parental neuroblasts have begun expressing a significant level of pros transcripts, suggesting a post-transcriptional control of pros expression. Our results provoke that in neuronal cell differentiation ganglion mother cells might play a pivotal role associating with the pros function.

Effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on rat AH66 hepatoma cells was studied with a reference to that of insulin and the epidermal growth factor (EGF). In a short term cell incubation, TPA and EGF caused an approximately 2-fold increase in the production of alpha-fetoprotein (AFP) and other acid-precipitable materials, while the same concentration of insulin brought a 3-fold increase. In a long term culture using a low serum medium, TPA as well as insulin and EGF caused remarkable proliferation of AH66 cells, but the increase in cell number was not accompanied by a proportional increase in the levels of AFP of the culture media. These biological effects of TPA, insulin and EGF appeared to resemble each other, and subsequent hormone binding studies showed that TPA inhibited 125I-EGF binding to its membrane receptors without affecting 125I-insulin binding. Scatchard analysis of TPA effect on EGF binding indicated that TPA altered the affinity of the membrane receptors for EGF without changing the total number of available receptors per cell. From these data, it is suggested that some of the biological effects of TPA on AH66 cells may result from alterations in the functions of cell membrane.

We isolated a protein complex containing major cytoskeletal components from the Triton shell of bovine erythrocytes. This protein complex, which we called the 26-S complex, consisted of three major components, spectrin, band-4.1 protein and actin, and one minor component, band-4.9 protein. The molar ratio of spectrin heterodimer:band 4.1:actin was determined by sodium dodecyl sulfate (SDS) gel electrophoresis to be about 1:2:2, approximately the same as that for the Triton shell. By electron microscopic examinations of rotary-shadowed specimens, it was revealed that the 26-S complex had a "spider-like" morphology with a central core and several spectrin heterodimers radiating from it. The number of spectrin arms in the complex was not constant but was in the range between 3 and 6. The complexes with five spectrin heterodimers were the most numerous. The results showed that the 26-S complex contained on the average five spectrin heterodimers, ten band-4.1 polypeptides and ten actin monomers. As judged from the formation of oligomeric 26-S complexes through spectrin arms, the central core of the complex presumably contains band 4.1 and actin. Supporting this conclusion, the central core acted as a nucleus for actin polymerization when the 26-S complex was mixed with G-actin under an actin-polymerizing condition. The 26-S complex could form large aggregates under a certain condition that spectrin was promoted to associate from dimer to tetramer. We conclude that the 26-S complex is the structural unit of the erythrocyte cytoskeleton.