The molecular and cellular hematopoietic stem cell specification niche.

Hematopoietic stem cells (HSCs) are a population of tissue-specific stem cells that reside in the bone marrow of adult mammals, where they self-renew and continuously regenerate the adult hematopoietic lineages over the life of the individual. Prominence as a stem cell model and clinical usefulness have driven interest in understanding the physiologic processes that lead to the specification of HSCs during embryonic development. High-efficiency directed differentiation of HSCs by the instruction of defined progenitor cells using sequentially defined instructive molecules and conditions remains impossible, indicating that comprehensive knowledge of the complete set of precursor intermediate identities and required inductive inputs remains incompletely understood. Recently, interest in the molecular and cellular microenvironment where HSCs are specified from endothelial precursors-the "specification niche"-has increased. Here we review recent progress in understanding these niche spaces across vertebrate phyla, as well as how a better characterization of the origin and molecular phenotypes of the niche cell populations has helped inform and complicate previous understanding of signaling required for HSC emergence and maturation.

Constitutively active CaMKII Drives B lineage acute lymphoblastic leukemia/lymphoma in tp53 mutant zebrafish.

Acute lymphoblastic leukemia/lymphoma (ALL) is the most common pediatric cancer and is a malignancy of T or B lineage lymphoblasts. Dysregulation of intracellular Ca2+ levels has been observed in patients with ALL, leading to improper activation of downstream signaling. Here we describe a new zebrafish model of B ALL, generated by expressing human constitutively active CaMKII (CA-CaMKII) in tp53 mutant lymphocytes. In this model, B cell hyperplasia in the kidney marrow and spleen progresses to overt leukemia/lymphoma, with only 29% of zebrafish surviving the first year of life. Leukemic fish have reduced productive genomic VDJ recombination in addition to reduced expression and improper splicing of ikaros1, a gene often deleted or mutated in patients with B ALL. Inhibiting CaMKII in human pre-B ALL cells induced cell death, further supporting a role for CaMKII in leukemogenesis. This research provides novel insight into the role of Ca2+-directed signaling in lymphoid malignancy and will be useful in understanding disease development and progression.

An NFIX-mediated regulatory network governs the balance of hematopoietic stem and progenitor cells during hematopoiesis.

The transcription factor (TF) nuclear factor I-X (NFIX) is a positive regulator of hematopoietic stem and progenitor cell (HSPC) transplantation. Nfix-deficient HSPCs exhibit a severe loss of repopulating activity, increased apoptosis, and a loss of colony-forming potential. However, the underlying mechanism remains elusive. Here, we performed cellular indexing of transcriptomes and epitopes by high-throughput sequencing (CITE-seq) on Nfix-deficient HSPCs and observed a loss of long-term hematopoietic stem cells and an accumulation of megakaryocyte and myelo-erythroid progenitors. The genome-wide binding profile of NFIX in primitive murine hematopoietic cells revealed its colocalization with other hematopoietic TFs, such as PU.1. We confirmed the physical interaction between NFIX and PU.1 and demonstrated that the 2 TFs co-occupy super-enhancers and regulate genes implicated in cellular respiration and hematopoietic differentiation. In addition, we provide evidence suggesting that the absence of NFIX negatively affects PU.1 binding at some genomic loci. Our data support a model in which NFIX collaborates with PU.1 at super-enhancers to promote the differentiation and homeostatic balance of hematopoietic progenitors.

Tissue-Specific Regulation of the Wnt/ß-Catenin Pathway by PAGE4 Inhibition of Tankyrase.

Spatiotemporal control of Wnt/ß-catenin signaling is critical for organism development and homeostasis. The poly-(ADP)-ribose polymerase Tankyrase (TNKS1) promotes Wnt/ß-catenin signaling through PARylation-mediated degradation of AXIN1, a component of the ß-catenin destruction complex. Although Wnt/ß-catenin is a niche-restricted signaling program, tissue-specific factors that regulate TNKS1 are not known. Here, we report prostate-associated gene 4 (PAGE4) as a tissue-specific TNKS1 inhibitor that robustly represses canonical Wnt/ß-catenin signaling in human cells, zebrafish, and mice. Structural and biochemical studies reveal that PAGE4 acts as an optimal substrate decoy that potently hijacks substrate binding sites on TNKS1 to prevent AXIN1 PARylation and degradation. Consistently, transgenic expression of PAGE4 in mice phenocopies TNKS1 knockout. Physiologically, PAGE4 is selectively expressed in stromal prostate fibroblasts and functions to establish a proper Wnt/ß-catenin signaling niche through suppression of autocrine signaling. Our findings reveal a non-canonical mechanism for TNKS1 inhibition that functions to establish tissue-specific control of the Wnt/ß-catenin pathway.

Ventromorphins: A New Class of Small Molecule Activators of the Canonical BMP Signaling Pathway.

Here, we describe three new small-molecule activators of BMP signaling found by high throughput screening of a library of ∼600 000 small molecules. Using a cell-based luciferase assay in the BMP4-responsive human cervical carcinoma clonal cell line, C33A-2D2, we identified three compounds with similar chemotypes that each ventralize zebrafish embryos and stimulate increased expression of the BMP target genes, bmp2b and szl. Because these compounds ventralize zebrafish embryos, we have termed them "ventromorphins." As expected for a BMP pathway activator, they induce the differentiation of C2C12 myoblasts to osteoblasts. Affymetrix RNA analysis confirmed the differentiation results and showed that ventromorphins treatment elicits a genetic response similar to BMP4 treatment. Unlike isoliquiritigenin (SJ000286237), a flavone that maximally activates the pathway after 24 h of treatment, all three ventromorphins induced SMAD1/5/8 phosphorylation within 30 min of treatment and achieved peak activity within 1 h, indicating that their responses are consistent with directly activating BMP signaling.

Pdgf signalling guides neural crest contribution to the haematopoietic stem cell specification niche.

Haematopoietic stem cells (HSCs) support maintenance of the haematopoietic and immune systems throughout the life of vertebrates, and are the therapeutic component of bone marrow transplants. Understanding native specification of HSCs, to uncover key signals that might help improve in vitro directed differentiation protocols, has been a long-standing biomedical goal. The current impossibility of specifying true HSCs in vitro suggests that key signals remain unknown. We speculated that such signals might be presented by surrounding 'niche' cells, but no such cells have been defined. Here we demonstrate in zebrafish, that trunk neural crest (NC) physically associate with HSC precursors in the dorsal aorta (DA) just prior to initiation of the definitive haematopoietic program. Preventing association of the NC with the DA leads to loss of HSCs. Our results define NC as key cellular components of the HSC specification niche that can be profiled to identify unknown HSC specification signals.

R-spondin 1 is required for specification of hematopoietic stem cells through Wnt16 and Vegfa signaling pathways.

Hematopoietic stem cells (HSCs) are the therapeutic component of bone marrow transplants, but finding immune-compatible donors limits treatment availability and efficacy. Recapitulation of endogenous specification during development is a promising approach to directing HSC specification in vitro, but current protocols are not capable of generating authentic HSCs with high efficiency. Across phyla, HSCs arise from hemogenic endothelium in the ventral floor of the dorsal aorta concurrent with arteriovenous specification and intersegmental vessel (ISV) sprouting, processes regulated by Notch and Wnt. We hypothesized that coordination of HSC specification with vessel patterning might involve modulatory regulatory factors such as R-spondin 1 (Rspo1), an extracellular protein that enhances ß-catenin-dependent Wnt signaling and has previously been shown to regulate ISV patterning. We find that Rspo1 is required for HSC specification through control of parallel signaling pathways controlling HSC specification: Wnt16/DeltaC/DeltaD and Vegfa/Tgfß1. Our results define Rspo1 as a key upstream regulator of two crucial pathways necessary for HSC specification.

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.

FGF signalling specifies haematopoietic stem cells through its regulation of somitic Notch signalling.

Haematopoietic stem cells (HSCs) derive from haemogenic endothelial cells of the primitive dorsal aorta (DA) during vertebrate embryogenesis. The molecular mechanisms governing this unique endothelial to haematopoietic transition remain unclear. Here, we demonstrate a novel requirement for fibroblast growth factor (FGF) signalling in HSC emergence. This requirement is non-cell-autonomous, and acts within the somite to bridge the Wnt and Notch signalling pathways. We previously demonstrated that Wnt16 regulates the somitic expression of two Notch ligands, deltaC (dlc) and deltaD (dld), whose combined function is required for HSC fate. How Wnt16 connects to Notch function has remained an open question. Our current studies demonstrate that FGF signalling, via FGF receptor 4 (Fgfr4), mediates a signal-transduction pathway between Wnt16 and Dlc, but not Dld, to regulate HSC specification. Our findings demonstrate that FGF signalling acts as a key molecular relay within the developmental HSC niche to instruct HSC fate.

Discrete Notch signaling requirements in the specification of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non-cell-autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium.

Signalling pathways that control vertebrate haematopoietic stem cell specification.

Haematopoietic stem cells (HSCs) are tissue-specific stem cells that replenish all mature blood lineages during the lifetime of an individual. Clinically, HSCs form the foundation of transplantation-based therapies for leukaemias and congenital blood disorders. Researchers have long been interested in understanding the normal signalling mechanisms that specify HSCs in the embryo, in part because recapitulating these requirements in vitro might provide a means to generate immune-compatible HSCs for transplantation. Recent embryological work has demonstrated the existence of previously unknown signalling requirements. Moreover, it is now clear that gene expression in the nearby somite is integrally involved in regulating the transition of the embryonic endothelium to a haemogenic fate. Here, we review current knowledge of the intraembryonic signals required for the specification of HSCs in vertebrates.

A somitic Wnt16/Notch pathway specifies haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are a self-renewing population of cells that continuously replenish all blood and immune cells during the lifetime of an individual. HSCs are used clinically to treat a wide array of diseases, including acute leukaemias and congenital blood disorders, but obtaining suitable numbers of cells and finding immune-compatible donors remain serious problems. These difficulties have led to an interest in the conversion of embryonic stem cells or induced pluripotent stem cells into HSCs, which is not possible using current methodologies. To accomplish this goal, it is critical to understand the native mechanisms involved in the specification of HSCs during embryonic development. Here we demonstrate in zebrafish that Wnt16 controls a novel genetic regulatory network required for HSC specification. Non-canonical signalling by Wnt16 is required for somitic expression of the Notch ligands deltaC (dlc) and deltaD (dld), and these ligands are, in turn, required for the establishment of definitive haematopoiesis. Notch signalling downstream of Dlc and Dld is earlier than, and distinct from, known cell-autonomous requirements for Notch, strongly suggesting that novel Notch-dependent relay signal(s) induce the first HSCs in parallel to other established pathways. Our results demonstrate that somite-specific gene expression is required for the production of haemogenic endothelium.

Crystal structure of a beta-catenin/APC complex reveals a critical role for APC phosphorylation in APC function.

The tumor suppressor adenomatous polyposis coli (APC) plays a critical role in the turnover of cytosolic beta-catenin, the key effector of the canonical Wnt signaling pathway. APC contains seven 20 amino acid (20 aa) beta-catenin binding repeats that are required for beta-catenin turnover. We have determined the crystal structure of beta-catenin in complex with a phosphorylated APC fragment containing two 20 aa repeats. Surprisingly, one single phosphorylated 20 aa repeat, together with its flanking regions, covers the entire structural groove of beta-catenin and may thus compete for beta-catenin binding with all other beta-catenin armadillo repeat partners. Our biochemical studies show that phosphorylation of the APC 20 aa repeats increases the affinity of the repeats for beta-catenin by 300- to 500-fold and the phosphorylated 20 aa repeats prevent beta-catenin binding to Tcf. Our work suggests that the phosphorylation of the APC 20 aa repeats could be a critical switch for APC function.

Crystal structure of a beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex.

The "beta-catenin destruction complex" is central to canonical Wnt/beta-catenin signaling. The scaffolding protein Axin and the tumor suppressor adenomatous polyposis coli protein (APC) are critical components of this complex, required for rapid beta-catenin turnover. We determined the crystal structure of a complex between beta-catenin and the beta-catenin-binding domain of Axin (Axin-CBD). The Axin-CBD forms a helix that occupies the groove formed by the third and fourth armadillo repeats of beta-catenin and thus precludes the simultaneous binding of other beta-catenin partners in this region. Our biochemical studies demonstrate that, when phosphorylated, the 20-amino acid repeat region of APC competes with Axin for binding to beta-catenin. We propose that a key function of APC in the beta-catenin destruction complex is to remove phosphorylated beta-catenin product from the active site.

The crystal structure of the beta-catenin/ICAT complex reveals the inhibitory mechanism of ICAT.

Beta-catenin is a multifunctional protein involved in both cell adhesion and transcriptional activation. Transcription mediated by the beta-catenin/Tcf complex is involved in embryological development and is upregulated in various cancers. We have determined the crystal structure at 2.5 A resolution of a complex between beta-catenin and ICAT, a protein that prevents the interaction between beta-catenin and Tcf/Lef family transcription factors. ICAT contains a 3-helix bundle that binds armadillo repeats 10-12 and a C-terminal tail that, similar to Tcf and E-cadherin, binds in the groove formed by armadillo repeats 5-9 of beta-catenin. We show that ICAT selectively inhibits beta-catenin/Tcf binding in vivo, without disrupting beta-catenin/cadherin interactions. Thus, it should be possible to design cancer therapeutics that inhibit beta-catenin-mediated transcriptional activation without interfering with cell adhesion.