Expression of neuritin during liver maturation and regeneration

Cell surface molecules are not only important for cell-cell interactions but also useful for a marker to define cell types and differentiation stages. Unlike hematopoietic system in which numerous such antigens have been identified, only a few cell surface molecules have been used to define differentiation stage of hepatocytes. In order to identify such cell surface molecules, we performed DNA microarray analysis using mRNA from fetal hepatocytes in E12.5 and E17.5 mice and cDNAs encoding a membrane protein were selected. Northern blot analysis was employed to confirm the genes upregulated during maturation of fetal hepatocytes and neuritin, a GPI-anchored protein, was found as a membrane protein expressed in hepatocytes, but not in nonparenchymal cells. Its expression increased along with liver development and the maximum expression was achieved from the neonatal to adult stage. The neuritin protein was localized in sinusoidal lumen of hepatocytes in adult liver. Partial hepatectomy transiently downregulated the expression of neuritin. The expression of neuritin mRNA in C/EBPalpha deficient liver was reduced to about 50% of that of wild type mice. Thus, neuritin expression is well correlated to the maturation of hepatocytes and can be a useful tool to define the differentiation stage of hepatocytes.     

Expression of the Cytoskeletal-Associated Protein Filamin in Adult Rat Organs

Filamin is a well-characterized actin-associated protein first isolated from chicken smooth muscle. Subsequently, this polypeptide and its nonmuscle homolog actin-binding protein have been shown to be expressed in avian muscle tissue, mammalian smooth muscle, mammalian macrophages and other blood cell types, as well as several cultured cell lines. In this report, the occurrence of this polypeptide in adult mammalian organs has been investigated. Immunoblot analysis using three anti-filamin monoclonal antibodies showed that this protein was largely detected in adult rat organs that possess a substantial smooth muscle component. Furthermore, the limited expression of filamin in smooth muscle tissue was corroborated by immunohistochemical analysis. In contrast to avian systems, filamin was never found in detectable quantities in either mammalian cardiac or skeletal muscle. Quantitative immunoblot analysis demonstrated that filamin amounts roughly correlated with the abundance of the smooth muscle component of a given organ, comprising as much as 16.5% of the total SDS-extractable protein in bovine aorta. Work in avian systems and cells in culture has suggested that filamin is a rather ubiquitous cytoskeletal element. By contrast, this work demonstrates that filamin is highly restricted in its expression in mammalian organ systems, in situ.     

Prion potency in stem cells biology

Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation, and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.     

The C. elegans heterochronic gene lin-46 affects developmental timing at two larval stages and encodes a relative of the scaffolding protein gephyrin

The succession of developmental events in the C. elegans larva is governed by the heterochronic genes. When mutated, these genes cause either precocious or retarded developmental phenotypes, in which stage-specific patterns of cell division and differentiation are either skipped or reiterated, respectively. We identified a new heterochronic gene, lin-46, from mutations that suppress the precocious phenotypes caused by mutations in the heterochronic genes lin-14 and lin-28. lin-46 mutants on their own display retarded phenotypes in which cell division patterns are reiterated and differentiation is prevented in certain cell lineages. Our analysis indicates that lin-46 acts at a step immediately downstream of lin-28, affecting both the regulation of the heterochronic gene pathway and execution of stage-specific developmental events at two stages: the third larval stage and adult. We also show that lin-46 is required prior to the third stage for normal adult cell fates, suggesting that it acts once to control fates at both stages, and that it affects adult fates through the let-7 branch of the heterochronic pathway. Interestingly, lin-46 encodes a protein homologous to MoeA of bacteria and the C-terminal domain of mammalian gephyrin, a multifunctional scaffolding protein. Our findings suggest that the LIN-46 protein acts as a scaffold for a multiprotein assembly that controls developmental timing, and expand the known roles of gephyrin-related proteins to development.     

Stemness,fusion and renewal of hematopoietic and embryonic stem cells

Development of replacement cell therapies awaits the identification of factors that regulate nuclear reprogramming and the mechanisms that control stem cell renewal and differentiation. Once such factors and signals will begin to be elucidated, new technologies will have to be envisaged where uniform differentiation of adult or embryonic stem cells along one differentiation pathway can be induced. Controlled differentiation of stem cells will require the engineering of niches and extracellular signal combinations that would amplify a particular signaling network and allow uniform and selective differentiation. Three recent advances in stem cell research open the possibility to approach engineering studies for cell replacement therapies. Fusion events between stem cells and adult cells or between adult and embryonic stem cells have been shown to result in altered fates and nuclear reprogramming of cell hybrids. Hematopoietic stem cells were shown to require Wnt signaling in order to renew. The purification of Wnt proteins would allow their use as exogenous purified cytokines in attempts to amplify stem cells before bone marrow transplantation. The homeodomain protein Nanog has been shown to be crucial for the embryonic stem cell renewal and pluripotency. However, the cardinal question of how stemness is preserved in the early embryo and adult stem cells remains opened.     

Region specific knock-out reveals distinct roles of chromatin modifiers in adult neurogenic niches

Histone methyltransferases (HMTs) are present in heterogeneous cell populations within the adult brain including neurogenic niches. Yet the question remains whether loss of HMTs and the resulting changes in histone methylation alter cell fate in a region-specific manner. We utilized stereotaxic injection of Cre recombinant protein into the adult neurogenic niches, the subventricular zone (SVZ) adjacent to the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus. We confirmed that Cre protein was enzymatically active in vivo and recombination events were restricted to the vicinity of injection areas. In this study, we focus on using Cre mediated recombination in mice harboring floxed HMT: enhancer of zeste homolog 2 (EZH2) or suppressor of variegation homolog (Suv4-20h). Injectable Cre protein successfully knocked out either EZH2 or Suv4-20h, allowing assessment of long-term effects in a region-specific fashion. We performed meso-scale imaging and flow cytometry for phenotype analysis and unbiased quantification. We demonstrated that regional loss of EZH2 affects the differentiation paradigm of neural stem progenitor cells as well as the maintenance of stem cell population. We further demonstrated that regional loss of Suv4-20h influences the cell cycle but does not affect stem cell differentiation patterns. Therefore, Cre protein mediated knock-out a given HMT unravel their distinguishable and important roles in adult neurogenic niches. This Cre protein-based approach offers tightly-controlled knockouts in multiple cell types simultaneously for studying diverse regulatory mechanisms and is optimal for region-specific manipulation within complex, heterogeneous brain architectures.     

NeuN, a neuronal specific nuclear protein in vertebrates.

A battery of monoclonal antibodies (mAbs) against brain cell nuclei has been generated by repeated immunizations. One of these, mAb A60, recognizes a vertebrate nervous system- and neuron-specific nuclear protein that we have named NeuN (Neuronal Nuclei). The expression of NeuN is observed in most neuronal cell types throughout the nervous system of adult mice. However, some major cell types appear devoid of immunoreactivity including cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells. NeuN can also be detected in neurons in primary cerebellar cultures and in retinoic acid-stimulated P19 embryonal carcinoma cells. Immunohistochemically detectable NeuN protein first appears at developmental timepoints which correspond with the withdrawal of the neuron from the cell cycle and/or with the initiation of terminal differentiation of the neuron. NeuN is a soluble nuclear protein, appears as 3 bands (46-48 x 10(3) M(r)) on immunoblots, and binds to DNA in vitro. The mAb crossreacts immunohistochemically with nervous tissue from rats, chicks, humans, and salamanders. This mAb and the protein recognized by it serve as an excellent marker for neurons in the central and peripheral nervous systems in both the embryo and adult, and the protein may be important in the determination of neuronal phenotype.     

DIFFERENTIATION AND DEDIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN SHORT- AND LONG-TERM CULTURES

The crystallin synthesis of rat lens cells in cell culture systems was studied in relevance to their terminal differentiation into lens fibers. SDS-gel electrophoresis combined with several immunological techniques showed that γ-crystallin is a fiber-specific lens protein and is not localized in the epithelium of either newborn or adult lenses. When lens epithelial cells of newborn rats were cultured in vitro , α-crystaIlin was detected in many, but not all, of cells cultured for 10 days. Cells with α-crystallin gradually changed their shape into a flattened filmy form and finally differentiated into lentoid bodies. The differentiation of lentoid bodies was also found in cultures of epithelial cells obtained from adult lenses. The molecular constitution of lentoid bodies was the same as that of lens fibers in situ . The differentiation of lentoid bodies occurred successively for 5 months in cultures of lens epithelial cells. Most of the proliferating cells, however, lost α-crystallin during the culture period. Thereafter, they did not show any sign of further differentiation into lens fibers. Four clonal lines were established from these cells. One protein which is specific to the lens epithelium and the neural retina in situ (tentatively named as β_u-crystallin) was maintained in all lines, suggesting that some specific properties of ocular cells remain in the lined cells.     

Altered differentiation and clustering of Sertoli cells in transgenic mice showing a Sertoli cell specific knockout of the connexin 43 gene

Histological analysis revealed that Sertoli cell specific knockout of the predominant testicular gap junction protein connexin 43 results in a spermatogenic arrest at the level of spermatogonia or Sertoli cell-only syndrome, intratubular cell clusters and still proliferating adult Sertoli cells, implying an important role for connexin 43 in the Sertoli and germ cell development. This study aimed to determine the (1) Sertoli cell maturation state, (2) time of occurrence and (3) composition, differentiation and fate of clustered cells in knockout mice. Using immunohistochemistry connexin 43 deficient Sertoli cells showed an accurate start of the mature markers androgen receptor and GATA-1 during puberty and a vimentin expression from neonatal to adult. Expression of anti-Muellerian hormone, as a marker of Sertoli cell immaturity, was finally down-regulated during puberty, but its disappearance was delayed. This observed extended anti-Müllerian hormone synthesis during puberty was confirmed by western blot and Real-Time PCR and suggests a partial alteration in the Sertoli cell differentiation program. Additionally, Sertoli cells of adult knockouts showed a permanent and uniform expression of GATA-1 at protein and mRNA level, maybe caused by the lack of maturing germ cells and missing negative feedback signals. At ultrastructural level, basally located adult Sertoli cells obtained their mature appearance, demonstrated by the tripartite nucleolus as a typical feature of differentiated Sertoli cells. Intratubular clustered cells were mainly formed by abnormal Sertoli cells and single attached apoptotic germ cells, verified by immunohistochemistry, TUNEL staining and transmission electron microscopy. Clusters first appeared during puberty and became more numerous in adulthood with increasing cell numbers per cluster suggesting an age-related process. In conclusion, adult connexin 43 deficient Sertoli cells seem to proliferate while maintaining expression of mature markers and their adult morphology, indicating a unique and abnormal intermediate phenotype with characteristics common to both undifferentiated and differentiated Sertoli cells.     

Pepsinogen C: a type 2 cell-specific protease

Pepsinogen C, also known as progastricsin or pepsinogen II, is an aspartic protease expressed primarily in gastric chief cells. Prior microarray studies of an in vitro model of type 2 cell differentiation indicated that pepsinogen C RNA was highly induced, comparable to surfactant protein RNA induction. Using second-trimester human fetal lung, third-trimester postnatal and adult lung, and a model of type 2 cell differentiation, we examined the specificity of pepsinogen C expression in lung. Pepsinogen C RNA and protein were only detected in >22 wk gestation samples of neonatal lung or in adult lung tissue. By immunohistochemistry and in situ hybridization, pepsinogen C expression was restricted to type 2 cells. Pepsinogen C expression was rapidly induced during type 2 cell differentiation and rapidly quenched with dedifferentiation of type 2 cells after withdrawal of hormones. In all samples, pepsinogen C expression occurred concomitantly with or in advance of processing of surfactant protein-B to its mature 8-kDa form. Our results indicate that pepsinogen C is a type 2 cell-specific marker that exhibits tight developmental regulation in vivo during human lung development, as well as during in vitro differentiation and dedifferentiation of type 2 cells.     

Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential.

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (beta-H1) globin, donor-derived erythroid cells in the recipient express only adult (beta-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.     

Identification of candidate regulators of embryonic stem cell differentiation by comparative phosphoprotein affinity profiling

Embryonic stem cells are a unique cell population capable both of self-renewal and of differentiation into all tissues in the adult organism. Despite the central importance of these cells, little information is available regarding the intracellular signaling pathways that govern self-renewal or early steps in the differentiation program. Embryonic stem cell growth and differentiation correlates with kinase activities, but with the exception of the JAK/STAT3 pathway, the relevant substrates are unknown. To identify candidate phosphoproteins with potential relevance to embryonic stem cell differentiation, a systems biology approach was used. Proteins were purified using phosphoprotein affinity columns, then separated by two-dimensional gel electrophoresis, and detected by silver stain before being identified by tandem mass spectrometry. By comparing preparations from undifferentiated and differentiating mouse embryonic stem cells, a set of proteins was identified that exhibited altered post-translational modifications that correlated with differentiation state. Evidence for altered post-translational modification included altered gel mobility, altered recovery after affinity purification, and direct mass spectra evidence. Affymetrix microarray analysis indicated that gene expression levels of these same proteins had minimal variability over the same differentiation period. Bioinformatic annotations indicated that this set of proteins is enriched with chromatin remodeling, catabolic, and chaperone functions. This set of candidate phosphoprotein regulators of stem cell differentiation includes products of genes previously noted to be enriched in embryonic stem cells at the mRNA expression level as well as proteins not associated previously with stem cell differentiation status.     

Subcellular Localization of p44/WDR77 Determines Proliferation and Differentiation of Prostate Epithelial Cells

The molecular mechanism that controls the proliferation and differentiation of prostate epithelial cells is currently unknown. We previously identified a 44-kDa protein (p44/wdr77) as an androgen receptor-interacting protein that regulates a set of androgen receptor target genes in prostate epithelial cells and prostate cancer. In this study, we found that p44 localizes in the cytoplasm of prostate epithelial cells at the early stage of prostate development when cells are proliferating, and its nuclear translocation is associated with cellular and functional differentiation in adult prostate tissue. We further demonstrated that cytoplasmic p44 protein is essential for proliferation of prostate epithelial cells, whereas nuclear p44 is required for cell differentiation and prostate- specific protein secretion. These studies suggest a novel mechanism by which proliferation and differentiation of prostate epithelial cells are controlled by p44’s location in the cell.     

The lens epithelium in ocular health and disease

The lens arises from invagination of head ectoderm during embryonic development and in the adult has a relatively simple structure, comprising just two cell types (epithelial and fibre cells). Its isolation from nerves and blood vessels in the adult make it a tractable model to investigate mechanisms that regulate epithelial cells. A major focus in lens research in the past 50 years has been on the differentiation of fibre cells from epithelial cells. Hence, there has been much interest in the role of signalling systems regulating fibre cell differentiation during development. In contrast, the signalling systems that control the formation and maintenance of the lens epithelium have, until recently, been largely ignored or incidental to studies on differentiation or cataract. One notable example has been the identification of signals that underlie epithelial-mesenchymal transition (EMT) that characterizes anterior subcapsular cataract (ASC) and posterior capsule opacification (PCO). Recent data indicate that normal epithelial phenotype is regulated by several key signalling systems, including receptor tyrosine kinase receptors acting via the MAPK and Akt pathways, Wnt, Notch as well as extracellular matrix cues and possibly the Sal-Warts-Hippo pathway. Here we have shifted emphasis onto molecular mechanisms that regulate the establishment, maintenance and function of the lens epithelium.