Medial edge epithelial cell fate during palatal fusion

To explain the disappearance of medial edge epithelial (MEE) cells during palatal fusion, programmed cell death, epithelial-mesenchymal transformation, and migration of these cells to the oral and nasal epithelia have been proposed. However, MEE cell death has not always been accepted as a mechanism involved in midline epithelial seam disappearance. Similarly, labeling of MEE cells with vital lipophilic markers has not led to a clear conclusion as to whether MEE cells migrate, transform into mesenchyme, or both. To clarify these controversies, we first utilized TUNEL techniques to detect apoptosis in mouse palates at the fusion stage and concomitantly analyzed the presence of macrophages by immunochemistry and confocal microscopy. Second, we in vitro infected the MEE with the replication-defective helper-free retroviral vector CXL, which carries the Escherichia coli lacZ gene, and analyzed beta-galactosidase activity in cells after fusion to follow their fate. Our results demonstrate that MEE cells die and transform into mesenchyme during palatal fusion and that dead cells are phagocytosed by macrophages. In addition, we have investigated the effects of the absence of transforming growth factor beta(3) (TGF-beta(3)) during palatal fusion. Using environmental scanning electron microscopy and TUNEL labeling we compared the MEE of the clefted TGF-beta(3) null and wild-type mice. We show that MEE cell death in TGF-beta(3) null palates is greatly reduced at the time of fusion, revealing that TGF-beta(3) has an important role as an inducer of apoptosis during palatal fusion. Likewise, the bulging cells observed on the MEE surface of wild-type mice prior to palatal shelf contact are very rare in the TGF-beta(3) null mutants. We hypothesize that these protruding cells are critical for palatal adhesion, being morphological evidence of increased cell motility/migration.     

Molecular and morphologic changes during the epithelial-mesenchymal transformation of palatal shelf medial edge epithelium in vitro.

The fate of the medial edge epithelial (MEE) cells during palatal fusion has been proposed to be either programmed cell death or epithelial-mesenchymal transformation. Vital cell labeling techniques were used to mark the MEE and observe their fate during palatal fusion in vitro. Fetal mouse palatal shelves were labeled with Dil and allowed to proceed through fusion while maintained in an organ culture system. The tissues were examined at several stages of palatal fusion for the distribution of Dil, presence of specific antigens and ultrastructural appearance of the cells. The MEE labeled with Dil occupied a midline position at all stages of palatal fusion. Initially the cells had keratin intermediate filaments and were separated from the underlying mesenchyme by an intact basement membrane. During the process of fusion the basement membrane was degraded and the Dil-labeled MEE were in contact with the mesenchymal-derived extracellular matrix. In the late stages of fusion the Dil-labeled MEE altered their cellular morphology, had vimentin intermediate filaments, and were not associated with an identifiable basement membrane. Dil-labeled cells, without an epithelial phenotype, remained present in the midline of the completely fused palate. The data indicate that the MEE did not die but underwent a phenotypic transformation to viable mesenchymal cell types, which were retained in the palatal mesenchyme.     

Mother-daughter precursor cell fate transformation after Cdc2 down-regulation in the Drosophila bristle lineage

The Drosophila bristle lineage is an excellent system in which to study how cell cycle and fate determination are synchronized in invariant cell lineages. In this model, five different cells arise from a single precursor cell, pI, after four asymmetric cell divisions. Cell diversity is achieved by the asymmetric segregation of cell determinants, such as Numb and Neuralized (Neur), resulting in differential activation of the Notch (N) pathway. We show that down-regulation of Cdc2, by over-expressing Tribbles, Dwee1, and Dmyt1 (three negative regulators of Cdc2) or by using thermo-sensitive Cdc2 mutant flies, delayed pI mitosis, and altered the polarity and the number of subsequent cell divisions. These modifications were associated with a mother-daughter cell fate transformation as the pI cell acquired the identity of the secondary precursor cell, pIIb. This type of change in cell identity only occurred when the N signaling pathway was inactive since ectopic N signaling transformed pI to pIIa-progeny fate. These transformations in cell identity suggest that, although synchronized, cell cycle and fate determination are independent phenomena in the bristle lineage.     

Cell lineage and determination of cell fate in ascidian embryos

A detailed cell lineage of ascidian embryos has been available since the turn of the century. This cell lineage was deduced from the segregation of pigmented egg cytoplasmic regions into particular blastomeres during embryogenesis. The invariant nature of the cell lineage, the segregation of specific egg cytoplasmic regions into particular blastomeres, and the autonomous development of most embryonic cells suggests that cell fate is determined primarily by cytoplasmic determinants. Modern studies have provided strong evidence for the existence of cytoplasmic determinants, especially in the primary muscle cells, yet the molecular identity, localization, and mode of action of these factors are still a mystery. Recent revisions of the classic cell lineage and demonstrations of the lack of developmental autonomy in certain embryonic cells suggest that induction may also be an important mechanism for the determination of cell fate in ascidians. There is strong evidence for the induction of neural tissue and indirect evidence for inductive interactions in the development of the secondary muscle cells. In contrast to the long-accepted dogma, specification of cell fate in ascidians appears to be established by a combination of cytoplasmic determinants and inductive cell interactions.     

Embryonic lineage tracing with Procr-CreER marks balanced hematopoietic stem cell fate during entire mouse lifespan

The functional heterogeneity of hematopoietic stem cells (HSCs) has been comprehensively investigated by single-cell transplantation assay. However, the heterogeneity regarding their physiological contribution remains an open question, especially for those with life-long hematopoietic fate of rigorous self-renewing and balanced differentiation capacities. In this study, we revealed that Procr expression was detected principally in phenotypical vascular endothelium co-expressing Dll4 and CD44 in the mid-gestation mouse embryos, and could enrich all the HSCs of the embryonic day 11.5 (E11.5) aorta-gonad-mesonephros (AGM) region. We then used a temporally restricted genetic tracing strategy to irreversibly label the Procr-expressing cells at E9.5. Interestingly, most labeled mature HSCs in multiple sites (such as AGM) around E11.5 were functionally categorized as lymphomyeloid-balanced HSCs assessed by direct transplantation. Furthermore, the labeled cells contributed to an average of 7.8% of immunophenotypically defined HSCs in E14.5 fetal liver (FL) and 6.9% of leukocytes in peripheral blood (PB) during one-year follow-up. Surprisingly, in aged mice of 24 months, the embryonically tagged cells displayed constant contribution to leukocytes with no bias to myeloid or lymphoid lineages. Altogether, we demonstrated, for the first time, the existence of a subtype of physiologically long-lived balanced HSCs as hypothesized, whose precise embryonic origin and molecular identity await further characterization.     

Lineage diversion of T cell receptor transgenic thymocytes revealed by lineage fate mapping

Background

The binding of the T cell receptor (TCR) to major histocompatibility complex (MHC) molecules in the thymus determines fates of TCRαβ lymphocytes that subsequently home to secondary lymphoid tissue. TCR transgenic models have been used to study thymic selection and lineage commitment. Most TCR transgenic mice express the rearranged TCRαβ prematurely at the double negative stage and abnormal TCRαβ populations of T cells that are not easily detected in non-transgenic mice have been found in secondary lymphoid tissue of TCR transgenic mice.

Methodology and Principal Findings

To determine developmental pathways of TCR-transgenic thymocytes, we used Cre-LoxP-mediated fate mapping and show here that premature expression of a transgenic TCRαβ diverts some developing thymocytes to a developmental pathway which resembles that of gamma delta cells. We found that most peripheral T cells with the HY-TCR in male mice have bypassed the RORγt-positive CD4⁺8⁺ (double positive, DP) stage to accumulate either as CD4⁻8⁻ (double negative, DN) or as CD8α⁺ T cells in lymph nodes or gut epithelium. Likewise, DN TCRαβ cells in lymphoid tissue of female mice were not derived from DP thymocytes.

Conclusion

The results further support the hypothesis that the premature expression of the TCRαβ can divert DN thymocytes into gamma delta lineage cells.     

Stem-cell dynamics and lineage topology from in vivo fate mapping in the hematopoietic system

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The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy.

Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.     

Glial cell lineage in vivo in the mouse cerebellum

Glial cells of the cerebellum originate from cells of the ventricular germinative layer, but their lineage has not been fully elucidated. For studying the glial cell lineage in vivo by retrovirus-mediated gene transfer, we introduced a marker retrovirus into the ventricular germinative layer of embryonic day 13 mice. In the resulting adult cerebella, virus-labeled glial cells were grouped in discrete clusters, and statistical analysis showed that these clusters represented clones in high probability. Of 71 of the virus-labeled glial clusters, 33 clusters were composed of astrocytes/Bergmann glia, 10 were composed of only white matter astrocytes, and 24 were composed of only oligodendrocytes. No glial clusters contained virus-labeled neurons. These results suggest that astrocytes/Bergmann glia, white matter astrocytes and oligodendrocytes immediately arise from separate glial precursors: these three glial lineages may diverge in the course of cerebellar development.     

From epithelium to neuroblasts to neurons: the role of cell interactions and cell lineage during insect neurogenesis

The grasshopper central nervous system is composed of a brain and a chain of segmental ganglia. Each hemiganglion contains about 1000 neurons, most of which can be individually identified by their unique morphology and synaptic connectivity. Shortly after gastrulation the ventral ectoderm becomes a neurogenic region. In each hemisegment, ca. 150 neurogenic ectodermal cells (nECs) give rise to a stereotyped pattern of 30 identified neuroblasts (NBs, neuronal stem cells); the remaining nECs become various non-neuronal cells or die. The 30 NBs then give rise to about 1000 neurons as each NB initiates an invariant lineage, generating a stereotyped chain of ganglion mother cells (GMCs), each of which in turn divides once to generate two identified neurons. We have used a laser microbeam or microelectrode to ablate individual cells in ovo and in vitro at various stages of embryogenesis to study how neuronal diversity and specificity are generated during development. Our results suggest that cell interactions between ca. 150 equivalent nECs allow 30 cells to enlarge into NBs, the dominant fate in a hierarchy; the NBs inhibit adjacent nECs and thus cause them to differentiate into various non-neuronal cells; each NB is assigned its unique identity according to its position of enlargement within the neurogenic epithelium; each NB then generates its characteristic chain of GMCs by an invariant cell lineage; and each GMC generates a pair of equivalent progeny, the fate of each individual neuron being determined by both its GMC of origin and interactions with its sibling.     

Clonal analysis of the Arabidopsis root confirms that position, not lineage, determines cell fate

 The cellular organization of the Arabidopsis thaliana (L.) Heynh. root meristem suggests that a regular pattern of cell divisions occurs in the root tip. Deviations from this pattern of division might be expected to disrupt the organization of cells and tissues in the root. A clonal analysis of the 3-d-old primary root meristem was carried out to determine if there is variability in division patterns, and if so to discover their effect on cellular organization in the root. Clones induced in the seedling meristem largely confirmed the predicted pattern of cell divisions. However, the cellular initials that normally give rise to the different cell files in the root were shown to exhibit some instability. For example, it was calculated that a lateral root cap/epidermal initial is displaced every 13 d. Furthermore, the existence of large marked clones that included more than two adjacent cell layers suggests that intrusive growth followed by cell division may occur at low frequency, perhaps in response to local cell deaths in the meristem. These findings support the view that even in plant organs with stereotypical cell division patterns, positional information is still the key determinant of cell fate. Received: 27 August 1999 / Accepted: 4 December 1999     

Cell lineage and cell fate specification in the embryonic CNS of Drosophila

The Drosophila CNS derives from a population of neural stem cells, called neuroblasts (NBs), which delaminate individually from the neurogenic region of the ectoderm. In the embryonic ventral nerve cord each NB can be uniquely identified and gives rise to a specific lineage consisting of neurons and/or glial cells. This 'NB identity' is dependent on the position of the progenitor cells in the neuroectoderm before delamination. The positional information is provided by the products of segment polarity and dorsoventral (D/V) patterning genes. Subsequently, 'cell fate genes' like huckebein (hkb) and eagle (eg) contribute to the generation of specific NB lineages. These genes act downstream of segment polarity and D/V patterning genes and regulate different processes such as the generation of glial cells and the determination of serotonergic neurons.     

Evaluation of effector cell fate and function by in vivo bioluminescence imaging

The effector functions of immune cells have typically been examined using assays that require sampling of tissues or cells to reveal specific aspects of an immune response (e.g., antigen-specificity, cytokine expression or killing of target cells). The outcome of an immune response in vivo, however, is not solely determined by a single effector function of a specific cell population, but is the result of numerous cellular and molecular interactions that occur in the complex environment of intact organ systems. These interactions influence survival, migration, and activation, as well as final effector function of a given population of cells. Efforts to reveal the cellular and molecular basis of biological processes have resulted in a number of technologies that combine molecular biology and imaging sciences that are collectively termed as Molecular Imaging. This emerging field has developed to reveal functional aspects of cells, genes, and proteins in real time in living animals and humans and embraces multiple modalities, including established clinical imaging methods such as magnetic resonance imaging, single photon emission computed tomography, and positron emission tomography, as well as novel methodologies specifically designed for research animals. Here, we highlight one of the newer modalities, in vivo bioluminescence imaging, as a method for evaluating effector T cell proliferation, migration, and function in model systems of malignant and non-malignant diseases.