Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries.

Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating klf2a levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed klf2a and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased klf2a levels and decreased vSMC association with arteries. We supported the small molecule data with in vivo genetic suppression of piezo1 and 2 in zebrafish, resulting in loss of transgelin+ vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific Piezo1 knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used in vitro modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.

Regulation of angiogenesis by endocytic trafficking mediated by cytoplasmic dynein 1 light intermediate chain 1.

Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, DYNC1LI1) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a dync1li1 zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. In vitro, LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. In vivo, endothelial-specific expression of constitutively active Rab11a leads to excessive angiogenesis, similar to the dync1li1 mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in rilpl1/2, the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis.

EGFR-dependent endocytosis of Wnt9a and Fzd9b promotes ß-catenin signaling during hematopoietic stem cell development in zebrafish.

Cell-to-cell communication through secreted Wnt ligands that bind to members of the Frizzled (Fzd) family of transmembrane receptors is critical for development and homeostasis. Wnt9a signals through Fzd9b, the co-receptor LRP5 or LRP6 (LRP5/6), and the epidermal growth factor receptor (EGFR) to promote early proliferation of zebrafish and human hematopoietic stem cells during development. Here, we developed fluorescently labeled, biologically active Wnt9a and Fzd9b fusion proteins to demonstrate that EGFR-dependent endocytosis of the ligand-receptor complex was required for signaling. In human cells, the Wnt9a-Fzd9b complex was rapidly endocytosed and trafficked through early and late endosomes, lysosomes, and the endoplasmic reticulum. Using small-molecule inhibitors and genetic and knockdown approaches, we found that Wnt9a-Fzd9b endocytosis required EGFR-mediated phosphorylation of the Fzd9b tail, caveolin, and the scaffolding protein EGFR protein substrate 15 (EPS15). LRP5/6 and the downstream signaling component AXIN were required for Wnt9a-Fzd9b signaling but not for endocytosis. Knockdown or loss of EPS15 impaired hematopoietic stem cell development in zebrafish. Other Wnt ligands do not require endocytosis for signaling activity, implying that specific modes of endocytosis and trafficking may represent a method by which Wnt-Fzd specificity is established.

Wnt regulation of hematopoietic stem cell development and disease.

Hematopoietic stem cells (HSCs) are multipotent stem cells that give rise to all cells of the blood and most immune cells. Due to their capacity for unlimited self-renewal, long-term HSCs replenish the blood and immune cells of an organism throughout its life. HSC development, maintenance, and differentiation are all tightly regulated by cell signaling pathways, including the Wnt pathway. Wnt signaling is initiated extracellularly by secreted ligands which bind to cell surface receptors and give rise to several different downstream signaling cascades. These are classically categorized either ß-catenin dependent (BCD) or ß-catenin independent (BCI) signaling, depending on their reliance on the ß-catenin transcriptional activator. HSC development, homeostasis, and differentiation is influenced by both BCD and BCI, with a high degree of sensitivity to the timing and dosage of Wnt signaling. Importantly, dysregulated Wnt signals can result in hematological malignancies such as leukemia, lymphoma, and myeloma. Here, we review how Wnt signaling impacts HSCs during development and in disease.

Machine learning for atherosclerotic tissue component classification in combined near-infrared spectroscopy intravascular ultrasound imaging: Validation against histology.

BACKGROUND AND AIMS: Accurate classification of plaque composition is essential for treatment planning. Intravascular ultrasound (IVUS) has limited efficacy in assessing tissue types, while near-infrared spectroscopy (NIRS) provides complementary information to IVUS but lacks depth information. The aim of this study is to train and assess the efficacy of a machine learning classifier for plaque component classification that relies on IVUS echogenicity and NIRS-signal, using histology as reference standard. METHODS: Matched NIRS-IVUS and histology images from 15 cadaveric human coronary arteries were analyzed (10 vessels were used for training and 5 for testing). Fibrous/pathological intimal thickening (F-PIT), early necrotic core (ENC), late necrotic core (LNC), and calcific tissue regions-of-interest were detected on histology and superimposed onto IVUS frames. The pixel intensities of these tissue types from the training set were used to train a J48 classifier for plaque characterization (ECHO-classification). To aid differentiation of F-PIT from necrotic cores, the NIRS-signal was used to classify non-calcific pixels outside yellow-spot regions as F-PIT (ECHO-NIRS classification). The performance of ECHO and ECHO-NIRS classifications were validated against histology. RESULTS: 262 matched frames were included in the analysis (162 constituted the training set and 100 the test set). The pixel intensities of F-PIT and ENC were similar and thus these two tissues could not be differentiated by echogenicity. With ENC and LNC as a single class, ECHO-classification showed good agreement with histology for detecting calcific and F-PIT tissues but had poor efficacy for necrotic cores (recall 0.59 and precision 0.29). Similar results were found when F-PIT and ENC were treated as a single class (recall and precision for LNC 0.78 and 0.33, respectively). ECHO-NIRS classification improved necrotic core and LNC detection, resulting in an increase of the overall accuracy of both models, from 81.4% to 91.8%, and from 87.9% to 94.7%, respectively. Comparable performance of the two models was seen in the test set where the overall accuracy of ECHO-NIRS classification was 95.0% and 95.5%, respectively. CONCLUSIONS: The combination of echogenicity with NIRS-signal appears capable of overcoming limitations of echogenicity, enabling more accurate characterization of plaque components.

Robust quantitative assessment of collagen fibers with picrosirius red stain and linearly polarized light as demonstrated on atherosclerotic plaque samples.

Collagen is an important component in maintaining structural integrity and functionality of tissues and is modulated in various biological processes. Its visualization and possible quantification using histopathological stains can be important for understanding disease progression or therapeutic response. Visualization of collagen fiber with the histological stain picrosirius red (PSR) is enhanced with polarized light and quantitative analysis is possible using circular polarizers. However, linear polarizers are more commonly available and easier to optically align. The objective of the present study is to demonstrate a novel image acquisition technique and analysis method using linearly polarized light. The proposed imaging technique is based on image acquisition at multiple slide rotation angles, which are co-registered to form a composite image used for quantitative analysis by pixel intensity or pixel counting. The technique was demonstrated on multiple human coronary samples with varying histopathologies and developed specifically to analyze cap collagen in atherosclerotic plaque. Pixel counting image analysis was found to be reproducible across serial tissue sections and across different users and sufficiently sensitive to detect differences in cap structural integrity that are likely relevant to prediction of rupture risk. The benefit of slide rotation angle under linear polarization to acquire images represents a feasible and practical implementation for expanding the general utility of PSR for quantitative analysis.

Zebrafish models of acute leukemias: Current models and future directions.

Acute myeloid leukemias (AML) and acute lymphoid leukemias (ALL) are heterogenous diseases encompassing a wide array of genetic mutations with both loss and gain of function phenotypes. Ultimately, these both result in the clonal overgrowth of blast cells in the bone marrow, peripheral blood, and other tissues. As a consequence of this, normal hematopoietic stem cell function is severely hampered. Technologies allowing for the early detection of genetic alterations and understanding of these varied molecular pathologies have helped to advance our treatment regimens toward personalized targeted therapies. In spite of this, both AML and ALL continue to be a major cause of morbidity and mortality worldwide, in part because molecular therapies for the plethora of genetic abnormalities have not been developed. This underscores the current need for better model systems for therapy development. This article reviews the current zebrafish models of AML and ALL and discusses how novel gene editing tools can be implemented to generate better models of acute leukemias. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cells and Disease Technologies > Perturbing Genes and Generating Modified Animals.

Selective activation of FZD7 promotes mesendodermal differentiation of human pluripotent stem cells.

WNT proteins are secreted symmetry breaking signals that interact with cell surface receptors of the FZD family to regulate a multitude of developmental processes. Studying selectivity between WNTs and FZDs has been hampered by the paucity of purified WNT proteins and by their apparent non-selective interactions with the FZD receptors. Here, we describe an engineered protein, called F7L6, comprised of antibody-derived single-chain variable fragments, that selectively binds to human FZD7 and the co-receptor LRP6. F7L6 potently activates WNT/ß-catenin signaling in a manner similar to Wnt3a. In contrast to Wnt3a, F7L6 engages only FZD7 and none of the other FZD proteins. Treatment of human pluripotent stem (hPS) cells with F7L6 initiates transcriptional programs similar to those observed during primitive streak formation and subsequent gastrulation in the mammalian embryo. This demonstrates that selective engagement and activation of FZD7 signaling is sufficient to promote mesendodermal differentiation of hPS cells.

Haematopoietic stem cell-dependent Notch transcription is mediated by p53 through the Histone chaperone Supt16h.

Haematopoietic stem and progenitor cells (HSPCs) have been the focus of developmental and regenerative studies, yet our understanding of the signalling events regulating their specification remains incomplete. We demonstrate that supt16h, a component of the Facilitates chromatin transcription (FACT) complex, is required for HSPC formation. Zebrafish supt16h mutants express reduced levels of Notch-signalling components, genes essential for HSPC development, due to abrogated transcription. Whereas global chromatin accessibility in supt16h mutants is not substantially altered, we observe a specific increase in p53 accessibility, causing an accumulation of p53. We further demonstrate that p53 influences expression of the Polycomb-group protein PHC1, which functions as a transcriptional repressor of Notch genes. Suppression of phc1 or its upstream regulator, p53, rescues the loss of both Notch and HSPC phenotypes in supt16h mutants. Our results highlight a relationship between supt16h, p53 and phc1 to specify HSPCs via modulation of Notch signalling.

Continuous Formation of Ultrathin, Strong Collagen Sheets with Tunable Anisotropy and Compaction.

The multiscale organization of protein-based fibrillar materials is a hallmark of many organs, but the recapitulation of hierarchal structures down to fibrillar scales, which is a requirement for withstanding physiological loading forces, has been challenging. We present a microfluidic strategy for the continuous, large-scale formation of strong, handleable, free-standing, multicentimeter-wide collagen sheets of unprecedented thinness through the application of hydrodynamic focusing with the simultaneous imposition of strain. Sheets as thin as 1.9 µm displayed tensile strengths of 0.5-2.7 MPa, Young's moduli of 3-36 MPa, and modulated the diffusion of molecules as a function of collagen nanoscale structure. Smooth muscle cells cultured on engineered sheets oriented in the direction of aligned collagen fibrils and generated coordinated vasomotor responses. The described biofabrication approach enables rapid formation of ultrathin collagen sheets that withstand physiologically relevant loads for applications in tissue engineering and regenerative medicine, as well as in organ-on-chip and biohybrid devices.

EGFR is required for Wnt9a-Fzd9b signalling specificity in haematopoietic stem cells.

Wnt signalling drives many processes in development, homeostasis and disease; however, the role and mechanism of individual ligand-receptor (Wnt-Frizzled (Fzd)) interactions in specific biological processes remain poorly understood. Wnt9a is specifically required for the amplification of blood progenitor cells during development. Using genetic studies in zebrafish and human embryonic stem cells, paired with in vitro cell biology and biochemistry, we determined that Wnt9a signals specifically through Fzd9b to elicit ß-catenin-dependent Wnt signalling that regulates haematopoietic stem and progenitor cell emergence. We demonstrate that the epidermal growth factor receptor (EGFR) is required as a cofactor for Wnt9a-Fzd9b signalling. EGFR-mediated phosphorylation of one tyrosine residue on the Fzd9b intracellular tail in response to Wnt9a promotes internalization of the Wnt9a-Fzd9b-LRP signalosome and subsequent signal transduction. These findings provide mechanistic insights for specific Wnt-Fzd signals, which will be crucial for specific therapeutic targeting and regenerative medicine.

Embryonic Immune Cells Remodel the Heart.

How the products of transient hematopoiesis in the yolk sac, dorsal aorta, and developing heart tube function at their sites of production is poorly understood. In this issue of Developmental Cell, Shigeta et al. (2019) elegantly demonstrate that macrophages derived from the heart tube contribute to local tissue remodeling during valve development.

Zebrafish snai2 mutants fail to phenocopy morphant phenotypes.

Snail2 is a zinc-finger transcription factor best known to repress expression of genes encoding cell adherence proteins to facilitate induction of the epithelial-to-mesenchymal transition. While this role has been best documented in the developmental migration of the neural crest and mesoderm, here we expand on previously reported preliminary findings that morpholino knock-down of snai2 impairs the generation of hematopoietic stem cells (HSCs) during zebrafish development. We demonstrate that snai2 morphants fail to initiate HSC specification and show defects in the somitic niche of migrating HSC precursors. These defects include a reduction in sclerotome markers as well as in the Notch ligands dlc and dld, which are known to be essential components of HSC specification. Accordingly, enforced expression of the Notch1-intracellular domain was capable of rescuing HSC specification in snai2 morphants. To parallel our approach, we obtained two mutant alleles of snai2. In contrast to the morphants, homozygous mutant embryos displayed no defects in HSC specification or in sclerotome development, and mutant fish survive into adulthood. However, when these homozygous mutants were injected with snai2 morpholino, HSCs were improperly specified. In summary, our morpholino data support a role for Snai2 in HSC development, whereas our mutant data suggest that Snai2 is dispensable for this process. Together, these findings further support the need for careful consideration of both morpholino and mutant phenotypes in studies of gene function.

Mechanisms of Wnt signaling and control.

The Wnt signaling pathway is a highly conserved system that regulates complex biological processes across all metazoan species. At the cellular level, secreted Wnt proteins serve to break symmetry and provide cells with positional information that is critical to the patterning of the entire body plan. At the organismal level, Wnt signals are employed to orchestrate fundamental developmental processes, including the specification of the anterior-posterior body axis, induction of the primitive streak and ensuing gastrulation movements, and the generation of cell and tissue diversity. Wnt functions extend into adulthood where they regulate stem cell behavior, tissue homeostasis, and damage repair. Disruption of Wnt signaling activity during embryonic development or in adults results in a spectrum of abnormalities and diseases, including cancer. The molecular mechanisms that underlie the myriad of Wnt-regulated biological effects have been the subject of intense research for over three decades. This review is intended to summarize our current understanding of how Wnt signals are generated and interpreted. This article is categorized under: Biological Mechanisms > Cell Signaling Developmental Biology > Stem Cell Biology and Regeneration.

Wnt Signaling in Hematological Malignancies.

Leukemia and lymphoma are a wide encompassing term for a diverse set of blood malignancies that affect people of all ages and result in approximately 23,000 deaths in the United States per year (Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7-30.). Hematopoietic stem cells (HSCs) are tissue-specific stem cells at the apex of the hierarchy that gives rise to all of the terminally differentiated blood cells, through progressively restricted progenitor populations, a process that is known to be Wnt-responsive. In particular, the progenitor populations are subject to uncontrolled expansion during oncogenic processes, namely the common myeloid progenitor and common lymphoid progenitor, as well as the myeloblast and lymphoblast. Unregulated growth of these cell-types leads to mainly three types of blood cancers (i.e., leukemia, lymphoma, and myeloma), which frequently exhibit deregulation of the Wnt signaling pathway. Generally, leukemia is caused by the expansion of myeloid progenitors, leading to an overproduction of white blood cells; as such, patients are unable to make sufficient numbers of red blood cells and platelets. Likewise, an overproduction of lymphocytes leads to clogging of the lymph system and impairment of the immune system in lymphomas. Finally, cancer of the plasma cells in the blood is called myeloma, which also leads to immune system failure. Within each of these three types of blood cancers, there are multiple subtypes, usually characterized by their timeline of onset and their cell type of origin. Of these, 85% of leukemias are encompassed by the four most common diseases, that is, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL); AML accounts for the majority of leukemia-related deaths (Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7-30.). Through understanding how HSCs are normally developed and maintained, we can understand how the normal functions of these pathways are disrupted during blood cancer progression; the Wnt pathway is important in regulation of both normal and malignant hematopoiesis. In this chapter, we will discuss the role of Wnt signaling in normal and aberrant hematopoiesis. Our understanding the relationship between Wnt and HSCs will provide novel insights into therapeutic targets.

Biallelic mutations in the 3' exonuclease TOE1 cause pontocerebellar hypoplasia and uncover a role in snRNA processing.

Deadenylases are best known for degrading the poly(A) tail during mRNA decay. The deadenylase family has expanded throughout evolution and, in mammals, consists of 12 Mg2+-dependent 3'-end RNases with substrate specificity that is mostly unknown. Pontocerebellar hypoplasia type 7 (PCH7) is a unique recessive syndrome characterized by neurodegeneration and ambiguous genitalia. We studied 12 human families with PCH7, uncovering biallelic, loss-of-function mutations in TOE1, which encodes an unconventional deadenylase. toe1-morphant zebrafish displayed midbrain and hindbrain degeneration, modeling PCH-like structural defects in vivo. Surprisingly, we found that TOE1 associated with small nuclear RNAs (snRNAs) incompletely processed spliceosomal. These pre-snRNAs contained 3' genome-encoded tails often followed by post-transcriptionally added adenosines. Human cells with reduced levels of TOE1 accumulated 3'-end-extended pre-snRNAs, and the immunoisolated TOE1 complex was sufficient for 3'-end maturation of snRNAs. Our findings identify the cause of a neurodegenerative syndrome linked to snRNA maturation and uncover a key factor involved in the processing of snRNA 3' ends.

CRISPR Guide RNA Validation In Vitro.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been applied to edit genomes in a wide variety of model systems. Although this process can be quite efficient, editing at precise locations in the genome remains difficult without a suitable single guide RNA (sgRNA). We have developed a method for screening sgRNA function in vitro, using reagents that most zebrafish laboratories are already using. The results from our in vitro assay correlate with function in vivo in every sgRNA that we have examined so far. When combined with endonucleases with alternative protospacer adjacent motif site specificities and alternative sgRNAs, this method will streamline genome editing at almost any locus.

Wnt9a Is Required for the Aortic Amplification of Nascent Hematopoietic Stem Cells.

All mature blood cell types in the adult animal arise from hematopoietic stem and progenitor cells (HSPCs). However, the developmental cues regulating HSPC ontogeny are incompletely understood. In particular, the details surrounding a requirement for Wnt/ß-catenin signaling in the development of mature HSPCs are controversial and difficult to consolidate. Using zebrafish, we demonstrate that Wnt signaling is required to direct an amplification of HSPCs in the aorta. Wnt9a is specifically required for this process and cannot be replaced by Wnt9b or Wnt3a. This proliferative event occurs independently of initial HSPC fate specification, and the Wnt9a input is required prior to aorta formation. HSPC arterial amplification occurs prior to seeding of secondary hematopoietic tissues and proceeds, in part, through the cell cycle regulator myca (c-myc). Our results support a general paradigm, in which early signaling events, including Wnt, direct later HSPC developmental processes.

Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells.

The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease.

Cdx1 and Cdx2 function as tumor suppressors.

In humans, colorectal cancer is often initiated through APC loss of function, which leads to crypt hyperplasia and polyposis driven by unrestricted canonical Wnt signaling. Such polyps typically arise in the colorectal region and are at risk of transforming to invasive adenocarcinomas. Although colorectal cancer is the third most common cause of cancer-related death worldwide, the processes impacting initiation, transformation, and invasion are incompletely understood. Murine APC(Min/+) mutants are often used to model colorectal cancers; however, they develop nonmetastatic tumors confined largely to the small intestine and are thus not entirely representative of the human disease. APC(Min/+) alleles can collaborate with mutations impacting other pathways to recapitulate some aspects of human colorectal cancer. To this end, we assessed APC(Min/+)-induced polyposis following somatic loss of the homeodomain transcription factor Cdx2, alone or with a Cdx1 null allele, in the adult gastrointestinal tract. APC(Min/+)-Cdx2 mutants recapitulated several aspects of human colorectal cancer, including an invasive phenotype. Notably, the concomitant loss of Cdx1 led to a significant increase in the incidence of tumors in the distal colon, relative to APC(Min/+)-Cdx2 offspring, demonstrating a previously unrecognized role for this transcription factor in colorectal tumorigenesis. These findings underscore previously unrecognized roles for Cdx members in intestinal tumorigenesis.