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Acquired genetic or cytogenetic alterations in a blood stem cell that confer clonal fitness promote its relative expansion leading to clonal hematopoiesis (CH). Despite a largely intact hematopoietic output, CH is associated with a heightened risk of progression to hematologic malignancies and with non-hematologic health manifestations, including cardiovascular disease and overall mortality. We focus on the evidence for the role of inflammation in establishing, maintaining and reciprocally being affected by CH. We describe the known pro-inflammatory signals associated with CH and preclinical studies that elucidated the cellular mechanisms involved. We review the evolving literature on early-onset CH in germline predisposition conditions and the possible role of immune dysregulation in this context.

The National Heart, Lung, and Blood Institute Progenitor Cell Translational Consortium Blood Progenitor Meeting was hosted virtually on November 5, 2020, with 93 attendees across 20 research groups. The purpose of this meeting was to exchange recent findings, discuss current efforts, and identify prospective opportunities in the field of hematopoietic stem and progenitor cell research and therapeutic discovery.

Summary There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.

Summary Studies of hematopoietic stem cell (HSC) development from pre-HSC-producing hemogenic endothelial cells (HECs) are hampered by the rarity of these cells and the presence of other cell types with overlapping marker expression profiles. We generated a Tg(Runx1-mKO2; Ly6a-GFP) dual reporter mouse to visualize hematopoietic commitment and study pre-HSC emergence and maturation. Runx1-mKO2 marked all intra-arterial HECs and hematopoietic cluster cells (HCCs), including pre-HSCs, myeloid- and lymphoid progenitors, and HSCs themselves. However, HSC and lymphoid potential were almost exclusively found in reporter double-positive (DP) cells. Robust HSC activity was first detected in DP cells of the placenta, reflecting the importance of this niche for (pre-)HSC maturation and expansion before the fetal liver stage. A time course analysis by single-cell RNA sequencing revealed that as pre-HSCs mature into fetal liver stage HSCs, they show signs of interferon exposure, exhibit signatures of multi-lineage differentiation gene expression, and develop a prolonged cell cycle reminiscent of quiescent adult HSCs., Highlights • A Runx1 and Ly6a dual reporter system identifies intra-arterial HECs • Lymphoid and HSC potential is restricted to dual reporter double-positive cells • The placenta is the first major niche for HSC maturation and expansion • Single-cell RNA-seq analyses reveal early acquisition of a quiescent HSC phenotype, Chen and colleagues generate a Runx1 and Ly6a dual reporter mouse that marks pre-HSCs from specification of hemogenic endothelial cells to their migration to placenta, where they mature and expand, before colonization of the fetal liver as mature HSCs. Single-cell RNA-seq analyses of maturing HSCs reveal progressive acquisition of a quiescent state reminiscent of adult bone marrow long-term repopulating HSCs.

Cancer has joined heart disease as the leading source of mortality in the US. In an era of organoids, patient-derived xenografts, and organs on a chip, model organisms continue to thrive with a combination of powerful genetic tools, rapid pace of discovery, and affordability. Model organisms enable the analysis of both the tumor and its associated microenvironment, aspects that are particularly relevant to our understanding of metastasis and drug resistance. In this Perspective, we explore some of the strengths of fruit flies and zebrafish for addressing fundamental cancer questions and how these two organisms can contribute to identifying promising therapeutic candidates.

Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well-understood. To this end, here we profiled gene expression in EPO-treated 32D pro-B cells and developing fetal liver erythroid cells to identify additional iron regulatory genes. We determined that FAM210B, a mitochondrial inner-membrane protein, is essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation. These defects were corrected with a lipid-soluble, small-molecule iron transporter, hinokitiol, in Fam210b-deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter but is required for adequate mitochondrial iron import to sustain heme synthesis and iron-sulfur cluster formation during erythroid differentiation. FAM210B was also required for maximal ferrochelatase activity in differentiating erythroid cells. We propose that FAM210B functions as an adaptor protein that facilitates the formation of an oligomeric mitochondrial iron transport complex, required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis. Collectively, our results reveal a critical mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism.

Monocytes/macrophages differentiating from bone marrow (BM) cells pulsed for 2 hours at 37°C with a stabilized derivative of prostaglandin E2, 16,16-dimethyl PGE2 (dmPGE2), migrated less efficiently toward a chemoattractant than monocytes/macrophages differentiated from BM cells pulsed with vehicle. To confirm that the effect on BM cells was long lasting and to replicate human BM transplantation, chimeric mice were established with donor BM cells pulsed for 2 hours with dmPGE2 before injection into marrow-ablated congenic recipient mice. After 12 weeks, when high levels (90%) of engraftment were obtained, regenerated BM-derived monocytes/macrophages differentiating in vitro or in vivo migrated inefficiently toward the chemokines colony-stimulating factor-1 (CSF-1) and chemokine (C-C motif) ligand 2 (CCL2) or thioglycollate, respectively. Our results reveal long-lasting changes to progenitor cells of monocytes/macrophages by a 2-hour dmPGE2 pulse that, in turn, limits the migration of their daughter cells to chemoattractants and inflammatory mediators.

Summary Hematopoietic stem/progenitor cells (HSPCs) are formed during ontogeny from hemogenic endothelium in the ventral wall of the dorsal aorta (VDA). Critically, the cellular mechanism(s) allowing HSPC egress and migration to secondary niches are incompletely understood. Matrix metalloproteinases (MMPs) are inflammation-responsive proteins that regulate extracellular matrix (ECM) remodeling, cellular interactions, and signaling. Here, inhibition of vascular-associated Mmp2 function caused accumulation of fibronectin-rich ECM, retention of runx1/cmyb+ HSPCs in the VDA, and delayed caudal hematopoietic tissue (CHT) colonization; these defects were absent in fibronectin mutants, indicating that Mmp2 facilitates endothelial-to-hematopoietic transition via ECM remodeling. In contrast, Mmp9 was dispensable for HSPC budding, being instead required for proper colonization of secondary niches. Significantly, these migration defects were mimicked by overexpression and blocked by knockdown of C-X-C motif chemokine-12 (cxcl12), suggesting that Mmp9 controls CHT homeostasis through chemokine regulation. Our findings indicate Mmp2 and Mmp9 play distinct but complementary roles in developmental HSPC production and migration., Graphical Abstract, Highlights • Mmp2 function is required for completion of EHT and HSPC egress from the VDA • Mmp2 controls EHT via remodeling fibronectin-rich extracellular matrix in the VDA • Mmp9 activity is necessary for migration between secondary hematopoietic niches • Mmp9 influences Cxcl12 signaling to control CHT vascularity and HSPC colonization, Theodore and colleagues illustrate essential yet distinct roles for inflammation-induced matrix metalloproteinase 2 (Mmp2) and Mmp9 in developmental hematopoiesis. Mmp2 facilitates hematopoietic stem and progenitor cell (HSPC) budding and egress from the ventral dorsal aorta by regulating extracellular matrix fibronectin accumulation; in contrast, Mmp9 modifies Cxcl12/Cxcr4 signaling to enable HSPC colonization of, localization within, and migration between, secondary hematopoietic niches.

The essential functions of polycomb repressive complex 1 (PRC1) in development and gene silencing are thought to involve long non-coding RNAs (lncRNAs), but few specific lncRNAs that guide PRC1 activity are known. We screened for lncRNAs, which co-precipitate with PRC1 from chromatin and found candidates that impact polycomb group protein (PcG)-regulated gene expression in vivo. A novel lncRNA from this screen, CAT7, regulates expression and polycomb group binding at the MNX1 locus during early neuronal differentiation. CAT7 contains a unique tandem repeat domain that shares high sequence similarity to a non-syntenic zebrafish analog, cat7l. Defects caused by interference of cat7l RNA during zebrafish embryogenesis were rescued by human CAT7 RNA, enhanced by interference of a PRC1 component, and suppressed by interference of a known PRC1 target gene, demonstrating cat7l genetically interacts with a PRC1. We propose a model whereby PRC1 acts in concert with specific lncRNAs and that CAT7/cat7l represents convergent lncRNAs that independently evolved to tune PRC1 repression at individual loci.

Understanding in-vivo mechanisms of erythropoiesis is critical for directed differentiation approaches to treat blood disorders. Zebrafish moonshine (mon) mutant embryos defective for the conserved transcriptional intermediary factor 1 gamma (tif1γ) do not specify enough erythroid progenitors. To elucidate the TIF1γ-mediated mechanisms in erythroid differentiation, we performed a chemical suppressor screen using 3,100 compounds and identified inhibitors of the essential mitochondrial pyrimidine synthesis enzyme dihydroorotate dehydrogenase (DHODH). Leflunomide as well as the structurally unrelated DHODH inhibitor brequinar rescue the formation of erythroid progenitors in 61% (38/62) and 68% (50/74) of mon embryos, respectively. Beyond changes in nucleotide metabolism, in-vivo metabolic analyses revealed low levels of TCA cycle metabolites which were functionally linked to a reduced oxygen consumption rate. In addition, an increased 2HG/αKG ratio was associated with higher histone methylation states at H3K27, H3K36 and H4K20 as assessed by quantitative targeted mass spectrometry, which may contribute to the erythroid differentiation block upon tif1γ loss. DHODH is the only pyrimidine de novo synthesis enzyme located in mitochondria and its activity is coupled to that of the electron transport chain (ETC) via coenzyme Q (CoQ). Rotenone, a potent ETC complex I inhibitor reversed the rescue by DHODH inhibition in mon embryos. Through parallel genome-wide transcriptome and chromatin immunoprecipitation analyses, we found that genes encoding CoQ metabolic enzymes are direct TIF1γ targets. Treatment with the CoQ analog decylubiquinone rescued erythroid progenitors in 26% (33/126) of mon embryos. Our work highlights the importance of transcription regulatory processes for tuning metabolism during lineage differentiation and could have therapeutic implications.

In development local signals induce hematopoietic stem cell (HSC) emergence, trafficking, and division. Using a brainbow color barcoding system, we previously found that zebrafish produce 20-30 HSCs in the embryonic aorta. Using spinning disk confocal microscopy, we imaged runx1+23:mCherry+ HSPCs in the fetal niche and found surprising interactions with mpeg1:GFP+ primitive macrophages. At any one time, 20-30% of HSPCs contacted macrophages and were either fully engulfed or had a piece of cytoplasm removed. Using the transgenic cell cycle marker FUCCI, we found 65% of HSPCs in G2M phase engaged with macrophages. To evaluate HSC clonality, we depleted macrophages in brainbow embryos and raised fish to adulthood. Embryonic macrophage depletion via the irf8 morpholino or clodronate liposomes significantly reduced the number of HSC clones in adulthood with an average of 14 clones vs 24.6 in controls (p = 0.0002). To better characterize the interaction between macrophages and HSPCs we performed few-cell proteomics. This identified 166 peptides enriched in macrophages which had recently engaged HSPCs, including three isoforms of calreticulin (calr). Normally ER bound, calr may become surface bound during stress, where it acts as a ‘come-eat-me’ signal. Antibody staining confirms surface calr on HSPCs and scRNA-seq identifies expression of lrp1ab, the canonical calr receptor, specifically in recently-engaged macrophages. Morpholino knockdown of calr isoforms reduces macrophage-HSPC interactions by up to 2-fold (p = 0.0008), and HSPCs overexpressing a non-ER bound form of calr are 4-fold more likely to engage macrophages (p

Despite sharing oncogenetic mutations, only a small number of cells within a given tissue will undergo malignant transformation. Biochemical and physical factors responsible for this cancer-initiation process are not well understood. Here we study biophysical differences of pre-melanoma and melanoma cells in a BRAF(V600E) / P53 zebrafish model. The AFM indentation technique was used to study the cancer-initiating cells while the surrounding melanocytes were the control. We observed a statistically significant decrease in the modulus of elasticity (the effective Young’s modulus) of cancer-initiating cells compared to the surrounding melanocytes. No significant differences in the pericellular coat surrounding cells were observed. These results contribute to a better understanding of the factors responsible for the initiation of cancer.

A recent publication identifies npas4l as the gene defective in the well-known cloche mutant that lacks most endothelial as well as hematopoietic cells. This work poses intriguing questions as to the genetic and molecular nature of the origin of hemato-vascular lineages during early embryogenesis.

SummaryCRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish, it allows the rapid generation of knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish.

International audience; Chronic inflammatory diseases are associated with altered hematopoiesis that could result in neutrophilia and anemia. Here we report that genetic or chemical manipulation of different inflammasome components altered the differentiation of hematopoietic stem and progenitor cells (HSPC) in zebrafish. Although the inflammasome was dispensable for the emergence of HSPC, it was intrinsically required for their myeloid differentiation. In addition, Gata1 transcript and protein amounts increased in inflammasome-deficient larvae, enforcing erythropoiesis and inhibiting myelopoiesis. This mechanism is evolutionarily conserved, since pharmacological inhibition of the inflammasome altered erythroid differentiation of human erythroleukemic K562 cells. In addition, caspase-1 inhibition rapidly upregulated GATA1 protein in mouse HSPC promoting their erythroid differentiation. Importantly, pharmacological inhibition of the inflammasome rescued zebrafish disease models of neutrophilic inflammation and anemia. These results indicate that the inflammasome plays a major role in the pathogenesis of neutrophilia and anemia of chronic diseases and reveal druggable targets for therapeutic interventions.

Summary Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor cells (HSPCs) has limited their characterization to in vitro assays. We report a strategy to respecify lineage-restricted CD34 + CD45 + myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engrafted in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34 + CD38 − cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, conferred engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription-factor-mediated engraftment of blood progenitors from human pluripotent cells.

The zebrafish is an important model for the study of development and disease. Its miniature size, large numbers and optical clarity make it advantageous for high-throughput in vivo phenotyping. In this review we will highlight how the zebrafish contributes to drug discovery by in vivo chemical screening and as a disease model.

Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.

Transcriptome analysis of adult hematopoietic stem cells (HSC) and their progeny has revealed mechanisms of blood differentiation and leukemogenesis, but a similar analysis of HSC development is lacking. Here, we acquired the transcriptomes of developing HSC purified from >2500 murine embryos and adult mice. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs undergoing specification, and definitive HSCs. We applied a network biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development and functionally validated candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knock-down in zebrafish embryos. Moreover, we found that HSCs from in vitro differentiated embryonic stem cells closely resemble definitive HSC, yet lack a Notch-signaling signature, likely accounting for their defective lymphopoiesis. Our analysis and web resource (http://hsc.hms.harvard.edu) will enhance efforts to identify regulators of HSC ontogeny and facilitate the engineering of hematopoietic specification.

Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.

Diamond Blackfan anemia (DBA) is a rare congenital anemia, with more than 50% of patients having mutations in a ribosomal protein. Evidence suggests that both translation and p53 activation play roles in mediating the hematopoietic phenotype. The reason for erythroid specificity of DBA is unclear. Several zebrafish models of DBA have been generated, and these models have already provided key information about disease pathogenesis. The zebrafish model is particularly amenable for studying blood development, allows for advanced imaging techniques, can be manipulated genetically, and is useful for high-throughput screening. By applying zebrafish approaches to the existing DBA models, we will be able to better understand the role of the ribosomal protein mutation in DBA and develop better treatments for this disease.

SummaryHematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable; however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2 treatment by using human and nonhuman primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34+ hCB after xenotransplantation. In nonhuman primate autologous transplantation, dmPGE2-treated CD34+ MPBSCs showed stable multilineage engraftment over 1 year postinfusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and nonhuman primates and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.

The ability of cells to count and remember their divisions could underlie many alterations that occur during development, aging, and disease. We tracked the cumulative divisional history of slow-cycling hematopoietic stem cells (HSCs) throughout adult life. This revealed a fraction of rarely dividing HSCs that contained all the long-term HSC (LT-HSC) activity within the aging HSC compartment. During adult life, this population asynchronously completes four traceable symmetric self-renewal divisions to expand its size before entering a state of dormancy. We show that the mechanism of expansion involves progressively lengthening periods between cell divisions, with long-term regenerative potential lost upon a fifth division. Our data also show that age-related phenotypic changes within the HSC compartment are divisional history dependent. These results suggest that HSCs accumulate discrete memory stages over their divisional history and provide evidence for the role of cellular memory in HSC aging.

Discrepancies in published results about the role of N-cadherin in hematopoietic stem cells have led to confusion in the field. Attempting to settle the disagreements and reach a consensus, we undertook a collective discussion approach. This process clarified a number of issues but left some questions still unresolved.

Long restricted to the field of developmental biology, the use of the zebrafish (Danio rerio) has extended to the study of human pathogenesis. Fostered by the rapid adaptation of new technologies, the design and analysis of fish models of human diseases have contributed important findings that are now making their way from aquariums to clinics. Here we outline the clinical relevance of the zebrafish as a model organism.

Hematopoietic Stem Cells (HSCs) are used clinically to treat human blood-related genetic diseases and leukemias, but the availability of this therapy is constrained by the limiting number of transplantable HSCs. Previous strategies to expand HSCs in vitro resulted in precocious differentiation and reduced transplant efficiency. In vivo analysis of the mechanisms utilized by fetal and adult niches to control HSCs can be mimicked for therapeutic use. This review summarizes the latest research on the in vivo HSC niche and the clinical applications of this knowledge to promote ex vivo expansion and direct de novo generation of HSCs.

Background & Aims The ParaHox transcription factor Cdx2 is an essential determinant of intestinal phenotype in mammals throughout development, influencing gut function, homeostasis, and epithelial barrier integrity. Cdx2 expression demarcates the zones of intestinal stem cell proliferation in the adult gut, with deregulated expression implicated in intestinal metaplasia and cancer. However, in vivo analysis of these prospective roles has been limited because inactivation of Cdx2 in mice leads to preimplantation embryonic lethality. We used the zebrafish, a valuable model for studying gut development, to generate a system to further understanding of the role of Cdx2 in normal intestinal function and in disease states. Methods We isolated and characterized the zebrafish cdx1b ortholog and analyzed its function by antisense morpholino gene knockdown. Results We showed that zebrafish Cdx1b replaces the role of Cdx2 in gut development. Evolutionary studies have indicated that the zebrafish cdx2 loci were lost following the genome-wide duplication event that occurred in teleosts. Zebrafish Cdx1b is expressed exclusively in the developing intestine during late embryogenesis and regulates intestinal cell proliferation and terminal differentiation. Conclusions This work established an in vivo system to explore further the activity of Cdx2 in the gut and its impact on processes such as inflammation and cancer.

The genes required for stem cell specification and lineage restriction during embryogenesis also play fundamental roles in adult tissue regeneration and cancer. This "development-regeneration-cancer" axis is exemplified by the vertebrate pigmentation system. Melanocytes exhibit almost unlimited self-renewal capacity during regenerative processes such as mammalian hair recoloration and zebrafish fin regeneration. Melanoma utilizes many regulatory signals and pathways required during ontogeny and regeneration. A discussion of these interconnections highlights how studies of stem cell function in embryonic and regenerative contexts can yield insights into melanoma biology.

Developmental signaling pathways hold the keys to unlocking the promise of adult tissue regeneration, and to inhibiting carcinogenesis. Patients with mutations in the Adenomatous Polyposis Coli (APC) gene are at increased risk of developing hepatoblastoma, an embryonal form of liver cancer, suggesting that Wnt affects hepatic progenitor cells. To elucidate the role of APC loss and enhanced Wnt activity in liver development, we examined APC mutant and wnt inducible transgenic zebrafish. APC(+/-) embryos developed enlarged livers through biased induction of hepatic gene programs and increased proliferation. Conversely, APC(-/-) embryos formed no livers. Blastula transplantations determined that the effects of APC loss were cell autonomous. Induction of wnt modulators confirmed biphasic consequences of wnt activation: endodermal pattern formation and gene expression required suppression of wnt signaling in early somitogenesis; later, increased wnt activity altered endodermal fate by enhancing liver growth at the expense of pancreas formation; these effects persisted into the larval stage. In adult APC(+/-) zebrafish, increased wnt activity significantly accelerated liver regeneration after partial hepatectomy. Similarly, liver regeneration was significantly enhanced in APC(Min/+) mice, indicating the conserved effect of Wnt pathway activation in liver regeneration across vertebrate species. These studies reveal an important and time-dependent role for wnt signaling during liver development and regeneration.

Establishment and maintenance of the blood system relies on self-renewing hematopoietic stem cells (HSCs) that normally reside in small numbers in the bone marrow niche of adult mammals. This Review describes the developmental origins of HSCs and the molecular mechanisms that regulate lineage-specific differentiation. Studies of hematopoiesis provide critical insights of general relevance to other areas of stem cell biology including the role of cellular interactions in development and tissue homeostasis, lineage programming and reprogramming by transcription factors, and stage- and age-specific differences in cellular phenotypes.

SummaryThe formation of blood in the embryo is dependent on bone morphogenetic protein (BMP), but how BMP signaling intersects with other regulators of hematopoietic development is unclear. Using embryonic stem (ES) cells, we show that BMP4 first induces ventral-posterior (V-P) mesoderm and subsequently directs mesodermal cells toward blood fate by activating Wnt3a and upregulating Cdx and Hox genes. When BMP signaling is blocked during this latter phase, enforced expression of either Cdx1 or Cdx4 rescues hematopoietic development, thereby placing BMP4 signaling upstream of the Cdx-Hox pathway. Wnt signaling cooperates in BMP-induced hemogenesis, and the Wnt effector LEF1 mediates BMP4 activation of Cdx genes. Our data suggest that BMP signaling plays two distinct and sequential roles during blood formation, initially as an inducer of mesoderm, and later to specify blood via activation of Wnt signaling and the Cdx-Hox pathway.

Titin (also called connectin) acts as a scaffold for signaling proteins in muscle and is responsible for establishing and maintaining the structure and elasticity of sarcomeres in striated muscle. Several human muscular dystrophies and cardiomyopathies have previously been linked to mutations in the titin gene. This study reports linkage of the runzel homozygous lethal muscular dystrophy in the zebrafish Danio rerio to a genomic interval containing the titin gene. Analysis of the genomic sequence suggests that zebrafish contain two adjacent titin loci. One titin locus lies within the genetic linkage interval and its expression is significantly reduced in runzel mutants by both immunofluorescence and protein electrophoresis. Morpholino downregulation of this same titin locus in wild-type embryos results in decreased muscle organization and mobility, phenocopying runzel mutants. Additional protein analysis demonstrates that, in wild-type zebrafish, titin isoform sizes are rapidly altered during the development of striated muscle, likely requiring a previously unrecognized need for vertebrate sarcomere remodeling to incorporate developmentally regulated titin isoforms. Decreases of affected titin isoforms in runzel mutants during this time correlate with a progressive loss of sarcomeric organization and suggest that the unaffected titin proteins are capable of sarcomerogenesis but not sarcomere maintenance. In addition, microarray analysis of the ruz transcriptome suggests a novel mechanism of dystrophy pathogenesis, involving mild increases in calpain-3 expression and upregulation of heat shock proteins. These studies should lead to a better understanding of titin's role in normal and diseased muscle.

SummaryThe ventral spinal cord generates multiple inhibitory and excitatory interneuron subtypes from four cardinal progenitor domains (p0, p1, p2, p3). Here we show that cell-cell interactions mediated by the Notch receptor play a critical evolutionarily conserved role in the generation of excitatory v2aIN and inhibitory v2bIN interneurons. Lineage-tracing experiments show that the v2aIN and v2bIN develop from genetically identical p2 progenitors. The p2 daughter cell fate is controlled by Delta4 activation of Notch receptors together with MAML factors. Cells receiving Notch signals activate a transcription factor code that specifies the v2bIN fate, whereas cells deprived of Notch signaling express another code for v2aIN formation. Thus, our study provides insight into the cell-extrinsic signaling that controls combinatorial transcription factor profiles involved in regulating the process of interneuron subtype diversification.

The lineage relationship between the blood cells found in the developmentally successive hematopoietic organs has remained elusive. In this issue of Immunity, Murayama et al. (2006) track the migration of nascent hematopoietic stem cells in zebrafish from their site of origin to a newly described intermediate location.

The hox genes play a central role in organogenesis and are implicated in the formation of hematopoietic stem cells (HSCs). The cdx genes encode homeodomain transcription factors that act as master regulators of the hox genes. In zebrafish, mutations in cdx4 cause a severe, but not complete, deficit in embryonic blood cells. Here, we report the expression and function of cdx1a, a zebrafish Cdx1 paralogue. Using morpholino-mediated knockdown of cdx1a in a cdx4 mutant background, we show that a deficiency in both cdx genes causes a severe perturbation of hox gene expression and a complete failure to specify blood. The hematopoietic defect in cdx-deficient embryos does not result from a general block in posterior mesoderm differentiation as endothelial cells and kidney progenitors are still formed in the doubly deficient embryos. In addition, cdx-deficient embryos display a significant reduction in runx1a+ putative HSCs in the zebrafish equivalent to the aorta–gonad–mesonephros (AGM) region. Overexpressing hoxa9a in cdx-deficient embryos rescues embryonic erythropoiesis in the posterior mesoderm as well as the formation of HSCs in the AGM region. Taken together, these results suggest that the cdx-hox pathway plays an essential role in the formation of both embryonic erythroid cells and definitive HSCs during vertebrate embryogenesis.

International audience; Calponin 2 (h2 calponin, CNN2) is an actin-binding protein implicated in cytoskeletal organization. We have found that the expression of calponin 2 is relatively restricted to vasculature from 16 to 30 h post-fertilization during zebrafish (Danio rerio) development. Forty-eight hours after injecting antisense morpholino oligos against calponin 2 into embryos at the 1-4-cell stage, zebrafish demonstrated various cardiovascular defects, including sluggish axial and head circulation, absence of circulation in intersegmental vessels and in the dorsal longitudinal anastomotic vessel, enlarged cerebral ventricles, and pericardial edema, in addition to an excess bending, spiraling tail and twisting of the caudal fin. Knockdown of calponin 2 in the Tg(fli1:EGFP)(y1) zebrafish line (in which a fli1 promoter drives vascular-specific enhanced green fluorescent protein expression) indicated that diminished calponin 2 expression blocked the proper migration of endothelial cells during formation of intersegmental vessels. In vitro studies showed that basic fibroblast growth factor-induced human umbilical vein endothelial cell migration was down-regulated by knockdown of calponin 2 expression using an antisense adenovirus, and overexpression of calponin 2 enhanced migration and hastened wound healing. These events were correlated with activation of mitogen-activated protein kinase; moreover, inhibition of this pathway blocked the promigratory effect of calponin 2. Collectively, these data suggest that calponin 2 plays an important role in the migration of endothelial cells both in vivo and in vitro and that its expression is critical for proper vascular development.

The Lmo2 transcription factor, a T-cell oncoprotein, is required for both hematopoiesis and angiogenesis. To investigate the fate of lmo2-expressing cells and the transcriptional regulation of lmo2 in vivo, we generated stable transgenic zebrafish that express green fluorescent protein (EGFP) or DsRed under the control of an lmo2 promoter. A 2.5-kb fragment contains the cis-regulatory elements required to recapitulate endogenous lmo2 expression in embryonic hematopoietic and vascular tissues. We further characterized embryonic Lmo2+ cells through transplantation into vlad tepes (vlt), an erythropoietic mutant. These Lmo2+ primitive wave donor cells differentiated into circulating hematopoietic cells and extended the life span of vlt recipients, but did not demonstrate long-term repopulation of the erythroid lineage. Promoter analysis identified a 174-bp proximal promoter that was sufficient to recapitulate lmo2 expression. This element contains critical ETS-binding sites conserved between zebrafish and pufferfish. Furthermore, we show that ets1 is coexpressed with lmo2, and overexpression experiments indicate that ets1 can activate the lmo2 promoter through this element. Our studies elucidate the transcriptional regulation of this key transcription factor, and provide a transgenic system for the functional analysis of blood and blood vessels in zebrafish.

Stem cells are defined by their capacity for self-renewal and multilineage differentiation, making them uniquely situated to treat a broad spectrum of human diseases. For example, because hematopoietic stem cells can reconstitute the entire blood system, bone marrow transplantation has long been used in the clinic to treat various diseases. Similarly, the transplantation of other tissue-specific stem cells, such as stem cells isolated from epithelial and neural tissues, can treat mouse disease models and human patients in which epithelial and neural cells are damaged. An alternative to tissue-specific stem cell therapy takes advantage of embryonic stem cells, which are capable of differentiating into any tissue type. Furthermore, nuclear transfer, the transfer of a post-mitotic somatic cell nucleus into an enucleated oocyte, creates a limitless source of autologous cells that, when combined with gene therapy, can serve as a powerful therapeutic tool.

Two distinct forms of zebrafish hepatocyte nuclear factor 1 (hnf1) were identified and referred to as hnf1alpha/tcf1 and hnf1beta/tcf2. Both hnf1 genes were shown to be expressed abundantly in liver, pancreas, gut and kidney. Zebrafish HNF1alpha and HNF1beta proteins contain all HNF1 signature domains including the dimerization domain, POU-like domain and atypical homeodomain. Sequence and phylogenetic analysis reveals that zebrafish hnf1alpha is closer to tetrapodian hnf1alpha than to tetrapodian hnf1beta and zebrafish hnf1beta is highly conserved with tetrapodian hnf1beta. Existences of hnf1alpha and hnf1beta in teleost zebrafish, tilapia and fugu suggest that hnf1 gene duplication might occur before the divergence of teleost and tetrapod ancestors. Zebrafish hnf1alpha and hnf1beta genes were mapped to linkage group LG8 and LG15 in T51 panel by RH mapping and are composed of 10 and 9 exons, respectively. Zebrafish hnf1beta gene with at least 11 genes in LG15 was identified to maintain the conserved synteny with those of human in chromosome 17 and those of mouse in chromosome 11. Our results indicate that distinct hnf1alpha and hnf1beta genes in teleosts had been evolved from the hnf1 ancestor gene of chordate.

Previously, we described the isolation and characterization of the first zebrafish neuropilin gene, which we now call nrp1a, and found its protein to be a mediator of vascular endothelial growth factor (VEGF)-dependent angiogenesis [Proc. Natl Acad. Sci. USA 99 (2002) 10470]. Subsequently, we have isolated three other full-length neuropilin genes (nrp1b, nrp2a, and nrp2b) and find that they map to independent zebrafish linkage groups. The nrp1s and nrp2s had differential spatio-temporal gene expression profiles with nrp1a being most prominent in the gut, brain, retina, hypochord, motorneurons, fin bud and mandibular cartilage, nrp1b in the brain, dorsal aorta, melanophores, ventral fin, and heart, nrp2a in the brain, retina, heart, and caudal vessels, and nrp2b in the brain, retina, gut, fin bud, melanophores, heart, and caudal vessels. In addition, we have identified an alternatively-spliced transcript of the nrp1b gene (denoted as nrp1b(s)) which is predicted to encode a soluble form of Nrp1b, containing only the a, b, and c extracellular domains. Transcript expression of nrp1b(s) was different from full-length nrp1b transcript, with prominence in the brain, developing mouth, heart, and fin bud. The NRP1s were tested for VEGF-binding ability. Both 125 kDa Nrp1a and 145 kDa Nrp1b bound 125I-labelled VEGFA165. In summary, two nrp1 and two nrp2 genes, with expression patterns similar to higher vertebrates, have been isolated from zebrafish.

For decades immunologists have relied heavily on the mouse model for their experimental designs. With the realization of the important role innate immunity plays in orchestrating immune responses, invertebrates such as worms and flies have been added to the repertoire. Here, we discuss the advent of the zebrafish as a powerful vertebrate model organism that promises to positively impact immunologic research.

Hemoglobin switching is a complex process by which distinct globin chains are produced during stages of development. In an effort to characterize the process of hemoglobin switching in the zebrafish model system, we have isolated and characterized several embryonic globin genes. The embryonic and adult globin genes are found in clusters in a head-to-head configuration. One cluster of embryonic and adult genes is localized to linkage group 3, whereas another embryonic cluster is localized on linkage group 12. Several embryonic globin genes demonstrate an erythroid-specific pattern of expression early during embryogenesis and later are downregulated as definitive hematopoiesis occurs. We utilized electrospray mass spectroscopy to correlate globin genes and protein expression in developing embryonic red cells. The mutation, zinfandel, has a hypochromic microcytic anemia as an embryo, but later recovers in adulthood. The zinfandel gene maps to linkage group 3 near the major globin gene locus, strongly suggesting that zinfandel represents an embryonic globin defect. Our studies are the first to systematically evaluate the embryonic globins in the zebrafish and will ultimately be useful in evaluating zebrafish mutants with defects in hemoglobin production and switching. © 2003 Elsevier Science (USA). All rights reserved.

The GC box binding transcription factor Sp3 both activates and represses transcription. We have found that Sp3 activity is regulated by SUMO-1 modification. Endogenous Sp3 is sumoylated and localized to the nuclear periphery and in nuclear dots. Removal of SUMO-1 from Sp3 by mutation of the SUMO acceptor lysines or expression of the SUMO-1 protease SuPr-1 converted Sp3 to a strong activator with a diffuse nuclear localization. Covalent attachment of SUMO-1 to Sp3 by gene fusion was sufficient to repress Sp3-dependent transcription and relocalize Sp3 to the nuclear periphery and nuclear dots. These studies reveal a direct effect of SUMO-1 modification on activity of a dual function transcription factor and provide a mechanism for functional specificity within the Sp transcription factor family.

In this issue of Cell, a study by N. Cho and coworkers provides insight into the role of vascular endothelial growth factor (VEGF) signaling in Drosophila hematopoiesis. Their work suggests that an ancestral function of VEGF was to guide blood cell migration and highlights the conservation of at least one aspect of VEGF signaling during evolution.

The specification of the erythroid lineage from hematopoietic stem cells requires the expression and activity of lineage-specific transcription factors. One transcription factor family that has several members involved in hematopoiesis is the Krüppel-like factor (KLF) family [1]. For example, erythroid KLF (EKLF) regulates β-globin expression during erythroid differentiation [2–6]. KLFs share a highly conserved zinc finger-based DNA binding domain (DBD) that mediates binding to CACCC-box and GC-rich sites, both of which are frequently found in the promoters of hematopoietic genes. Here, we identified a novel Xenopus KLF gene, neptune, which is highly expressed in the ventral blood island (VBI), cranial ganglia, and hatching and cement glands. neptune expression is induced in response to components of the BMP-4 signaling pathway in injected animal cap explants. Similar to its family member, EKLF, Neptune can bind CACCC-box and GC-rich DNA elements. We show that Neptune cooperates with the hematopoietic transcription factor XGATA-1 to enhance globin induction in animal cap explants. A fusion protein comprised of Neptune's DBD and the Drosophila engrailed repressor domain suppresses the induction of globin in ventral marginal zones and in animal caps. These studies demonstrate that Neptune is a positive regulator of primitive erythropoiesis in Xenopus.