Cell Lineage Analysis Research Articles

True monolayer cell culture in a confined 3D microenvironment enables lineage informatics

There is a need for methods to (1) track cells continuously to generate lineage trees; (2) culture cells in in vivo-like microenvironments; and (3) measure many biological parameters simultaneously and noninvasively. Herein, we present a novel imaging culture chamber that facilitates "lineage informatics," a lineage-centric approach to cytomics. We cultured cells in a confined monolayer using a novel "gap chamber" that produces images with confocal-like qualities using standard DIC microscopy. Lineage and other cytometric data were semiautomatically extracted from image sets of neural stem and progenitor cells and analyzed using lineage informatics. Cells imaged in the chamber every 3 min could be tracked for at least 6 generations allowing for the construction of extensive lineage trees with multiparameter data sets at hundreds of time points for each cell. The lineage informatics approach reveals relationships between lineage, phenotype, and microenvironment. Mass transfer characteristics and 3D geometry make the chamber more in vivo-like than traditional culture systems. The gap chamber allows cells to be cultured, imaged, and tracked in true monolayers permitting detailed informatics analysis of cell lineage, phenotype, and fate determinants. The chamber is biomimetic and straightforward to build and use, and should find many applications in long-term cell imaging.

A 4D-microscopic analysis of the germ band in the isopod crustacean Porcellio scaber (Malacostraca, Peracarida)—developmental and phylogenetic implications

Malacostracan crustaceans have evolved a conserved stereotyped cell division pattern in the post-naupliar germ band. This cleavage pattern is unique in arthropods investigated so far, and allows a combined analysis of gene expression and cell lineage during segmentation and organ development at the level of individual cells. To investigate the cell lineage in the germ band of the isopod Porcellio scaber, we used a 4D-microscopy system, which enables us to analyse every cell event in the living embryo. The study was combined with the analysis of the expression of the gene engrailed (en) at different stages of germ band formation. Our findings confirm the results of earlier investigations of the cell division pattern in the posterior part of the isopod germ band. Furthermore, we can show that in the anterior region, in contrast to the posterior part, cleavage directions are variable and cell sorting takes place-similar to other arthropod germ bands. Additionally, the gene expression pattern of en in this region is not as regular as in the post-naupliar germ band, and only later becomes regulated into its characteristic stripe pattern. The comparison of the cell lineage of P. scaber with that of other malacostracan crustaceans shows an enhancement in the velocity of cell divisions relative to the arrangement of these cells in rows in the isopod germ band. The striking similarity of the formation of the genealogical units in the anterior part suggests a sister group relationship between the peracarid taxa Tanaidacea and Isopoda.

Anteroposterior patterning in the limb and digit specification: Contribution of mouse genetics

The limb has been a privileged object of investigation and reflection for scientists over the past two centuries and continues to provide a heuristic framework to analyze vertebrate development. Recently, accumulation of new data has significantly changed our view on the mechanisms of limb patterning, in particular along the anterior-posterior axis. These data have led us to revisit the mode of action of the zone of polarizing activity. They shed light on the molecular and cellular mechanisms of patterning linked to the Shh-Gli3 signaling pathway and give insights into the mechanism of activation of these cardinal factors, as well as the consequences of their activity. These new data are in good part the result of systematic Application of tools used in contemporary mouse molecular genetics. These have extended the power of mouse genetics by introducing mutational strategies that allow fine-tuned modulation of gene expression, interchromosomal deletions and duplication. They have even made the mouse embryo amenable to cell lineage analysis that used to be the realm of chick embryos. In this review, we focus on the data acquired over the last five years from the analysis of mouse limb development and discuss new perspectives opened by these results.

Formation of a functional thymus initiated by a postnatal epithelial progenitor cell

The thymus is essential for the generation of self-tolerant effector and regulatory T cells. Intrathymic T-cell development requires an intact stromal microenvironment, of which thymic epithelial cells (TECs) constitute a major part. For instance, cell-autonomous genetic defects of forkhead box N1 (Foxn1) and autoimmune regulator (Aire) in thymic epithelial cells cause primary immunodeficiency and autoimmunity, respectively. During development, the thymic epithelial rudiment gives rise to two major compartments, the cortex and medulla. Cortical TECs positively select T cells, whereas medullary TECs are involved in negative selection of potentially autoreactive T cells. It has long been unclear whether these two morphologically and functionally distinct types of epithelial cells arise from a common bi-potent progenitor cell and whether such progenitors are still present in the postnatal period. Here, using in vivo cell lineage analysis in mice, we demonstrate the presence of a common progenitor of cortical and medullary TECs after birth. To probe the function of postnatal progenitors, a conditional mutant allele of Foxn1 was reverted to wild-type function in single epithelial cells in vivo. This led to the formation of small thymic lobules containing both cortical and medullary areas that supported normal thymopoiesis. Thus, single epithelial progenitor cells can give rise to a complete and functional thymic microenvironment, suggesting that cell-based therapies could be developed for thymus disorders.

Regeneration of the intestinal epithelia: Regulation of bone marrow-derived epithelial cell differentiation towards secretory lineage cells

The intestinal epithelia consists of four lineages of differentiated cells, all of which arise from stem cells residing in the intestinal crypt. For proper regeneration from epithelial damage, both expansion of the epithelial cell number and appropriate regulation of lineage differentiation from the remaining stem cells are thought to be required. In a series of studies, we have shown that bone-marrow derived cells could promote the regeneration of damaged epithelia in the human intestinal tract. Donor-derived epithelial cells substantially repopulated the gastrointestinal tract of bone-marrow transplant recipients during epithelial regeneration after graft-versus-host disease. Furthermore, precise analysis of epithelial cell lineages revealed that during epithelial regeneration, secretory lineage epithelial cells that originated from bone-marrow significantly increased in number. These findings may lead to a novel therapy to repair damaged intestinal epithelia using bone marrow cells, and provide an alternative therapy for refractory inflammatory bowel diseases.

Lectin Histochemistry of Taste Buds in the Circumvallate Papilla of the Rat

In mammals, taste buds are localized mostly within two regions, i.e. lingual and palatal epithelium. In the lingual epithelium, taste buds are localized in three lingual papillae; fungiform, foliate and circumvallate papillae. In the palatal epithelium, taste buds are present in the nasoincisor papillae and soft palate. In addition, few taste buds are recognized at the laryngeal surface of epiglottis. Taste buds contain 50–80 elongated specialized epithelial cells (taste cells) and proliferative basal cells (progenitor cells). Ultrastructually, taste cells have traditionally been classified into two types of cells; dark and light cells. Dark cells or type I cells have an electron-dense cytoplasm and thought to be supporting cells. Light cells are characterized by the presence of electron-lucent cytoplasm and further subdivided into two types based on the presence of synaptic vesicles. Light cells without apparent synaptic vesicles are called type II cells, while those with synaptic vesicles are termed as type III cells. Progenitor cells are located at the basal portion of the taste buds. There are at least two theories for cell lineage of the taste buds; each type of cells in taste buds has individual progenitor cells (multi cell line theory) and all types of cells are derived from the same progenitor cell (one cell line theory). Several histochemical studies to demonstrate the expression of bioactive substances in specific cell type of taste cells for the analysis of cell lineage of the taste buds. Lectins are useful for histochemistry and have been used in several areas to identify particular cell populations on the base of different ability to bind with high specificity to different carbohydrate epitopes of cell membrane. Although several lectin histochemical studies have been carried out to the taste buds to map the overall distribution of different kinds of lectins in the different location of taste buds in various species (Witt and Reutter, 1988; Witt and Miller, 1992; Zeng et al., 1995; Ohnishi et al., 2000; Kano et al., 2001), few studies are conducted on the relation between lectin binding patterns and specific cell types within the taste buds. The present presentation focused on the lectin binding pattern of rat circumvallate papilla with special references to the relation with type II cells, i.e. α-gustducin-immunoreactive cells. Ulex europaeus agglutinin-I (UEA-I) reacts strongly with α(1, 2)linked fucose. In the taste buds of the circumvallate papilla, UEA-I was found to bind with the cell membrane of taste buds (Figure 1A, compared with Figure 1C). Within the taste buds, UEA-I bound to almost all individual cells in the taste buds. Most blood group H type 2 antigen (AbH) immunoreactive cells co-expressed UEA-I (Figure 1B). In addition to the taste buds, UEA-I binds occasionally to the keratinized layer of the trench wall of the circumvallate papilla and in the lingual epithelium. Although lectin from Lotus tetragonolobus is also specific for L-fucose, it did not, however, bind to the taste bud cells. Peanut agglutinin (PNA) is specific for terminal β-galactose and binds to the membrane of rounded-shaped cells at the basal portion of taste buds. Spindle-shaped cells within the taste buds were devoid of PNA binding. In the trench wall epithelium, PNA strongly labeled the membrane of cells from basal cell layer to granular cell layer. Cells at keratinized cell layer lack PNA bindings. In the lingual epithelium, strong bindings for PNA were also detected from basal cell layer to granular cell layer. Jacalin is purified from jackfruit and specific for D-galactose. Similar to PNA, jacalin binds the membrane of cells located from basal cell layer to granular layer in the trench wall epithelium. Keratinized cells lacked in jacalin binding. Some rounded cells at the basal portion of the taste buds were also found to bind to jacalin. PNA bound to membrane of some rounded-shaped cells that does not react with jacalin. Within the taste buds, some spindle-shaped cells reacted with both wheat germ agglutinin (WGA) and succinyl WGA. The number of cells binding to WGA is slightly smaller than that binding to succinyl WGA. Many cells binding to WGA and succinyl WGA displayed immunoreactivity for α-gustducin; Not all cells lebeled with WGA or succinyl WGA showed α-gustducin immunoreactivity and vise versa. Dolichos bifforus agglutinin (DBA) binds to the mostly apical portion of the spindle-shaped cells within the taste buds of circumvallate papilla. Occasionally, DBA also binds to the cytoplasm of the basal portion of taste buds. The number of cells binding to DBA is low compared to cells binding to WGA or succinyl WGA. Most of cells binding with DBA did not show immunoreactivity for α-gustducin. The present lectin histochemical analysis of taste cells showed that some lectins bind specifically to certain types of cells in the rat circumvallate papilla.

Clonality analysis of cell lineages in acute myeloid leukemia with inversion 16

Bone marrow from 5 patients with acute myeloid leukemia with inversion 16, inv(16), was studied by a combination of fluorescence activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) to establish the extent of cell-lineage involvement of inv(16). In all cases, interphase FISH demonstrated the inv(16) in 80–92% of blasts, monocytes, granulocytes, and a proportion of B-cells (21–41%). In contrast, inv(16) was not detectable above threshold levels in mature T-cells. The current study provides a direct evidence for the clonal involvement of myeloid lineage cells and B-lymphocytes and suggests that T-cells are not part of the malignant clone in this disease.

The fate of isolated blastomeres with respect to germ cell formation in the amphipod crustacean Parhyale hawaiensis

Germ cells may be specified through the localization of germ line determinants to specific cells in early embryogenesis, or by inductive signals from neighboring cells to germ cell precursors in later embryogenesis. Such determinants can be produced and localized during or after oogenesis, either autonomously by oocytes or by associated nutritive cells. In Drosophila, each oocyte is connected to nurse cells by cytoplasmic bridges, and determinants synthesized in nurse cells are transported through these bridges to the oocyte. However, the Drosophila model may not be applicable to all arthropods, since in many species of all four extant arthropod classes, gametogenesis functions without nurse cells. In this paper, I use immunodetection of Vasa protein to study germ cell development in the amphipod crustacean Parhyale hawaiensis, a species whose ovaries lack nurse cells and whose eggs lack obvious polarity. Previous cell lineage analyses have shown that all three germ layers and the germ line are exclusively specified by third cleavage. In the present study, I use a molecular marker to follow germ cell development during P. hawaiensis embryogenesis. I determine the capacity of individual blastomeres to form germ cells by isolating blastomeres at early cleavage stages and provide experimental evidence for localized germ cell determinants at the two-cell stage in P. hawaiensis. These experiments indicate that many aspects of early amphipod development, including timing and symmetry of cell division, the transition from holoblastic to superficial cleavage, and possibly some gastrulation movements, are cell autonomous following first cleavage.

Genetic lineage of poorly differentiated gastric carcinoma with a tubular component analysed by comparative genomic hybridization.

Analysis of cell lineage is based on the use of genetic markers inherent to the lineage to be analysed. The breakpoints of unbalanced translocations, and the pattern of chromosomal loss/gain determined by comparative genomic hybridization (CGH), have been previously used to demonstrate lineages in diffuse-type gastric carcinoma. Signet ring cell carcinoma was shown to progress to poorly differentiated adenocarcinoma, and early diffuse-type gastric carcinoma to advanced diffuse-type gastric carcinoma. The present study focuses on poorly differentiated adenocarcinoma with a tubular component to clarify its derivation. CGH and array CGH were applied to DNA extracted from multiple portions of individual tumours and amplified by degenerate oligonucleotide-primed (DOP) PCR and the changes common to the samples in each tumour (stemline changes) were compared between the tumours with and those without a tubular component. Within individual tumours, the samples from the tubular component and those from the other components had common stemline changes and a very similar frequency pattern of chromosomal changes, indicating their common derivation. Frequent stemline changes were 8q+, 7p+, 3q+, 20q+, and 10p+, and these were different from those in the tumours without a tubular component. It was noticed that there were two subgroups in the tumours with a tubular component: one with 5p+, 6p+, 7p+, and 10p+, and the other without these changes. The latter had cytogenetic and clinicopathological features similar to those of the tumours without a tubular component. Analysis of the clonal evolution process by constructing dendrograms for each tumour gave results consistent with the notion that the latter subgroup may derive from signet ring cell carcinoma and the former from tubular adenocarcinoma.

The cell lineage of the polyplacophoran, Chaetopleura apiculata: variation in the spiralian program and implications for molluscan evolution

Polyplacophorans, or chitons, are an important group of molluscs, which are argued to have retained many plesiomorphic features of the molluscan body plan. Polyplacophoran trochophore larvae posses several features that are distinctly different from those of their sister trochozoan taxa, including modifications of the ciliated prototrochal cells, the postrochal position of the larval eyes or ocelli, epidermal calcareous spicules, and a collection of serially reiterated epidermal shell plates. Despite these differences, chitons demonstrate a canonical pattern of equal spiral cleavage shared by other spiralian phyla that permits the identification of homologous cells across this animal clade. Cell lineage analysis using intracellular labeling on one chiton species, Chaetopleura apiculata, shows that the ocelli are generated from different lineal precursors (second-quartet micromeres: 2a, 2c) compared to those in all other spiralians studied to date (first-quartet micromeres: 1a, 1c). This situation implies that significant changes have also occurred in terms of the inductive interactions that control eye development in the spiralians. Although radical departures from the spiralian developmental program are seen in some molluscs (i.e., cephalopods), the findings presented here indicate that important changes can occur even within the highly constrained framework of the spiral cleavage program. Among spiralians, variation has been reported for the origin of the anterior, sensory, apical organ, which arises from the 1c and 1d micromeres in C. apiculata. The prototroch of C. apiculata consists of two to three irregular rows of ciliated cells but arise from 1q and 2q daughters, similar to that of Ischnochiton rissoi, as well as the gastropod, Patella vulgata. Despite certain early claims, there is no supporting evidence that any of the shell plates arise pretrochally in C. apiculata. The first seven of eight definitive shell plates that arise in the larva originate from shell secreting grooves in the postrochal region (derived from 2c, 2d, 3d). Earlier descriptions indicate that the eighth plate arises later at metamorphosis, and as this is formed posteriorly, it too forms in the postrochal region. On the other hand, epidermal spicules originate from both pretrochal and postrochal cells (1a,1d, 2a, 2c, 3c, 3d). The significance of these observations is discussed in light of various hypotheses concerning the origin of the conchiferan shell. This study reveals conservation, as well as evolutionary novelty, in the assignment of specific cell fates in the spiralians.

Frimousse – a spontaneous ascidian mutant with anterior ectodermal fate transformation

Among vertebrates, the cellular and molecular mechanisms of neural induction appear largely divergent [1], thus providing little indication of the ancestral chordate mode. Ascidians, which belong to a sub-phylum of the chordates (Urochordata) long separated from the lineage leading to the vertebrates, may provide insight to these questions [2,3]. We have investigated neural specification in ascidians using a genetic approach. Ascidians are hermaphrodites, and the ability of ascidians of the genus Ciona to self-fertilize provides a rapid means for identifying recessive zygotic mutations [4,5]. We screened Ciona intestinalis for naturally occurring mutations, which can be detected in a large percentage of the wild population One such mutant is frimousse (frm). In the ascidian larva, the sensory vesicle, the putative homolog of the vertebrate foreand midbrain [6], is the most prominent feature of the anterior CNS, and contains two pigmented sensory organs, the ocellus and the otolith, which are sensitive to light and gravity, respectively (Figure 1A). The frimousse (frm) mutant was initially identified in larvae of a self-fertilized brood due to the absence of the pigmented sensory structures in ~25% of individuals (Figure 1B). The founder was subsequently crossed to wild-type individuals, and the mutant line has been maintained for over six generations without a change in phenotype. Conservationists have good reason to ring alarm bells about the decline in numbers of many species worldwide. Most are linked to the increasing intrusion from human activity, but one decline has proved a major puzzle: all around the world there appears to be a decline in numbers of amphibians and often in habitats with minimal human influence. A new study has looked at the falling numbers of the mountain yellow-legged frog (Rana muscosa) in the Sierra Nevada in California. Reporting in the Proceedings of the National Academy of Sciences (published online) a researcher has for once got some good news. Not only does he determine one major factor in the frog population's decline, he describes how to reverse it. The key factor was thought by Vance Vredenburg, at the University of California, Berkeley, to be the widespread introduction of both the rainbow trout and brook trout into lakes in these mountains where the frogs live. His study experimentally manipulated the presence and absence of these two fish species. From 1996 to 2003, the introduced trout were removed from five lakes in a remote protected area, and 16 nearby lakes were used as controls, eight with introduced trout and eight without. To determine the vulnerable life stages, rainbow trout were placed in cages in three lakes containing amphibians. The study found that removal of introduced trout resulted in rapid recovery of frog populations and in the caging experiment, tadpoles were found to be vulnerable to trout predation. The results suggest that the widespread introduction of these fish has had a major impact on frog populations in this region of California, but that removal of them can lead to a rapid and dramatic reversal in the amphibians' fortunes. Other observations in Costa Rica, Spain and field experiments in Australia implicate introduced trout in the decline of local frog populations, but this study has shown for this frog species what has actually been happening and what can be done. Trout out

A Two-Step Mechanism for Myotome Formation in Chick

The study of the morphogenetic cell movements underlying myotome formation in the chick embryo has led to the emergence of highly controversial models. Here we report a real-time cell lineage analysis of myotome development using electroporation of a GFP reporter in newly formed chick somites. Confocal analysis of cell movements demonstrates that myotome formation involves two sequential steps. In a first phase, incremental myotome growth results from a contribution of myocytes derived solely from the medial border of the dermomyotome. In a second phase, myocytes are produced from all four borders of the dermomyotome. The relative distribution of myocytes demonstrates that the medial and the lateral borders of the somite generate exclusively epaxial and hypaxial muscles. This analysis also identified five myotomal regions, characterized by the origin of the myocytes that constitute them. Together, our results provide a comprehensive model describing the morphogenesis of the early myotome in higher vertebrates.

Somatic transgenesis using retroviral vectors in the chicken embryo.

The avian embryo is an excellent model system for experimental studies because of its accessibility and ease of microsurgical manipulations. While the complete chicken genome sequence will soon be determined, a comprehensive germ cell transmission-based genetic approach is not available for this animal model. Several techniques of somatic cell transgenesis have been developed in the past decade. Of these, the retroviral shuttle vector system provides both (1) stable integration of exogenous genes into the host cell genome, and (2) constant expression levels in a target cell population over the course of development. This review summarizes retroviral vectors available for the avian model and outlines the uses of retroviral-mediated gene transfer for cell lineage analysis as well as functional studies of genes and proteins in the chick embryo.

Nestin expression in pancreatic exocrine cell lineages

Expression of nestin has been suggested to be a characteristic of pancreatic islet stem cells. To determine whether nestin is indeed expressed in such putative cells during embryonic development, or in the adult pancreas after injury, we performed a cell lineage analysis using two independent lines of transgenic mice encoding Cre recombinase under the control of rat nestin cis-regulatory sequences, each crossed with loxP-bearing R26R mice. F1 animals produced the reporter molecule β-galactosidase only upon Cre-mediated recombination, thus solely in cells using (or having used) the transgenic nestin promoter. In early pancreatic primordia, β-galactosidase was observed in mesenchymal and epithelial cells. At later developmental stages or in adults, vast clusters of acinar cells and few ductal cells were labeled, in addition to fibroblasts and vascular cells, but no endocrine cells were tagged by β-galactosidase. This correlated with the transient expression, observed with an anti-nestin antibody, of endogenous nestin in about 5% of epithelial cells during development (whether in cord-forming arrangements or in nascent acini), and in vascular and mesenchymal structures. After partial pancreatectomy, there was a transient increase of the number of anti-nestin-labeled endothelial cells, but again, no endocrine cells bore β-galactosidase. Together, these findings show that nestin is expressed in the pancreatic exocrine cell lineage, and suggest that consistent nestin expression is not a major feature of islet endocrine progenitor cells.

Myotome meanderings. Cellular morphogenesis and the making of muscle.

The formation of muscles within the vertebrate embryo is a tightly orchestrated and complex undertaking. Beyond the initial specification of cells to become muscle are several complex cellular movements and migrations, which lead to the positioning of muscle precursors at specific locations within the embryo. The consequent differentiation, elongation and striation of these cells results in the formation of individual muscles. Investigation of the in vivo morphogenesis of individual vertebrate muscle cells has only recently begun, and is being approached through the use of sophisticated cell labelling and lineage analysis techniques. However, a consensus about the mechanisms involved has yet to be achieved. This review outlines vertebrate embryonic muscle formation in chick, fish and mice, focusing on the embryonic myotome, which generates both the axial musculature and the appendicular muscle of the fins and limbs. We highlight the points of consensus about, and the complexity of, this developmental system, and propose an evolutionary context for the basis of these understandings.

Analysis of cell lineage in two- and four-cell mouse embryos

Compared with other animals, the embryos of mammals are considered to have a highly regulative mode of development. However, recent studies have provided a strong correlation between the first cleavage plane and the future axis of the blastocyst, but it is still unclear how the early axes of the preimplantation embryo reflect the future body axes that emerge after implantation. We have carried out lineage tracing during mouse embryogenesis using the Cre-loxP system, which allowed us to analyze cell fates over a long period of development. We used a transgenic mouse strain, CAG-CAT-Z as a reporter line. The descendants of the manipulated blastomere heritably express beta-galactosidase. We examined the distribution of descendants of a single blastomere in the 8.5-day embryo after labeling at the two-cell and four-cell stages. The derivatives of one blastomere in the two-cell embryo randomly mix with cells originating from the second blastomere in all cell layers examined. Thus we find cells from different blastomeres intermingled and localized randomly along the body axis. The results of labeling experiments performed in the four-cell stage embryo fall into three categories. In the first, the labeled cells were intermingled with non-labeled cells in a manner similar to that seen after labeling at the two-cell stage. In the second, labeled cells were distributed only in the extra-embryonic ectoderm layers. Finally in the third category, labeled cells were seen only in the embryo proper and the extra-embryonic mesoderm. Manipulated embryos analyzed at the blastocyst stage showed localized distribution of the descendants of a single blastomere. These results suggest that incoherent clonal growth and drastic cell mixing occurs in the early mouse embryo after the blastocyst stage. The first cell specification event, i.e., partitioning cell fate between the inner cell mass and trophectoderm, can occur between the two-cell and four-cell stage, yet the cell fate is not determined.

Essential embryonic roles of the CKI-1 cyclin-dependent kinase inhibitor in cell-cycle exit and morphogenesis in C. elegans

Following a phase of rapid proliferation, cells in developing embryos must decide when to cease division and then whether to survive and differentiate or instead undergo programmed death. In screens for genes that regulate embryonic patterning of the endoderm in Caenorhabditis elegans, we identified overlapping chromosomal deletions that define a gene required for these decisions. These deletions result in embryonic hyperplasia in multiple somatic tissues, excessive numbers of cell corpses, and profound defects in morphogenesis and differentiation. However, cell-cycle arrest of the germline is unaffected. Cell lineage analysis of these mutants revealed that cells that normally stop dividing earlier than their close relatives instead undergo an extra round of division. These deletions define a genomic region that includes cki-1 and cki-2, adjacent genes encoding members of the Cip/Kip family of cyclin-dependent kinase inhibitors. cki-1 alone can rescue the cell proliferation, programmed cell death, and differentiation and morphogenesis defects observed in these mutants. In contrast, cki-2 is not capable of significantly rescuing these phenotypes. RNA interference of cki-1 leads to embryonic lethality with phenotypes similar to, or more severe than, the deletion mutants. cki-1 and -2 gene reporters show distinct expression patterns; while both are expressed at around the time that embryonic cells exit the cell cycle, cki-2 also shows marked expression starting early in embryogenesis, when rapid cell division occurs. Our findings demonstrate that cki-1 activity plays an essential role in embryonic cell cycle arrest, differentiation and morphogenesis, and suggest that it may be required to suppress programmed cell death or engulfment of cell corpses.

Apoptosis-mediated cell death within the ovarian polar cell lineage ofDrosophila melanogaster

Polar cells have been described as pairs of specific follicular cells present at each pole of Drosophila egg chambers. They are required at different stages of oogenesis for egg chamber formation and establishment of both the anteroposterior and planar polarities of the follicular epithelium. We show that definition of polar cell pairs is a progressive process since early stage egg chambers contain a cluster of several polar cell marker-expressing cells at each pole, while as of stage 5, they contain invariantly two pairs of such cells. Using cell lineage analysis, we demonstrate that these pre-polar cell clusters have a polyclonal origin and derive specifically from the polar cell lineage, rather than from that giving rise to follicular cells. In addition, selection of two polar cells from groups of pre-polar cells occurs via an apoptosis-dependent mechanism and is required for correct patterning of the anterior follicular epithelium of vitellogenic egg chambers.

Cell lineage analysis of the amphipod crustacean Parhyale hawaiensis reveals an early restriction of cell fates.

In the amphipod crustacean, Parhyale hawaiensis, the first few embryonic cleavages are total and generate a stereotypical arrangement of cells. In particular, at the eight-cell stage there are four macromeres and four micromeres, and each of these cells is uniquely identifiable. We describe our studies of the cell fate pattern of these eight blastomeres, and find that the eight clones resulting from these cells set up distinct cell lineages that differ in terms of proliferation, migration and cell fate. Remarkably, the cell fate of each blastomere is restricted to a single germ layer. The ectoderm originates from three of the macromeres, while the remaining macromere generates the visceral mesoderm. Two of the micromeres generate the somatic mesoderm, a third micromere generates the endoderm and the fourth micromere generates the germline. These findings demonstrate for the first time a total cleavage pattern in an arthropod which results in an invariant cell fate of the blastomeres, but notably, the cell lineage pattern of Parhyale reported shows no clear resemblance to those found in spiralians, nematodes or deuterostomes. Finally, the techniques we have developed for the analysis of Parhyale development suggest that this arthropod may be particularly useful for future functional analyses of crustacean development.

Gene expression markers for Caenorhabditiselegans vulval cells

The analysis of cell fate patterning during the vulval development of Caenorhabditis elegans has relied mostly on the direct observation of cell divisions and cell movements (cell lineage analysis). However, reconstruction of the developing vulva from EM serial sections has suggested seven different cell types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), many of which cannot be distinguished based on such observations. Here we report the vulval expression of seven genes, egl-17, cdh-3, ceh-2, zmp-1, B0034.1, T04B2.6 and F47B8.6 based on gfp, cfp and yfp (green fluorescent protein and color variants) reporter fusions. Each gene expresses in a specific subset of vulval cells, and is therefore useful as a marker for vulval cell fates. Together, expressions of markers distinguish six cell types, and reveal a strict temporal control of gene expression in the developing vulva.