Kazuko Keino-Masu

Conversion of biliary system to pancreatic tissue in Hes1 -deficient mice

The biliary system, pancreas and liver all develop from the nearby foregut at almost the same time in mammals. The molecular mechanisms that determine the identity of each organ in this complex area are unknown. Hes1 encodes the basic helix-loop-helix protein Hes1 (ref. 1), which represses positive basic helix-loop-helix genes such as Neurog3 (ref. 3). Expression of Hes1 is controlled by the evolutionarily conserved Notch pathway. Hes1 operates as a general negative regulator of endodermal endocrine differentiation, and defects in Notch signaling lead to accelerated pancreatic endocrine differentiation. Mutations in JAG1, encoding a Notch ligand, cause the Alagille syndrome in humans, characterized by poor development of the biliary system, suggesting that the Notch pathway is also involved in normal biliary development. Here we show that Hes1 is expressed in the extrahepatic biliary epithelium throughout development and that Hes1-deficient mice have gallbladder agenesis and severe hypoplasia of extrahepatic bile ducts. Biliary epithelium in Hes1−/− mice ectopically expresses the proendocrine gene Neurog3 (refs. 12,13), differentiates into endocrine and exocrine cells and forms acini and islet-like structures in the mutant bile ducts. Thus, biliary epithelium has the potential for pancreatic differentiation and Hes1 determines biliary organogenesis by preventing the pancreatic differentiation program, probably by directly repressing transcription of Neurog3.

Identification of a novel nonlysosomal sulphatase expressed in the floor plate, choroid plexus and cartilage

Background: Sulphated glycosaminoglycans (GAGs) attached to proteoglycan core proteins are implicated in cell adhesion, motility and morphogenesis. Variable sulphation patterns, which are thought to be important for regulating proteoglycan function, are generated by sequential reactions during GAG biosynthesis. However, the mechanism by which such diversity is generated remains unclear.

Ccd1, a Novel Protein with a DIX Domain, Is a Positive Regulator in the Wnt Signaling during Zebrafish Neural Patterning

Wnt signaling plays a crucial role in directing cell differentiation, polarity, and growth. In the canonical pathway, Wnt receptors activate Dishevelled (Dvl), which then blocks the degradation of a key signal transducer, beta-catenin, leading to the nuclear accumulation of beta-catenin and induction of Wnt target genes through TCF/LEF family transcription factors. Here we identified a novel zebrafish gene encoding Ccd1, which possesses a DIX (Dishevelled-Axin) domain. DIX domains are essential for the signal transduction of two major Wnt downstream mediators, Dvl and Axin. Ccd1 formed homomeric and heteromeric complexes with Dvl and Axin and activated TCF-dependent transcription in vitro. In addition, overexpression of ccd1 in zebrafish embryos led to a reduction in the size of the eyes and forebrain (posteriorization), as seen with wnt8 overexpression, whereas a dominant-negative ccd1 (DN-ccd1) caused the opposite phenotype. Furthermore, the Wnt activation phenotype induced by ccd1 was inhibited by the expression of axin1 or DN-ccd1, and the wnt8 overexpression phenotype was rescued by DN-ccd1, suggesting that Ccd1 functions downstream of the Wnt receptor and upstream of Axin. These results indicate that Ccd1 is a novel positive regulator in this Wnt signaling pathway during zebrafish neural patterning.

The N-terminal hydrophobic sequence of autotaxin (ENPP2) functions as a signal peptide.

Autotaxin, also known as ENPP2, was originally isolated from the culture medium of melanoma cells as a cell‐motility promoting protein. It regulates cell growth, motility, and angiogenesis through the production of lysophosphatidic acid and sphingosine 1‐phosphate. Because autotaxin shows overall structural similarity to the well‐characterized PC‐1, it has been assumed to be a type II transmembrane protein that is expressed on the cell surface and is released into the extracellular space after proteolytic cleavage. We found, however, that while autotaxin was efficiently secreted into the extracellular space both in vitro and in vivo, it was expressed neither on the surfaces of autotaxin‐transfected cells nor on those of the autotaxin‐expressing choroid plexus epithelium cells. N‐terminal sequencing of the secreted autotaxin revealed that it was cleaved at two N‐terminal sites that match the consensus sequences for cleavage by a signal peptidase and furin. In addition, when translated in vitro, autotaxin was co‐translationally translocated into microsome membranes, and its N‐terminal 3‐kDa fragment corresponding to a signal sequence was cleaved. These data demonstrate that the N‐terminal hydrophobic sequence of autotaxin functions as a signal peptide, not as a transmembrane segment, and thus autotaxin is synthesized as a secreted protein.

Autotaxin/Lysophospholipase D-mediated Lysophosphatidic Acid Signaling Is Required to Form Distinctive Large Lysosomes in the Visceral Endoderm Cells of the Mouse Yolk Sac

Autotaxin, a lysophospholipase D encoded by the Enpp2 gene, is an exoenzyme that produces lysophosphatidic acid in the extracellular space. Lysophosphatidic acid acts on specific G protein-coupled receptors, thereby regulating cell growth, migration, and survival. Previous studies have revealed that Enpp2−/− mouse embryos die at about embryonic day (E) 9.5 because of angiogenic defects in the yolk sac. However, what cellular defects occur in Enpp2−/− embryos and what intracellular signaling pathways are involved in the phenotype manifestation remain unknown. Here, we show that Enpp2 is required to form distinctive large lysosomes in the yolk sac visceral endoderm cells. From E7.5 to E9.5, Enpp2 mRNA is abundantly expressed in the visceral endoderm cells. In Enpp2−/− mouse embryos, lysosomes in the visceral endoderm cells are fragmented. By using a whole embryo culture system combined with specific pharmacological inhibitors for intracellular signaling molecules, we show that lysophosphatidic acid receptors and the Rho-Rho-associated coiled-coil containing protein kinase (ROCK)-LIM kinase pathway are required to form large lysosomes. In addition, electroporation of dominant negative forms of Rho, ROCK, or LIM kinase also leads to the size reduction of lysosomes in wild-type visceral endoderm cells. In Enpp2−/− visceral endoderm cells, the steady-state levels of cofilin phosphorylation and actin polymerization are reduced. In addition, perturbations of actin turnover dynamics by actin inhibitors cytochalasin B and jasplakinolide result in the defect in lysosome formation. These results suggest that constitutive activation of the Rho-ROCK-LIM kinase pathway by extracellular production of lysophosphatidic acid by the action of autotaxin is required to maintain the large size of lysosomes in visceral endoderm cells.

Activation of protein kinase A by nitric oxide in cultured dorsal root ganglion neurites of the rat, examined by a fluorescence probe, ARII

To study the roles of nitric oxide (NO) in growth of nerve fibers, (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamine (NOR3), an NO-donor, was applied to cultured dorsal root ganglion (DRG) neurites from a micropipette. Ejection of a small volume of 1 mM NOR3 solution (not more than 1 pl/s) from a micropipette to terminal branches of neurites caused enlargement of the neurites, and often, elongation of their growth cones. This neurite enlargement was blocked by inhibitors for soluble guanylate cyclase. The neurite enlargement did not occur when protein kinase A (PKA) was inhibited. To prove that NOR3 activated PKA, we introduced a fluorescence peptide probe, ARII that reduces its fluorescence by activated PKA, to monitor PKA activity in DRG neurites. ARII fluorescence was reduced by NOR3, which was not observed when PKA was inhibited by its specific inhibitors. These indicated that PKA was indeed activated by NO. To examine whether the PKA activation is due to inhibition of phosphodiesterase III (PDE III) by cyclic GMP, we applied PDE III-specific inhibitors and found that the inhibitions activated PKA. Since PKA regulates various neuronal functions, our finding that NO activates PKA is important to understand roles of NO in nerve fibers.

Heparan sulfate 6-O-endosulfatases, Sulf1 and Sulf2, regulate glomerular integrity by modulating growth factor signaling

Glomerular integrity and functions are maintained by growth factor signaling. Heparan sulfate, the major component of glomerular extracellular matrixes, modulates growth factor signaling, but its roles in glomerular homeostasis are unknown. We investigated the roles of heparan sulfate 6-O-endosulfatases, sulfatase (Sulf)1 and Sulf2, in glomerular homeostasis. Both Sulf1 and Sulf2 were expressed in the glomeruli of wild-type (WT) mice. Sulf1 and Sulf2 double-knockout (DKO) mice showed glomerular hypercellularity, matrix accumulation, mesangiolysis, and glomerular basement membrane irregularity. Platelet-derived growth factor (PDGF)-B and PDGF receptor-β were upregulated in Sulf1 and Sulf2 DKO mice compared with WT mice. Glomeruli from Sulf1 and Sulf2 DKO mice in vitro stimulated by either PDGF-B, VEGF, or transforming growth factor-β similarly showed reduction of phospho-Akt, phospho-Erk1/2, and phospho-Smad2/3, respectively. Since glomerular lesions in Sulf1 and Sulf2 DKO mice were reminiscent of diabetic nephropathy, we examined the effects of Sulf1 and Sulf2 gene disruption in streptozotocin-induced diabetes. Diabetic WT mice showed an upregulation of glomerular Sulf1 and Sulf2 mRNA by in situ hybridization. Diabetic DKO mice showed significant increases in albuminuria and serum creatinine and an acceleration of glomerular pathology without glomerular hypertrophy; those were associated with a reduction of glomerular phospho-Akt. In conclusion, Sulf1 and Sulf2 play indispensable roles to maintain glomerular integrity and protective roles in diabetic nephropathy, probably by growth factor modulation.

Sulfatase 2 Modulates Fate Change from Motor Neurons to Oligodendrocyte Precursor Cells through Coordinated Regulation of Shh Signaling with Sulfatase 1

Sulfatases (Sulfs) are a group of endosulfatases consisting of Sulf1 and Sulf2, which specifically remove sulfate from heparan sulfate proteoglycans. Although several studies have shown that Sulf1 acts as a regulator of sonic hedgehog (Shh) signaling during embryonic ventral spinal cord development, the detailed expression pattern and function of Sulf2 in the spinal cord remains to be determined. In this study, we found that Sulf2 also modulates the cell fate change from motor neurons (MNs) to oligodendrocyte precursor cells (OPCs) by regulating Shh signaling in the mouse ventral spinal cord in coordination with Sulf1. In the mouse, Sulf mRNAs colocalize with Shh mRNA and gradually expand dorsally from embryonic day (E) 10.5 to E12.5, following strong Patched1 signals (a target gene of Shh signaling). This coordinated expression pattern led us to hypothesize that in the mouse, strong Shh signaling is induced when Shh is released by Sulf1/2, and this strong Shh signaling subsequently induces the dorsal expansion of Shh and Sulf1/2 expression. Consistent with this hypothesis, in the ventral spinal cord of Sulf1 knockout (KO) or Sulf2 KO mice, the expression patterns of Shh and Patched1 differed from that in wild-type mice. Moreover, the position of the pMN and p3 domains were shifted ventrally, MN generation was prolonged, and OPC generation was delayed at E12.5 in both Sulf1 KO and Sulf2 KO mice. These results demonstrated that in addition to Sulf1, Sulf2 also plays an important and overlapping role in the MN-to-OPC fate change by regulating Shh signaling in the ventral spinal cord. However, neither Sulf1 nor Sulf2 could compensate for the loss of the other in the developing mouse spinal cord. In vitro studies showed no evidence of an interaction between Sulf1 and Sulf2 that could increase sulfatase activity. Furthermore, Sulf1/2 double heterozygote and Sulf1/2 double KO mice exhibited phenotypes similar to the Sulf1 KO and Sulf2 KO mice. These results indicate that there is a threshold for sulfatase activity (which is likely reflected in the dose of Shh) required to induce the MN-to-OPC fate change, and Shh signaling requires the coordinated activity of Sulf1 and Sulf2 in order to reach that threshold in the mouse ventral spinal cord.

Expression of the heparan sulfate 6‐O‐endosulfatases, Sulf1 and Sulf2, in the avian and mammalian inner ear suggests a role for sulfation during inner ear development

Background: Inner ear morphogenesis is tightly regulated by the temporally and spatially coordinated action of signaling ligands and their receptors. Ligand–receptor interactions are influenced by heparan sulfate proteoglycans (HSPGs), cell surface molecules that consist of glycosaminoglycan chains bound to a protein core. Diversity in the sulfation pattern within glycosaminoglycan chains creates binding sites for numerous cell signaling factors, whose activities and distribution are modified by their association with HSPGs. Results: Here we describe the expression patterns of two extracellular 6‐O‐endosulfatases, Sulf1 and Sulf2, whose activity modifies the 6‐O‐sulfation pattern of HSPGs. We use in situ hybridization to determine the temporal and spatial distribution of transcripts during the development of the chick and mouse inner ear. We also use immunocytochemistry to determine the cellular localization of Sulf1 and Sulf2 within the sensory epithelia. Furthermore, we analyze the organ of Corti in Sulf1/Sulf2 double knockout mice and describe an increase in the number of mechanosensory hair cells. Conclusions: Our results suggest that the tuning of intracellular signaling, mediated by Sulf activity, plays an important role in the development of the inner ear. Developmental Dynamics 244:168–180, 2015.

A unique mouse model for investigating the properties of amyotrophic lateral sclerosis-associated protein TDP-43, by in utero electroporation

TDP-43 is a discriminative protein that is found as intracellular aggregations in the neurons of the cerebral cortex and spinal cord of patients with amyotrophic lateral sclerosis (ALS); however, the mechanisms of neuron loss and its relation to the aggregations are still unclear. In this study, we generated a useful model to produce TDP-43 aggregations in the motor cortex using in utero electroporation on mouse embryos. The plasmids used were full-length TDP-43 and C-terminal fragments of TDP-43 (wild-type or M337V mutant) tagged with GFP. For the full-length TDP-43, both wild-type and mutant, electroporated TDP-43 localized mostly in the nucleus, and though aggregations were detected in embryonic brains, they were very rarely observed at P7 and P21. In contrast, TDP-43 aggregations were generated in the brains electroporated with the C-terminal TDP-43 fragments as previously reported in in vitro experiments. TDP-43 protein was distributed diffusely-not only in the nucleus, but also in the cytoplasm-and the inclusion bodies were ubiquitinated and included phosphorylated TDP-43, which reflects the human pathology of ALS. This model using in utero electroporation of pathogenic genes into the brain of the mouse will likely become a useful model for studying ALS and also for evaluation of agents for therapeutic purpose, and may be applicable to other neurodegenerative diseases, as well.