Developmental Fate Decisions Research Articles

Histone Deacetylase 7 Regulates Cell Survival and TCR Signaling in CD4/CD8 Double-Positive Thymocytes

CD4/CD8 double-positive thymocytes express the transcriptional repressor histone deacetylase (HDAC)7, a class IIa HDAC that is exported from the cell nucleus after TCR engagement. Through signal-dependent nuclear export, class IIa HDACs such as HDAC7 mediate signal-dependent changes in gene expression that are important to developmental fate decisions in multiple tissues. We report that HDAC7 is exported from the cell nucleus during positive selection in mouse thymocytes and that it regulates genes mediating the coupling between TCR engagement and downstream events that determine cell survival. Thymocytes lacking HDAC7 are inefficiently positively selected due to a severely shortened lifespan and exhibit a truncated repertoire of TCR Jα segments. The expression of multiple important mediators and modulators of the response to TCR engagement is altered in HDAC7-deficient thymocytes, resulting in increased tonic MAPK activity that contributes to the observed loss of viability. Remarkably, the activity of protein kinase D, the kinase that mediates nuclear export of HDAC7 in response to TCR signaling, is also increased in HDAC7-deficient thymocytes, suggesting that HDAC7 nuclear export governs a self-sustaining autoexcitatory loop. These experiments add to the understanding of the life/death decision in thymic T cell development, define a novel function for class IIa HDACs, and point to a novel feed-forward mechanism whereby these molecules regulate their own state and mediate stable developmental transitions.

Switching on Flowers: Transient LEAFY Induction Reveals Novel Aspects of the Regulation of Reproductive Development in Arabidopsis

Developmental fate decisions in cell populations fundamentally depend on at least two parameters: a signal that is perceived by the cell and the intrinsic ability of the cell to respond to the signal. The same regulatory logic holds for phase transitions in the life cycle of an organism, for example the switch to reproductive development in flowering plants. Here we have tested the response of the monocarpic plant species Arabidopsis thaliana to a signal that directs flower formation, the plant-specific transcription factor LEAFY (LFY). Using transient steroid-dependent LEAFY (LFY) activation in lfy null mutant Arabidopsis plants, we show that the plant’s competence to respond to the LFY signal changes during development. Very early in the life cycle, the plant is not competent to respond to the signal. Subsequently, transient LFY activation can direct primordia at the flanks of the shoot apical meristem to adopt a floral fate. Finally, the plants acquire competence to initiate the flower-patterning program in response to transient LFY activation. Similar to a perennial life strategy, we did not observe reprogramming of all primordia after perception of the transient signal, instead only a small number of meristems responded, followed by reversion to the prior developmental program. The ability to initiate flower formation and to direct flower patterning in response to transient LFY upregulation was dependent on the known direct LFY target APETALA1 (AP1). Prolonged LFY or activation could alter the developmental gradient and bypass the requirement for AP1. Prolonged high AP1 levels, in turn, can also alter the plants’ competence. Our findings shed light on how plants can fine-tune important phase transitions and developmental responses.

Human Vascular Progenitor Cells Can Guide Mesodermal Lineage Choice of Mesenchymal Stem and Progenitor Cells After Co-Transplantation In Vivo.

AbstractAbstract 939 Background:Multilineage differentiation potential of mesenchymal stem and progenitor cells (MSPCs) make them attractive candidates for tissue regeneration purposes. Guiding the differentiation of MSPCs towards single lineages would facilitate their application for targeted therapies in vivo. We have previously shown that MSPCs are essential for endothelial colony-forming progenitor cell (ECFC)-derived patent vessel formation in vivo* [Blood 2009; 113 (26):6716-25]. Preliminary data indicate that the ratio of co-applied cells can change mesenchymal lineage differentiation from vascular support towards either osteogenesis with subsequent bone marrow (BM) ingrowth or chondrogenesis. We hypothesized that environmental conditioning by ECFCs plays an instructive role during the developmental fate decision of MSPCs in vivo. Methods:MSPCs as well as ECFCs were isolated from adult BM, white adipose tissue (WAT), umbilical cord blood (UCB) and perivascular cord tissue**[J Vis Exp. 2009;(32) pii: 1525]. Proliferation potential and clonogenicity were monitored. Phenotype was analyzed by flow cytometry and immune cytochemistry. Cell function was studied in differentiation assays and during vascular network assembly in vitro. Models for in vivo human vessel as compared to bone, BM or cartilage formation were established in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). Non-invasive imaging was performed using computed tomography (CT), magnetic resonance (MRI) and near-infrared fluorescence imaging to elucidate the time course of heterotopic tissue development. Immune histochemistry was applied for morphologic studies of organogenesis. Results:Baseline analysis confirmed MSPC and ECFC purity, immune phenotype and sustained proliferation potential. We could show that human BM-derived MSPCs are capable of forming bone in vivo. Osteogenic differentiation and heterotopic ossicle formation was followed by attraction of mouse hematopoiesis and the establishment of entire murine BM including red and white blood cells and megakaryocytes within a human endosteal niche. Co-transplanted human ECFCs could instruct the MSPCs to differentiate also into pericytes or chondrocytes in vivo, depending on the applied MSPC/ECFC cell ratio. Non-invasive imaging and histological staining revealed that ectopic organogenesis had already started after 2–4 weeks and was stable during the observation period of 20 weeks. Non-BM-derived populations, although phenotypically identical, invariably lacked the capacity to build bone and marrow environment in this model in vivo. Conclusion:These data indicate that human ECFCs can instruct MSPCs and induce developmental fate decisions early in the time course of organ regeneration after transplantation. We suppose that effective regenerative stem cell therapy in vivo requires more than the injection of one single cell population. For vascular repair as compared to bone and marrow environment reconstitution our model is a promising tool to study the therapeutic applicability and risk profile of such ECFC/MSPC-based transplantation strategies. Disclosures:No relevant conflicts of interest to declare.

Developmental transition of Plasmodium sporozoites into liver-stage forms is regulated by the RNA binding protein Pumilio 2

Developmental and cell fate decisions in many organisms are frequently made on a post-transcriptional level that involves RNA binding proteins and mechanisms of translational repression and regulation of translation of key mRNAs. In the unicellular eukaryote Plasmodium, one of the most dramatic life-cycle dependent changes in cell morphology and function occurs during transmission from the mosquito to the human host. In the mosquito salivary glands Plasmodium sporozoites remain slender, motile and infectious for several days/weeks. At this stage, Plasmodium parasites are on stand-by, awaiting injection onto the mammalian host. Immedietaly fter transmission and liver cell invasion, a remarkable differentiation process is initiated. During this process, each parasite rapidly transforms into a round, stationary exo-erythrocytic form (EEF) that expands into thousands of infectious merozoites to be released into the blood stream. Here we reveal a Plasmodium homolog of the RNA binding protein Pumilio as a key regulator of the sporozoite to EEF transition. In the absence of Pumilio-2 (Puf2), Plasmodium berghei sporozoites initiate early stage EEF development inside mosquito salivary glands, in the absence of the appropriate environmental cues. Mutant salivary gland sporozoites present reduced motility but also significantly reduced ability to invade and infect their mammalian host cells, both in vitro and in vivo. Global expression profiling confirmed that transgenic parasites exhibit genome-wide transcriptional adaptations that are typical for Plasmodium intra-hepatic development. Our data, demonstrates that Puf2 is a key player in the developmental control involved in the transition between sporozoite and EEF, implying that transformation of salivary gland-resident sporozoites into early liver stage parasites is in fact prevented by a post-translational mechanism.

Myelodysplastic Syndromes Are Characterized by Gene Expression Changes in Hematopoietic Stem Cells and Alterations in Hematopoietic Stem Cell and Myeloid Progenitor Composition.

Abstract 1762Poster Board I-788The myelodysplastic syndromes (MDS) represent a heterogeneous group of disorders characterized by peripheral cytopenias due to impaired hematopoietic differentiation. To date, most data characterizing the immature hematopoietic compartment in MDS have relied on evaluation of CD34+ bone marrow cells, which are a heterogeneous population containing a predominance of oligo- and unilineage-potent progenitors and few hematopoietic stem cells (HSC). In this study we show that MDS are disorders of HSC, evidenced by the presence of recurrent cytogenetic alterations, including -5q, -7, and -20q, in highly purified HSC (Lin-CD34+CD38-CD90+CD45RA-) by FISH. Because MDS HSC harbor cytogenetic changes, we sought to better characterize the molecular basis of MDS HSC function by performing whole transcriptome analysis of highly purified HSC and committed myeloid progenitor populations from low-risk (n=8) and high-risk (n=2) MDS patients. When compared to control HSC from healthy patients (n=10), MDS HSC showed broad transcriptional changes. Using the significance analysis of microarrays (SAM) algorithm and Ingenuity Pathways Analysis software, we identified 3,258 differentially expressed genes (FDR < 0.1) with increased expression of genes positively associated with cell growth and proliferation (p < 0.001) and increased expression of inflammatory response genes (p < 0.015). Interestingly, while MDS common myeloid progenitors (CMP, Lin-CD34+CD38+CD123+CD45RA-) showed increased expression of cell death-related genes when compared to normal CMP (p < 0.001), neither MDS HSC nor multipotent progenitors (MPP, Lin-CD34+CD38-CD90-CD45RA-) showed significant differential expression of these genes when compared to their normal counterparts. To assess the cellular and developmental correlates of HSC/committed progenitor transcriptional changes, we evaluated by flow cytometry the frequency of HSC and committed myeloid progenitors in bone marrow aspirates from 35 low-risk MDS, 6 high-risk MDS and 32 healthy patient samples (range 4-84 yo). Low-risk MDS bone marrow samples showed significantly increased numbers of HSC compared to normal bone marrow samples (+3-fold change, p < 0.03). In addition, myeloid progenitor composition was frequently altered in low-risk MDS patients, with decreased percentages of granulocyte-macrophage progenitors (GMP, Lin-CD34+CD38+CD123+CD45RA+) when expressed as a percentage of total myeloid progenitors [including GMP, CMP and megakaryocyte-erythroid progenitors (MEP, Lin-CD34+CD38+CD123loCD45RA-)] (-2.3-fold change, p < 1e-6). This altered myeloid progenitor profile was highly specific to MDS, even when MDS patient samples were compared to a group of control bone marrow samples from non-MDS patients exhibiting at least one cytopenia (n=34, p < 1e-5), allowing for the distinction of MDS samples from non-MDS cytopenias with 0.89 sensitivity and 0.89 specificity. Together, these data indicate that MDS HSC exhibit significantly altered gene expression profiles and suggest that gene expression changes in MDS HSC induce the altered developmental fate decisions and transcriptional changes observed in MDS committed myeloid progenitors. These data also demonstrate that the changes in MDS myeloid progenitor composition may provide a novel, flow cytometric method for distinguishing MDS from other hematologic conditions that mimic MDS. Finally, these studies indicate that molecular characterization of MDS phenotypes may require evaluation of purified hematopoietic progenitors in order to account for the differential effect of MDS-associated changes on specific hematopoietic progenitor populations. DisclosuresWeissman:Amgen: Equity Ownership; Cellerant Inc.: Founder; Stem Cells Inc.: Equity Ownership, Founder; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

Oct4 dependence of chromatin structure within the extended Nanog locus in ES cells

Embryonic stem (ES) cells offer insight into early developmental fate decisions, and their controlled differentiation may yield vast regenerative potential. The molecular determinants supporting ES cell self-renewal are incompletely understood. The homeodomain proteins Nanog and Oct4 are essential for mouse ES cell self-renewal. Using a high-throughput approach, we discovered DNaseI hypersensitive sites and potential regulatory elements along a 160-kb region of the genome that includes GDF3, Dppa3, and Nanog. We analyzed gene expression, chromatin occupancy, and higher-order chromatin structure throughout this gene locus and found that expression of the reprogramming factor Oct4 is required to maintain its integrity.

Human cord blood-derived neural stem cell line—Possible implementation in studying neurotoxicity

Neural stem cell line developed from human umbilical cord blood (HUCB-NSC) [Bużańska et al., 2003. Journal of Neurochemistry 85, 33] is an ethically uncontroversial source of stem cells, able to differentiate into neuronal, astrocytic and oligodendroglial lineages. Developmental fate decisions of HUCB-NSC can be experimentally manipulated in vitro by the presence of trophic factors, mitogenes and neuromorphogenes, but can also be influenced by neurotoxins. In this report two-dimensional (2-D) and three-dimensional (3-D) HUCB-NSC cultures are introduced as useful models for testing developmental neurotoxicity. For 2-D culture models we established a standardized method for the assessment of the growth rate and cell differentiation in 96-well plates. The proliferative capacity of the HUCB-NSC was monitored by the MTT test while their ability to differentiate into neural-like cells by immunocytochemistry of β-tubulin III and MAP-2 for neurons, GFAP and S-100β for astrocytes and GalC for oligodendrocytes. The 3-D culture of HUCB-NSC is represented by neurospheres. Proliferation and migration of the intermediate precursors from attached neurospheres are shown to be controlled and altered by various growth factors and further modulated by the extracellular matrix component—fibronectin. Thus, neurospheres derived from the HUCB-NSC line can represent a suitable model of the activation of dormant stem cells residing in their niche, and can be used for neurotoxic studies.

A model Notch response element detects Suppressor of Hairless-dependent molecular switch.

Cell-cell signaling mediated by Notch is critical during many different developmental processes for the specification or restriction of cell fates. Currently, the only known transduction pathway involves a DNA binding protein, Suppressor of Hairless [Su(H)] in Drosophila and CBF1 in mammals, and results in the direct activation of target genes. It has been proposed that in the absence of Notch, Su(H)/CBF1 acts as a repressor and is converted into an activator through interactions with the Notch intracellular domain [1--4]. Recently, we have also suggested that the activation of specific target genes requires synergy between Su(H) and other transcriptional activators [5]. Here we have designed an assay that allows us to directly test these hypotheses in vivo. Our results clearly demonstrate that Su(H) is able to function as the core of a molecular switch, repressing transcription in the absence of Notch and activating in the presence of Notch. In its capacity as an activator, Su(H) can cooperate synergistically with a DNA-bound transcription factor, Grainyhead. These interactions indicate a simple model for Notch target-gene regulation that could explain the precision of gene activation elicited by Notch signaling in different developmental fate decisions.

The Human T Cell Leukemia/Lymphotropic Virus Type 1 Tax Protein Represses MyoD-dependent Transcription by Inhibiting MyoD-binding to the KIX Domain of p300: A POTENTIAL MECHANISM FOR Tax-MEDIATED REPRESSION OF THE TRANSCRIPTIONAL ACTIVITY OF BASIC HELIX-LOOP-HELIX FACTORS

The human T cell leukemia/lymphotropic virus type 1 (HTLV-1) Tax protein strongly activates viral and cellular gene transcription. It mainly functions by interacting with cellular transcription factors and the KIX domain of the p300/CBP coactivators. Tax can also repress the transcription of cellular genes through the basic helix-loop-helix (bHLH) protein family. To investigate the molecular mechanisms of this Tax-mediated inhibition, we analyzed its effect on the transcriptional activity of the myogenic MyoD protein, which was used as a paradigm of bHLH factors. In this study, we show that overexpression of the p300 coactivator in transient transfection assays was sufficient to rescue MyoD repression by Tax. Furthermore, an N-terminal domain of p300 (amino acids 379-654) containing the region of KIX serving as the Tax binding site was found, when overexpressed, to potentiate Tax-mediated transactivation of HTLV-1 proviral as well as MyoD-dependent transcription, and to antagonize the inhibition by Tax of the transcriptional activity of MyoD. These results revealing the presence of an N-terminal MyoD binding site were confirmed by in vitro protein-protein interaction assays that demonstrate that MyoD binds to the KIX domain of p300 and that Tax competes with MyoD binding in a nonreciprocal manner. These observations provide evidence that Tax binding to the KIX domain of p300 prevents bHLH proteins from contacting this N-terminal domain of the coactivator, thus resulting in their transcriptional repression. As bHLH proteins are implicated in many developmental fate decisions, especially during thymopoiesis, Tax-mediated inhibition of their transcriptional activity may contribute to the induction of HTLV-1-linked leukemogenesis.

Protein Stability and Domain Topology Determine the Transcriptional Activity of the Mammalian Glial Cells Missing Homolog, GCMb

The glial cells missing (GCM) family of transcription factors consists of Drosophila GCM and the mammalian proteins GCMa and GCMb. They are expressed in a highly restricted manner during development and are known or assumed to be important regulators of developmental fate decisions. As the biochemical properties of GCMb have not been studied so far, we have undertaken a detailed structure-function analysis of the mouse GCMb (mGCMb) protein. DNA-binding specificity was very similar to that of other GCM proteins. Nevertheless, mGCMb was only a weak transcriptional activator in a number of different tissue culture systems. Interestingly, this was not due to an intrinsic absence of transactivation potential. In effect, we were able to identify two separate transactivation domains within mGCMb, one carboxyl-terminally adjacent to the DNA-binding domain and the second within the extreme carboxyl terminus. Activity of both transactivation domains was, however, modulated by an inhibitory region unique to mGCMb and located between the two transactivation domains. Furthermore, pulse-chase experiments proved that the mGCMb protein has a half-life approximately four times shorter than mGCMa. Introduction of the above mentioned inhibitory domain of mGCMb into mGCMa shortened the half-life of mGCMa to a value typical of mGCMb with a concomitant reduction in transactivation potential. Given the strong correlation between protein stability and transactivation potential, functional differences between the two mammalian GCM homologs are likely due to differences in stability with a single inhibitory region in mGCMb being involved in the reduction of both.

Isolation of a novel chick homolog of Serrate and its coexpression with C-Notch-1 in chick development.

Intercellular signaling mediated by the transmembrane proteins, Notch as receptor and its ligands, Delta and Serrate, plays essential roles in the developmental fate decision of many cell types in Drosophila. The Notch genes are highly conserved both in invertebrates and in vertebrates, suggesting that Notch pathway regulates cell fate decisions during vertebrates development. Notch, Delta and Serrate homologs in chicken have been cloned (Henrique et al., Nature 375: 787-790, 1995; Myat et al., Dev. Biol. 174: 233-247, 1996). We isolated a novel chick homolog of Drosophila Serrate, named C-Serrate-2, and examined its expression patterns during the early chick development using whole-mount in situ hybridization. C-Serrate-2 transcripts were detected in several tissues including the forebrain, the myotome and the apical ectodermal ridge (AER) of the limb bud of a 4-day-old chick embryo. In most of the regions where C-Serrate-2 was expressed, C-Notch-1 was also expressed. Our observations suggest that Serrate-2-Notch-1 signaling plays a role in a variety of morphogeneses during the chick development.

Divide and confer: how worm embryo cells specialize.

Nashville, Tennessee—The animal embryo faces a daunting task as it implements the architectural plans, inscribed in its DNA, for building a complex, multicelled body from the original single-celled zygote. Only for the fruit fly do researchers have a good idea of how early embryonic cells get the marching orders that tell them what cells and tissues they will eventually produce. But at the national meeting of the Society for Developmental Biology here 3 weeks ago, researchers presented a recent flurry of new results on the tiny worm Caenorhabditis elegans . The new insights they provide into how early developmental fate decisions are made in this organism led one researcher to comment that “ C. elegans is entering the heyday that Drosophila entered years ago.”

Identification of novel protein kinases expressed in the myocardium of the developing mouse heart

In Drosophila and Caenorhabditis, signal transduction pathways initiated by the activation of receptor-protein tyrosine kinases can mediate developmental fate decisions. In order to examine whether similar mechanisms are employed during mammalian embryogenesis, we undertook a search for novel protein kinases expressed during heart development in the mouse. The primitive mouse heart is formed between 7.75 and 8.5 days post cotium (dpc) and consists of myocardial ad endocardial cells. A reverse transcriptase polymerase chain reaction-based approach was used to amplify protein kinase specific products from cDNAs obtained from 8.5 dpc heart tissue. Twenty independent PCR products corresponding to either protein serine/threonine or tyrosine kinases were identified. In this report, we describe the characterization of two of the genes corresponding to the novel PCR products (designated Hek2 and msk). Hek2 encodes the mouse ortholog of human HEK2, a recently identified member of the eph receptor-protein tyrosine kinase gene family. Prior to and at the time of heart formation (7.5–8.0 dpc), Hek2 is expressed in the cranial (rostral) region of the embryo from which a subpopulation of cells will give rise to the rudimentary heart. Between 8.0 and 9.5 dpc, Hek2 mRNA expression is observed in myocardial cells, head mesenchyme and paraxial mesoderm. Hek2 transcripts are not detected in endocardial cells. After 9.5 dpc, Hek2 expression is downregulated. msk (for myocardial SNF1-like kinase) encodes a putative protein serine/threonine kinase most similar to the yeast gene SNF1. msk mRNA expression is restricted to myocardial cells and their progenitors in the 7.75–8.5 dpc developing heart. Subsequently, msk mRNA expression is rapidly downregulated. the patterns of Hek2 and msk expression suggest that these protein kinases may function during development of the primitive heart.