At the onset of mammalian neurogenesis, neuroepithelial (NE) cells switch from proliferative to neuron-generating divisions. Understanding the molecular basis of this switch requires the ability to distinguish between these two types of division. Here we show that in the mouse ventricular zone, expression of the mRNA of the antiproliferative gene TIS21 (PC3, BTG2) (i) starts at the onset of neurogenesis, (ii) is confined to a subpopulation of NE cells that increases in correlation with the progression of neurogenesis, and (iii) is not detected in newborn neurons. Expression of the TIS21 mRNA in the NE cells occurs transiently during the cell cycle, i.e., in the G 1 phase. In contrast to the TIS21 mRNA, the TIS21 protein persists through the division of NE cells and is inherited by the neurons, where it remains detectable during neuronal migration and the initial phase of differentiation. Our observations indicate that the TIS21 gene is specifically expressed in those NE cells that, at their next division, will generate postmitotic neurons, but not in proliferating NE cells. Using TIS21 as a marker, we find that the switch from proliferative to neuron-generating divisions is initiated in single NE cells rather than in synchronized neighboring cells.Neuroepithelial (NE) cells are the progenitors of all neurons and macroglial cells of the mammalian central nervous system (CNS). At the onset of CNS neurogenesis, NE cells are thought to switch from symmetric proliferative divisions (two NE daughter cells) to asymmetric neuron-generating divisions (one NE daughter cell, one postmitotic neuron) (1-4). Although several classes of molecules, including growth factors (5) and the products of neurogenic genes (6), have been implicated in this switch, its precise molecular mechanism is unknown. A major problem lies in the fact that during neurogenesis, proliferative and neuron-generating divisions of NE cells coexist (1-3). It is therefore essential to be able to distinguish between proliferating and neuron-generating NE cells. Here we investigate, for the early phase of neurogenesis in the mouse CNS, whether the product of the antiproliferative gene TIS21 (PC3, BTG2) (7-10), that is expressed in the neuroepithelium in correlation with neurogenesis (11), is a specific marker of neuron-generating NE cells. METHODS Morphology.Cryosections. For all combined in situ hybridization (ISH)/immunoperoxidase stainings and for some immunofluorescence, cryosections (5-6 m) were prepared from embryos (either BrdUrd-labeled or unlabeled) fixed overnight at 4°C in 4% paraformaldehyde/4% sucrose in PBS.Polyester Sections. In most double immunofluorescence experiments, polyester sections (6 m) were used. Embryos were fixed overnight at 4°C in 4% paraformaldehyde/0.1% glutaraldehyde in PBS, dehydrated in ethanol, and embedded in polyester wax (BDH) (12).Combined ISH/immunoperoxidase staining on cryosections was performed as described (13). Digoxygenin-labeled sense and antisense TIS21 riboprobes, used at 500 ng RNA per ml of hybridiza...

ABSTRACT

Defense mechanisms are psychological factors that influence emotional distress and quality of life. There are a number of measures assessing the construct of defense mechanisms, but only few available instruments reflect the gold-standard theoretical hierarchical organization of defenses. We report on the development of a novel 30 item self-report questionnaire, the DMRS-SR-30, based on the parent instrument, the Defense Mechanism Rating Scales (DMRS). This study tested preliminary reliability and validity of the Italian version of the DMRS-SR-30. We first extracted 30 items from the DMRS Q-sort version (DMRS-Q) and adapted them for a self-reported format. We then applied the DMRS quantitative scoring algorithms to provide proportional scores for the 28 individual defenses and summary scores for seven defense levels and overall defensive functioning (ODF) scores. A dynamic interview was used for assessing participant’s defense mechanisms with the observer-rated DMRS and DMRS-Q. We examined internal consistency of the scales along with criterion, concurrent, convergent and discriminant validity among participants (N = 94) who completed the DMRS-SR-30, SCL-90, BDI, and IES-R. Results showed very good internal consistency for ODF (Cronbach’s alpha = .890) and the high adaptive defense level, whereas some subscales with few items had lower values. Correlation analyses between DMRS-SR-30 and the two DMRS-based observer-rated measures showed very good criterion and concurrent validity for ODF and moderate to high for defense levels subscales. Correlations between the DMRS-SR-30 ODF and SCL-90 GSI, BDI and IES=R (r = −.456, r= −.540, r = −.402, respectively, all p <.001), indicated good convergent validity. Despite the well-known limitations of self-report methods of psychodynamic phenomena, self-report measures are highly practicable for assessing large samples. The DMRS-SR-30 is the first self-assessed measure describing the whole hierarchy of 28 defense mechanisms and providing scores for ODF, defensive categories, defense levels, and individual defenses. Preliminary examination of the Italian version of the DMRS-SR-30 showed promising results of internal consistency, criterion and concurrent validity, and convergent validity and of the measure. Further validation is needed to confirm these findings and explore other aspects of validity and reliability.

We investigated the fate of human cord blood CD133+ hematopoietic stem cells (HSC) transplanted intravenously (IV) into irradiated nod-scid mice previously made deaf by ototoxic treatment with kanamycin and/ or intense noise, to verify whether HSC engraft the cochlea and contribute to inner ear restoration, in vivo. We tested the presence of HLA.DQalpha1 by PCR, used for traceability of engrafted cells, finding evidence that HSC migrated to various host tissues, including the organ of Corti (OC). By histology, antibody and lectin-staining analysis, we confirmed that HSC IV transplantation in mice previously damaged by ototoxic agents correlated with the repair process and stimulation ex novo of morphological recovery in the inner ear, while the cochlea of control oto-injured, nontransplanted mice remained seriously damaged. Dual color FISH analysis also provided evidence of positive engraftment in the inner ear and in various mouse tissues, also revealing small numbers of heterokaryons, probably derived from fusion of donor with endogenous cells, for up to 2 months following transplantation. These observations offer the first evidence that transplanted human HSC migrating to the inner ear of oto-injured mice may provide conditions for the resumption of deafened cochlea, emerging as a potential strategy for inner ear rehabilitation.

Rhesus monkey embryonic stem cells (ESCs) (R366.4), cultured on a three-dimensional (3D) collagen matrix with or without human neonatal foreskin fibroblasts (HPI.1) as feeder cells, or embedded in the collagen matrix, formed complex tubular or spherical gland-like structures and differentiated into phenotypes characteristic of neural, epithelial and endothelial lineages. Here, we analysed the production of endogenous extracellular matrix (ECM) proteins, cell-cell adhesion molecules, cell-surface receptors, lectins and their glycoligands, by differentiating ESCs, forming a micro-environment, a niche, able to positively influence cell behaviour. The expression of some of these molecules was modulated by HPI.1 cells while others were unaffected. We hypothesized that both soluble factors and the niche itself were critical in directing growth and/or differentiation of ESCs in this 3D environment. Creating such an appropriate experimental 3D micro-environment, further modified by ESCs and modulated by exogenous soluble factors, may constitute a template for adequate culture systems in developmental biology studies concerning differentiation of stem cells.