Defined Microenvironments Trigger In Vitro Gastrulation in Human Pluripotent Stem Cells

Gastrulation is a stage in embryo development where three germ layers arise to dictate the human body plan. In vitro models of gastrulation have been demonstrated by treating pluripotent stem cells with soluble morphogens to trigger differentiation. However, in vivo gastrulation is a multistage process coordinated through feedback between soluble gradients and biophysical forces, with the multipotent epiblast transforming to the primitive streak followed by germ layer segregation. Here, the authors show how constraining pluripotent stem cells to hydrogel islands triggers morphogenesis that mirrors the stages preceding in vivo gastrulation, without the need for exogenous supplements. Within hours of initial seeding, cells display a contractile phenotype at the boundary, which leads to enhanced proliferation, yes-associated protein (YAP) translocation, epithelial to mesenchymal transition, and emergence of SRY-box transcription factor 17 (SOX17)+ T/BRACHYURY+ cells. Molecular profiling and pathway analysis reveals a role for mechanotransduction-coupled wingless-type (WNT) signaling in orchestrating differentiation, which bears similarities to processes observed in whole organism models of development. After two days, the colonies form multilayered aggregates, which can be removed for further growth and differentiation. This approach demonstrates how materials alone can initiate gastrulation, thereby providing in vitro models of development and a tool to support organoid bioengineering efforts.Pallavi Srivastava, Sara Romanazzo, Chantal Kopecky, Stephanie Nemec, Jake Ireland, Thomas G. Molley, Kang Lin, Pavithra B. Jayathilaka, Elvis Pandzic, Avani Yeola, Vashe Chandrakanthan, John Pimanda, and Kristopher Kilia

A novel role for Lyl1 in primitive erythropoiesis

Stem cell leukemia (Scl or Tal1) and lymphoblastic leukemia 1 (Lyl1) encode highly related members of the basic helix-loop-helix family of transcription factors that are co-expressed in the erythroid lineage. Previous studies have suggested that Scl is essential for primitive erythropoiesis. However, analysis of single-cell RNA-seq data of early embryos showed that primitive erythroid cells express both Scl and Lyl1. Therefore, to determine whether Lyl1 can function in primitive erythropoiesis, we crossed conditional Scl knockout mice with mice expressing a Cre recombinase under the control of the Epo receptor, active in erythroid progenitors. Embryos with 20% expression of Scl from E9.5 survived to adulthood. However, mice with reduced expression of Scl and absence of Lyl1 (double knockout; DKO) died at E10.5 because of progressive loss of erythropoiesis. Gene expression profiling of DKO yolk sacs revealed loss of Gata1 and many of the known target genes of the SCL-GATA1 complex. ChIP-seq analyses in a human erythroleukemia cell line showed that LYL1 exclusively bound a small subset of SCL targets including GATA1. Together, these data show for the first time that Lyl1 can maintain primitive erythropoiesis

VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

Angiogenesis is the process by which new blood vessels arise from existing ones by the budding out of endothelial cell capillaries from the luminal side of blood vessels. Blood vessel formation is essential for organ development during embryogenesis and is associated with several physiological and pathological processes, such as wound healing and tumor development. The VE-statin/egfl7 gene is specifically expressed in endothelial cells during embryonic development and in the adult. We studied here the regulatory mechanisms that control this tissue-specific expression. RT-qPCR analyses showed that the specificity of expression of VE-statin/egfl7 in endothelial cells is not shared with its closest neighbor genes notch1 and agpat2 on the mouse chromosome 2. Chromatin-immunoprecipitation analysis of histone modifications at the VE-statin/egfl7 locus showed that the chromatin is specifically opened in endothelial cells, but not in fibroblasts at the transcription start sites. A 13 kb genomic fragment of promoter was cloned and analyzed by gene reporter assays which showed that two conserved regions are important for the specific expression of VE-statin/egfl7 in endothelial cells; a −8409/−7563 enhancer and the −252/+38 region encompassing the exon-1b transcription start site. The latter contains essential GATA and ETS-binding sites, as assessed by linker-scanning analysis and site-directed mutagenesis. An analysis of expression of the ETS and GATA transcription factors showed that Erg, Fli-1 and GATA-2 are the most highly expressed factors in endothelial cells. Erg and GATA-2 directly control the expression of the endogenous VE-statin/egfl7 while Fli-1 probably exerts an indirect control, as assessed by RNA interference and chromatin immunoprecipitation. This first detailed analysis of the mechanisms that govern the expression of the VE-statin/egfl7 gene in endothelial cells pinpoints the specific importance of ETS and GATA factors in the specific regulation of genes in this cell lineage

Integrative Genomics Identifies the Molecular Basis of Resistance to Azacitidine Therapy in Myelodysplastic Syndromes

© 2017 The Author(s) Myelodysplastic syndromes and chronic myelomonocytic leukemia are blood disorders characterized by ineffective hematopoiesis and progressive marrow failure that can transform into acute leukemia. The DNA methyltransferase inhibitor 5-azacytidine (AZA) is the most effective pharmacological option, but only ∼50% of patients respond. A response only manifests after many months of treatment and is transient. The reasons underlying AZA resistance are unknown, and few alternatives exist for non-responders. Here, we show that AZA responders have more hematopoietic progenitor cells (HPCs) in the cell cycle. Non-responder HPC quiescence is mediated by integrin α5 (ITGA5) signaling and their hematopoietic potential improved by combining AZA with an ITGA5 inhibitor. AZA response is associated with the induction of an inflammatory response in HPCs in vivo. By molecular bar coding and tracking individual clones, we found that, although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders

BB0172, a Borrelia burgdorferi Outer Membrane Protein That Binds Integrin Α3Β1

Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi infection. Upon infection, some B. burgdorferi genes are upregulated, including members of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) protein family, which facilitate B. burgdorferi adherence to extracellular matrix components of the host. Comparative genome analysis has revealed a new family of B. burgdorferi proteins containing the von Willebrand factor A (vWFA) domain. In the present study, we characterized the expression and membrane association of the vWFA domain-containing protein BB0172 by using in vitro transcription/translation systems in the presence of microsomal membranes and with detergent phase separation assays. Our results showed evidence of BB0172 localization in the outer membrane, the orientation of the vWFA domain to the extracellular environment, and its function as a metal ion-dependent integrin-binding protein. This is the first report of a borrelial adhesin with a metal ion-dependent adhesion site (MIDAS) motif that is similar to those observed in eukaryotic integrins and has a similar function

Ginger Stimulates Hematopoiesis via Bmp Pathway in Zebrafish

) has been widely used in traditional medicine; however, to date there is no scientific research documenting the potential of ginger to stimulate hematopoiesis. expression in the caudal hematopoietic tissue area. We further confirmed that Bmp/Smad pathway mediates this hematopoiesis promoting effect of ginger by using the Bmp-activated Bmp type I receptor kinase inhibitors dorsomorphin, LND193189 and DMH1.Our study provides a strong foundation to further evaluate the molecular mechanism of ginger and its bioactive components during hematopoiesis and to investigate their effects in adults. Our results will provide the basis for future research into the effect of ginger during mammalian hematopoiesis to develop novel erythropoiesis promoting agents

Genome-Wide Analysis of Transcriptional Reprogramming in Mouse Models of Acute Myeloid Leukaemia

Acute leukaemias are commonly caused by mutations that corrupt the transcriptional circuitry of haematopoietic stem/progenitor cells. However, the mechanisms underlying large-scale transcriptional reprogramming remain largely unknown. Here we investigated transcriptional reprogramming at genome-scale in mouse retroviral transplant models of acute myeloid leukaemia (AML) using both gene-expression profiling and ChIP-sequencing. We identified several thousand candidate regulatory regions with altered levels of histone acetylation that were characterised by differential distribution of consensus motifs for key haematopoietic transcription factors including Gata2, Gfi1 and Sfpi1/Pu.1. In particular, downregulation of Gata2 expression was mirrored by abundant GATA motifs in regions of reduced histone acetylation suggesting an important role in leukaemogenic transcriptional reprogramming. Forced re-expression of Gata2 was not compatible with sustained growth of leukaemic cells thus suggesting a previously unrecognised role for Gata2 in downregulation during the development of AML. Additionally, large scale human AML datasets revealed significantly higher expression of GATA2 in CD34+ cells from healthy controls compared with AML blast cells. The integrated genome-scale analysis applied in this study represents a valuable and widely applicable approach to study the transcriptional control of both normal and aberrant haematopoiesis and to identify critical factors responsible for transcriptional reprogramming in human cancer

Mapping and Functional Characterisation of a CTCF-Dependent Insulator Element at the 3′ Border of the Murine Scl Transcriptional Domain

The Scl gene encodes a transcription factor essential for haematopoietic development. Scl transcription is regulated by a panel of cis-elements spread over 55 kb with the most distal 3′ element being located downstream of the neighbouring gene Map17, which is co-regulated with Scl in haematopoietic cells. The Scl/Map17 domain is flanked upstream by the ubiquitously expressed Sil gene and downstream by a cluster of Cyp genes active in liver, but the mechanisms responsible for delineating the domain boundaries remain unclear. Here we report identification of a DNaseI hypersensitive site at the 3′ end of the Scl/Map17 domain and 45 kb downstream of the Scl transcription start site. This element is located at the boundary of active and inactive chromatin, does not function as a classical tissue-specific enhancer, binds CTCF and is both necessary and sufficient for insulator function in haematopoietic cells in vitro. Moreover, in a transgenic reporter assay, tissue-specific expression of the Scl promoter in brain was increased by incorporation of 350 bp flanking fragments from the +45 element. Our data suggests that the +45 region functions as a boundary element that separates the Scl/Map17 and Cyp transcriptional domains, and raise the possibility that this element may be useful for improving tissue-specific expression of transgenic constructs

A Single cis Element Maintains Repression of the Key Developmental Regulator Gata2

In development, lineage-restricted transcription factors simultaneously promote differentiation while repressing alternative fates. Molecular dissection of this process has been challenging as transcription factor loci are regulated by many trans-acting factors functioning through dispersed cis elements. It is not understood whether these elements function collectively to confer transcriptional regulation, or individually to control specific aspects of activation or repression, such as initiation versus maintenance. Here, we have analyzed cis element regulation of the critical hematopoietic factor Gata2, which is expressed in early precursors and repressed as GATA-1 levels rise during terminal differentiation. We engineered mice lacking a single cis element −1.8 kb upstream of the Gata2 transcriptional start site. Although Gata2 is normally repressed in late-stage erythroblasts, the −1.8 kb mutation unexpectedly resulted in reactivated Gata2 transcription, blocked differentiation, and an aberrant lineage-specific gene expression pattern. Our findings demonstrate that the −1.8 kb site selectively maintains repression, confers a specific histone modification pattern and expels RNA Polymerase II from the locus. These studies reveal how an individual cis element establishes a normal developmental program via regulating specific steps in the mechanism by which a critical transcription factor is repressed