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1.  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.
PMCID: PMC3814331  PMID: 24204299
2.  Notch and Prospero Repress Proliferation following Cyclin E Overexpression in the Drosophila Bristle Lineage 
PLoS Genetics  2009;5(8):e1000594.
Understanding the mechanisms that coordinate cell proliferation, cell cycle arrest, and cell differentiation is essential to address the problem of how “normal” versus pathological developmental processes take place. In the bristle lineage of the adult fly, we have tested the capacity of post-mitotic cells to re-enter the cell cycle in response to the overexpression of cyclin E. We show that only terminal cells in which the identity is independent of Notch pathway undergo extra divisions after CycE overexpression. Our analysis shows that the responsiveness of cells to forced proliferation depends on both Prospero, a fate determinant, and on the level of Notch pathway activity. Our results demonstrate that the terminal quiescent state and differentiation are regulated by two parallel mechanisms acting simultaneously on fate acquisition and cell cycle progression.
Author Summary
Despite substantial progress that has been made, we still know little about how single precursor cells undergo a limited number of cell divisions before arrest. Discovering the mechanisms by which terminal cells maintain cell division arrest is essential for understanding “normal” development, as well as the origin of pathological deregulations. Using the bristle cell lineage, a model system widely employed to analye cell identity acquisition, we observed that only two out of four terminal cells in this lineage are unable to re-enter the cell cycle and proliferate. Our study shows that in these cells, cell division arrest is maintained by the action of the transcription factor Prospero and the signalling pathway Notch. Since both of these factors also control cell identity in this lineage, this finding demonstrates that common elements acting simultaneously and in parallel regulate the terminal quiescent state and differentiation. This system provides a unique animal model in which to understand how the mechanisms involved in cell fate acquisition and those controlling cell division intermingle to produce cell lineages resulting in terminal cells in the right number and at the right place and time.
PMCID: PMC2715135  PMID: 19662164
3.  Systems-level quantification of division timing reveals a common genetic architecture controlling asynchrony and fate asymmetry 
Molecular Systems Biology  2015;11(6):814.
Coordination of cell division timing is crucial for proper cell fate specification and tissue growth. However, the differential regulation of cell division timing across or within cell types during metazoan development remains poorly understood. To elucidate the systems-level genetic architecture coordinating division timing, we performed a high-content screening for genes whose depletion produced a significant reduction in the asynchrony of division between sister cells (ADS) compared to that of wild-type during Caenorhabditis elegans embryogenesis. We quantified division timing using 3D time-lapse imaging followed by computer-aided lineage analysis. A total of 822 genes were selected for perturbation based on their conservation and known roles in development. Surprisingly, we find that cell fate determinants are not only essential for establishing fate asymmetry, but also are imperative for setting the ADS regardless of cellular context, indicating a common genetic architecture used by both cellular processes. The fate determinants demonstrate either coupled or separate regulation between the two processes. The temporal coordination appears to facilitate cell migration during fate specification or tissue growth. Our quantitative dataset with cellular resolution provides a resource for future analyses of the genetic control of spatial and temporal coordination during metazoan development.
PMCID: PMC4501849  PMID: 26063786
asynchrony of cell division; automated lineaging; C. elegans; cell cycle length; cell division timing
4.  E3 ubiquitin ligases promote progression of differentiation during C. elegans embryogenesis 
Developmental biology  2014;398(2):267-279.
Regulated choice between cell fate maintenance and differentiation provides decision points in development to progress toward more restricted cell fates or to maintain the current one. C. elegans embryogenesis follows an invariant cell lineage where cell fate is generally more restricted upon each cell division. EMS is a progenitor cell in the four-cell embryo that gives rise to the endomesoderm. We recently found that when ubiquitin-mediated protein degradation is compromised, the anterior daughter of EMS, namely MS, reiterates the EMS fate. This observation demonstrates an essential function of ubiquitin-mediated protein degradation in driving the progression of EMS-to-MS differentiation. Here we report a genome-wide screen of the ubiquitin pathway and extensive lineage analyses. The results suggest a broad role of E3 ligases in driving differentiation progression. First, we identified three substrate-binding proteins for two CRL (Cullin-RING ubiquitin Ligase) E3 complexes that promote the progression from the EMS fate to MS, namely LIN-23/β-TrCP and FBXB-3 for the CRL1/SCF complex and ZYG-11/ZYG-11B for the CRL2 complex. Genetic analyses suggest these E3 ligases function through a multifunctional protein OMA-1 and the endomesoderm lineage specifier SKN-1 to drive differentiation. Second, we found that depletion of components of the CRL1/SCF complex induces fate reiteration in all major founder cell lineages. These data suggest that regulated choice between self-renewal and differentiation is widespread during C. elegans embryogenesis as in organisms with regulative development, and ubiquitin-mediated protein degradation drives the choice towards differentiation. Finally, bioinformatic analysis of time series gene expression data showed that expression of E3 genes is transiently enriched during time windows of developmental stage transitions. Transcription factors show similar enrichment, but not other classes of regulatory genes. Based on these findings we propose that ubiquitin-mediated protein degradation, like many transcription factors, function broadly as regulators driving developmental progression during embryogenesis in C. elegans.
PMCID: PMC4314322  PMID: 25523393
differentiation; self-renewal; ubiquitin ligase; cell lineage; endomesoderm; C. elegans
5.  Plectus - a stepping stone in embryonic cell lineage evolution of nematodes 
EvoDevo  2012;3:13.
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.
The study was conducted using 4-D lineage analysis, 3-D modeling of developing embryos and laser-induced ablation of individual blastomeres.
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.
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.
PMCID: PMC3464786  PMID: 22748136
Nematode; embryogenesis; cell lineage; cell specification; evolution; developmental system drift; Plectus; C. elegans
6.  Dynamic interaction networks in a hierarchically organized tissue 
We have integrated gene expression profiling with database and literature mining, mechanistic modeling, and cell culture experiments to identify intercellular and intracellular networks regulating blood stem cell self-renewal.Blood stem cell fate in vitro is regulated non-autonomously by a coupled positive–negative intercellular feedback circuit, composed of megakaryocyte-derived stimulatory growth factors (VEGF, PDGF, EGF, and serotonin) versus monocyte-derived inhibitory factors (CCL3, CCL4, CXCL10, TGFB2, and TNFSF9).The antagonistic signals converge in a core intracellular network focused around PI3K, Raf, PLC, and Akt.Model simulations enable functional classification of the novel endogenous ligands and signaling molecules.
Intercellular (between cell) communication networks are required to maintain homeostasis and coordinate regenerative and developmental cues in multicellular organisms. Despite the recognized importance of intercellular networks in regulating adult stem and progenitor cell fate, the specific cell populations involved, and the underlying molecular mechanisms are largely undefined. Although a limited number of studies have applied novel bioinformatic approaches to unravel intercellular signaling in other cell systems (Frankenstein et al, 2006), a comprehensive analysis of intercellular communication in a stem cell-derived, hierarchical tissue network has yet to be reported.
As a model system to explore intercellular communication networks in a hierarchically organized tissue, we cultured human umbilical cord blood (UCB)-derived stem and progenitor cells in defined, minimal cytokine-supplemented liquid culture (Madlambayan et al, 2006). To systematically explore the molecular and cellular dynamics underlying primitive progenitor growth and differentiation, gene expression profiles of primitive (lineage negative; Lin−) and mature (lineage positive; Lin+) populations were generated during phases of stem cell expansion versus depletion. Parallel phenotypic and subproteomic experiments validated that mRNA expression correlated with complex measures of proteome activity (protein secretion and cell surface expression). Using a curated list of secreted ligand–receptor interactions and published expression profiles of purified mature blood populations, we implemented a novel algorithm to reconstruct the intercellular signaling networks established between stem cells and multi-lineage progeny in vitro. By correlating differential expression patterns with stem cell growth, we predict cell populations, pathways, and secreted ligands associated with stem cell self-renewal and differentiation (Figure 3A).
We then tested the correlative predictions in a series of cell culture experiments. UCB progenitor cell cultures were supplemented with saturating amounts of 18 putative regulatory ligands, or cocultured with purified mature blood lineages (megakaryocytes, monocytes, and erythrocytes), and analyzed for effects on total cell, progenitor, and primitive progenitor growth. At the primitive progenitor level, 3/5 novel predicted stimulatory ligands (EGF, PDGFB, and VEGF) displayed significant positive effects, 5/7 predicted inhibitory factors (CCL3, CCL4, CXCL10, TNFSF9, and TGFB2) displayed negative effects, whereas only 1/5 non-correlated ligand (CXCL7) displayed an effect. Also consistent with predictions from gene expression data, megakaryocytes and monocytes were found to stimulate and inhibit primitive progenitor growth, respectively, and these effects were attributable to differential secretome profiles of stimulatory versus inhibitory ligands.
Cellular responses to external stimuli, particularly in heterogeneous and dynamic cell populations, represent complex functions of multiple cell fate decisions acting both directly and indirectly on the target (stem cell) populations. Experimentally distinguishing the mode of action of cytokines is thus a difficult task. To address this we used our previously published interactive model of hematopoiesis (Kirouac et al, 2009) to classify experimentally identified regulatory ligands into one of four distinct functional categories based on their differential effects on cell population growth. TGFB2 was classified as a proliferation inhibitor, CCL4, CXCL10, SPARC, and TNFSF9 as self-renewal inhibitors, CCL3 a proliferation stimulator, and EGF, VEGF, and PDGFB as self-renewal stimulators.
Stem and progenitor cells exposed to combinatorial extracellular signals must propagate this information through intracellular molecular networks, and respond appropriately by modifying cell fate decisions. To explore how our experimentally identified positive and negative regulatory signals are integrated at the intracellular level, we constructed a blood stem cell self-renewal signaling network through extensive literature curation and protein–protein interaction (PPI) network mapping. We find that signal transduction pathways activated by the various stimulatory and inhibitory ligands converge on a limited set of molecular control nodes, forming a core subnetwork enriched for known regulators of self-renewal (Figure 6A). To experimentally test the intracellular signaling molecules computationally predicted as regulators of stem cell self-renewal, we obtained five small molecule antagonists against the kinases Phosphatidylinositol 3-kinase (PI3K), Raf, Akt, Phospholipase C (PLC), and MEK1. Liquid cultures were supplemented with the five molecules individually, and resultant cell population outputs compared against model simulations to deconvolute the functional effects on proliferation (and survival) versus self-renewal. This analysis classifies inhibition of PI3K and Raf activity as selectively targeting self-renewal, PLC as selectively targeting survival, and Akt as selectively targeting proliferation; MEK inhibition appears non-specific for these processes.
This represents the first systematic characterization of how cell fate decisions are regulated non-autonomously through lineage-specific interactions with differentiated progeny. The complex intercellular communication networks can be approximated as an antagonistic positive–negative feedback circuit, wherein progenitor expansion is modulated by a balance of megakaryocyte-derived stimulatory factors (EGF, PDGF, VEGF, and possibly serotonin) versus monocyte-derived inhibitory factors (CCL3, CCL4, CXCL10, TGFB2, and TNFSF9). This complex milieu of endogenous regulatory signals is integrated and processed within a core intracellular signaling network, resulting in modulation of cell-level kinetic parameters (proliferation, survival, and self-renewal). We reconstruct a stem cell associated intracellular network, and identify PI3K, Raf, Akt, and PLC as functionally distinct signal integration nodes, linking extracellular and intracellular signaling. These findings lay the groundwork for novel strategies to control blood stem cell self-renewal in vitro and in vivo.
Intercellular (between cell) communication networks maintain homeostasis and coordinate regenerative and developmental cues in multicellular organisms. Despite the importance of intercellular networks in stem cell biology, their rules, structure and molecular components are poorly understood. Herein, we describe the structure and dynamics of intercellular and intracellular networks in a stem cell derived, hierarchically organized tissue using experimental and theoretical analyses of cultured human umbilical cord blood progenitors. By integrating high-throughput molecular profiling, database and literature mining, mechanistic modeling, and cell culture experiments, we show that secreted factor-mediated intercellular communication networks regulate blood stem cell fate decisions. In particular, self-renewal is modulated by a coupled positive–negative intercellular feedback circuit composed of megakaryocyte-derived stimulatory growth factors (VEGF, PDGF, EGF, and serotonin) versus monocyte-derived inhibitory factors (CCL3, CCL4, CXCL10, TGFB2, and TNFSF9). We reconstruct a stem cell intracellular network, and identify PI3K, Raf, Akt, and PLC as functionally distinct signal integration nodes, linking extracellular, and intracellular signaling. This represents the first systematic characterization of how stem cell fate decisions are regulated non-autonomously through lineage-specific interactions with differentiated progeny.
PMCID: PMC2990637  PMID: 20924352
cellular networks; hematopoiesis; intercellular signaling; self-renewal; stem cells
7.  C. elegans Nucleostemin Is Required for Larval Growth and Germline Stem Cell Division 
PLoS Genetics  2008;4(8):e1000181.
The nucleolus has shown to be integral for many processes related to cell growth and proliferation. Stem cells in particular are likely to depend upon nucleolus-based processes to remain in a proliferative state. A highly conserved nucleolar factor named nucleostemin is proposed to be a critical link between nucleolar function and stem-cell–specific processes. Currently, it is unclear whether nucleostemin modulates proliferation by affecting ribosome biogenesis or by another nucleolus-based activity that is specific to stem cells and/or highly proliferating cells. Here, we investigate nucleostemin (nst-1) in the nematode C. elegans, which enables us to examine nst-1 function during both proliferation and differentiation in vivo. Like mammalian nucleostemin, the NST-1 protein is localized to the nucleolus and the nucleoplasm; however, its expression is found in both differentiated and proliferating cells. Global loss of C. elegans nucleostemin (nst-1) leads to a larval arrest phenotype due to a growth defect in the soma, while loss of nst-1 specifically in the germ line causes germline stem cells to undergo a cell cycle arrest. nst-1 mutants exhibit reduced levels of rRNAs, suggesting defects in ribosome biogenesis. However, NST-1 is generally not present in regions of the nucleolus where rRNA transcription and processing occurs, so this reduction is likely secondary to a different defect in ribosome biogenesis. Transgenic studies indicate that NST-1 requires its N-terminal domain for stable expression and both its G1 GTPase and intermediate domains for proper germ line function. Our data support a role for C. elegans nucleostemin in cell growth and proliferation by promoting ribosome biogenesis.
Author Summary
Stem cells are carefully poised between the alternate fates of proliferation and differentiation. The regulation of this choice is a complex one that occurs on many different levels. One major influence controlling this choice derives signals emanating from the nucleolus, which serves dual roles as the site of ribosome biogenesis and as a repository for sequestered key regulatory factors. The nucleolar GTPase nucleostemin has recently been identified as a potential link between stem cell proliferation and nucleolar function, but its exact role in the nucleolus has not been directly addressed in a metazoan. Here, we use the model organism C. elegans to investigate the function of nucleostemin in both differentiated cells and proliferating stem cells. We show that nucleostemin probably acts to regulate ribosome biogenesis, and through this process controls cell proliferation. We also suggest that, at least in C. elegans, the function of nucleostemin is not restricted to proliferating stem cells, but that it also functions in differentiated cells to control cell growth. Our study highlights the complexity of the role of the nucleolus in regulation of cell growth and division.
PMCID: PMC2515194  PMID: 18725931
8.  A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans 
PLoS Genetics  2011;7(6):e1002109.
One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo.
Author Summary
The generation of a neuron from a fertilized egg requires a multi-step cascade of molecules acting from within and outside that cell to direct it towards a neuronal fate, rather than, say, a muscle cell. These cascades are not fully understood. In this study we systematically eliminate the function of almost all genes in the C. elegans genome, one by one, to determine what it takes to build a neuron. We identified 245 genes that affect the development of a specific sensory neuron pair, e.g. the neurons were not generated or the neurons were generated but the terminal fate was not correctly specified. We characterize in more detail the transcription factor hlh-14, which we find is required to generate multiple neurons, and the COMPASS histone methyltransferase complex, which we find to have a surprisingly specific role in the specification of a molecular and functional left-right asymmetry in this sensory neuron pair. Our study represents the first genome-wide analysis of a single neuronal cell fate decision. Further characterization of the genes identified here will enhance our understanding, and thus our capacity for treatment and prevention, of human neurological disorders.
PMCID: PMC3116913  PMID: 21698137
9.  Functional Dissection of Caenorhabditis elegans CLK-2/TEL2 Cell Cycle Defects during Embryogenesis and Germline Development 
PLoS Genetics  2009;5(4):e1000451.
CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2–null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development.
Author Summary
PI3K-related protein kinases (PIKKs) ATM and ATR are essential upstream components of DNA damage signalling pathways, while TOR-1 acts as a nutrient sensor. CLK-2/TEL2 is a conserved gene initially implicated in budding yeast telomere length regulation and uncovered in the same genetic screen as the yeast TEL1 ATM like kinase. CLK-2/TEL2 was first implicated in DNA damage response signalling by C. elegans genetics, a function confirmed in yeast and human cells. In addition, CLK-2/TEL2 is essential for cellular and organismal survival from yeasts to vertebrates, but the essential phenotypes were not defined. A direct interaction between CLK-2/TEL2 and all PI3K-related protein kinases and the reduction of PIKK protein levels upon CLK-2/TEL2 depletion lead to the widely discussed notion that CLK-2/TEL2 mutants might phenocopy PIKK depletion phenotypes. We take advantage of embryonic lineage analysis and germline cytology to dissect developmental and cell cycle related functions of CLK-2. CLK-2 depletion does not phenocopy PIKK kinase depletion. We rather link CLK-2 to multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development. Furthermore, we implicate CLK-2 in a distinct cell lineage decision and show that its depletion leads to a novel germline cell cycle arrest phenotype.
PMCID: PMC2660272  PMID: 19360121
10.  Transcriptome Analysis of the Arabidopsis Megaspore Mother Cell Uncovers the Importance of RNA Helicases for Plant Germline Development 
PLoS Biology  2011;9(9):e1001155.
Germ line specification is a crucial step in the life cycle of all organisms. For sexual plant reproduction, the megaspore mother cell (MMC) is of crucial importance: it marks the first cell of the plant “germline” lineage that gets committed to undergo meiosis. One of the meiotic products, the functional megaspore, subsequently gives rise to the haploid, multicellular female gametophyte that harbours the female gametes. The MMC is formed by selection and differentiation of a single somatic, sub-epidermal cell in the ovule. The transcriptional network underlying MMC specification and differentiation is largely unknown. We provide the first transcriptome analysis of an MMC using the model plant Arabidopsis thaliana with a combination of laser-assisted microdissection and microarray hybridizations. Statistical analyses identified an over-representation of translational regulation control pathways and a significant enrichment of DEAD/DEAH-box helicases in the MMC transcriptome, paralleling important features of the animal germline. Analysis of two independent T-DNA insertion lines suggests an important role of an enriched helicase, MNEME (MEM), in MMC differentiation and the restriction of the germline fate to only one cell per ovule primordium. In heterozygous mem mutants, additional enlarged MMC-like cells, which sometimes initiate female gametophyte development, were observed at higher frequencies than in the wild type. This closely resembles the phenotype of mutants affected in the small RNA and DNA-methylation pathways important for epigenetic regulation. Importantly, the mem phenotype shows features of apospory, as female gametophytes initiate from two non-sister cells in these mutants. Moreover, in mem gametophytic nuclei, both higher order chromatin structure and the distribution of LIKE HETEROCHROMATIN PROTEIN1 were affected, indicating epigenetic perturbations. In summary, the MMC transcriptome sets the stage for future functional characterization as illustrated by the identification of MEM, a novel gene involved in the restriction of germline fate.
Author Summary
Germline specification is a key step in sexual reproduction. In plants, the reproductive lineage or “germline” doesn't arise early in development, as it does in animals; rather, the germline is specified during flower development. In the female reproductive organs of the flower, a single sporophytic cell in each ovule is selected and differentiates into a megaspore mother cell (MMC), which will undergo meiosis. Despite the importance of the specification of the MMC as the first committed cell of the germline lineage, little is known about the genetic and molecular bases of this process. We performed a cell-type-specific transcriptome analysis of Arabidopsis MMCs using laser-assisted microdissection and microarrays. Statistical data analysis comparing these results with the transcriptomes of 71 other types of cells and tissues revealed the importance of translational control pathways and RNA helicases for plant germline development, a feature reminiscent of the animal germline. We further characterized a novel MMC-enriched RNA helicase, called MNEME, and showed that it plays important roles in MMC differentiation and the restriction of the plant germline to only one cell per ovule. This example illustrates the usefulness of our transcriptome dataset for the identification of novel candidate genes involved in this crucial step of plant reproduction.
PMCID: PMC3176755  PMID: 21949639
11.  A Mutation of cdc-25.1 Causes Defects in Germ Cells But Not in Somatic Tissues in C. elegans 
Molecules and cells  2009;28(1):43-48.
By screening C. elegans mutants for severe defects in germline proliferation, we isolated a new loss-of-function allele of cdc-25.1, bn115. bn115 and another previously identified loss-of-function allele nr2036 do not exhibit noticeable cell division defects in the somatic tissues but have reduced numbers of germ cells and are sterile, indicating that cdc-25.1 functions predominantly in the germ line during postembryonic development, and that cdc-25.1 activity is probably not required in somatic lineages during larval development. We analyzed cell division of germ cells and somatic tissues in bn115 homozygotes with germline-specific anti-PGL-1 immunofluorescence and GFP transgenes that express in intestinal cells, in distal tip cells, and in gonadal sheath cells, respectively. We also analyzed the expression pattern of cdc-25.1 with conventional and quantitative RT-PCR. In the presence of three other family members of cdc-25 in C. elegans, defects are observed only in the germ line but not in the somatic tissues in cdc-25.1 single mutants, and cdc-25.1 is expressed predominantly, if not exclusively, in the germ line during postembryonic stages. Our findings indicate that the function of cdc-25.1 is unique in the germ line but likely redundant with other members in the soma.
PMCID: PMC2908335  PMID: 19533027
bn115; Caenorhabditis elegans; CDC-25.1; germline proliferation; somatic gonadal tissues
12.  The Arabidopsis Receptor Kinase ZAR1 Is Required for Zygote Asymmetric Division and Its Daughter Cell Fate 
PLoS Genetics  2016;12(3):e1005933.
Asymmetric division of zygote is critical for pattern formation during early embryogenesis in plants and animals. It requires integration of the intrinsic and extrinsic cues prior to and/or after fertilization. How these cues are translated into developmental signals is poorly understood. Here through genetic screen for mutations affecting early embryogenesis, we identified an Arabidopsis mutant, zygotic arrest 1 (zar1), in which zygote asymmetric division and the cell fate of its daughter cells were impaired. ZAR1 encodes a member of the RLK/Pelle kinase family. We demonstrated that ZAR1 physically interacts with Calmodulin and the heterotrimeric G protein Gβ, and ZAR1 kinase is activated by their binding as well. ZAR1 is specifically expressed micropylarly in the embryo sac at eight-nucleate stage and then in central cell, egg cell and synergids in the mature embryo sac. After fertilization, ZAR1 is accumulated in zygote and endosperm. The disruption of ZAR1 and AGB1 results in short basal cell and an apical cell with basal cell fate. These data suggest that ZAR1 functions as a membrane integrator for extrinsic cues, Ca2+ signal and G protein signaling to regulate the division of zygote and the cell fate of its daughter cells in Arabidopsis.
Author Summary
Flowering plants are featured as double fertilization, a process that the egg cell and the central cell of embryo sac fuse with a sperm and give rise to a diploid zygote and a triploid primary endosperm cell, respectively. The zygote develops into embryo after cell division and differentiation, and starts a new trip of next generation. Meanwhile, the primary endosperm cell proceeds nuclear division to generate a syncytium and develops into endosperm after cellularization. Embryo development initiates from asymmetric division of zygote. A small apical cell and a long basal cell are produced after the first zygotic division, which establishes the pattern of an early embryo. To unveil the molecular mechanism controlling zygote asymmetric division, we screened our Ac/Ds insertion lines for mutations controlling early embryogenesis, one of the mutations zygotic arrest 1 (zar1) was reported here. In zar1, zygote was either arrested after elongation or displayed symmetric division. The mutation also had a slight impact on the apical- and basal-cell fates manifested by the mis-expression of the cell-lineage specific markers. ZAR1 encodes a member of the RLK/Pelle kinase family, and interacts physically with Calmodulin and the heterotrimeric G protein Gβ, both in vitro and in vivo. These data suggest that ZAR1 might act as an integrator for intracellular Ca2+ and heterotrimeric G protein signaling with extracellular signals during early zygote development. Interestingly, complete loss of Gβ or ZAR1 function displayed very weak phenotype. This suggests that there might be genetic redundancy and plasticity during early embryogenesis. More studies are needed to dissect the complexity of early embryo development in plants.
PMCID: PMC4807781  PMID: 27014878
13.  Exdpf Is a Key Regulator of Exocrine Pancreas Development Controlled by Retinoic Acid and ptf1a in Zebrafish 
PLoS Biology  2008;6(11):e293.
Both endocrine and exocrine pancreatic cells arise from pancreatic-duodenal homeobox 1 (pdx1)-positive progenitors. The molecular mechanisms controlling cell fate determination and subsequent proliferation, however, are poorly understood. Unlike endocrine cells, less is known about exocrine cell specification. We report here the identification and characterization of a novel exocrine cell determinant gene, exocrine differentiation and proliferation factor (exdpf), which is highly expressed in the exocrine cell progenitors and differentiated cells of the developing pancreas in zebrafish. Knockdown of exdpf by antisense morpholino caused loss or significant reduction of exocrine cells due to lineage-specific cell cycle arrest but not apoptosis, whereas the endocrine cell mass appeared normal. Real-time PCR results demonstrated that the cell cycle arrest is mediated by up-regulation of cell cycle inhibitor genes p21Cip, p27Kip, and cyclin G1 in the exdpf morphants. Conversely, overexpression of exdpf resulted in an overgrowth of the exocrine pancreas and a severe reduction of the endocrine cell mass, suggesting an inhibitory role for exdpf in endocrine cell progenitors. We show that exdpf is a direct target gene of pancreas-specific transcription factor 1a (Ptf1a), a transcription factor critical for exocrine formation. Three consensus Ptf1a binding sites have been identified in the exdpf promoter region. Luciferase assay demonstrated that Ptf1a promotes transcription of the exdpf promoter. Furthermore, exdpf expression in the exocrine pancreas was lost in ptf1a morphants, and overexpression of exdpf successfully rescued exocrine formation in ptf1a-deficient embryos. Genetic evidence places expdf downstream of retinoic acid (RA), an instructive signal for pancreas development. Knocking down exdpf by morpholino abolished ectopic carboxypeptidase A (cpa) expression induced by RA. On the other hand, exdpf mRNA injection rescued endogenous cpa expression in embryos treated with diethylaminobenzaldehyde, an inhibitor of RA signaling. Moreover, exogenous RA treatment induced anterior ectopic expression of exdpf and trypsin in a similar pattern. Our study provides a new understanding of the molecular mechanisms controlling exocrine cell specification and proliferation by a novel gene, exdpf. Highly conserved in mammals, the expression level of exdpf appears elevated in several human tumors, suggesting a possible role in tumor pathogenesis.
Author Summary
The pancreas is a vital organ comprising endocrine and exocrine components. Both endocrine and exocrine cells derive from a common pool of progenitors present in the gut endoderm during embryogenesis. The molecular mechanisms regulating cell fate decisions and lineage-specific proliferation are not fully understood. In this work, we report the characterization of a novel gene, exocrine differentiation and proliferation factor (exdpf), as a regulator for exocrine cell fate and differentiation/proliferation. We show that it is a direct target of the transcription factor pancreas-specific transcription factor 1a (Ptf1a), which is expressed in progenitors that give rise to all pancreatic cell types. We find that a deficiency of exdpf results in a severe reduction of exocrine size due to defects in cell proliferation. Consistent with this finding, overexpression of exdpf leads to an increase of exocrine size and a decrease of endocrine size, suggesting a possible change in fate of the endocrine progenitors. The human ortholog of exdpf is highly conserved and its expression level appears elevated in several cancers, including hepatic and pancreatic cancers, implying a possible role in pathogenesis of these malignancies.
The zebrafishexdpf, a novel regulator of pancreatic exocrine cell fate, is essential for exocrine cell differentiation and proliferation.
PMCID: PMC2586380  PMID: 19067490
14.  Evolution of embryonic development in nematodes 
EvoDevo  2011;2:18.
Nematodes can be subdivided into basal Enoplea (clades 1 and 2) and more derived Chromadorea (clades 3 to 12). Embryogenesis of Caenorhabditis elegans (clade 9) has been analyzed in most detail. Their establishment of polarity and asymmetric cleavage requires the differential localization of PAR proteins. Earlier studies on selected other nematodes revealed that embryonic development of nematodes is more diverse than the essentially invariant development of C. elegans and the classic study object Ascaris had suggested. To obtain a more detailed picture of variations and evolutionary trends we compared embryonic cell lineages and pattern formation in embryos of all 12 nematode clades.
The study was conducted using 4-D microscopy and 3-D modeling of developing embryos.
We found dramatic differences compared to C. elegans in Enoplea but also considerable variations among Chromadorea. We discovered 'Polarity Organizing Centers' (POCs) that orient cleavage spindles along the anterior-posterior axis in distinct cells over consecutive cell generations. The resulting lineally arranged blastomeres represent a starting point for the establishment of bilateral symmetry within individual lineages. We can discern six different early cleavage types and suggest that these variations are due to modifications in the activity of the POCs in conjunction with changes in the distribution of PAR proteins. In addition, our studies indicate that lineage complexity advanced considerably during evolution, that is we observe trends towards an increase of somatic founder cells, from monoclonal to polyclonal lineages and from a variable (position-dependent) to an invariable (lineage-dependent) way of cell fate specification. In contrast to the early phase of embryogenesis, the second half ('morphogenesis') appears similar in all studied nematodes. Comparison of early cleavage between the basal nematode Tobrilus stefanskii and the tardigrade Hypsibius dujardini revealed surprising similarities indicating that the presence of POCs is not restricted to nematode embryos.
The pattern of cleavage, spatial arrangement and differentiation of cells diverged dramatically during the history of the phylum Nematoda without corresponding changes in the phenotype. While in all studied representatives the same distinctive developmental steps need to be taken, cell behavior leading to these is not conserved.
PMCID: PMC3195109  PMID: 21929824
nematode; embryogenesis; cell lineage; polarity; symmetry formation; cell specification; evolution; Tobrilus; Prionchulus; C. elegans
15.  A Complete Developmental Sequence of a Drosophila Neuronal Lineage as Revealed by Twin-Spot MARCM 
PLoS Biology  2010;8(8):e1000461.
Labeling every neuron in a lineage in the fruit fly olfactory system reveals that every cell is born with a pre-determined cell fate that is invariant and dependent upon neuron birth order
Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain.
Author Summary
A brain consists of numerous, potentially individually unique neurons that derive from a limited number of progenitors. It has been shown in various model organisms that specific neurons arise in a lineage made by a repeatedly renewing progenitor at specific times of development. However, except in the worm C. elegans, the stereotype of neural development has never been examined in sufficient detail to account for every single neuron derived from a common progenitor. Here we applied a sophisticated genetic mosaic system to mark single neurons in the adult Drosophila brain and simultaneously reveal in which lineage a targeted neuron had arisen and when along the lineage it was made. We have identified each neuron in a lineage of olfactory projection neurons. There are a remarkable 40 types of neurons within this lineage born over two epochs. Strikingly, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death, such that every neuron type has a unique and invariant cell count. Sequencing an entire neuronal lineage provides definitive evidence for origin-dependent neuron type specification. It further permits a systematic characterization of neuron types for comprehensive circuitry mapping.
PMCID: PMC2927434  PMID: 20808769
16.  Fate Specification and Tissue-specific Cell Cycle Control of the Caenorhabditis elegans Intestine 
Molecular Biology of the Cell  2010;21(5):725-738.
The Caenorhabditis elegans β-TrCP orthologue LIN-23 of maternal origin regulates a progressive decline of CDC-25.1 abundance over several embryonic cell-cycles and specifies cell number of one tissue, the embryonic intestine.
Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein β-transducin repeat-containing protein (β-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans β-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant.
PMCID: PMC2828960  PMID: 20053685
17.  The lin-35/Rb and RNAi pathways cooperate to regulate a key cell cycle transition in C. elegans 
The Retinoblastoma gene product (Rb) has been shown to regulate the transcription of key genes involved in cell growth and proliferation. Consistent with this, mutations in Rb are associated with numerous types of cancer making it a critical tumour suppressor gene. Its function is conferred through a large multiprotein complex that exhibits a dual function in both activation and repression of gene targets. In C. elegans, the Rb orthologue lin-35 functions redundantly with other transcriptional regulators to appropriately specify both vulval and pharyngeal cell fates.
In C. elegans the intestinal cells must alter their cell cycle from the mitotic cell divisions typical of embryogenesis to karyokinesis and then endoreplication, which facilitates growth during larval development. While screening for genes that affect the ability of the intestinal cells to appropriately make this cell cycle transition during post-embryonic development, we isolated mutants that either compromise this switch and remain mononucleate, or cause these cells to undergo multiple rounds of nuclear division. Among these mutants we identified a novel allele of lin-35/Rb, while we also found that the components of the synMuv B complex, which are involved in vulval specification, are also required to properly regulate the developmentally-controlled cell cycle transition typical of these intestinal cells during larval development. More importantly, our work uncovered a role for certain members of the pathways involved in RNAi in mediating the efficient transition between these cell cycle programs, suggesting that lin-35/Rb cooperates with these RNAi components. Furthermore, our findings suggest that met-2, a methyltransferase as well as hpl-1 and hpl-2, two C. elegans homologues of the heterochromatin protein HP1 are also required for this transition.
Our findings are consistent with lin-35/Rb, synMuv and RNAi components cooperating, probably through their additive effects on chromatin modification, to appropriately modulate the expression of genes that are required to switch from the karyokinesis cell cycle to endoreplication; a highly specified growth pathway in the intestinal epithelium. The lin-35/Rb repressor complex may be required to initiate this process, while components of the RNAi machinery positively reinforce this repression.
PMCID: PMC1877806  PMID: 17466069
18.  The Function of a Spindle Checkpoint Gene bub-1 in C. elegans Development 
PLoS ONE  2009;4(6):e5912.
The serine/threonine kinase BUB1 (Budding Uninhibited by Benzimidazole 1) was originally identified in yeast as a checkpoint protein, based on its mutant's incapacity of delaying the cell cycle in response to loss of microtubules. Our understanding of its function is primarily from studies carried out in yeast S. cerevisiae. It has been shown that it is a component of the mitotic spindle checkpoint and regulates the separation of sister chromatids through its downstream molecules. However, its roles in multi-cellular organisms remain unclear.
Methods and Findings
In nematode C. elegans, rapid cell divisions primarily occur in embryos and in germline of postembryonic larvae and adults. In addition, a select set of cells undergo a few rounds of cell division postembryonically. One common phenotype associated with impaired cell division is described as Stu (Sterile and Uncoordinated) [1], [2]. We conducted a genetic screen for zygotic mutants that displayed Stu phenotype in C. elegans. We isolated seven Stu mutants that fell into five complementation groups. We report here that two mutations, FanWang5 (fw5) and FanWang8 (fw8) affect the bub-1 gene, a homolog of yeast BUB1. Both mutant alleles of fw5 and fw8 exhibited variable behavioral defects, including developmental arrest, uncoordination and sterility. The number of postembryonically born neurons in the ventral cord decreased and their axon morphology was abnormal. Also, the decrease of neurons in the ventral cord phenotype could not be suppressed by a caspase-3 loss-of-function mutant. In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages. We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans. Studies in yeast have shown that BUB1 functions as a spindle checkpoint protein by regulating the anaphase promoting complex/cyclosome (APC/C). We performed double mutant analysis and observed that bub-1 genetically interacted with several downstream genes, including fzy-1/CDC20, mat-2/APC1 and emb-27/APC6.
Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically. Further, our genetic analysis is consistent with that the function of bub-1 in C. elegans is likely similar to its yeast and mammalian homologs.
PMCID: PMC2691579  PMID: 19526056
19.  Temporal Transcriptional Profiling of Somatic and Germ Cells Reveals Biased Lineage Priming of Sexual Fate in the Fetal Mouse Gonad 
PLoS Genetics  2012;8(3):e1002575.
The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process, because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates.
Author Summary
How cells diverge from a common progenitor and adopt specific fates is still poorly understood. We analyzed gene expression profiles in the distinct cell lineages of the gonad over the period when sex determination occurs. The undifferentiated progenitor cells expressed genes characteristic of both sexual fates, explaining the plasticity of the gonadal cells to differentiate as male or female cell types. The establishment of sex-specific fate in both the germ cells and somatic cells involved activation of some genes; but, importantly, we show that an active repression of genes associated with the alternative pathway is also a characteristic of cell fate commitment. Although germ cell progenitors expressed genes associated with both possible fates, genes characteristic of the male fate were over-represented in the progenitors, giving them a male bias. However, in somatic cell progenitors, which control sex determination, genes associated with the female fate were over-represented. These results suggest an explanation for why the female fate is the developmental default for the gonad, and they advance our understanding of how complex transcriptional networks regulate fate determination during organ development.
PMCID: PMC3305395  PMID: 22438826
20.  How Variable Clones Build an Invariant Retina 
Neuron  2012;75(5):786-798.
A fundamental question in developmental neuroscience is how a collection of progenitor cells proliferates and differentiates to create a brain of the appropriate size and cellular composition. To address this issue, we devised lineage-tracing assays in developing zebrafish embryos to reconstruct entire retinal lineage progressions in vivo and thereby provide a complete quantitative map of the generation of a vertebrate CNS tissue from individual progenitors. These lineage data are consistent with a simple model in which the retina is derived from a set of equipotent retinal progenitor cells (RPCs) that are subject to stochastic factors controlling lineage progression. Clone formation in mutant embryos reveals that the transcription factor Ath5 acts as a molecular link between fate choice and mode of cell division, giving insight into the elusive molecular mechanisms of histogenesis, the conserved temporal order by which neurons of different types exit the cell cycle.
► Method for full live lineage tracing of retinal cells in vivo ► Demonstration that retinal clone growth is representative of retinal growth ► A stochastic model accurately predicts clone growth from equipotent progenitors ► Links between mode of cell division and cell fate help explain histogenesis
A key question in developmental neuroscience is how a collection of progenitors proliferates and differentiates to create a brain of the consistent size and composition. He et al. use lineage tracing to reconstruct the full retinal lineages in vivo and propose a model for stochastic control of lineage progression.
PMCID: PMC3485567  PMID: 22958820
21.  An incoherent regulatory network architecture that orchestrates B cell diversification in response to antigen signaling 
B cell receptor signaling controls the expression of IRF-4, a transcription factor required for B cell differentiation. This study shows that IRF-4 regulates divergent B cell fates via a ‘kinetic-control' mechanism that determines the duration of a transient developmental state.
The intensity of signaling through the B cell receptor controls the level of expression of IRF-4, a transcription factor required for B cell differentiation. The rate of IRF-4 production dictates the extent of antibody gene diversification that B cells undergo upon antigen encounter before differentiating into antibody-secreting plasma cells.Computational modeling and experimental analyses substantiate a model, whereby IRF-4 regulates B cell fate trajectories via a ‘kinetic-control' mechanism.Kinetic control is a process by which B cells pass through an obligate state of variable duration that sets the degree of cellular diversification prior to their terminal differentiation.An incoherent regulatory network architecture, within which IRF-4 is embedded, is the basis for realization of kinetic control.
The generation of a diverse set of pathogen-specific antibodies, with differing affinities and effector functions, by B lymphocytes is essential for efficient protection from many microorganisms. Antibody gene diversification in B cells is mediated by two molecular processes termed class-switch recombination and somatic hypermutation (CSR/SHM) (F1A). The former enables the generation of antibodies with the same antigen-binding specificity, but different effector domains, whereas the latter results in a repertoire of antibodies with a range of affinities for a given antigen containing the same effector domain. CSR/SHM occurs in antigen-activated B cells before their terminal differentiation into plasma cells. The transcription factor IRF-4 is required for CSR/SHM as well as plasma-cell differentiation, with its highest levels of expression being necessary for the latter. IRF-4 acts in the context of a network of regulators that include Blimp-1, Pax5, Bach2 and Bcl-6 (F1B). Despite extensive characterization of these individual factors, how the network responds to sensing of antigen by the B cell antigen receptor (BCR, antibody molecule expressed on cell surface) to regulate the extent of antibody gene diversification and plasma-cell differentiation remains to be addressed.
To address this issue, we assemble a computational model. The model reveals two contrasting scenarios that can underlie B cell fate dynamics. In one case, the initial rate of IRF-4 production controls a binary cell fate choice that involves either going to the CSR/SHM state or to the plasma-cell state; the time spent in the CSR state is relatively insensitive to the initial rate of IRF-4 production (herein called ‘basic bistability'). In the other case, IRF-4 drives all cells through a transient CSR/SHM state, but the initial rate of IRF-4 production sets its duration (‘kinetic control'). Both scenarios predict that increasing the initial rate of IRF-4 production favors the generation of plasma cells at the expense of CSR/SHM, but they differ fundamentally with respect to the underlying gene expression patterns.
To distinguish between these two scenarios experimentally, we utilize two different genetic models. The first involves the B1-8i transgenic mouse whose B cells express a rearranged V187.2 VDJ Ig heavy chain gene segment that is specific for the hapten nitrophenol (NP). The second is a newly developed mouse model that allows exogenous control of IRF-4 expression in naive primary B cells using a tet-inducible allele. Using these models, we show that (i) BCR signal strength sets the initial rate of IRF-4 accumulation and (ii) the concentration of IRF-4 is sensed by an incoherent gene regulatory network architecture to regulate the extent of CSR/SHM prior to plasma-cell differentiation. Our results are consistent with the ‘kinetic-control model' in which the levels of BCR-induced IRF-4 expression control the duration of an obligate CSR/SHM state that enables B cell diversification before terminal differentiation into plasma cells. Evidence for the transient CSR/SHM state is corroborated by both patterns of gene expression and the presence of AID-dependent mutations in individual non-switched plasmablasts.
Our results provide a molecular framework for understanding how B cells balance the competing demands for Ig CSR and SHM with the secretion of antibodies during humoral immune responses. The key feature of the network architecture that allows IRF-4 to coordinate the two competing states of gene expression in a temporal manner is that it simultaneously but asymmetrically activates both sides of a bistable mutual repression circuit. Because the two effects of the primary regulator antagonize each other, we describe the circuit as being based on an ‘incoherent' regulatory motif. Other incoherent regulatory motifs in varied biological systems are also associated with the acquisition of transient cell states, and we consider how the kinetic-control mechanism proposed by us could more generally serve to translate developmental cues into elaborate morphogenetic patterns.
The B-lymphocyte lineage is a leading system for analyzing gene regulatory networks (GRNs) that orchestrate distinct cell fate transitions. Upon antigen recognition, B cells can diversify their immunoglobulin (Ig) repertoire via somatic hypermutation (SHM) and/or class switch DNA recombination (CSR) before differentiating into antibody-secreting plasma cells. We construct a mathematical model for a GRN underlying this developmental dynamic. The intensity of signaling through the Ig receptor is shown to control the bimodal expression of a pivotal transcription factor, IRF-4, which dictates B cell fate outcomes. Computational modeling coupled with experimental analysis supports a model of ‘kinetic control', in which B cell developmental trajectories pass through an obligate transient state of variable duration that promotes diversification of the antibody repertoire by SHM/CSR in direct response to antigens. More generally, this network motif could be used to translate a morphogen gradient into developmental inductive events of varying time, thereby enabling the specification of distinct cell fates.
PMCID: PMC3130558  PMID: 21613984
BCR signal strength; bistability; gene regulatory network; ghost of a fixed point; Irf4
22.  Cell Cycle–Dependent Differentiation Dynamics Balances Growth and Endocrine Differentiation in the Pancreas 
PLoS Biology  2015;13(3):e1002111.
Organogenesis relies on the spatiotemporal balancing of differentiation and proliferation driven by an expanding pool of progenitor cells. In the mouse pancreas, lineage tracing at the population level has shown that the expanding pancreas progenitors can initially give rise to all endocrine, ductal, and acinar cells but become bipotent by embryonic day 13.5, giving rise to endocrine cells and ductal cells. However, the dynamics of individual progenitors balancing self-renewal and lineage-specific differentiation has never been described. Using three-dimensional live imaging and in vivo clonal analysis, we reveal the contribution of individual cells to the global behaviour and demonstrate three modes of progenitor divisions: symmetric renewing, symmetric endocrinogenic, and asymmetric generating a progenitor and an endocrine progenitor. Quantitative analysis shows that the endocrine differentiation process is consistent with a simple model of cell cycle–dependent stochastic priming of progenitors to endocrine fate. The findings provide insights to define control parameters to optimize the generation of β-cells in vitro.
During the development of the pancreas, the asymmetric or symmetric differentiation of progenitor cells into endocrine cells results from stochastic priming of mitotic progenitors at different stages of the cell cycle.
Author Summary
In order to form organs of the right size and cell composition, progenitor cells must balance their proliferation and their differentiation into functional cell types. Here we study how individual progenitor cells in the developing pancreas execute their choices to either expand their pool or differentiate into hormone-producing endocrine cells. Using live microscopy to track the genetically marked progeny of single cells, we reveal that after they divide, individual cells generate either two progenitors, two cells on the endocrine path, or one progenitor and one cell on the endocrine path. Quantitative analysis shows that endocrine differentiation is largely stochastic and that the probability of progenitor cell differentiation by the end of mid-gestation is about 20%. We propose a model in which the production of a progenitor and a differentiated cell in the pancreas results from the stochastic induction of differentiation in one daughter after cell division, rather than the unequal partitioning of molecules between two daughters at the time of division, as observed in the nervous system. Furthermore, when two daughters become endocrine cells, this results from the induction of differentiation followed by cell division—rather than two independent induction events. This model may be applicable to other organs and provides insights to optimize the generation of β-cells in vitro for diabetes therapy.
PMCID: PMC4364879  PMID: 25786211
23.  Spatial Pattern Dynamics of 3D Stem Cell Loss of Pluripotency via Rules-Based Computational Modeling 
PLoS Computational Biology  2013;9(3):e1002952.
Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into cells from all germ lineages, making them a potentially robust cell source for regenerative medicine therapies, but difficulties in predicting and controlling ESC differentiation currently limit the development of therapies and applications from such cells. A common approach to induce the differentiation of ESCs in vitro is via the formation of multicellular aggregates known as embryoid bodies (EBs), yet cell fate specification within EBs is generally considered an ill-defined and poorly controlled process. Thus, the objective of this study was to use rules-based cellular modeling to provide insight into which processes influence initial cell fate transitions in 3-dimensional microenvironments. Mouse embryonic stem cells (D3 cell line) were differentiated to examine the temporal and spatial patterns associated with loss of pluripotency as measured through Oct4 expression. Global properties of the multicellular aggregates were accurately recapitulated by a physics-based aggregation simulation when compared to experimentally measured physical parameters of EBs. Oct4 expression patterns were analyzed by confocal microscopy over time and compared to simulated trajectories of EB patterns. The simulations demonstrated that loss of Oct4 can be modeled as a binary process, and that associated patterns can be explained by a set of simple rules that combine baseline stochasticity with intercellular communication. Competing influences between Oct4+ and Oct4− neighbors result in the observed patterns of pluripotency loss within EBs, establishing the utility of rules-based modeling for hypothesis generation of underlying ESC differentiation processes. Importantly, the results indicate that the rules dominate the emergence of patterns independent of EB structure, size, or cell division. In combination with strategies to engineer cellular microenvironments, this type of modeling approach is a powerful tool to predict stem cell behavior under a number of culture conditions that emulate characteristics of 3D stem cell niches.
Author Summary
Pluripotent embryonic stem cells can differentiate into all cell types making up the adult body; however, this process occurs in a complex three dimensional environment with many different parameters present that are capable of influencing cell fate decisions. A model that can accurately predict the strengths of factors influencing cell fate would allow examination of spatial and temporal patterns of cell phenotype. For this study, we focused on the earliest fate transition that occurs in 3D clusters of embryonic stem cells by monitoring the presence of a transcription factor (Oct4) associated with stem cell pluripotency. After experimentally classifying patterns that arise en route to a fully differentiated aggregate in vitro, we constructed a computational model to deduce how stem cells integrate cues from their surrounding environment to give rise spatial patterns. We used a rules-based modeling approach in which information exchanged by cells with their nearest neighbors regulated cell fate decisions. This parsimonious model captured the spatial dynamics of early cell lineage commitment in a 3D multicellular structure. In combination with strategies to modulate cellular environments, our model provides a flexible tool for elucidating the extra- and intercellular interactions regulating spatially organized differentiation of stem cells in 3D.
PMCID: PMC3597536  PMID: 23516345
24.  Cleavage pattern and fate map of the mesentoblast, 4d, in the gastropod Crepidula: a hallmark of spiralian development 
EvoDevo  2012;3:21.
Animals with a spiral cleavage program, such as mollusks and annelids, make up the majority of the superphylum Lophotrochozoa. The great diversity of larval and adult body plans in this group emerges from this highly conserved developmental program. The 4d micromere is one of the most conserved aspects of spiralian development. Unlike the preceding pattern of spiral divisions, cleavages within the 4d teloblastic sublineages are bilateral, representing a critical transition towards constructing the bilaterian body plan. These cells give rise to the visceral mesoderm in virtually all spiralians examined and in many species they also contribute to the endodermal intestine. Hence, the 4d lineage is an ideal one for studying the evolution and diversification of the bipotential endomesodermal germ layer in protostomes at the level of individual cells. Little is known of how division patterns are controlled or how mesodermal and endodermal sublineages diverge in spiralians. Detailed modern fate maps for 4d exist in only a few species of clitellate annelids, specifically in glossiphoniid leeches and the sludge worm Tubifex. We investigated the 4d lineage in the gastropod Crepidula fornicata, an established model system for spiralian biology, and in a closely related direct-developing species, C. convexa.
High-resolution cell lineage tracing techniques were used to study the 4d lineage of C. fornicata and C. convexa. We present a new nomenclature to name the progeny of 4d, and report the fate map for the sublineages up through the birth of the first five pairs of teloblast daughter cells (when 28 cells are present in the 4d sublineage), and describe each clone’s behavior during gastrulation and later stages as these undergo differentiation. We identify the precise origin of the intestine, two cells of the larval kidney complex, the larval retractor muscles and the presumptive germ cells, among others. Other tissues that arise later in the 4d lineage include the adult heart, internal foot tissues, and additional muscle and mesenchymal cells derived from later-born progeny of the left and right teloblasts. To test whether other cells can compensate for the loss of these tissues (that is, undergo regulation), specific cells were ablated in C. fornicata.
Our results present the first fate map of the 4d micromere sublineages in a mollusk. The fate map reveals that endodermal and mesodermal fates segregate much later than previously thought. We observed little evidence of regulation between sublineages, consistent with a lineage-driven cell specification process. Our results provide a framework for comparisons with other spiralians and lay the groundwork for investigation of the molecular mechanisms of endomesoderm formation, germ line segregation and bilateral differentiation in Crepidula.
PMCID: PMC3724503  PMID: 22992254
25.  Live cell imaging reveals marked variability in myoblast proliferation and fate 
Skeletal Muscle  2013;3:10.
During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear.
We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line.
Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls.
Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated.
PMCID: PMC3712004  PMID: 23638706
Live cell imaging; Single cell analysis; Cell death; Insulin-like growth factors

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