The first lineage determination in mammals.

This review describes in detail the morphological, cytoskeletal and gene expression events leading to the gene regulatory network bifurcation point of trophoblast and inner cell mass cells in a variety of mammalian preimplantation embryos. The interrelated processes of compaction and polarity establishment are discussed in terms of how they affect YAP/WWTR activity and the location and fate of cells. Comparisons between mouse, human, cattle, pig and rabbit embryos suggest a conserved role for YAP/WWTR signalling in trophoblast induction in eutherian animals though the mechanisms for, and timing of, YAP/WWTR activation differs among species. Downstream targets show further differences, with the trophoblast marker GATA3 being a direct target in all examined mammals, while CDX2-positive and SOX2-negative regulation varies.

Microenvironmental and cell intrinsic factors governing human cDC2 differentiation and monocyte reprogramming.

cDC2s occur abundantly in peripheral tissues and arise from circulating blood cDC2s. However, the factors governing cDC2 differentiation in tissues, especially under inflammatory conditions, remained poorly defined. We here found that psoriatic cDC2s express the efferocytosis receptor Axl and exhibit a bone morphogenetic protein (BMP) and p38MAPK signaling signature. BMP7, strongly expressed within the lesional psoriatic epidermis, cooperates with canonical TGF-ß1 signaling for inducing Axl+cDC2s from blood cDC2s in vitro. Moreover, downstream induced p38MAPK promotes Axl+cDC2s at the expense of Axl+CD207+ Langerhans cell differentiation from blood cDC2s. BMP7 supplementation allowed to model cDC2 generation and their further differentiation into LCs from CD34+ hematopoietic progenitor cells in defined serum-free medium. Additionally, p38MAPK promoted the generation of another cDC2 subset lacking Axl but expressing the non-classical NFkB transcription factor RelB in vitro. Such RelB+cDC2s occurred predominantly at dermal sites in the inflamed skin. Finally, we found that cDC2s can be induced to acquire high levels of the monocyte lineage identity factor kruppel-like-factor-4 (KLF4) along with monocyte-derived DC and macrophage phenotypic characteristics in vitro. In conclusion, inflammatory and psoriatic epidermal signals instruct blood cDC2s to acquire phenotypic characteristics of several tissue-resident cell subsets.

Distinct subpopulations of DN1 thymocytes exhibit preferential γδ T lineage potential.

The αß and γδ T cell lineages both differentiate in the thymus from common uncommitted progenitors. The earliest stage of T cell development is known as CD4-CD8- double negative 1 (DN1), which has previously been shown to be a heterogenous mixture of cells. Of these, only the CD117+ fraction has been proposed to be true T cell progenitors that progress to the DN2 and DN3 thymocyte stages, at which point the development of the αß and γδ T cell lineages diverge. However, recently, it has been shown that at least some γδ T cells may be derived from a subset of CD117- DN thymocytes. Along with other ambiguities, this suggests that T cell development may not be as straightforward as previously thought. To better understand early T cell development, particularly the heterogeneity of DN1 thymocytes, we performed a single cell RNA sequence (scRNAseq) of mouse DN and γδ thymocytes and show that the various DN stages indeed comprise a transcriptionally diverse subpopulations of cells. We also show that multiple subpopulations of DN1 thymocytes exhibit preferential development towards the γδ lineage. Furthermore, specific γδ-primed DN1 subpopulations preferentially develop into IL-17 or IFNγ-producing γδ T cells. We show that DN1 subpopulations that only give rise to IL-17-producing γδ T cells already express many of the transcription factors associated with type 17 immune cell responses, while the DN1 subpopulations that can give rise to IFNγ-producing γδ T cell already express transcription factors associated with type 1 immune cell responses.

The miR-424(322)/503 gene cluster regulates pro- versus anti-inflammatory skin DC subset differentiation by modulating TGF-ß signaling.

Transforming growth factor ß (TGF-ß) family ligands are key regulators of dendritic cell (DC) differentiation and activation. Epidermal Langerhans cells (LCs) require TGF-ß family signaling for their differentiation, and canonical TGF-ß1 signaling secures a non-activated LC state. LCs reportedly control skin inflammation and are replenished from peripheral blood monocytes, which also give rise to pro-inflammatory monocyte-derived DCs (moDCs). By studying mechanisms in inflammation, we previously screened LCs versus moDCs for differentially expressed microRNAs (miRNAs). This revealed that miR-424/503 is the most strongly inversely regulated (moDCs > LCs). We here demonstrate that miR-424/503 is induced during moDC differentiation and promotes moDC differentiation in human and mouse. Inversely, forced repression of miR-424 during moDC differentiation facilitates TGF-ß1-dependent LC differentiation. Mechanistically, miR-424/503 deficiency in monocyte/DC precursors leads to the induction of TGF-ß1 response genes critical for LC differentiation. Therefore, the miR-424/503 gene cluster plays a decisive role in anti-inflammatory LC versus pro-inflammatory moDC differentiation from monocytes.

Wwc2 Is a Novel Cell Division Regulator During Preimplantation Mouse Embryo Lineage Formation and Oogenesis.

Formation of the hatching mouse blastocyst marks the end of preimplantation development, whereby previous cell cleavages culminate in the formation of three distinct cell lineages (trophectoderm, primitive endoderm and epiblast). We report that dysregulated expression of Wwc2, a genetic paralog of Kibra/Wwc1 (a known activator of Hippo-signaling, a key pathway during preimplantation development), is specifically associated with cell autonomous deficits in embryo cell number and cell division abnormalities. Division phenotypes are also observed during mouse oocyte meiotic maturation, as Wwc2 dysregulation blocks progression to the stage of meiosis II metaphase (MII) arrest and is associated with spindle defects and failed Aurora-A kinase (AURKA) activation. Oocyte and embryo cell division defects, each occurring in the absence of centrosomes, are fully reversible by expression of recombinant HA-epitope tagged WWC2, restoring activated oocyte AURKA levels. Additionally, clonal embryonic dysregulation implicates Wwc2 in maintaining the pluripotent epiblast lineage. Thus, Wwc2 is a novel regulator of meiotic and early mitotic cell divisions, and mouse blastocyst cell fate.

Lineage Decision-Making within Normal Haematopoietic and Leukemic Stem Cells.

To produce the wide range of blood and immune cell types, haematopoietic stem cells can "choose" directly from the entire spectrum of blood cell fate-options. Affiliation to a single cell lineage can occur at the level of the haematopoietic stem cell and these cells are therefore a mixture of some pluripotent cells and many cells with lineage signatures. Even so, haematopoietic stem cells and their progeny that have chosen a particular fate can still "change their mind" and adopt a different developmental pathway. Many of the leukaemias arise in haematopoietic stem cells with the bulk of the often partially differentiated leukaemia cells belonging to just one cell type. We argue that the reason for this is that an oncogenic insult to the genome "hard wires" leukaemia stem cells, either through development or at some stage, to one cell lineage. Unlike normal haematopoietic stem cells, oncogene-transformed leukaemia stem cells and their progeny are unable to adopt an alternative pathway.

AMPKß1 and AMPKß2 define an isoform-specific gene signature in human pluripotent stem cells, differentially mediating cardiac lineage specification.

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism that phosphorylates a wide range of proteins to maintain cellular homeostasis. AMPK consists of three subunits: α, ß, and γ. AMPKα and ß are encoded by two genes, the γ subunit by three genes, all of which are expressed in a tissue-specific manner. It is not fully understood, whether individual isoforms have different functions. Using RNA-Seq technology, we provide evidence that the loss of AMPKß1 and AMPKß2 lead to different gene expression profiles in human induced pluripotent stem cells (hiPSCs), indicating isoform-specific function. The knockout of AMPKß2 was associated with a higher number of differentially regulated genes than the deletion of AMPKß1, suggesting that AMPKß2 has a more comprehensive impact on the transcriptome. Bioinformatics analysis identified cell differentiation as one biological function being specifically associated with AMPKß2. Correspondingly, the two isoforms differentially affected lineage decision toward a cardiac cell fate. Although the lack of PRKAB1 impacted differentiation into cardiomyocytes only at late stages of cardiac maturation, the availability of PRKAB2 was indispensable for mesoderm specification as shown by gene expression analysis and histochemical staining for cardiac lineage markers such as cTnT, GATA4, and NKX2.5. Ultimately, the lack of AMPKß1 impairs, whereas deficiency of AMPKß2 abrogates differentiation into cardiomyocytes. Finally, we demonstrate that AMPK affects cellular physiology by engaging in the regulation of hiPSC transcription in an isoform-specific manner, providing the basis for further investigations elucidating the role of dedicated AMPK subunits in the modulation of gene expression.

Are Leukaemic Stem Cells Restricted to a Single Cell Lineage?

Cancer-stem-cell theory states that most, if not all, cancers arise from a stem/uncommitted cell. This theory revolutionised our view to reflect that cancer consists of a hierarchy of cells that mimic normal cell development. Elegant studies of twins who both developed acute lymphoblastic leukaemia in childhood revealed that at least two genomic insults are required for cancer to develop. These 'hits' do not appear to confer a growth advantage to cancer cells, nor do cancer cells appear to be better equipped to survive than normal cells. Cancer cells created by investigators by introducing specific genomic insults generally belong to one cell lineage. For example, transgenic mice in which the LIM-only 2 (LMO2,associated with human acute T-lymphoblastic leukaemia) and BCR-ABLp210 (associated with human chronic myeloid leukaemia) oncogenes were active solely within the haematopoietic stem-cell compartment developed T-lymphocyte and neutrophil lineage-restricted leukaemia, respectively. This recapitulated the human form of these diseases. This 'hardwiring' of lineage affiliation, either throughout leukaemic stem cell development or at a particular stage, is different to the behaviour of normal haematopoietic stem cells. While normal cells directly commit to a developmental pathway, they also remain versatile and can develop into a terminally differentiated cell that is not part of the initial lineage. Many cancer stem cells do not have this versatility, and this is an essential difference between normal and cancer stem cells. In this report, we review findings that support this notion.

PGC-1α Controls Skeletal Stem Cell Fate and Bone-Fat Balance in Osteoporosis and Skeletal Aging by Inducing TAZ.

Aberrant lineage specification of skeletal stem cells (SSCs) contributes to reduced bone mass and increased marrow adipose tissue (MAT) in osteoporosis and skeletal aging. Although master regulators of osteoblastic and adipogenic lineages have been identified, little is known about factors that are associated with MAT accumulation and osteoporotic bone loss. Here, we identify peroxisome-proliferator-activated receptor γ coactivator 1-α (PGC-1α) as a critical switch of cell fate decisions whose expression decreases with aging in human and mouse SSCs. Loss of PGC-1α promoted adipogenic differentiation of murine SSCs at the expense of osteoblastic differentiation. Deletion of PGC-1α in SSCs impaired bone formation and indirectly promoted bone resorption while enhancing MAT accumulation. Conversely, induction of PGC-1α attenuated osteoporotic bone loss and MAT accumulation. Mechanistically, PGC-1α maintains bone and fat balance by inducing TAZ. Our results suggest that PGC-1α is a potentially important therapeutic target in the treatment of osteoporosis and skeletal aging.

CD4 Helper and CD8 Cytotoxic T Cell Differentiation.

A fundamental question in developmental immunology is how bipotential thymocyte precursors generate both CD4+ helper and CD8+ cytotoxic T cell lineages. The MHC specificity of αß T cell receptors (TCRs) on precursors is closely correlated with cell fate-determining processes, prompting studies to characterize how variations in TCR signaling are linked with genetic programs establishing lineage-specific gene expression signatures, such as exclusive CD4 or CD8 expression. The key transcription factors ThPOK and Runx3 have been identified as mediating development of helper and cytotoxic T cell lineages, respectively. Together with increasing knowledge of epigenetic regulators, these findings have advanced our understanding of the transcription factor network regulating the CD4/CD8 dichotomy. It has also become apparent that CD4+ T cells retain developmental plasticity, allowing them to acquire cytotoxic activity in the periphery. Despite such advances, further studies are necessary to identify the molecular links between TCR signaling and the nuclear machinery regulating expression of ThPOK and Runx3.

Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells - Tools for analyzing dynamics of cell cycle, migration, and lineage selection.

Neural stem cell (NSC) cultures have been considered technically challenging for time-lapse analysis due to high motility, photosensitivity, and growth at confluent densities. We have tested feasibility of long-term live-cell time-lapse analysis for NSC migration and differentiation studies. Here, we describe a method to study the dynamics of cell cycle, migration, and lineage selection in cultured multipotent mouse or human NSCs using single-cell tracking during a long-term, 7-14 day live-cell time-lapse analysis. We used in-house made PDMS inserts with five microwells on a glass coverslip petri-dish to constrain NSC into the area of acquisition during long-term live-cell imaging. In parallel, we have defined image acquisition settings for single-cell tracking of cell cycle dynamics using Fucci-reporter mouse NSC for 7 days as well as lineage selection and migration using human NSC for 14 days. Overall, we show that adjustments of live-cell analysis settings can extend the time period of single-cell tracking in mouse or human NSC from 24-72 h up to 7-14 days and potentially longer. However, we emphasize that experimental use of repeated fluorescence imaging will require careful consideration of controls during acquisition and analysis.

Identification of Chloride Intracellular Channel Protein 3 as a Novel Gene Affecting Human Bone Formation.

Osteoporosis is a common skeletal disorder characterized by low bone mass leading to increased bone fragility and fracture susceptibility. The bone building cells, osteoblasts, are derived from mesenchymal stromal cells (MSCs); however, with increasing age osteogenic differentiation is diminished and more adipocytes are seen in the bone marrow, suggesting a shift in MSC lineage commitment. Identification of specific factors that stimulate osteoblast differentiation from human MSCs may deliver therapeutic targets to treat osteoporosis. The aim of this study was to identify novel genes involved in osteoblast differentiation of human bone marrow-derived MSCs (hMSCs). We identified the gene chloride intracellular channel protein 3 (CLIC3) to be strongly upregulated during MSC-derived osteoblast differentiation. Lentiviral overexpression of CLIC3 in hMSCs caused a 60% increase of matrix mineralization. Conversely, knockdown of CLIC3 in hMSCs using two short-hairpin RNAs (shRNAs) against CLIC3 resulted in a 69% to 76% reduction in CLIC3 mRNA expression, 53% to 37% less alkaline phosphatase (ALP) activity, and 78% to 88% less matrix mineralization compared to scrambled control. Next, we used an in vivo human bone formation model in which hMSCs lentivirally transduced with the CLIC3 overexpression construct were loaded onto a scaffold (hydroxyapatite-tricalcium-phosphate), implanted under the skin of NOD-SCID mice, and analyzed for bone formation 8 weeks later. CLIC3 overexpression led to a 15-fold increase in bone formation (0.33% versus 5.05% bone area relative to scaffold). Using a Clic3-His-tagged pull-down assay and liquid chromatography-mass spectrometry (LS/MS)-based proteomics analysis in lysates of osteogenically differentiated hMSCs, we showed that CLIC3 interacts with NIMA-related kinase 9 (NEK9) and phosphatidylserine synthase 1 (PTDSS1) in vitro, and this finding was supported by immunofluorescent analysis. In addition, inhibition of NEK9 or PTDSS1 gene expression by shRNAs inhibited osteoblast differentiation and mineralization. In conclusion, we successfully identified CLIC3 to be a lineage-specific gene regulating osteoblast differentiation and bone formation through its interaction with NEK9 and PTDSS1. © The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Human skin dendritic cell fate is differentially regulated by the monocyte identity factor Kruppel-like factor 4 during steady state and inflammation.

BACKGROUND: Langerhans cell (LC) networks play key roles in immunity and tolerance at body surfaces. LCs are established prenatally and can be replenished from blood monocytes. Unlike skin-resident dermal DCs (dDCs)/interstitial-type DCs and inflammatory dendritic epidermal cells appearing in dermatitis/eczema lesions, LCs lack key monocyte-affiliated markers. Inversely, LCs express various epithelial genes critical for their long-term peripheral tissue residency. OBJECTIVE: Dendritic cells (DCs) are functionally involved in inflammatory diseases; however, the mechanisms remained poorly understood. METHODS: In vitro differentiation models of human DCs, gene profiling, gene transduction, and immunohistology were used to identify molecules involved in DC subset specification. RESULTS: Here we identified the monocyte/macrophage lineage identity transcription factor Kruppel-like factor 4 (KLF4) to be inhibited during LC differentiation from human blood monocytes. Conversely, KLF4 is maintained or induced during dermal DC and monocyte-derived dendritic cell/inflammatory dendritic epidermal cell differentiation. We showed that in monocytic cells KLF4 has to be repressed to allow their differentiation into LCs. Moreover, respective KLF4 levels in DC subsets positively correlate with proinflammatory characteristics. We identified epithelial Notch signaling to repress KLF4 in monocytes undergoing LC commitment. Loss of KLF4 in monocytes transcriptionally derepresses Runt-related transcription factor 3 in response to TGF-ß1, thereby allowing LC differentiation marked by a low cytokine expression profile. CONCLUSION: Monocyte differentiation into LCs depends on activation of Notch signaling and the concomitant loss of KLF4.

Studying lineage decision-making in vitro: emerging concepts and novel tools.

Correct and timely lineage decisions are critical for normal embryonic development and homeostasis of adult tissues. Therefore, the search for fundamental principles that underlie lineage decision-making lies at the heart of developmental biology. Here, we review attempts to understand lineage decision-making as the interplay of single-cell heterogeneity and gene regulation. Fluctuations at the single-cell level are an important driving force behind cell-state transitions and the creation of cell-type diversity. Gene regulatory networks amplify such fluctuations and define stable cell types. They also mediate the influence of signaling inputs on the lineage decision. In this review, we focus on insights gleaned from in vitro differentiation of embryonic stem cells. We discuss emerging concepts, with an emphasis on transcriptional regulation, dynamical aspects of differentiation, and functional single-cell heterogeneity. We also highlight some novel tools to study lineage decision-making in vitro.