Flow-induced protein kinase A–CREB pathway acts via BMP signaling to promote HSC emergence

Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta–gonad–mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)–cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA–CREB and BMP pathways in isolated AGM VE-cadherin⁺ cells from mid-gestation embryos, we demonstrate that PKA–CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA–CREB–BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.Embryonic hematopoietic development in the mouse proceeds through defined stages. The first hematopoietic cells of the erythroid lineage develop in the extraembryonic yolk sac at embryonic day 7.5 (E7.5; Moore and Metcalf, 1970). At E9, hematopoietic stem cell (HSC) activity from the yolk sac and paraaortic splanchnopleura (PSp) can be detected when transplanted into neonatal mice (Yoder et al., 1997; Arora et al., 2014). HSCs that engraft lethally irradiated adult recipients emerge in the aorta–gonad–mesonephros (AGM) region at E10-11 (Medvinsky and Dzierzak, 1996; North et al., 2002). These HSCs later colonize additional organs required for adult hematopoiesis and possess the ability to reconstitute multiple hematopoietic lineages.The development of the murine circulatory system at E8.5 coincides with the development of more definitive hematopoietic compartments, including HSCs and the lymphoid lineage. Accordingly, recent studies have linked biomechanical forces, such as blood flow–induced shear stress, to hematopoietic development (Adamo et al., 2009; North et al., 2009). In these studies, genetic mutants lacking intravascular circulation were used to demonstrate the reduction in hematopoietic emergence in the AGM and nitric oxide signaling was implicated in blood flow–dependent AGM hematopoiesis (Adamo et al., 2009; North et al., 2009; Wang et al., 2011). Although chimeric analysis demonstrated a cell autonomous requirement for nitric oxide signaling in zebrafish (North et al., 2009), whether this pathway directly promotes hematopoiesis remains an open question due to the vasodilatory effect of nitric oxide donors and their effects on smooth muscle. Apart from these observations, signaling pathways responsible for flow-dependent hematopoietic induction have not been characterized.Activation of protein kinase A (PKA) and its downstream target cAMP response element-binding protein (CREB) by exogenous shear stress has been observed in diverse cell types, including chondrocytes and osteocytes (Cherian et al., 2003; Ogawa et al., 2014). The classic mechanism of PKA activation involves the binding of a ligand to a G protein–coupled receptor and activation of adenylyl cyclase, which converts ATP into the second messenger cyclic AMP (cAMP). The binding of cAMP to PKA releases catalytic subunits that phosphorylate CREB in the nucleus. In differentiating mouse embryonic stem cells (mESCs), PKA–CREB has been linked to endothelial and hematopoietic differentiation via binding of CREB to the Etv2 promoter, which up-regulates pro-hematopoietic factors such as Gata2 and Scl/Tal1 (Yamamizu et al., 2012). Moreover, the PKA–CREB signaling pathway has been explored in the context of the prostaglandin E₂ signaling pathway in zebrafish, where it promotes AGM hematopoiesis via activation of the Wnt pathway (Goessling et al., 2009). However, whether this pathway is conserved in the mouse is unclear, especially given conflicting reports on Wnt signaling in AGM hematopoiesis (Ruiz-Herguido et al., 2012; Chanda et al., 2013). Prostaglandin E₂ also directly activates several pathways including PI3K–AKT and ERK–MAPK, which makes it difficult to conclude that PKA–CREB is the sole mediator of the pro-hematopoietic effects of this molecule (Alfranca et al., 2006). Given the shear-responsiveness of the PKA–CREB pathway and its implication in early embryonic hematopoiesis in other species, we investigated the possible role of shear stress–activated PKA–CREB signaling during AGM hematopoiesis in the mouse.We first verified that this pathway is activated by shear stress in VE-cadherin⁺ endothelial cells and present in the murine AGM, specifically in the cells lining the dorsal aorta. We then conducted a bioinformatics-based screen using microarray data on CREB overexpression and CREB chromatin immunoprecipitation-sequencing (ChIP-Seq) data using data available at Encyclopedia of DNA Elements (ENCODE) and elsewhere to identify regulators of CREB function in hematopoietic cells (Esparza et al., 2008; Jolma et al., 2010; Pencovich et al., 2011; Raney et al., 2011; Trompouki et al., 2011; Martens et al., 2012). Using insight gained from bioinformatics, we discover that the bone morphogenetic protein (BMP) signaling pathway acts downstream of PKA–CREB signaling in regulating AGM hematopoiesis. Finally, we show that this is a blood flow–dependent pathway by demonstrating the abrogation of PKA–CREB–BMP signaling axis in Ncx1-null embryos, which lack a heartbeat and intravascular flow. Our data thus document a blood-flow dependent pathway regulating hematopoietic development.     

Zebrafish: a genetic approach in studying hematopoiesis

The zebrafish (Danio rerio) has emerged in recent years as an exciting animal model system for studying vertebrate organ development and, in particular, the development of the hematopoietic system. The combined advantages of developmental biology and genetic screens for mutations in zebrafish have provided insights into early events in hematopoiesis and identified several genes required for normal blood development in vertebrates. As a result of the large-scale mutagenesis screens for developmental mutants, several zebrafish mutants with defects in blood development have been recovered. This review discusses how these blood mutations in zebrafish have given new perspectives on hematopoietic development.     

Cooperative Effects of Growth Factors Involved in the Induction of Hematopoietic Mesoderm

Hematopoietic induction occurs on the ventral side ofXenopus gastrulae and is thought to be triggered by the growthfactor bone morphogenetic protein 4 (BMP-4). To characterize thisprocess, we developed a quantitative and sensitive assay for theinduction of erythroid cells from totipotent ectoderm of the embryo.When high doses of BMP-4 were used in this explant assay, few erythroid cells were detected. In contrast, large numbers of differentiated erythroid cells were induced when ectoderm was treated with BMP-4 andthe mesoderm inducers, activin, or fibroblast growthfactor (FGF). Ectopic expression of GATA-1 also induced abundanterythroid cells in ectoderm treated with bFGF. This induction oferythroid cells by GATA-1 was blocked by coexpression with a dominantnegative BMP-4 receptor, showing that GATA-1 requires the BMP signaling cascade to function. These results suggest that BMP-4 requires mesoderminduction to generate a program of gene expression, which regulates thespecification of hematopoietic mesoderm by GATA factors.     

Estrogen Activation of G-Protein–Coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-Kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes

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Effects of lethal irradiation in zebrafish and rescue by hematopoietic cell transplantation

The study of hematopoiesis has been greatly facilitated by transplantation of blood cell populations into recipient animals. Efficient engraftment of donor cells generally requires ablation of the host hematopoietic system. The zebrafish has recently emerged as a developmental and genetic system to study hematopoiesis. To enable the study of hematopoietic stem cell (HSC) biology, immune cell function, and leukemogenesis in zebrafish, we have developed hematopoietic cell transplantation (HCT) into adult recipient animals conditioned by gamma irradiation. Dose-response experiments showed that the minimum lethal dose (MLD) of 40 Gy led to the specific ablation of hematolymphoid cells and death by 14 days after irradiation. Sublethal irradiation doses of 20 Gy predominantly ablated lymphocytes and permitted transplantation of a lethal T-cell leukemia. Finally, transplantation of hematopoietic cells carrying transgenes yielding red fluorescent erythrocytes and green fluorescent leukocytes showed that HCT is sufficient to rescue the MLD, that recipient hematolymphoid tissues were repopulated by donor-derived cells, and that donor blood cell lineages can be independently visualized in living recipients. Together, these results establish transplantation assays to test for HSC function and oncogenic transformation in zebrafish.     

SPRED1 deletion confers resistance to MAPK inhibition in melanoma

Functional evaluation of genetic lesions can discover a role in cancer initiation and progression and help develop novel therapeutic strategies. We previously identified the negative MAPK regulator SPRED1 as a novel tumor suppressor in KIT-driven melanoma. Here, we show that SPRED1 is also frequently deleted in human melanoma driven by mutant BRAF. We found that SPRED1 inactivation in human melanoma cell lines and primary zebrafish melanoma conferred resistance to BRAF^V600E inhibition in vitro and in vivo. Mechanistically, SPRED1 loss promoted melanoma cell proliferation under mutant BRAF inhibition by reactivating MAPK activity. Consistently, biallelic deletion of SPRED1 was observed in a patient whose melanoma acquired resistance to MAPK-targeted therapy. These studies combining work in human cells and in vivo modeling in zebrafish demonstrate a new mechanism of resistance to BRAF^V600E inhibition in melanoma.     

Site‐directed zebrafish transgenesis into single landing sites with the phiC31 integrase system

Background: Linear DNA‐based and Tol2‐mediated transgenesis are powerful tools for the generation of transgenic zebrafish. However, the integration of multiple copies or transgenes at random genomic locations complicates comparative transgene analysis and makes long‐term transgene stability unpredictable with variable expression. Targeted, site‐directed transgene integration into pre‐determined genomic loci can circumvent these issues. The phiC31 integrase catalyzes the unidirectional recombination reaction between heterotypic attP and attB sites and is an efficient platform for site‐directed transgenesis. Results: We report the implementation of the phiC31 integrase‐mediated attP/attB recombination for site‐directed zebrafish transgenics of attB‐containing transgene vectors into single genomic attP landing sites. We generated Tol2‐based single‐insertion attP transgenic lines and established their performance in phiC31 integrase‐catalyzed integration of an attB‐containing transgene vector. We found stable germline transmission into the next generation of an attB reporter transgene in 34% of all tested animals. We further characterized two functional attP landing site lines and determined their genomic location. Our experiments also demonstrate tissue‐specific transgene applications as well as long‐term stability of phiC31‐mediated transgenes. Conclusions: Our results establish phiC31 integrase‐controlled site‐directed transgenesis into single, genomic attP sites as space‐, time‐, and labor‐efficient zebrafish transgenesis technique. The described reagents are available for distribution to the zebrafish community. Developmental Dynamics 242:949–963, 2013. © 2013 Wiley Periodicals, Inc.