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1.  AceTree: a tool for visual analysis of Caenorhabditis elegans embryogenesis 
BMC Bioinformatics  2006;7:275.
Background
The invariant lineage of the nematode Caenorhabditis elegans has potential as a powerful tool for the description of mutant phenotypes and gene expression patterns. We previously described procedures for the imaging and automatic extraction of the cell lineage from C. elegans embryos. That method uses time-lapse confocal imaging of a strain expressing histone-GFP fusions and a software package, StarryNite, processes the thousands of images and produces output files that describe the location and lineage relationship of each nucleus at each time point.
Results
We have developed a companion software package, AceTree, which links the images and the annotations using tree representations of the lineage. This facilitates curation and editing of the lineage. AceTree also contains powerful visualization and interpretive tools, such as space filling models and tree-based expression patterning, that can be used to extract biological significance from the data.
Conclusion
By pairing a fast lineaging program written in C with a user interface program written in Java we have produced a powerful software suite for exploring embryonic development.
doi:10.1186/1471-2105-7-275
PMCID: PMC1501046  PMID: 16740163
2.  Using machine learning to speed up manual image annotation: application to a 3D imaging protocol for measuring single cell gene expression in the developing C. elegans embryo 
BMC Bioinformatics  2010;11:84.
Background
Image analysis is an essential component in many biological experiments that study gene expression, cell cycle progression, and protein localization. A protocol for tracking the expression of individual C. elegans genes was developed that collects image samples of a developing embryo by 3-D time lapse microscopy. In this protocol, a program called StarryNite performs the automatic recognition of fluorescently labeled cells and traces their lineage. However, due to the amount of noise present in the data and due to the challenges introduced by increasing number of cells in later stages of development, this program is not error free. In the current version, the error correction (i.e., editing) is performed manually using a graphical interface tool named AceTree, which is specifically developed for this task. For a single experiment, this manual annotation task takes several hours.
Results
In this paper, we reduce the time required to correct errors made by StarryNite. We target one of the most frequent error types (movements annotated as divisions) and train a support vector machine (SVM) classifier to decide whether a division call made by StarryNite is correct or not. We show, via cross-validation experiments on several benchmark data sets, that the SVM successfully identifies this type of error significantly. A new version of StarryNite that includes the trained SVM classifier is available at http://starrynite.sourceforge.net.
Conclusions
We demonstrate the utility of a machine learning approach to error annotation for StarryNite. In the process, we also provide some general methodologies for developing and validating a classifier with respect to a given pattern recognition task.
doi:10.1186/1471-2105-11-84
PMCID: PMC2838868  PMID: 20146825
3.  Evolutionary comparisons reveal a positional switch for spindle pole oscillations in Caenorhabditis embryos 
The Journal of Cell Biology  2013;201(5):653-662.
In one-cell stage embryos of different worm species, a conserved positional switch controls the onset of mitotic spindle oscillations, whereas the maximum amplitude of oscillations is determined by the time spent in the oscillating phase.
During the first embryonic division in Caenorhabditis elegans, the mitotic spindle is pulled toward the posterior pole of the cell and undergoes vigorous transverse oscillations. We identified variations in spindle trajectories by analyzing the outwardly similar one-cell stage embryo of its close relative Caenorhabditis briggsae. Compared with C. elegans, C. briggsae embryos exhibit an anterior shifting of nuclei in prophase and reduced anaphase spindle oscillations. By combining physical perturbations and mutant analysis in both species, we show that differences can be explained by interspecies changes in the regulation of the cortical Gα–GPR–LIN-5 complex. However, we found that in both species (1) a conserved positional switch controls the onset of spindle oscillations, (2) GPR posterior localization may set this positional switch, and (3) the maximum amplitude of spindle oscillations is determined by the time spent in the oscillating phase. By investigating microevolution of a subcellular process, we identify new mechanisms that are instrumental to decipher spindle positioning.
doi:10.1083/jcb.201210110
PMCID: PMC3664713  PMID: 23690175
4.  Grandparental stem cells in leech segmentation: differences in CDC42 expression are correlated with an alternating pattern of blast cell fates 
Developmental biology  2009;336(1):112-121.
Embryonic segmentation in clitellate annelids (oligochaetes and leeches) is a cell lineage-driven process. Embryos of these worms generate a posterior growth zone consisting of 5 bilateral pairs of identified segmentation stem cells (teloblasts), each of which produces a column of segmental founder cells (blast cells). Each blast cell generates a lineage-specific clone via a stereotyped sequence of cell divisions, which are typically unequal both in terms of the relative size of the sister cells and in the progeny to which they give rise. In two of the five teloblast lineages, including the ventralmost, primary neurogenic (N) lineage, the blast cells adopt two different fates, designated nf and ns, in exact alternation within the blast cell column; this is termed a grandparental stem cell lineage. To lay groundwork for investigating unequal divisions in the leech Helobdella, we have surveyed the H. robusta genome for genes encoding orthologs of the Rho family GTPases, including the rho, rac and cdc42 sub-families, which are known to be involved in multiple processes involving cell polarization in other systems. We find that, in contrast to most other known systems the Helobdella genome contains two cdc42 orthologs, one of which is expressed at higher levels in the ns blast cells than in nf blast cells. We also demonstrate that the asymmetric divisions of the primary nf and ns blast cells are regulated by the polarized distribution of the activated form of the Cdc42 protein, rather than by the overall level of expression. Our results provide the first molecular insights into the mechanisms of the grandparental stem cell lineages, a novel, yet evolutionarily ancient stem cell division pattern. Our results also provide an example in which asymmetries in the distribution of Cdc42 activity, rather than in the overall levels of Cdc42 protein, are important regulating unequal divisions in animal cells.
doi:10.1016/j.ydbio.2009.09.006
PMCID: PMC2783548  PMID: 19747476
5.  A quantitative model of normal C. elegans embryogenesis and its disruption after stress 
Developmental biology  2012;374(1):12-23.
The invariant lineage of Caenorhabditis elegans has powerful potential for quantifying developmental variability in normal and stressed embryos. Previous studies of division timing by automated lineage tracing suggested that variability in cell cycle timing is low in younger embryos, but manual lineage tracing of specific lineages suggested that variability may increase for later divisions. We developed improved automated lineage tracing methods that allow routine lineage tracing through the last round of embryonic cell divisions and we applied these methods to trace the lineage of 18 wild-type embryos. Cell cycle lengths, division axes and cell positions are remarkably consistent among these embryos at all stages, with only slight increases in variability later in development. The resulting quantitative 4-dimensional model of embryogenesis provides a powerful reference dataset to identify defects in mutants or in embryos that have experienced environmental perturbations. We also traced the lineages of embryos imaged at higher temperatures to quantify the decay in developmental robustness under temperature stress. Developmental variability increases modestly at 25°C compared with 22°C and dramatically at 26°C, and we identify homeotic transformations in a subset of embryos grown at 26°C. The deep lineage tracing methods provide a powerful tool for analysis of normal development, gene expression and mutants and we provide a graphical user interface to allow other researchers to explore the average behavior of arbitrary cells in a reference embryo.
doi:10.1016/j.ydbio.2012.11.034
PMCID: PMC3548946  PMID: 23220655
robustness; microscopy; cell lineage; image analysis; C. elegans; stress
6.  Control of Cell Cycle Timing during C. elegans Embryogenesis 
Developmental biology  2008;318(1):65-72.
As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first eight out of ten rounds of embryonic cell division in C. elegans, we identified synchronous and invariantly ordered divisions that are tightly associated with fate differentiation. Our results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. We then set out to identify the pace-setting mechanisms in different fates. Our results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P3 (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. Our results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution.
doi:10.1016/j.ydbio.2008.02.054
PMCID: PMC2442716  PMID: 18430415
statistics; single cell; fate differentiation; cdc25; Skp1-related
7.  Nematode Postembryonic Cell Lineages 
Journal of Nematology  1982;14(2):240-248.
The complete postembryonic ceil lineages of the free-living nentatodes Caenorhabditis elegans and Panagrellus redivivus are known. Postembryonic cell divisions lead to substantial increases in the number of cells and, in most cases, in the number of types of cells in the neuronal, muscular, hypodermal, and digestive systems. The patterns of postembyronic cell divisions are essentially invariant and generate a fixed number of progeny cells of strictly specified fates. Cell fates depend upon both lineage history and cell-cell interactions: lineage limits the developmental potential of each cell and, for certain cells, cell-cell interactions specify which of a small number of alternative potential fates is acquired. Relatively simple differences in cell lineage account for some of the striking differences in gross morphology both between sexes and between species. Genetic studies indicate that these cell lineage differences reflect one or a few relatively simple mutational events. Interspecific differences in cell lineage are likely to be good indicators of evolutionary distance and may be helpful in defining taxonomic relationships. Both the techniques utilized in, and the information acquired from, studies of cell lineages in C. elegans and P. redivivus may prove useful to other hematologists.
PMCID: PMC2618157  PMID: 19295705
Caenorhabditis elegans; Panagrellus redivivus; anatomy; development; taxonomy; evolution
8.  Neural Stem Cells in Drosophila: Molecular Genetic Mechanisms Underlying Normal Neural Proliferation and Abnormal Brain Tumor Formation 
Stem Cells International  2012;2012:486169.
Neural stem cells in Drosophila are currently one of the best model systems for understanding stem cell biology during normal development and during abnormal development of stem cell-derived brain tumors. In Drosophila brain development, the proliferative activity of neural stem cells called neuroblasts gives rise to both the optic lobe and the central brain ganglia, and asymmetric cell divisions are key features of this proliferation. The molecular mechanisms that underlie the asymmetric cell divisions by which these neuroblasts self-renew and generate lineages of differentiating progeny have been studied extensively and involve two major protein complexes, the apical complex which maintains polarity and controls spindle orientation and the basal complex which is comprised of cell fate determinants and their adaptors that are segregated into the differentiating daughter cells during mitosis. Recent molecular genetic work has established Drosophila neuroblasts as a model for neural stem cell-derived tumors in which perturbation of key molecular mechanisms that control neuroblast proliferation and the asymmetric segregation of cell fate determinants lead to brain tumor formation. Identification of novel candidate genes that control neuroblast self-renewal and differentiation as well as functional analysis of these genes in normal and tumorigenic conditions in a tissue-specific manner is now possible through genome-wide transgenic RNAi screens. These cellular and molecular findings in Drosophila are likely to provide valuable genetic links for analyzing mammalian neural stem cells and tumor biology.
doi:10.1155/2012/486169
PMCID: PMC3377361  PMID: 22737173
9.  Lineage programming: navigating through transient regulatory states via binary decisions 
Summary
Lineage based mechanisms are widely used to generate cell type diversity in both vertebrates and invertebrates. For the past few decades, the nematode C. elegans has served as a primary model system to study this process due to its fixed and well characterized cell lineage. Recent studies conducted at the level of single cells and individual cis-regulatory elements suggest a general model by which cellular diversity is generated in this organism. During its developmental history a cell passes through multiple transient regulatory states characterized by the expression of specific sets of transcription factors. The transition from one state to another is driven by a general binary decision mechanism acting at each successive division in a reiterative manner and ending up with the activation of the terminal differentiation program upon terminal division. A similar cell fate specification system seems to play a role in generating cellular diversity in the nervous system of more complex organisms such as Drosophila and vertebrates.
doi:10.1016/j.gde.2010.04.010
PMCID: PMC2944227  PMID: 20537527
transcription factors; cell fate; lineage; asymmetric division; Wnt; C. elegans
10.  Automated analysis of embryonic gene expression with cellular resolution in C. elegans 
Nature methods  2008;5(8):703-709.
We describe a system that permits the automated analysis of reporter gene expression in Caenorhabditis elegans with cellular resolution continuously during embryogenesis and demonstrate its utility by defining the expression patterns of reporters for several embryonically expressed transcription factors. The invariant cell lineage permits the automated alignment of multiple expression profiles, allowing the direct comparison of the expression of different genes' reporters. We have also used the system to monitor perturbations to normal development involving changes both in cell division timing and in cell fate. Systematic application could reveal the gene activity of each cell throughout development.
doi:10.1038/nmeth.1228
PMCID: PMC2553703  PMID: 18587405
11.  Use of transgenic mice to map cis-acting elements in the intestinal fatty acid binding protein gene (Fabpi) that control its cell lineage- specific and regional patterns of expression along the duodenal-colonic and crypt-villus axes of the gut epithelium 
The Journal of Cell Biology  1992;119(1):27-44.
The mouse intestinal epithelium is able to establish and maintain complex lineage-specific, spatial, and temporal patterns of gene expression despite its rapid and continuous renewal. A multipotent stem cell located near the base of each intestinal crypt gives rise to progeny which undergo amplification and allocation to either enterocytic, Paneth cell, goblet cell, or enteroendocrine cell lineages. Differentiation of these four lineages occurs during their geographically ordered migration along the crypt-villus axis. Gut stem cells appear to have a "positional address" which is manifested by differences in the differentiation programs of their lineal descendants along the duodenal-colonic (cephalocaudal) axis. We have used the intestinal fatty acid binding protein gene (Fabpi) as a model to identify cis-acting elements which regulate cell- and region-specific patterns of gene expression in the gut. Nucleotides -1178 to +28 of rat Fabpi direct a pattern of expression of a reporter (human growth hormone [hGH]) which mimics that of mouse Fabpi (a) steady-state levels of hGH mRNA are highest in the distal jejunum of adult transgenic mice and fall progressively toward both the duodenum and the mid-colon; and (b) hGH is confined to the enterocytic lineage and first appears as postmitotic, differentiating cells exit the crypt and migrate to the base of small intestinal villi or their colonic homologs, the surface epithelial cuffs. Nucleotides -103 to +28, which are highly conserved in rat, mouse and human Fabpi, are able to correctly initiate transgene expression in late fetal life, restrict hGH to the enterocytic lineage, and establish an appropriate cephalocaudal gradient of reporter expression. This cephalocaudal gradient is also influenced by cis- acting elements located between nucleotides -1178 and -278, and -277 and -185 that enhance and suppress (respectively) expression in the ileum and colon and by element(s) located upstream of nucleotide -277 that are needed to sustain high levels of hGH production after weaning. Nucleotides -277 to -185 contain part of a domain conserved between the three orthologous Fabpi genes (nucleotides -240 to -159), a 24-bp element (nucleotides -212 to -188) that binds nuclear factors present in colonic but not small intestinal epithelial cells, and a portion of a CCAAT/enhancer binding protein footprint (C/EBP alpha, nucleotides - 188 to -167). Removal of nucleotides -277 to -185 (yielding I-FABP-184 to +28/hGH+3) results in inappropriate expression of hGH in proliferating and nonproliferating epithelial cells located in the mid and upper portions of duodenal, jejunal, ileal, and colonic crypts without affecting the "shape" of the cephalocaudal gradient of transgene expression.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC2289640  PMID: 1527171
12.  Caenorhabditis elegans EVL-14/PDS-5 and SCC-3 Are Essential for Sister Chromatid Cohesion in Meiosis and Mitosis 
Molecular and Cellular Biology  2003;23(21):7698-7707.
Sister chromatid cohesion is fundamental for the faithful transmission of chromosomes during both meiosis and mitosis. Proteins involved in this process are highly conserved from yeasts to humans. In screenings for sterile animals with abnormal vulval morphology, mutations in the Caenorhabditis elegans evl-14 and scc-3 genes were isolated. Defects in cell divisions were observed in germ line as well as in vulval and somatic gonad lineages. Through positional cloning of these genes, we have shown that EVL-14 and SCC-3 are likely the only C. elegans homologs of the yeast sister chromatid cohesion proteins Pds5 and Scc3, respectively. Both evl-14 and scc-3 mutants displayed defects in the meiotic germ line. In evl-14 mutants, synaptonemal complexes (SCs) were detectable but more than the usual six DAPI (4′,6′-diamidino-2-phenylindole)-positive structures were seen at diakinesis, suggesting that EVL-14/PDS-5 is important for the maintenance of sister chromatid cohesion in late prophase. In scc-3 mutant animals, normal SCs were not visible and ∼24 DAPI-positive structures were seen at diakinesis, indicating that SCC-3 is necessary for sister chromatid cohesion. Immunostaining revealed that localization of REC-8, a homolog of the yeast meiotic cohesin subunit Rec8, to the chromosomes depends on the presence of SCC-3 but not that of EVL-14/PDS-5. scc-3 RNA interference (RNAi)-treated embryos were 100% lethal and displayed defects in cell divisions. evl-14 RNAi caused a range of phenotypes. These results indicate that EVL-14/PDS-5 and SCC-3 have functions in both mitosis and meiosis.
doi:10.1128/MCB.23.21.7698-7707.2003
PMCID: PMC207601  PMID: 14560015
13.  Formation of the embryonic-abembryonic axis of the mouse blastocyst: relationships between orientation of early cleavage divisions and pattern of symmetric/asymmetric divisions 
Development (Cambridge, England)  2008;135(5):953-962.
Setting aside pluripotent cells that give rise to the future body is a central cell fate decision in mammalian development. It requires some blastomeres divide asymmetrically to direct cells to the inside of the embryo. Despite its importance, it is unknown whether the decision to divide symmetrically versus asymmetrically shows any spatial or temporal pattern; whether it is lineage-dependent or occurs at random; or whether it influences the orientation of the embryonic-abembryonic axis. To address these questions, we developed time-lapse microscopy to enable a complete 3D analysis of the origins, fates and divisions of all cells from the 2- to 32-cell blastocyst stage. This showed how in the majority of embryos individual blastomeres give rise to distinct blastocyst regions. Tracking the division orientation of all cells revealed a spatial and temporal relationship between symmetric and asymmetric divisions and how this contributes to the generation of inside and outside cells and so embryo patterning. We found that the blastocyst cavity, defining the abembryonic pole, forms where symmetric divisions predominate. Tracking cell ancestry indicated that the pattern of symmetric/asymmetric divisions of a blastomere can be influenced by its origin in relation to the animal-vegetal axis of the zygote. Thus, it appears that the orientation of the embryonic-abembryonic axis is anticipated by earlier cell division patterns. Together our results suggest that two steps influence allocation of cells to the blastocyst. The first step involving orientation of 2- to 4-cell divisions along the animal-vegetal axis can affect the second step, the establishment of inside and outside cell populations by asymmetric 8-32-cell divisions.
doi:10.1242/dev.014316
PMCID: PMC2655627  PMID: 18234722
14.  Plectus - a stepping stone in embryonic cell lineage evolution of nematodes 
EvoDevo  2012;3:13.
Background
Recent studies have challenged the widespread view that the pattern of embryogenesis found in Caenorhabditis elegans (clade 9) is characteristic of nematodes in general. To understand this still largely unexplored landscape of developmental events, we set out to examine more distantly related nematodes in detail for temporospatial differences in pattern formation and cell specification. Members of the genus Plectus (clade 6) seem to be suitable candidates to show variety, with certain idiosyncratic features during early development and the convenient availability of cultivatable species.
Methods
The study was conducted using 4-D lineage analysis, 3-D modeling of developing embryos and laser-induced ablation of individual blastomeres.
Results
Detailed cell lineage studies of several Plectus species reveal that pattern formation and cell fate assignment differ markedly from C. elegans. Descendants of the first somatic founder cell S1 (AB) - but not the progeny of other founder cells - demonstrate extremely variable spatial arrangements illustrating that here distinct early cell-cell interactions between invariant partners, as found in C. elegans, cannot take place. Different from C. elegans, in Plectus alternative positional variations among early S1 blastomeres resulting in a ‘situs inversus’ pattern, nevertheless give rise to adults with normal left-right asymmetries. In addition, laser ablations of early blastomeres uncover inductions between variable cell partners.
Conclusions
Our results suggest that embryonic cell specification in Plectus is not correlated with cell lineage but with position. With this peculiarity, Plectus appears to occupy an intermediate position between basal nematodes displaying a variable early development and the C. elegans-like invariant pattern. We suggest that indeterminate pattern formation associated with late, position-dependent fate assignment represents a plesiomorphic character among nematodes predominant in certain basal clades but lost in derived clades. Thus, the behavior of S1 cells in Plectus can be considered an evolutionary relict in a transition phase between two different developmental strategies.
doi:10.1186/2041-9139-3-13
PMCID: PMC3464786  PMID: 22748136
Nematode; embryogenesis; cell lineage; cell specification; evolution; developmental system drift; Plectus; C. elegans
15.  Polarized Myosin Produces Unequal-Size Daughters During Asymmetric Cell Division 
Science (New York, N.Y.)  2010;330(6004):677-680.
Asymmetric positioning of the mitotic spindle before cytokinesis can produce different-sized daughter cells that have distinct fates. Here, we found an asymmetric division in the Caenorhabditis elegans Q neuroblast lineage that began with a centered spindle but generated different-sized daughters, the smaller (anterior) of which underwent apoptosis. During this division, more myosin II accumulated anteriorly, suggesting that asymmetric contractile forces might produce different-sized daughters. Indeed, partial inactivation of anterior myosin by chromophore-assisted laser inactivation created a more symmetric division and allowed the survival and differentiation of the anterior daughter. Thus, the balance of myosin activity on the two sides of a dividing cell can govern the size and fate of the daughters.
doi:10.1126/science.1196112
PMCID: PMC3032534  PMID: 20929735
16.  In Vitro Differentiation of Embryonic and Adult Stem Cells into Hepatocytes: State of the Art 
Stem Cells (Dayton, Ohio)  2009;27(3):577-605.
Stem cells are a unique source of self-renewing cells within the human body. Before the end of the last millennium, adult stem cells, in contrast to their embryonic counterparts, were considered to be lineage-restricted cells or incapable of crossing lineage boundaries. However, the unique breakthrough of muscle and liver regeneration by adult bone marrow stem cells at the end of the 1990s ended this long-standing paradigm. Since then, the number of articles reporting the existence of multipotent stem cells in skin, neuronal tissue, adipose tissue, and bone marrow has escalated, giving rise, both in vivo and in vitro, to cell types other than their tissue of origin. The phenomenon of fate reprogrammation and phenotypic diversification remains, though, an enigmatic and rare process. Understanding how to control both proliferation and differentiation of stem cells and their progeny is a challenge in many fields, going from preclinical drug discovery and development to clinical therapy. In this review, we focus on current strategies to differentiate embryonic, mesenchymal(-like), and liver stem/progenitor cells into hepatocytes in vitro. Special attention is paid to intracellular and extracellular signaling, genetic modification, and cell-cell and cell-matrix interactions. In addition, some recommendations are proposed to standardize, optimize, and enrich the in vitro production of hepatocyte-like cells out of stem/progenitor cells.
doi:10.1634/stemcells.2008-0963
PMCID: PMC2729674  PMID: 19056906
Adult stem cells; Embryonic stem cells; Hepatocytes; In vitro protocols; Differentiation
17.  Cortical dynein is critical for proper spindle positioning in human cells 
The Journal of Cell Biology  2012;199(1):97-110.
Dynein is anchored at the plasma membrane by a ternary complex comprising NuMA–LGN–Gα and thus ensures correct spindle positioning
Correct spindle positioning is fundamental for proper cell division during development and in stem cell lineages. Dynein and an evolutionarily conserved ternary complex (nuclear mitotic apparatus protein [NuMA]–LGN–Gα in human cells and LIN-5–GPR-1/2–Gα in Caenorhabditis elegans) are required for correct spindle positioning, but their relationship remains incompletely understood. By analyzing fixed specimens and conducting live-imaging experiments, we uncovered that appropriate levels of ternary complex components are critical for dynein-dependent spindle positioning in HeLa cells and C. elegans embryos. Moreover, using mutant versions of Gα in both systems, we established that dynein acts at the membrane to direct spindle positioning. Importantly, we identified a region within NuMA that mediates association with dynein. By using this region to target dynein to the plasma membrane, we demonstrated that the mere presence of dynein at that location is sufficient to direct spindle positioning in HeLa cells. Overall, we propose a model in which the ternary complex serves to anchor dynein at the plasma membrane to ensure correct spindle positioning.
doi:10.1083/jcb.201203166
PMCID: PMC3461507  PMID: 23027904
18.  The Wnt/β-catenin asymmetry pathway patterns the atonal ortholog lin-32 to diversify cell fate in a C. elegans sensory lineage 
Each sensory ray of the C. elegans male tail comprises three distinct neuroglial cell types. These three cells descend from a single progenitor, the ray precursor cell, through several rounds of asymmetric division called the ray sublineage. Ray development requires the conserved atonal-family bHLH gene lin-32, which specifies the ray neuroblast and promotes the differentiation of its progeny. However, the mechanisms that allocate specific cell fates among these progeny is unknown. Here we show that the distribution of LIN-32 during the ray sublineage is markedly asymmetric, localizing to anterior daughter cells in two successive cell divisions. The anterior-posterior patterning of LIN-32 expression and of differentiated ray neuroglial fates is brought about by the Wnt/β-catenin asymmetry pathway, including the Wnt ligand LIN-44, its receptor LIN-17, and downstream components LIT-1 (NLK), SYS-1 (β-catenin) and POP-1 (TCF). LIN-32 asymmetry itself has an important role in patterning ray cell fates, as the failure to silence lin-32 expression in posterior cells disrupts development of this branch of the ray sublineage. Together, our results illustrate a mechanism whereby the regulated function of a proneural-class gene in a single neural lineage can both specify a neural precursor and actively pattern the fates of its progeny. Moreover, they reveal a central role for the Wnt/β-catenin asymmetry pathway in patterning neural and glial fates in a simple sensory lineage.
doi:10.1523/JNEUROSCI.6504-10.2011
PMCID: PMC3183998  PMID: 21917811
19.  Coordination of Cell Proliferation and Cell Fate Determination by CES-1 Snail 
PLoS Genetics  2013;9(10):e1003884.
The coordination of cell proliferation and cell fate determination is critical during development but the mechanisms through which this is accomplished are unclear. We present evidence that the Snail-related transcription factor CES-1 of Caenorhabditis elegans coordinates these processes in a specific cell lineage. CES-1 can cause loss of cell polarity in the NSM neuroblast. By repressing the transcription of the BH3-only gene egl-1, CES-1 can also suppress apoptosis in the daughters of the NSM neuroblasts. We now demonstrate that CES-1 also affects cell cycle progression in this lineage. Specifically, we found that CES-1 can repress the transcription of the cdc-25.2 gene, which encodes a Cdc25-like phosphatase, thereby enhancing the block in NSM neuroblast division caused by the partial loss of cya-1, which encodes Cyclin A. Our results indicate that CDC-25.2 and CYA-1 control specific cell divisions and that the over-expression of the ces-1 gene leads to incorrect regulation of this functional ‘module’. Finally, we provide evidence that dnj-11 MIDA1 not only regulate CES-1 activity in the context of cell polarity and apoptosis but also in the context of cell cycle progression. In mammals, the over-expression of Snail-related genes has been implicated in tumorigenesis. Our findings support the notion that the oncogenic potential of Snail-related transcription factors lies in their capability to, simultaneously, affect cell cycle progression, cell polarity and apoptosis and, hence, the coordination of cell proliferation and cell fate determination.
Author Summary
Animal development is a complex process and requires the coordination in space and time of various processes. These processes include the controlled production of cells, also referred to as ‘cell proliferation’, and the adoption by cells of specific fates, also referred to as ‘cell fate determination’. The observation that uncontrolled cell proliferation and cell fate determination contribute to conditions such as cancer, demonstrates that a precise coordination of these processes is not only important for development but for the prevention of disease throughout life. Snail-related transcription factors have previously been shown to be involved in the regulation of cell proliferation and cell fate determination. For example, the Caenorhabditis elegans Snail-related protein CES-1 affects cell fate determination in a specific cell lineage, the NSM (neurosecretory motorneuron) lineage. We now present evidence that CES-1 also controls cell proliferation in this lineage. Within a short period of time, CES-1 therefore coordinates cell proliferation and cell fate determination in one and the same lineage. Based on this finding, we propose that CES-1 is an important coordinator that is involved in the precise control - in space (NSM lineage) and time (<150 min) - of processes that are critical for animal development.
doi:10.1371/journal.pgen.1003884
PMCID: PMC3814331  PMID: 24204299
20.  Pristionchus pacificus, a nematode with only three juvenile stages, displays major heterochronic changes relative to Caenorhabditis elegans. 
The nematode Pristionchus pacificus (Diplogastridae) has been described as a satellite organism for a functional comparative approach to the model organism Caenorhabditis elegans because genetic, molecular, and cell-biological tools can be used in a similar way in both species. Here we show that P. pacificus has three juvenile stages, instead of the usual four found in other nematodes. Embryogenesis is lengthened and many developmental events that take place during the first juvenile stage in C. elegans occur during late embryogenesis in P. pacificus. Video imaging and transmission electron microscopy revealed no embryonic moult. The timing of later developmental events relative to the moults differs between P. pacificus and C. elegans. In addition, the post-embryonic blast-cell divisions display a specific change in timing between the two species, resulting in heterochrony between different cell lineages, such as vulval and gonadal lineages. Developmental events appear to come into register during the last larval stage. Thus, differences in developmental timing between P. pacificus and C. elegans represent a deep heterochronic change. We designate the three juvenile stages of P. pacificus as J1 to J3. Comparison with other species of the family Diplogastridae indicates that this pattern represents an apomorphic character for the monophylum Diplogastridae.
PMCID: PMC1690183  PMID: 10501036
21.  Structural Complexity of Early Embryos: A Study on the Nematode Caenorhabditis elegans 
Journal of Biological Physics  2001;27(2-3):257-283.
For analytical studies on the dynamics of gene expression, gene expressioncontrol and cellular interactions, the nematode Caenorhabditis elegans[C. elegans] is at present one of the best suited models [1–4].In this organism the genetic map and sequence is known [5], moreover theconstancy of its lineage tree allows a complete description of cellularclones giving rise to embryos. These characteristics have fostered detailedstudies on several aspects of development for this organism. Quantitativestudies of cellular movement, through time lapse cinematography of gastrulation, allows the description of cellular migrations giving rise to the final embryonic structure. In perspective, these studies coupledwith: genetic analysis, patterns of gene expression obtained throughmolecular techniques or other methods, open up the possibility of dynamicalstudies at the organismic scale. This possibility implies, first of all,a study of partitioning of space, and raise several problems in order todefine basic conceptual tools to be used in such studies. One of the mainproblems to handle in this respect is the definition of embryonic structurein a quantitative way. We will show that this aspect is a more generalcase of distance geometry approaches, as defined in protein folding studies.In this paper we discuss measures of the complexity for embryonal body plans,at the end of grastrulation. These can be applied to studies on the dynamicsof gene expression and phylogenetic studies with further experiments orsimulations.
doi:10.1023/A:1013178514397
PMCID: PMC3456589  PMID: 23345748
Body plan; Caenorhabditis elegans; complexity; development; dynamical systems; gastrulation; genetic networks; protein folding
22.  Cytoplasmic Dynein Light Intermediate Chain Is Required for Discrete Aspects of Mitosis in Caenorhabditis elegans 
Molecular Biology of the Cell  2001;12(10):2921-2933.
We describe phenotypic characterization of dli-1, the Caenorhabditis elegans homolog of cytoplasmic dynein light intermediate chain (LIC), a subunit of the cytoplasmic dynein motor complex. Animals homozygous for loss-of-function mutations in dli-1 exhibit stochastic failed divisions in late larval cell lineages, resulting in zygotic sterility. dli-1 is required for dynein function during mitosis. Depletion of the dli-1 gene product through RNA-mediated gene interference (RNAi) reveals an early embryonic requirement. One-cell dli-1(RNAi) embryos exhibit failed cell division attempts, resulting from a variety of mitotic defects. Specifically, pronuclear migration, centrosome separation, and centrosome association with the male pronuclear envelope are defective in dli-1(RNAi) embryos. Meiotic spindle formation, however, is not affected in these embryos. DLI-1, like its vertebrate homologs, contains a putative nucleotide-binding domain similar to those found in the ATP-binding cassette transporter family of ATPases as well as other nucleotide-binding and -hydrolyzing proteins. Amino acid substitutions in a conserved lysine residue, known to be required for nucleotide binding, confers complete rescue in a dli-1 mutant background, indicating this is not an essential domain for DLI-1 function.
PMCID: PMC60145  PMID: 11598181
23.  Multiplex Genetic Fate Mapping Reveals a Novel Route of Neocortical Neurogenesis, Which Is Altered in the Ts65Dn Mouse Model of Down Syndrome 
While several major classes of neocortical neural precursor cells have been identified, the lineal relationships and molecular profiles of these cells are still largely unknown. Furthermore, the individual contribution of each cell class to neocortical growth during normal development and in neurodevelopmental disorders has not been determined. Using a novel fate-mapping approach, we demonstrate that precursors in the embryonic ventricular (VZ) and subventricular zones (SVZ), which give rise to excitatory neurons, are divided into distinct subtypes based on lineage profile, morphology, and transcription factor expression in vivo. Using this technique, we show that short neural precursors are a unique class of VZ intermediate progenitors derived from radial glial cells and are distinct from the multipolar Tbr2(+) intermediate progenitors, which divide in the SVZ. To test whether these multiple groups of intermediate progenitors are redundant or whether they are necessary for proper neocortical growth, we measured precursor cell diversity in the Ts65Dn mouse model of Down syndrome (DS), which exhibits reduced neurogenesis and postnatal microcephaly. We report that SNP generation is markedly reduced in the Ts65Dn VZ during mid-neurogenesis, indicating that faulty specification of this progenitor pool is a central component of the neocortical abnormality in DS. Together, these findings demonstrate that neocortical neurons are produced via multiple indirect routes during embryonic development and that these parallel streams of neurogenesis collectively contribute to the proper growth and development of the neocortex.
doi:10.1523/JNEUROSCI.5380-12.2013
PMCID: PMC3785112  PMID: 23516277
24.  On Hemangioblasts in Chicken 
PLoS ONE  2007;2(11):e1228.
Hemangioblasts are bi-potential precursors for blood and endothelial cells (BCs and ECs). Existence of the hemangioblast in vivo by its strict definition, i.e. a clonal precursor giving rise to these two cell types after division, is still debated. Using a combination of mitotic figure analysis, cell labeling and long-term cell tracing, we show that, in chicken, cell division does not play a major role during the entire ventral mesoderm differentiation process after gastrulation. One eighth of cells do undergo at least one round of division, but mainly give rise to daughter cells contributing to the same lineage. Approximately 7% of the dividing cells that contribute to either the BC or EC lineage meet the criteria of true hemangioblasts, with one daughter cell becoming a BC and the other an EC. Our data suggest that hemangioblast-type generation of BC/EC occurs, but is not used as a major mechanism during early chicken development. It remains unclear, however, whether hemangioblast-like progenitor cells play a more prominent role in later development.
doi:10.1371/journal.pone.0001228
PMCID: PMC2080762  PMID: 18043736
25.  Regulation of the Number of Cell Division Rounds by Tissue-Specific Transcription Factors and Cdk Inhibitor during Ascidian Embryogenesis 
PLoS ONE  2014;9(3):e90188.
Mechanisms that regulate the number of cell division rounds during embryogenesis have remained largely elusive. To investigate this issue, we used the ascidian, which develops into a tadpole larva with a small number of cells. The embryonic cells divide 11.45 times on average from fertilization to hatching. The number of cell division rounds varies depending on embryonic lineages. Notochord and muscle consist of large postmitotic cells and stop dividing early in developing embryos. Here we show that conversion of mesenchyme to muscle cell fates by inhibition of inductive FGF signaling or mis-expression of a muscle-specific key transcription factor for muscle differentiation, Tbx6, changed the number of cell divisions in accordance with the altered fate. Tbx6 likely activates a putative mechanism to halt cell division at a specific stage. However, precocious expression of Tbx6 has no effect on progression of the developmental clock itself. Zygotic expression of a cyclin-dependent kinase inhibitor, CKI-b, is initiated in muscle and then in notochord precursors. CKI-b is possibly downstream of tissue-specific key transcription factors of notochord and muscle. In the two distinct muscle lineages, postmitotic muscle cells are generated after 9 and 8 rounds of cell division depending on lineage, but the final cell divisions occur at a similar developmental stage. CKI-b gene expression starts simultaneously in both muscle lineages at the 110-cell stage, suggesting that CKI-b protein accumulation halts cell division at a similar stage. The difference in the number of cell divisions would be due to the cumulative difference in cell cycle length. These results suggest that muscle cells do not count the number of cell division rounds, and that accumulation of CKI-b protein triggered by tissue-specific key transcription factors after cell fate determination might act as a kind of timer that measures elapsed time before cell division termination.
doi:10.1371/journal.pone.0090188
PMCID: PMC3946487  PMID: 24608898

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