Naoko Oshima

Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver†

The adult liver progenitor cells appear in response to several types of pathological liver injury, especially when hepatocyte replication is blocked. These cells are histologically identified as cells that express cholangiocyte markers and proliferate in the portal area of the hepatic lobule. Although these cells play an important role in liver regeneration, the precise characterization that determines these cells as self‐renewing bipotent primitive hepatic cells remains to be shown. Here we attempted to isolate cells that express a cholangiocyte marker from the adult mouse liver and perform single cell‐based analysis to examine precisely bilineage differentiation potential and self‐renewing capability of these cells. Based on the results of microarray analysis and immunohistochemistry, we used an antibody against CD133 and isolate CD133+ cells via flow cytometry. We then cultured and propagated isolated cells in a single cell culture condition and examined their potential for proliferation and differentiation in vitro and in vivo. Isolated cells that could form large colonies (LCs) in culture gave rise to both hepatocytes and cholangiocytes as descendants, while maintaining undifferentiated cells by self‐renewing cell divisions. The clonogenic progeny of an LC‐forming cell is capable of reconstituting hepatic tissues in vivo by differentiating into fully functional hepatocytes. Moreover, the deletion of p53 in isolated LC‐forming cells resulted in the formation of tumors with some characteristics of hepatocellular carcinoma and cholangiocarcinoma upon subcutaneous injection into immunodeficient mutant mice. These data provide evidence for the stem cell‐like capacity of isolated and clonally cultured CD133+ LC‐forming cells. Conclusion: Our method for prospectively isolating hepatic progenitor cells from the adult mouse liver will facilitate study of their roles in liver regeneration and carcinogenesis. (HEPATOLOGY 2008;48:1964‐1978.)

Kif26b, a kinesin family gene, regulates adhesion of the embryonic kidney mesenchyme.

The kidney develops through reciprocal interactions between two precursor tissues: the metanephric mesenchyme and the ureteric bud. We previously demonstrated that the zinc finger protein Sall1 is essential for ureteric bud attraction toward the mesenchyme. Here, we show that Kif26b, a kinesin family gene, is a downstream target of Sall1 and that disruption of this gene causes kidney agenesis because of impaired ureteric bud attraction. In the Kif26b-null metanephros, compact adhesion between mesenchymal cells adjacent to the ureteric buds and the polarized distribution of integrin α8 were impaired, resulting in failed maintenance of Gdnf, a critical ureteric bud attractant. Overexpression of Kif26b in vitro caused increased cell adhesion through interactions with nonmuscle myosin. Thus, Kif26b is essential for kidney development because it regulates the adhesion of mesenchymal cells in contact with ureteric buds.

Nardilysin regulates axonal maturation and myelination in the central and peripheral nervous system

Axonal maturation and myelination are essential processes for establishing an efficient neuronal signaling network. We found that nardilysin (N-arginine dibasic convertase, also known as Nrd1 and NRDc), a metalloendopeptidase enhancer of protein ectodomain shedding, is a critical regulator of these processes. Nrd1−/− mice had smaller brains and a thin cerebral cortex, in which there were less myelinated fibers with thinner myelin sheaths and smaller axon diameters. We also found hypomyelination in the peripheral nervous system (PNS) of Nrd1−/− mice. Neuron-specific overexpression of NRDc induced hypermyelination, indicating that the level of neuronal NRDc regulates myelin thickness. Consistent with these findings, Nrd1−/− mice had impaired motor activities and cognitive deficits. Furthermore, NRDc enhanced ectodomain shedding of neuregulin1 (NRG1), which is a master regulator of myelination in the PNS. On the basis of these data, we propose that NRDc regulates axonal maturation and myelination in the CNS and PNS, in part, through the modulation of NRG1 shedding.

A murine model of neonatal diabetes mellitus in Glis3-deficient mice.

Glis3 is a member of the Gli‐similar subfamily. GLIS3 mutations in humans lead to neonatal diabetes, hypothyroidism, and cystic kidney disease. We generated Glis3‐deficient mice by gene‐targeting. The Glis3 −/− mice had significant increases in the basal blood sugar level during the first few days after birth. The high levels of blood sugar are attributed to a decrease in the Insulin mRNA level in the pancreas that is caused by impaired islet development and the subsequent impairment of Insulin‐producing cell formation. The pancreatic phenotypes indicate that the Glis3‐deficient mice are a model for GLIS3 mutation and diabetes mellitus in humans.

Targeted deletion of the murine corneodesmosin gene delineates its essential role in skin and hair physiology

Controlled proteolytic degradation of specialized junctional structures, corneodesmosomes, by epidermal proteases is an essential process for physiological desquamation of the skin. Corneodesmosin (CDSN) is an extracellular component of corneodesmosomes and, although considerable debate still exists, genetic studies have suggested that the CDSN gene in the major psoriasis-susceptibility locus (PSORS1) may be responsible for susceptibility to psoriasis, a human skin disorder characterized by excessive growth and aberrant differentiation of keratinocytes. CDSN is also expressed in the inner root sheath of hair follicles, and a heterozygous nonsense mutation of the CDSN gene in humans is associated with scalp-specific hair loss of poorly defined etiology. Here, we have investigated the pathogenetic roles of CDSN loss of function in the development of skin diseases by generating a mouse strain with targeted deletion of the Cdsn gene. Cdsn-deficient mouse skin showed detachment of the stratum corneum from the underlying granular layer and/or detachment within the upper granular layers due to the disrupted integrity of the corneodesmosomes. When grafted onto immunodeficient mice, Cdsn-deficient skin showed rapid hair loss together with epidermal abnormalities resembling psoriasis. These results underscore the essential roles of CDSN in hair physiology and suggest functional relevance of CDSN gene polymorphisms to psoriasis susceptibility.

Involvement of the Reck tumor suppressor protein in maternal and embryonic vascular remodeling in mice

BackgroundDevelopmental angiogenesis proceeds through multiple morphogenetic events including sprouting, intussusception, and pruning. Mice lacking the membrane-anchored metalloproteinase regulator Reck die in utero around embryonic day 10.5 with halted vascular development; however, the mechanisms by which this phenotype arises remain unclear.ResultsWe found that Reck is abundantly expressed in the cells associated with blood vessels undergoing angiogenesis or remodelling in the uteri of pregnant female mice. Some of the Reck-positive vessels show morphological features consistent with non-sprouting angiogenesis. Treatment with a vector expressing a small hairpin RNA against Reck severely disrupts the formation of blood vessels with a compact, round lumen. Similar defects were found in the vasculature of Reck-deficient or Reck conditional knockout embryos.ConclusionsOur findings implicate Reck in vascular remodeling, possibly through non-sprouting angiogenesis, in both maternal and embyornic tissues.

Tsukushi is required for anterior commissure formation in mouse brain.

The anterior commissure (AC) is one of the important commissure projections in the brain that conveys information from one side of the nervous system to the other. During development, the axons from the anterior AC (aAC) and the posterior AC (pAC) course in the same dorsoventral plane and converge into a common fascicle for midline crossing. Previously, we reported that Tsukushi (TSK), a member of the secreted small leucine rich repeat proteoglycan family, functions as a key coordinator of multiple pathways outside of cells through the regulation of an extracellular signaling network. Here, we show evidence that TSK is critical for the formation of the AC. In mice lacking TSK, the aAC and the pAC axons fail to cross the midline, leading to an almost total absence of the AC in adult mice. DiI labeling indicated that the aAC axons grew out from the anterior olfactory nucleus and migrated along normal pathways but never crossed the midline. Therefore, we have uncovered a crucial role for TSK for AC formation in the mouse brain.

Phospholipase C-eta2 is highly expressed in the habenula and retina.

Phospholipase C (PLC), a key enzyme involved in phosphoinositide turnover, hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate two second messengers, inositol 1,4,5-triphosphate and diacylglycerol. PLCeta2 (PLCeta2), a neuron-specific isozyme of PLC, is abundantly expressed in the postnatal brain, suggesting the importance of PLCeta2 in the formation and maintenance of the neuronal network in the postnatal brain. However, the detailed expression patterns of PLCeta2 in the brain and other neuronal tissues remain to be clarified. Here, we generated PLCeta2 knockout/LacZ knockin (plch2(lacZ)(/)(lacZ)) mice-the first mice to lack full-length PLCeta2. Although the plch2(lacZ)(/)(lacZ) mice exhibited no obvious abnormalities, the LacZ reporter revealed unexpected and abundant expressions of PLCeta2 in the habenula and retina. We confirmed these PLCeta2 expression patterns by in situ hybridization and immunohistochemical analyses. In the retina, strong PLCeta2 expression was detected in the photoreceptor (rod/cone), outer nuclear layer, outer plexiform layer, and inner nuclear layer, suggesting that PLCeta2 is expressed in rods and cones, and also in horizontal, bipolar, and amacrine cells, but not in ganglion cells. Interestingly PLCeta2 exhibited a dynamic expression pattern during postnatal retinal development, strongly suggesting that this isozyme might be involved in the development and maturation of the retina. Since both the habenula and retina are thought to play important roles in the regulation of circadian rhythms, our results suggest that PLCeta2 may be involved in the function of habenula and retina.

Ttyh1, a Ca2+-binding protein localized to the endoplasmic reticulum, is required for early embryonic development

Using comprehensive genetic studies on neuronal stem/progenitors cells through genome‐wide screening with oligonucleotide arrays, we identified an endoplasmic reticulum (ER) ‐resident protein, Tweety homologue 1 (ttyh1). Ttyh1 encodes a glycosylated protein composed of five predicted transmembrane segments and a C‐terminus that is enriched in negatively charged residues capable of Ca2+ binding. Ttyh1‐containing membranes changed to segmented tubuloreticular structures during mitosis, suggesting that the ER‐containing Ttyh1 could be responsible for Ca2+ sequestration and Ca2+ concentration regulation during mitosis. Ttyh1 inactivation in mice resulted in early embryonic lethality before organization of the nervous system, revealing that ttyh1 is essential in murine embryonic development. Our findings indicate that Ttyh1 plays an indispensable role during mitosis in early embryogenesis, possibly by maintaining Ca2+ homeostasis in the ER. Developmental Dynamics 239:2233–2245, 2010.

Slingshot-3 dephosphorylates ADF/cofilin but is dispensable for mouse development.

Actin‐depolymerizing factor (ADF) and cofilin constitute a family of key regulators of actin filament dynamics. ADF/cofilin is inactivated by phosphorylation at Ser‐3 by LIM‐kinases and reactivated by dephosphorylation by Slingshot (SSH) family phosphatases. Defects in LIM kinases or ADF/cofilin have been implicated in morbidity in human or mice; however, the roles of mammalian SSH in vivo have not been addressed. In this study, we examined the endogenous expression of each mouse SSH member in various cell lines and tissues, and showed that SSH‐3L protein was strongly expressed in epithelial cells. Our structure–function analysis of SSH‐3L suggested the possibility that the C‐tail unique to SSH‐3L negatively regulates the catalytic activity of this phosphatase. Furthermore we made ssh‐3 knockout mice to examine its potential in vivo roles. Unexpectedly, ssh‐3 was not essential for viability, fertility, or development of epithelial tissues; and ssh‐3 did not genetically modify the corneal disorder of the corn1/ADF/destrin mutant. genesis 46:246–255, 2008.