Identification of CD44 as a cell surface marker for Müller glia precursor cells

In the retina, both neurons and glia differentiate from a common progenitor population. CD44 cell surface antigen is a hyaluronic acid receptor expressed on mature Müller glial cells. We found that in the developing mouse retina, expression of CD44 was transiently observed at or around birth in a subpopulation of c-kit-positive retinal progenitor cells. During in vitro culture, purified CD44/c-kit-positive retinal progenitor cells exclusively differentiated into Müller glial cells and not into neurons, suggesting that CD44 marks a subpopulation of retinal progenitor cells that are fated to become glia. Over-expression of CD44 inhibited the extension of processes by Müller glial cells and neurons. Notch signaling is known to be involved in the specification of retinal progenitors into a glial fate. Activation of Notch signaling increased the number of CD44-positive cells, and treatment with the Notch signal inhibitor, DAPT, at early, but not later, stages of retinal development abolished both CD44-positive cells and Müller glial cells. Together, CD44 was identified as an early cell surface marker of the Müller glia lineage, and Notch signalling was involved in commitment of retinal progenitor cells to CD44 positive Müller glial precursor cells.     

Lineage analysis of transplanted individual cells in embryos of Drosophila melanogaster

Summary A method is presented which allows the study of the progeny of single cells during Drosophila embryogenesis. Cells from various larval anlagen of donor embryos labelled with a lineage tracer are individually transplanted from defined positions into similar, or different, positions in unlabelled hosts. The clones produced by these cells can be seen in whole mounts or in sections of fixed material, when using a histochemical marker (i.e. HRP), and/or in living embryos, when using fluorescent lineage tracers. The characteristics of the clones disclose lineage parameters, such as division patterns, morphogenetic movements and differentiation. The method is especially useful for testing the respective roles of positional information and cell lineage on the commitment of progenitor cells by transplanting these cells into heterotopic positions or into hosts of different genotypes.     

Notch Signaling Functions as a Cell-Fate Switch between the Endothelial and Hematopoietic Lineages

Recent studies have begun to elucidate how the endothelial lineage is specified from the nascent mesoderm [1] and [2]. However, the molecular mechanisms which regulate this process remain largely unknown. We hypothesized that Notch signaling might play an important role in specifying endothelial progenitors from the mesoderm, given that this pathway acts as a bipotential cell-fate switch on equipotent progenitor populations in other settings [3] and [4]. We found that zebrafish embryos with decreased levels of Notch signaling exhibited a significant increase in the number of endothelial cells, whereas embryos with increased levels of Notch signaling displayed a reduced number of endothelial cells. Interestingly, there is a concomitant gain of endothelial cells and loss of erythrocytes in embryos with decreased Notch activity, without an effect on cell proliferation or apoptosis. Lineage-tracing analyses indicate that the ectopic endothelial cells in embryos with decreased Notch activity originate from mesodermal cells that normally produce erythrocyte progenitors. Taken together, our data suggest that Notch signaling negatively regulates the number of endothelial cells by limiting the number of endothelial progenitors within the mesoderm, probably functioning as a cell-fate switch between the endothelial and the hematopoietic lineages.     

Stem-loop binding protein is required for retinal cell proliferation,neurogenesis, and intraretinal axon pathfinding in zebrafish

In the developing retina, neurogenesis and cell differentiation are coupled with cell proliferation. However, molecular mechanisms that coordinate cell proliferation and differentiation are not fully understood. In this study, we found that retinal neurogenesis is severely delayed in the zebrafish stem-loop binding protein (slbp) mutant. SLBP binds to a stem-loop structure at the 3′-end of histone mRNAs, and regulates a replication-dependent synthesis and degradation of histone proteins. Retinal cell proliferation becomes slower in the slbp1 mutant, resulting in cessation of retinal stem cell proliferation. Although retinal stem cells cease proliferation by 2 days postfertilization (dpf) in the slbp mutant, retinal progenitor cells in the central retina continue to proliferate and generate neurons until at least 5 dpf. We found that this progenitor proliferation depends on Notch signaling, suggesting that Notch signaling maintains retinal progenitor proliferation when faced with reduced SLBP activity. Thus, SLBP is required for retinal stem cell maintenance. SLBP and Notch signaling are required for retinal progenitor cell proliferation and subsequent neurogenesis. We also show that SLBP1 is required for intraretinal axon pathfinding, probably through morphogenesis of the optic stalk, which expresses attractant cues. Taken together, these data indicate important roles of SLBP in retinal development.     

Cell lineage in the rat cerebral cortex: a study using retroviral-mediated gene transfer

We have used a retroviral vector that codes for the bacterial enzyme beta-galactosidase to study cell lineage in the rat cerebral cortex. This vector has been used to label progenitor cells in the cerebral cortices of rat embryos during the period of neurogenesis. When these embryos are allowed to develop to adulthood, the clones of cells derived from the marked progenitor cells can be identified histochemically. In this way, we can ask what are the lineage relationships between different neural cell types. From these studies, we conclude that there are two distinct types of progenitor cells in the developing cortex. One generates only grey matter astrocytes, whereas the second gives rise to neurones - both pyramidal and nonpyramidal - and to another class of cells that we have tentatively identified as glial cells of the white matter. We have also been able to address the question of how neurones are dispersed in the cortex during histogenesis. It had been previously hypothesized that clonally related neurones migrated radially to form columns in the mature cortex. However, we find that clones of neurones do not form radial columns; rather, they tend to occupy the same or neighbouring cortical laminae and to be spread over several hundreds of micrometers of cortex in the horizontal dimension. This spread occurs in both mediolateral and rostrocaudal directions.     

Stem cells in the eye

In the adult organism, all tissue renewal and regeneration depends ultimately on somatic stem cells, and the eye is no exception. The importance of limbal stem cells in the maintenance of the corneal epithelium has long been recognised, and such cells are now used clinically for repair of a severely damaged cornea. The slow cycling nature of lens epithelial cells and their ability to terminally differentiate into fiber cells are suggestive of a stem cell lineage. Furthermore, recent studies have identified progenitor cells in the retina and ocular vasculature which may have important implications in health and disease. Although the recent literature has become flooded with articles discussing aspects of stem cells in a variety of tissues our understanding of stem cell biology, especially in the eye, remains limited. For instance, there is no definitive marker for ocular stem cells despite a number of claims in the literature, the patterns of stem cell growth and amplification are poorly understood and the microenvironments important for stem cell regulation and differentiation pathways are only now being elucidated. A greater understanding of ocular stem cell biology is essential if the clinical potential for stem cells is to be realised. For instance; How do we treat stem cell deficiencies? How do we use stem cells to regenerate damaged retinal tissue? How do we prevent stem cell lineages contributing to retinal vascular disease? This review will briefly consider the principal stem cells in the mature eye but will focus in depth on limbal stem cells and corneal epithelium. It will further discuss their role in pathology and their potential for therapeutic intervention.     

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.     

Cultivation and differentiation characteristics of human limbal progenitor cells

BACKGROUND: The aim of this study was to investigate whether limbal progenitor cells can be cultured, expanded and differentiated in vitro not only to enter corneal differentiation but also towards RPE (retinal pigment epithelium) characteristics. METHODS: A 3mm broad strip of human corneoscleral limbal tissue was digested enzymatically and cells were set into cell culture. Differentiation status and characteristics, proliferation and phagocytotic activity were assessed by immunocytochemical staining in combination with digital and confocal microscopy. RESULTS: Immunocytological analysis revealed expression of Nestin and p63 marker suggesting progenitor cell properties. Mitotic activity was demonstrated by BrdU (bromodesoxyuridine) uptake. Upon consecutive passages, corneal differentiation markers were predominantly expressed. Phagocytotic activity was demonstrated via uptake of FITC (fluorescein isothiocyanate) labelled latex beads. RPE markers Bestrophin and Cytokeratin 8/18 as well as glial marker GFAP and neuronal marker MAP with respective controls were negative indicating no differentiation towards characteristics of retinal pigment epithelium or neural and glial lineage. CONCLUSIONS: The results suggest that isolation and cultivation of proliferating and phagocytotic cells from the human corneal limbus was achieved which showed characteristics of both progenitor and differentiated corneal cells. No evidence was found for the hypothesis of spontaneous differentiation potential towards RPE lineage or neuronal characteristics, providing evidence of the inherent directional capacity of limbal progenitor cells.     

Sox5 Functions as a Fate Switch in Medaka Pigment Cell Development

Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor).     

Origin of non-cardiac endothelial cells from an Isl1+ lineage

The cardiovascular system consists of many cell types with distinct embryonic origins. Cells from an Islet1 (Isl1)-expressing progenitor population make a substantial contribution to the developing heart. We reasoned that cells derived from Isl1-expressing progenitors might contribute more widely to the cardiovascular system. We show that cells derived from an Isl1-expressing progenitor lineage make a wide contribution to the systemic vasculature and that embryos conditionally deficient for Rac1 within this cell population develop defects in the non-cardiac vasculature. These data define new roles for Isl1 in the developing embryo and demonstrate a contribution of Isl1-expressing progenitors to vascular endothelium in vivo.