Sequential specification of neurons and glia by developmentally regulated extracellular factors

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.     

The development of cell lineages: A sequential model

Abstract. The concept of cell lineage and the empirical characterization of specific lineages provide valuable insight into the problems of developmental biology. Of central interest is the decision-making process that results in the diversification of cell lines. Studies of the haemopoietic system, in which stem cells can be committed to one of at least six pathways of differentiation, have suggested that the restriction of differentiation potentials is a progressive and stochastic process. We have recently proposed an alternative model which hypothesizes that lineage potentials during haemopoiesis are expressed individually and in a predetermined sequence as progenitor cells mature. The model first arises from experimental studies which show that both normal myeloid progenitor cells and a human promyeloid cell line, which are able to differentiate towards either neutrophils or monocytes, express these potentials sequentially in culture. The close linear relationship between other haemopoietic progenitor cells is inferred from collective data from studies of bipotent progenitor cells and of haemopoietic proliferative disorders. If the development of haemopoietic cell lineages shows a tendency to follow a particular program, such a mechanism is likely to operate throughout development. In this paper we consider the evidence in favour of programmed events within progenitor cells implementing diversification, and the implications of predetermined and restricted pathways of embryonic development.     

Widespread tangential dispersion and extensive cell death during early neurogenesis in the mouse neocortex

The development of the mammalian neocortex requires radial and tangential migration of cells. Radial migration of differentiated neurons from the ventricular zone (VZ) is well established. It is hypothesised that an earlier phase of tangential migration of mitotically active cells lays down a widespread periodically spaced set of progenitors that generate radial arrays of postmitotic neurons. We use a transgenic cell lineage marker to label and observe the behaviour of progenitors before and during the early stages of neurogenesis. Using optical projection tomography (OPT), we show that individual progenitor cells generate many radially arrayed columns of periodically spaced cells. Column positions indicate the paths taken by these progenitor cells as they migrate, often over long distances, through the proliferative zone. Clonally related cells can be distributed in both hemispheres, suggesting progenitor cells cross the midline in the anterior neural plate. We observe a dramatic and rapid decline in the number of labelled clones after E13.5, indicating that there is extensive cell death at this time.     

E2F4 modulates differentiation and gene expression in hematopoietic progenitor cells during commitment to the lymphoid lineage

The E2F4 protein is involved in gene repression and cell cycle exit, and also has poorly understood effects in differentiation. We analyzed the impact of E2F4 deficiency on early steps in mouse hematopoietic development, and found defects in early hematopoietic progenitor cells that were propagated through common lymphoid precursors to the B and T lineages. In contrast, the defects in erythromyeloid precursor cells were self-correcting over time. This suggests that E2F4 is important in early stages of commitment to the lymphoid lineage. The E2F4-deficient progenitor cells showed reduced expression of several key lymphoid-lineage genes, and overexpression of two erythromyeloid lineage genes. However, we did not detect effects on cell proliferation. These findings emphasize the significance of E2F4 in controlling gene expression and cell fate.     

Adult reserve stem cells and their potential for tissue engineering

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories, have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher order. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve, precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including, tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50–70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.     

Fast quantification of immunohistochemistry tissue microarrays in lung carcinoma

Tissue microarrays (TMAs) are an effective tool for high-throughput molecular analysis of tissues to help identify new diagnostic and prognostic markers and targets in human cancers. We have developed a fully automated method for rapid, continuous and quantitative analysis of TMAs based on immunohistochemistry. The method deals with complex and varying tissue architectures, segments tumour cells from normal cells, conducts cell compartmentalisation, identifies nuclei and cytoplasm and produces three different continuous measurements of marker expression levels within tumour cell nuclei, tumour cell cytoplasm and total tumour cell protein expression. We have demonstrated this method using three independent protein markers (BAK, BAX and a novel biomarker, named KS) over 7 TMAs, involving 2 BAK stained TMAs with 229 tumour tissue cores, 2 BAX stained TMAs with 229 tumour tissue cores and 3 KS stained TMAs with 373 tumour cores of lung carcinomas. We validated the automated method, showing that the automated scoring is significantly correlated with the pathologist-based scoring.     

Neurogenin 3 and the enteroendocrine cell lineage in the adult mouse small intestinal epithelium

It is thought that small intestinal epithelial stem cell progeny, via Notch signaling, yield a Hes1-expressing columnar lineage progenitor and an Atoh1 (also known as Math1)-expressing common progenitor for all granulocytic lineages including enteroendocrine cells, one of the body's largest populations of endocrine cells. Because Neurogenin 3 (Neurog3) null mice lack enteroendocrine cells, Neurog3-expressing progenitors derived from the common granulocytic progenitor are thought to produce the enteroendocrine lineage, although more recent work indicates that Neurog3+ progenitors also contribute to non-enteroendocrine lineages. We aimed to test this model and better characterize the progenitors leading from the stem cells to the enteroendocrine lineage. We investigated clones derived from enteroendocrine precursors and found no evidence of a common granulocytic progenitor that routinely yields all granulocytic lineages. Rather, enteroendocrine cells are derived from a short-lived bipotential progenitor whose offspring, probably via Notch signaling, yield a Neurog3+ cell committed to the enteroendocrine lineage and a progenitor committed to the columnar lineage. The Neurog3+ cell population is heterogeneous; only about 1/3 are slowly cycling progenitors, the rest are postmitotic cells in early stages of enteroendocrine differentiation. No evidence was found that Neurog3+ cells contribute to non-enteroendocrine lineages. Revised lineage models for the small intestinal epithelium are introduced.     

Formation and regeneration of the endocrine pancreas

The elaboration of the pancreas from epithelial buds to the intricate organ requires complex patterning information that controls fundamental cellular processes such as differentiation and proliferation of pancreatic progenitor cells. During pancreatic organogenesis, endocrine cells are generated from a population of pancreatic progenitor cells. The progenitor cells during the early development simultaneously receive multiple signals, some mitogenic and some inducing differentiation. These extrinsic signals are interpreted through an intrinsic mechanism that either commits the progenitor cell to the mitotic cell cycle or leads to exit from the cell cycle in order to differentiate. The endocrine cells that differentiate from progenitor cells are postmitotic, and direct lineage tracing analyses indicate that a population of progenitor cells persists throughout embryogenesis to allow the differentiation of new endocrine cells. At the end of embryogenesis an early postnatal period is characterized by high rates of beta cell proliferation leading to massive increases in beta cell mass. The beta cell mass expansion considerably slows down in adult animals, though variations in insulin demand due to physiological and pathological states such as pregnancy and obesity can lead to adaptive changes in the beta cells that include hyperplasia, hypertrophy, and increased insulin synthesis and secretion. Deciphering the mechanisms that regulate the plasticity of beta cell mass can be an important step in developing effective strategies to treat diabetes.     

Cell lineage analysis of the mammalian female germline

Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.     

Stochastic branching model for hemopoietic progenitor cell differentiation

We present algebraic expressions describing the predictions of a stochastic branching model for differentiation of hemopoietic progenitor cells. The model assumes that there is a fixed probability, p (0 less than or equal to p less than or equal to 1), that commitment to a differentiative event occurs per progenitor cell division for each daughter cell. The model describes properties of in vitro hemopoietic cell differentiation including the population structure at the time the first progenitor cell becomes committed, the number of committed progenitor cells engendered by a single progenitor cell, and the probability of eventual commitment of all daughter cells derived from a single progenitor or stem cell. Application of the model to experimental data obtained from erythroid cultures suggests that the observed data can be explained by the stochastic branching model alone without making the deterministic assumption that there is a differentiative hierarchy in the lineage of the progenitors of erythropoiesis (BFU-E). The qualitative and quantitative aspects of the proposed stochastic model are discussed in conjunction with other analogous stochastic branching models.     

Enforced expression of EBF in hematopoietic stem cells restricts lymphopoiesis to the B cell lineage

Mice deficient in early B cell factor (EBF) are blocked at the progenitor B cell stage prior to immunoglobulin gene rearrangement. The EBF-dependent block in B cell development occurs near the onset of B-lineage commitment, which raises the possibility that EBF may act instructively to specify the B cell fate from uncommitted, multipotential progenitor cells. To test this hypothesis, we transduced enriched hematopoietic progenitor cells with a retroviral vector that coexpressed EBF and the green fluorescent protein (GFP). Mice reconstituted with EBF-expressing cells showed a near complete absence of T lymphocytes. Spleen and peripheral blood samples were >95 and 90% GFP+EBF+ mature B cells, respectively. Both NK and lymphoid-derived dendritic cells were also significantly reduced compared with control-transplanted mice. These data suggest that EBF can restrict lymphopoiesis to the B cell lineage by blocking development of other lymphoid-derived cell pathways.     

Detection of platelet-derived growth factor-alpha (PDGF-A) protein in cells of Leydig lineage in the postnatal rat testis

Platelet-derived growth factor-A (PDGF-A) is a locally produced growth factor in the rat testis secreted by both Sertoli cells and Leydig cells. It has been suggested that PDGF-A may be involved in modulation of testosterone production and may be essential to Leydig cell differentiation, however it is not known at what stage of differentiation PDGF-A begins to be expressed in the cells of Leydig lineage in the postnatal rat testis. Therefore, the objectives of this research were to determine at what postnatal age and in which cell type is PDGF-A first expressed in cells of the adult Leydig cell lineage, and does PDGF-A expression coincide with expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), an indicator of steroid hormone synthesis. Male Sprague Dawley rats of postnatal day 1, 7, 9-14, 21, 28, 40, 60, and 90 were used (n=6). Animals were euthanized and their testicles removed, fixed in Bouin's solution, embedded in paraffin, and 5 micrometers sections were prepared. Immunolocalization of PDGF-A and 3beta-HSD was carried out using a peroxidase-streptavidin-biotin method. PDGF-A was first detected in cells of the Leydig cell lineage at postnatal day 10 in progenitor cells, which were surrounding the seminiferous tubules (peritubular). These cells were confirmed to be the progenitor cells and not the mesenchymal or any other spindle-shaped cells in the testis interstitium by immunolocalization of 3beta-HSD and PDGF-A in the cells in adjacent sections of testis tissue from rats of postnatal days 10-14. After postnatal day 10, PDGF-A was continued to be expressed in subsequent cells of the Leydig lineage through day 90 (adult), however, was not present in peritubular mesenchymal precursor cells of the Leydig cell lineage or any other spindle-shaped cells in the testis interstitium at any tested age. These results revealed that PDGF-A first appears in Leydig progenitor cells in the postnatal rat testis at the onset of mesenchymal cell differentiation into progenitor cells at postnatal day 10 and suggest that a functional role(s) of PDGF-A in postnatally differentiated Leydig cells in the rat testis is established at the time of the onset of postnatal Leydig stem cell differentiation. It is suggested that the significance of the first expression of PDGF-A in the Leydig progenitor cells may be associated with inducing cell proliferation and migration of this cell away from the peritubular region during Leydig cell differentiation.     

The origin of pre-neoplastic metaplasia in the stomach: chief cells emerge from the Mist

The digestive-enzyme secreting, gastric epithelial chief (zymogenic) cell is remarkable and underappreciated. Here, we discuss how all available evidence suggests that mature chief cells in the adult, mammalian stomach are postmitotic, slowly turning over cells that arise via a relatively long-lived progenitor, the mucous neck cell, The differentiation of chief cells from neck cells does not involve cell division, and the neck cell has its own distinct pattern of gene expression and putative physiological function. Thus, the ontogeny of the normal chief cell lineage exemplifies transdifferentiation. Furthermore, under pathophysiogical loss of acid-secreting parietal cell, the chief cell lineage can itself trasndifferentiate into a mucous cell metaplasia designated Spasmolytic Polypeptide Expressing Metaplasia (SPEM). Especially in the presence of inflammation, this metaplastic lineage can regain proliferative capacity and, in humans may also further differentiate into intestinal metaplasia. The results indicate that gastric fundic lineages display remarkable plasticity in both physiological ontogeny and pathophysiological pre-neoplastic metaplasia.     

Vertebrate neural stem cell segmentation, tracking and lineaging with validation and editing

This protocol and the accompanying software program called LEVER (lineage editing and validation) enable quantitative automated analysis of phase-contrast time-lapse images of cultured neural stem cells. Images are captured at 5-min intervals over a period of 5-15 d as the cells proliferate and differentiate. LEVER automatically segments, tracks and generates lineage trees of the stem cells from the image sequence. In addition to generating lineage trees capturing the population dynamics of clonal development, LEVER extracts quantitative phenotypic measurements of cell location, shape, movement and size. When available, the system can include biomolecular markers imaged using fluorescence. It then displays the results to the user for highly efficient inspection and editing to correct any errors in the segmentation, tracking or lineaging. To enable high-throughput inspection, LEVER incorporates features for rapid identification of errors and for learning from user-supplied corrections to automatically identify and correct related errors.     

Epigenetic cues modulating the generation of cell‐type diversity in the cerebral cortex

The cerebral cortex is composed of a large variety of distinct cell‐types including projection neurons, interneurons, and glial cells which emerge from distinct neural stem cell lineages. The vast majority of cortical projection neurons and certain classes of glial cells are generated by radial glial progenitor cells in a highly orchestrated manner. Recent studies employing single cell analysis and clonal lineage tracing suggest that neural stem cell and radial glial progenitor lineage progression are regulated in a profound deterministic manner. In this review we focus on recent advances based mainly on correlative phenotypic data emerging from functional genetic studies in mice. We establish hypotheses to test in future research and outline a conceptual framework how epigenetic cues modulate the generation of cell‐type diversity during cortical development.