CLASP2 safeguards hematopoietic stem cell properties during mouse and fish development.

Hematopoietic stem cells (HSCs) express a large variety of cell surface receptors that are associated with acquisition of self-renewal and multipotent properties. Correct expression of these receptors depends on a delicate balance between cell surface trafficking, recycling, and degradation and is controlled by the microtubule network and Golgi apparatus, whose roles have hardly been explored during embryonic/fetal hematopoiesis. Here we show that, in the absence of CLASP2, a microtubule-associated protein, the overall production of HSCs is reduced, and the produced HSCs fail to self-renew and maintain their stemness throughout mouse and zebrafish development. This phenotype can be attributed to decreased cell surface expression of the hematopoietic receptor c-Kit, which originates from increased lysosomal degradation in combination with a reduction in trafficking to the plasma membrane. A dysfunctional Golgi apparatus in CLASP2-deficient HSCs seems to be the underlying cause of the c-Kit expression and signaling imbalance.

Recent Advances in Developmental Hematopoiesis: Diving Deeper With New Technologies.

The journey of a hematopoietic stem cell (HSC) involves the passage through successive anatomical sites where HSCs are in direct contact with their surrounding microenvironment, also known as niche. These spatial and temporal cellular interactions throughout development are required for the acquisition of stem cell properties, and for maintaining the HSC pool through balancing self-renewal, quiescence and lineage commitment. Understanding the context and consequences of these interactions will be imperative for our understanding of HSC biology and will lead to the improvement of in vitro production of HSCs for clinical purposes. The aorta-gonad-mesonephros (AGM) region is in this light of particular interest since this is the cradle of HSC emergence during the embryonic development of all vertebrate species. In this review, we will focus on the developmental origin of HSCs and will discuss the novel technological approaches and recent progress made to identify the cellular composition of the HSC supportive niche and the underlying molecular events occurring in the AGM region.

Multispecies RNA tomography reveals regulators of hematopoietic stem cell birth in the embryonic aorta.

The defined location of a stem cell within a niche regulates its fate, behavior, and molecular identity via a complex extrinsic regulation that is far from being fully elucidated. To explore the molecular characteristics and key components of the aortic microenvironment, where the first hematopoietic stem cells are generated during development, we performed genome-wide RNA tomography sequencing on zebrafish, chicken, mouse, and human embryos. The resulting anterior-posterior and dorsal-ventral transcriptional maps provided a powerful resource for exploring genes and regulatory pathways active in the aortic microenvironment. By performing interspecies comparative RNA sequencing analyses and functional assays, we explored the complexity of the aortic microenvironment landscape and the fine-tuning of various factors interacting to control hematopoietic stem cell generation, both in time and space in vivo, including the ligand-receptor couple ADM-RAMP2 and SVEP1. Understanding the regulatory function of the local environment will pave the way for improved stem cell production in vitro and clinical cell therapy.

Proinflammatory Signals as Fuel for the Fire of Hematopoietic Stem Cell Emergence.

Hematopoietic stem cells (HSCs) have the extraordinary ability to both self-renew and generate all mature blood cell lineages. The ability to produce or expand patient-derived HSCs in vitro would greatly improve the outcome for patients with blood disorders that are currently treated with allogeneic HSC transplantation. Many laboratories have been working to identify the signals required for HSC emergence in their native environments to apply this knowledge in vitro. Recently, several signals traditionally known to underlie classical inflammation have emerged as essential regulators of HSC development. In this review we synthesize the findings that have established inflammatory cues as key regulators of HSC development.

Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo.

The adult blood system is established by hematopoietic stem cells (HSCs), which arise during development from an endothelial-to-hematopoietic transition of cells comprising the floor of the dorsal aorta. Expression of aortic runx1 has served as an early marker of HSC commitment in the zebrafish embryo, but recent studies have suggested that HSC specification begins during the convergence of posterior lateral plate mesoderm (PLM), well before aorta formation and runx1 transcription. Further understanding of the earliest stages of HSC specification necessitates an earlier marker of hemogenic endothelium. Studies in mice have suggested that GATA2 might function at early stages within hemogenic endothelium. Two orthologs of Gata2 exist in zebrafish: gata2a and gata2b. Here, we report that gata2b expression initiates during the convergence of PLM, becoming restricted to emerging HSCs. We observe Notch-dependent gata2b expression within the hemogenic subcompartment of the dorsal aorta that is in turn required to initiate runx1 expression. Our results indicate that Gata2b functions within hemogenic endothelium from an early stage, whereas Gata2a functions more broadly throughout the vascular system.

Control of epithelial cell migration and invasion by the IKKß- and CK1α-mediated degradation of RAPGEF2.

Epithelial cell migration is crucial for the development and regeneration of epithelial tissues. Aberrant regulation of epithelial cell migration has a major role in pathological processes such as the development of cancer metastasis and tissue fibrosis. Here, we report that in response to factors that promote cell motility, the Rap guanine exchange factor RAPGEF2 is rapidly phosphorylated by I-kappa-B-kinase-ß and casein kinase-1α and consequently degraded by the proteasome via the SCF(ßTrCP) ubiquitin ligase. Failure to degrade RAPGEF2 in epithelial cells results in sustained activity of Rap1 and inhibition of cell migration induced by HGF, a potent metastatic factor. Furthermore, expression of a degradation-resistant RAPGEF2 mutant greatly suppresses dissemination and metastasis of human breast cancer cells. These findings reveal a molecular mechanism regulating migration and invasion of epithelial cells and establish a key direct link between IKKß and cell motility controlled by Rap-integrin signaling.

Regulation of E2F1 by the von Hippel-Lindau tumour suppressor protein predicts survival in renal cell cancer patients.

Biallelic mutations of the von Hippel-Lindau (VHL) gene are the most common cause of sporadic and inherited renal cell carcinoma (RCC). Loss of VHL has been reported to affect cell proliferation by deregulating cell cycle-associated proteins. We report that the VHL gene product (pVHL) inhibits E2F1 expression at both mRNA and protein level in zebrafish and human RCC cells, while loss of VHL increases E2F1 expression in patient kidney tumour tissue and RCC cells, resulting in a delay of cell cycle progression. RCCs from von Hippel-Lindau patients with known germline VHL mutations express significantly more E2F1 compared to sporadic RCCs with either clear-cell (cc) or non-cc histology. Analysis of 138 primary RCCs reveals that E2F1 expression is significantly higher in tumours with a diameter ≤7 cm and with a favourable American Joint Committee on Cancer (AJCC) stage. The expression of E2F1 in RCC significantly correlates with p27 expression, suggesting that increased expression of E2F1 in RCC induces tumour cell senescence via p27. Cox regression analysis shows significant prediction of E2F1 expression for disease-free survival and overall survival, implying that E2F1 expression in kidney tumour is a novel prognostic factor for patients with RCC.

Loss of Pten promotes angiogenesis and enhanced vegfaa expression in zebrafish.

Angiogenesis, the emergence of vessels from an existing vascular network, is pathologically associated with tumor progression and is of great interest for therapeutic intervention. PTEN is a frequently mutated tumor suppressor and has been linked to the progression of many types of tumors, including hemangiosarcomas in zebrafish. Here, we report that mutant zebrafish embryos lacking functional Pten exhibit enhanced angiogenesis, accompanied by elevated levels of phosphorylated Akt (pAkt). Inhibition of phosphoinositide 3-kinase (PI3K) by LY294002 treatment and application of sunitinib, a widely used anti-angiogenic compound, suppressed enhanced angiogenesis in Pten mutants. Vegfaa has a crucial role in angiogenesis and vegfaa expression was upregulated in embryos lacking functional Pten. Interestingly, vegfaa expression was also upregulated in hemangiosarcomas from haploinsufficient adult zebrafish Pten mutants. Elevated vegfaa expression in mutant embryos lacking functional Pten was suppressed by LY294002. Surprisingly, sunitinib treatment dramatically enhanced vegfaa expression in Pten mutant embryos, which might account for tumor relapse in human patients who are treated with sunitinib. Combined treatment with suboptimal concentrations of sunitinib and LY294002 rescued enhanced angiogenesis in pten mutant embryos without the dramatic increase in vegfaa expression, suggesting a new approach for therapeutic intervention in VEGFR-signaling-dependent tumors.

HIF proteins connect the RB-E2F factors to angiogenesis.

Recently, we showed that E2F7 and E2F8 (E2F7/8) are critical regulators of angiogenesis through transcriptional control of VEGFA in cooperation with HIF. (1) Here we investigate the existence of other novel putative angiogenic E2F7/8-HIF targets, and discuss the role of the RB-E2F pathway in regulating angiogenesis during embryonic and tumor development.

E2F7 and E2F8 promote angiogenesis through transcriptional activation of VEGFA in cooperation with HIF1.

The E2F family of transcription factors plays an important role in controlling cell-cycle progression. While this is their best-known function, we report here novel functions for the newest members of the E2F family, E2F7 and E2F8 (E2F7/8). We show that simultaneous deletion of E2F7/8 in zebrafish and mice leads to severe vascular defects during embryonic development. Using a panel of transgenic zebrafish with fluorescent-labelled blood vessels, we demonstrate that E2F7/8 are essential for proper formation of blood vessels. Despite their classification as transcriptional repressors, we provide evidence for a molecular mechanism through which E2F7/8 activate the transcription of the vascular endothelial growth factor A (VEGFA), a key factor in guiding angiogenesis. We show that E2F7/8 directly bind and stimulate the VEGFA promoter independent of canonical E2F binding elements. Instead, E2F7/8 form a transcriptional complex with the hypoxia inducible factor 1 (HIF1) to stimulate VEGFA promoter activity. These results uncover an unexpected link between E2F7/8 and the HIF1-VEGFA pathway providing a molecular mechanism by which E2F7/8 control angiogenesis.