John S. Hawkins

Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition

Changes in cell fate and identity are essential for endothelial-to-haematopoietic transition (EHT), an embryonic process that generates the first adult populations of haematopoietic stem cells (HSCs) from hemogenic endothelial cells. Dissecting EHT regulation is a critical step towards the production of in vitro derived HSCs. Yet, we do not know how distinct endothelial and haematopoietic fates are parsed during the transition. Here we show that genes required for arterial identity function later to repress haematopoietic fate. Tissue-specific, temporally controlled, genetic loss of arterial genes (Sox17 and Notch1) during EHT results in increased production of haematopoietic cells due to loss of Sox17-mediated repression of haematopoietic transcription factors (Runx1 and Gata2). However, the increase in EHT can be abrogated by increased Notch signalling. These findings demonstrate that the endothelial haematopoietic fate switch is actively repressed in a population of endothelial cells, and that derepression of these programs augments haematopoietic output.

Bacterial CRISPR: Accomplishments and Prospects

In this review we briefly describe the development of CRISPR tools for genome editing and control of transcription in bacteria. We focus on the Type II CRISPR/Cas9 system, provide specific examples for use of the system, and highlight the advantages and disadvantages of CRISPR versus other techniques. We suggest potential strategies for combining CRISPR tools with high-throughput approaches to elucidate gene function in bacteria.

Emergence of hematopoietic stem and progenitor cells involves a Chd1-dependent increase in total nascent transcription

Significance Adult hematopoietic stem and progenitor cells (HSPCs) develop from a small number of specialized endothelial cells in the embryo. Very little is known about how this process, known as the endothelial-to-hematopoietic transition, is regulated. In this paper, we used mouse genetic knockout models to establish Chd1 as the first chromatin remodeler, to our knowledge, shown to regulate this transition. Chd1 is not required in the endothelium prior to the transition, nor in the blood system after the transition. We found that the emergence of HSPCs involves an increase in total nascent transcription that is dependent on Chd1. These results reveal a new paradigm of regulation of a developmental transition by modulation of transcriptional output that may be relevant in other stem/progenitor cell contexts. Lineage specification during development involves reprogramming of transcriptional states, but little is known about how this is regulated in vivo. The chromatin remodeler chomodomain helicase DNA-binding protein 1 (Chd1) promotes an elevated transcriptional output in mouse embryonic stem cells. Here we report that endothelial-specific deletion of Chd1 leads to loss of definitive hematopoietic progenitors, anemia, and lethality by embryonic day (E)15.5. Mutant embryos contain normal numbers of E10.5 intraaortic hematopoietic clusters that express Runx1 and Kit, but these clusters undergo apoptosis and fail to mature into blood lineages in vivo and in vitro. Hematopoietic progenitors emerging from the aorta have an elevated transcriptional output relative to structural endothelium, and this elevation is Chd1-dependent. In contrast, hematopoietic-specific deletion of Chd1 using Vav-Cre has no apparent phenotype. Our results reveal a new paradigm of regulation of a developmental transition by elevation of global transcriptional output that is critical for hemogenesis and may play roles in other contexts.

Single-cell resolution of morphological changes in hemogenic endothelium

Endothelial-to-hematopoietic transition (EHT) occurs within a population of hemogenic endothelial cells during embryogenesis, and leads to the formation of the adult hematopoietic system. Currently, the prospective identification of specific endothelial cells that will undergo EHT, and the cellular events enabling this transition, are not known. We set out to define precisely the morphological events of EHT, and to correlate cellular morphology with the expression of the transcription factors RUNX1 and SOX17. A novel strategy was developed to allow for correlation of immunofluorescence data with the ultrastructural resolution of scanning electron microscopy. The approach can identify single endothelial cells undergoing EHT, as identified by the ratio of RUNX1 to SOX17 immunofluorescence levels, and the morphological changes associated with the transition. Furthermore, this work details a new technical resource that is widely applicable for correlative analyses of single cells in their native tissue environments. Summary: Correlative microscopy (scanning electron microscopy and SOX17/RUNX1 immunofluorescence) reveals morphological changes during human and mouse endothelial to hematopoietic transition at the single-cell level.

Targeted Transcriptional Repression in Bacteria Using CRISPR Interference (CRISPRi).

Clustered regularly interspersed short palindromic repeats (CRISPR) interference (CRISPRi) is a powerful technology for sequence-specifically repressing gene expression in bacterial cells. CRISPRi requires only a single protein and a custom-designed guide RNA for specific gene targeting. In Escherichia coli, CRISPRi repression efficiency is high (~300-fold), and there are no observable off-target effects. The method can be scaled up as a general strategy for the repression of many genes simultaneously using multiple designed guide RNAs. Here we provide a protocol for efficient guide RNA design, cloning, and assay of the CRISPRi system in E. coli. In principle, this protocol can be used to construct CRISPRi systems for gene repression in other species of bacteria.

Let-7 microRNA-dependent control of leukotriene signaling regulates the transition of hematopoietic niche in mice

Hematopoietic stem and progenitor cells arise from the vascular endothelium of the dorsal aorta and subsequently switch niche to the fetal liver through unknown mechanisms. Here we report that vascular endothelium-specific deletion of mouse Drosha (DroshacKO), an enzyme essential for microRNA biogenesis, leads to anemia and death. A similar number of hematopoietic stem and progenitor cells emerge from Drosha-deficient and control vascular endothelium, but DroshacKO-derived hematopoietic stem and progenitor cells accumulate in the dorsal aorta and fail to colonize the fetal liver. Depletion of the let-7 family of microRNAs is a primary cause of this defect, as it leads to activation of leukotriene B4 signaling and induction of the α4β1 integrin cell adhesion complex in hematopoietic stem and progenitor cells. Inhibition of leukotriene B4 or integrin rescues maturation and migration of DroshacKO hematopoietic stem and progenitor cells to the fetal liver, while it hampers hematopoiesis in wild-type animals. Our study uncovers a previously undefined role of innate leukotriene B4 signaling as a gatekeeper of the hematopoietic niche transition.Hematopoietic stem and progenitor cells are generated first from the vascular endothelium of the dorsal aorta and then the fetal liver but what regulates this switch is unknown. Here, the authors show that changing miRNA biogenesis and leukotriene B4 signaling in mice modulates this switch in the niche.

Mobile-CRISPRi: Enabling Genetic Analysis of Diverse Bacteria

The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. CRISPRi, a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs (sgRNAs), has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to human. However, the difficulty of establishing effective CRISPRi systems in non-model bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish “Mobile-CRISPRi”, a suite of CRISPRi systems that combine modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in Proteobacteria and Firmicutes at the individual gene scale by examining drug-gene synergies and at the library scale by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microbe interactions.

Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming

BackgroundNon-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the “Yamanaka reprogramming factors” (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluripotent state. However, the reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors.MethodsWe modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for reprogramming efficiency and for the ability to enrich for stoichiometric ratios of factors.ResultsThe reengineered vectors allow for cell sorting based on reprogramming factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the reprogramming factor dosage.ConclusionsTogether, these modified vectors are a useful tool for understanding the reprogramming process and improving induced pluripotent stem cell isolation efficiency.